Thioredoxins [, , , ]are small disulphide-containing redox proteins that have been found in all the kingdoms of living organisms. Thioredoxin serves as a general protein disulphide oxidoreductase. It interacts with a broad range of proteins by a redox mechanism based on reversible oxidation of two cysteine thiol groups to a disulphide, accompanied by the transfer of two electrons and two protons. The net result is the covalent interconversion of a disulphide and a dithiol. In the NADPH-dependent protein disulphide reduction, thioredoxin reductase (TR) catalyses the reduction of oxidised thioredoxin (trx) by NADPH using FAD and its redox-active disulphide; reduced thioredoxin then directly reduces the disulphide in the substrate protein [].Thioredoxin is present in prokaryotes and eukaryotes and the sequence around the redox-active disulphide bond is well conserved. All thioredoxins contain a cis-proline located in a loop preceding β-strand 4, which makes contact with the active site cysteines, and is important for stability and function []. Thioredoxin belongs to a structural family that includes glutaredoxin, glutathione peroxidase, bacterial protein disulphide isomerase DsbA, and the N-terminal domain of glutathione transferase []. Thioredoxins have a beta-alpha unit preceding the motif common to all these proteins.A number of eukaryotic proteins contain domains evolutionary related to thioredoxin, most of them are protein disulphide isomerases (PDI). PDI () [, , ]is an endoplasmic reticulum multi-functional enzyme that catalyses the formation and rearrangement of disulphide bonds during protein folding []. All PDI contains two or three (ERp72) copies of the thioredoxin domain, each of which contributes to disulphide isomerase activity, but which are functionally non-equivalent []. Moreover, PDI exhibits chaperone-like activity towards proteins that contain no disulphide bonds, i.e. behaving independently of its disulphide isomerase activity []. The various forms of PDI which are currently known are:PDI major isozyme; a multifunctional protein that also function as the beta subunit of prolyl 4-hydroxylase (), as a component of oligosaccharyl transferase (), as thyroxine deiodinase (), as glutathione-insulin transhydrogenase () and as a thyroid hormone-binding proteinERp60 (ER-60; 58 Kd microsomal protein). ERp60 was originally thought to be a phosphoinositide-specific phospholipase C isozyme and later to be a protease.ERp72.ERp5.Bacterial proteins that act as thiol:disulphide interchange proteins that allows disulphide bond formation in some periplasmic proteins also contain a thioredoxin domain. These proteins include:Escherichia coli DsbA (or PrfA) and its orthologs in Vibrio cholerae (TtcpG) and Haemophilus influenzae (Por).E. coli DsbC (or XpRA) and its orthologues in Erwinia chrysanthemi and H. influenzae.E. coli DsbD (or DipZ) and its H. influenzae orthologue.E. coli DsbE (or CcmG) and orthologues in H. influenzae.Rhodobacter capsulatus (Rhodopseudomonas capsulata) (HelX), Rhiziobiacae (CycY and TlpA).This entry represents a conserved site found in the thioredoxin domain. This site contains two cysteines that form the redox-active disulphide bond.
The CRISPR-Cas system is a prokaryotic defense mechanism against foreign genetic elements. The key elements of this defense system are the Cas proteins and the CRISPR RNA. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are a family of DNA direct repeats separated by regularly sized non-repetitive spacer sequences that are found in most bacterial and archaeal genomes []. CRISPRs appear to provide acquired resistance against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain sequences complementary to antecedent mobile elements and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA).The defense reaction is divided into three stages. In the adaptation stage, the invader DNA is cleaved, and a piece of it is selected to be integrated as a new spacer into the CRISPR locus, where it is stored as an identity tag for future attacks by this invader. During the second stage (the expression stage), the CRISPR RNA (pre-crRNA) is transcribed and subsequently processed into the mature crRNAs. In the third stage (the interference stage), Cas proteins, together with crRNAs, identify and degrade the invader [, , ].The CRISPR-Cas systems have been sorted into three major classes. In CRISPR-Cas types I and III, the mature crRNA is generally generated by a member of the Cas6 protein family. Whereas in system III the Cas6 protein acts alone, in some class I systems it is part of a complex of Cas proteins known as Cascade (CRISPR-associated complex for antiviral defense). The Cas6 protein is an endoribonuclease necessary for crRNA production whereas the additional Cas proteins that form the Cascade complex are needed for crRNA stability []. This entry represents a shared N-terminal region, of about 43 amino acids in length, found in a number of Cas proteins. This region is widely distributed and was designated Cas5. Proteins containing this region are generally 210 to 265 amino acids in length and show little or no homology between their C-terminal regions. The best characterised protein in this entry is DevS () from Myxococcus xanthus, a Cas protein that appears to participate in a species-specific developmental pathway. Cas5 is found within a cluster of three cas genes associated with CRISPR structures in many bacterial species, named cas1B, cas5 and cas6 [].
G protein-coupled receptors (GPCRs) constitute a vast protein family that encompasses a wide range of functions, including various autocrine, paracrine and endocrine processes. They show considerable diversity at the sequence level, on the basis of which they can be separated into distinct groups []. The term clan can be used to describe the GPCRs, as they embrace a group of families for which there are indications of evolutionary relationship, but between which there is no statistically significant similarity in sequence []. The currently known clan members include rhodopsin-like GPCRs (Class A, GPCRA), secretin-like GPCRs (Class B, GPCRB), metabotropic glutamate receptor family (Class C, GPCRC), fungal mating pheromone receptors (Class D, GPCRD), cAMP receptors (Class E, GPCRE) and frizzled/smoothened (Class F, GPCRF) [, , , , ]. GPCRs are major drug targets, and are consequently the subject of considerable research interest. It has been reported that the repertoire of GPCRs for endogenous ligands consists of approximately 400 receptors in humans and mice []. Most GPCRs are identified on the basis of their DNA sequences, rather than the ligand they bind, those that are unmatched to known natural ligands are designated by as orphan GPCRs, or unclassified GPCRs [].The rhodopsin-like GPCRs (GPCRA) represent a widespread protein family that includes hormone, neurotransmitter and light receptors, all of which transduce extracellular signals through interaction with guanine nucleotide-binding (G) proteins. Although their activating ligands vary widely in structure and character, the amino acid sequences of the receptors are very similar and are believed to adopt a common structural framework comprising 7 transmembrane (TM) helices [, , ].Thrombin is a coagulation protease that activates platelets, leukocytes, endothelial and mesenchymal cells at sites of vascular injury, acting partlythrough an unusual proteolytically activated GPCR []. Gene knockout experiments have provided definitive evidence for a second thrombin receptorin mouse platelets and have suggested tissue-specific roles for differentthrombin receptors. Because the physiological agonist at the receptor wasoriginally unknown, it was provisionally named protease-activated receptor(PAR) []. At least 4 PAR subtypes have now been characterised. Thus, the thrombin and PAR receptors constitute a fledgling receptor family that shares a novel proteolytic activation mechanism [].The human thrombin receptor, designated protease-activated receptor 3 (PAR3),has been cloned and characterised []. PAR3 can mediate thrombin-triggered phosphoinositide hydrolysis and is expressed in a variety of tissues, including human bone marrow and mouse megakaryocytes, making it a candidate for the sought-after second platelet thrombin receptor. PAR3 provides a new tool for understanding thrombin signalling and a possible target for therapeutics designed selectively to block thrombotic, inflammatory andproliferative responses to thrombin.
The Ubiquitin Interacting Motif (UIM), or 'LALAL-motif', is a stretch of about 20 amino acid residues, which was first described in the 26S proteasome subunit PSD4/RPN-10 that is known to recognise ubiquitin [, ]. In addition, the UIM is found, often in tandem or triplet arrays, in a variety of proteins either involved in ubiquitination and ubiquitin metabolism, or known to interact with ubiquitin-like modifiers. Among the UIM proteins are two different subgroups of the UBP (ubiquitin carboxy-terminal hydrolase) family of deubiquitinating enzymes, one F-box protein, one family of HECT-containing ubiquitin-ligases (E3s) from plants, and several proteins containing ubiquitin-associated UBA and/or UBX domains []. In most of these proteins, the UIM occurs in multiple copies and in association with other domains such as UBA (), UBX (), ENTH, EH (), VHS (), SH3 (), HECT (), VWFA (), EF-hand calcium-binding, WD-40 (), F-box (), LIM (), protein kinase (), ankyrin (), PX (), phosphatidylinositol 3- and 4-kinase (), C2 (), OTU (), dnaJ (), RING-finger () or FYVE-finger (). UIMs have been shown to bind ubiquitin and to serve as a specific targeting signal important for monoubiquitination. Thus, UIMs may have several functions in ubiquitin metabolism each of which may require different numbers of UIMs [, , ]. The UIM is unlikely to form an independent folding domain. Instead, based on the spacing of the conserved residues, the motif probably forms a short α-helix that can be embedded into different protein folds []. Some proteins known to contain an UIM are listed below: Eukaryotic PSD4/RPN-10/S5, a multi-ubiquitin binding subunit of the 26S proteasome. Vertebrate Machado-Joseph disease protein 1 (Ataxin-3), which acts as a histone-binding protein that regulates transcription; defects in Ataxin-3 cause the neurodegenerative disorder Machado-Joseph disease (MJD).Vertebrate epsin and epsin2. Vertebrate hepatocyte growth factor-regulated tyrosine kinase substrate (HRS). Mammalian epidermal growth factor receptor substrate 15 (EPS15), which is involved in cell growth regulation. Mammalian epidermal growth factor receptor substrate EPS15R. Drosophila melanogaster (Fruit fly) liquid facets (lqf), an epsin. Yeast VPS27 vacuolar sorting protein, which is required for membrane traffic to the vacuole.
Wnt proteins constitute a large family of secreted molecules that are involved in intercellular signalling during development. The name derives from the first 2 members of the family to be discovered: int-1 (mouse) and wingless (Drosophila) []. It is now recognised that Wnt signalling controls many cell fate decisions in a variety of different organisms, including mammals []. Wnt signalling has been implicated in tumourigenesis, early mesodermal patterning of the embryo, morphogenesis of the brain and kidneys, regulation of mammary gland proliferation and Alzheimer's disease [, ].Wnt-mediated signalling is believed to proceed initially through binding to cell surface receptors of the frizzled family; the signal is subsequently transduced through several cytoplasmic components to B-catenin, which enters the nucleus and activates the transcription of several genes important indevelopment []. Several non-canonical Wnt signalling pathways have also been elucidated that act independently of B-catenin. Canonical and noncanonical Wnt signaling branches are highly interconnected, and cross-regulate each other [].Members of the Wnt gene family are defined by their sequence similarity to mouse Wnt-1 and Wingless in Drosophila. They encode proteins of ~350-400 residues in length, with orthologues identified in several, mostly vertebrate, species. Very little is known about the structure of Wnts as they are notoriously insoluble, but they share the following features characteristics of secretory proteins: a signal peptide, several potential N-glycosylation sites and 22 conserved cysteines []that are probably involved in disulphide bonds. The Wnt proteins seem to adhere to the plasma membrane of the secreting cells and are therefore likely to signal over only few cell diameters. Fifteen major Wnt gene families have been identified in vertebrates, with multiple subtypes within some classes.Wnt inhibitory factor-1 (WIF-1) is a secreted protein that binds to Wnt proteins and inhibits their activities. It was first identified as an EST from human retina; ortholgues have since been identified in mouse, rat, Xenopus and zebrafish. WIF-1 proteins comprise an N-terminal signal sequence, a family-specific WIF domain, five epidermal growth factor (EGF) repeats and a hydrophilic C terminus. The interaction of Wnt proteins with WIF-1 and other inhibitors, such as frizzled-related protein, is thought to fine tune their activity [].
Tubby, an autosomal recessive mutation, mapping to mouse chromosome 7, was recently found to be the result of a splicing defect in a novel gene with unknown function. This mutation maps to the tub gene [, ]. The mouse tubby mutation is the cause of maturity-onset obesity, insulin resistance and sensory deficits. By contrast with the rapid juvenile-onset weight gain seen in diabetes (db) and obese (ob) mice, obesity in tubby mice develops gradually, and strongly resembles the late-onset obesity observed in the human population. Excessive deposition of adipose tissue culminates in a two-fold increase of body weight. Tubby mice also suffer retinal degeneration and neurosensory hearing loss. The tripartite character of the tubby phenotype is highly similar to human obesity syndromes, such as Alstrom and Bardet-Biedl. Although these phenotypes indicate a vital role for tubby proteins, no biochemical function has yet been ascribed to any family member [], although it has been suggested that the phenotypic features of tubby mice may be the result of cellular apoptosis triggered by expression of the mutated tub gene. TUB is the founding-member of the tubby-like proteins, the TULPs. TULPs are found in multicellular organisms from both the plant and animal kingdoms. Ablation of members of this protein family cause disease phenotypes that are indicative of their importance in nervous-system function and development [].Mammalian TUB is a hydrophilic protein of ~500 residues. The N-terminal () portion of the protein is conserved neither in length nor sequence, but, in TUB, contains the nuclear localisation signal and may have transcriptional-activation activity. The C-terminal 250 residues are highly conserved. The C-terminal extremity contains a cysteine residue that might play an important role in the normal functioning of these proteins. The crystal structure of the C-terminal core domain from mouse tubby has been determined to 1.9A resolution. This domain is arranged as a 12-stranded, all anti-parallel, closed β-barrel that surrounds a central alpha helix, (which is at the extreme carboxyl terminus of the protein) that forms most of the hydrophobic core. Structural analyses suggest that TULPs constitute a unique family of bipartite transcription factors [].This superfamily represents the tubby C-terminal domain and the structurally related LURP1-like domain.
This entry represents the N-terminal PH domain of FGD1.In general, FGDs (including FGD1, FGD2, FGD3 and FGD4/Frabin) have a RhoGEF (DH) domain, followed by an N-terminal PH domain, a FYVE domain and a C-terminal PH domain. All FGDs are guanine nucleotide exchange factors that activates the Rho GTPase Cdc42, an important regulator of membrane trafficking. The RhoGEF domain is responsible for GEF catalytic activity, while the N-terminal PH domain is involved in intracellular targeting of the DH domain []. Mutations in the FGD1 gene are responsible for the X-linked disorder known as faciogenital dysplasia (FGDY) []. Both FGD1 and FGD3 are targeted by the ubiquitin ligase SCF(FWD1/beta-TrCP) upon phosphorylation of two serine residues in its DSGIDS motif and subsequently degraded by the proteasome. However, FGD1 and FGD3 induced significantly different morphological changes in HeLa Tet-Off cells and while FGD1 induced long finger-like protrusions, FGD3 induced broad sheet-like protrusions when the level of GTP-bound Cdc42 was significantly increased by the inducible expression of FGD3. They also reciprocally regulated cell motility in inducibly expressed in HeLa Tet-Off cells, FGD1 stimulated cell migration while FGD3 inhibited it. FGD1 and FGD3 therefore play different roles to regulate cellular functions, even though their intracellular levels are tightly controlled by the same destruction pathway through SCF(FWD1/beta-TrCP) [, ].PH domains have diverse functions, but in general are involved in targeting proteins to the appropriate cellular location or in the interaction with a binding partner []. They share little sequence conservation, but all have a common fold, which is electrostatically polarized. Less than 10% of PH domains bind phosphoinositide phosphates (PIPs) with high affinity and specificity []. PH domains are distinguished from other PIP-binding domains by their specific high-affinity binding to PIPs with two vicinal phosphate groups: PtdIns(3,4)P2, PtdIns(4,5)P2 or PtdIns(3,4,5)P3 which results in targeting some PH domain proteins to the plasma membrane []. A few display strong specificity in lipid binding. Any specificity is usually determined by loop regions or insertions in the N terminus of the domain, which are not conserved across all PH domains. PH domains are found in cellular signaling proteins such as serine/threonine kinase, tyrosine kinases, regulators of G-proteins, endocytotic GTPases, adaptors, as well as cytoskeletal associated molecules and in lipid associated enzymes [].
This entry represents the C-terminal PH domain of FGD1-4.In general, FGDs (including FGD1, FGD2, FGD3 and FGD4/Frabin) have a RhoGEF (DH) domain, followed by an N-terminal PH domain, a FYVE domain and a C-terminal PH domain. All FGDs are guanine nucleotide exchange factors that activates the Rho GTPase Cdc42, an important regulatorof membrane trafficking. The RhoGEF domain is responsible for GEF catalytic activity, while the N-terminal PH domain is involved in intracellular targeting of the DH domain []. Mutations in the FGD1 gene are responsible for the X-linked disorder known as faciogenital dysplasia (FGDY) []. Both FGD1 and FGD3 are targeted by the ubiquitin ligase SCF(FWD1/beta-TrCP) upon phosphorylation of two serine residues in its DSGIDS motif and subsequently degraded by the proteasome. However, FGD1 and FGD3 induced significantly different morphological changes in HeLa Tet-Off cells and while FGD1 induced long finger-like protrusions, FGD3 induced broad sheet-like protrusions when the level of GTP-bound Cdc42 was significantly increased by the inducible expression of FGD3. They also reciprocally regulated cell motility in inducibly expressed in HeLa Tet-Off cells, FGD1 stimulated cell migration while FGD3 inhibited it. FGD1 and FGD3 therefore play different roles to regulate cellular functions, even though their intracellular levels are tightly controlled by the same destruction pathway through SCF(FWD1/beta-TrCP) [, ].PH domains have diverse functions, but in general are involved in targeting proteins to the appropriate cellular location or in the interaction with a binding partner []. They share little sequence conservation, but all have a common fold, which is electrostatically polarized. Less than 10% of PH domains bind phosphoinositide phosphates (PIPs) with high affinity and specificity []. PH domains are distinguished from other PIP-binding domains by their specific high-affinity binding to PIPs with two vicinal phosphate groups: PtdIns(3,4)P2, PtdIns(4,5)P2 or PtdIns(3,4,5)P3 which results in targeting some PH domain proteins to the plasma membrane []. A few display strong specificity in lipid binding. Any specificity is usually determined by loop regions or insertions in the N terminus of the domain, which are not conserved across all PH domains. PH domains are found in cellular signaling proteins such as serine/threonine kinase, tyrosine kinases, regulators of G-proteins, endocytotic GTPases, adaptors, as well as cytoskeletal associated molecules and in lipid associated enzymes [].
The CRM domain is an ~100-amino acid RNA-binding domain. The name chloroplast RNA splicing and ribosome maturation (CRM) has been suggested to reflect the functions established for the four characterised members of the family: Zea mays (Maize) CRS1 (), CAF1 () and CAF2 () proteins and the Escherichia coli protein YhbY (). The CRM domain is found in eubacteria, archaea, and plants. The CRM domain is represented as a stand-alone protein in archaea and bacteria, and in single- and multi-domain proteins in plants. It has been suggested that prokaryotic CRM proteins existed as ribosome-associated proteins prior to the divergence of archaea and bacteria, and that they were co-opted in the plant lineage as RNA binding modules by incorporation into diverse protein contexts. Plant CRM domains are predicted to reside not only in the chloroplast, but also in the mitochondrion and the nucleo/cytoplasmic compartment. The diversity of the CRM domain family in plants suggests a diverse set of RNA targets [, ].The CRM domain is a compact alpha/beta domain consisting of a four-stranded beta sheet and three alpha helices with an α-β-α-β-α-β-beta topology. The beta sheet face is basic, consistent with a role in RNA binding. Proximal to the basic beta sheet face is another moiety that could contribute to nucleic acid recognition. Connecting strand beta1 and helix alpha2 is a loop with a six amino acid motif, GxxG flanked by large aliphatic residues, within which one 'x' is typically a basic residue []. Escherichia coli YhbY is associated with pre-50S ribosomal subunits, which implies a function in ribosome assembly. GFP fused to a single-domain CRM protein from maize localises to the nucleolus, suggesting that an analogous activity may have been retained in plants []. A CRM domain containing protein in plant chloroplasts has been shown to function in group I and II intron splicing []. In vitro experiments with an isolated maize CRM domain have shown it to have RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes []. YhbY has a fold similar to that of the C-terminal domain of translation initiation factor 3 (IF3C), which binds to 16S rRNA in the 30S ribosome [].
This entry represents the DOT1 domain.The Dot1 protein (Dot1p) is an histone-lysine N-methyltransferase (EC2.1.1.43) that methylates lysine 79 (Lys-79) of histone H3. It was firstidentified as a Disruptor Of Telomeric silencing in yeast where Dot1p isimplicated in gene silencing and localization of the Silent InformationRegulator (SIR) complex; in higher eukaryotes the methylation carried out bythis enzyme may be used for differentiating chromatin domains. Unlike otherhistone-lysine methyltransferases (HKMTs), Dot1p displays a Rossmann-like(Class I) S-adenosyl-L-methyionine (SAM)-dependent MT foldwhile other HKMTs contain the SET domain and hence belong toa whole different structural class [, ].Whereas most HKMTs, such as Suvar3-9 methylate Lys on the N-terminal tails ofhistones that stick out from the nucleosome, Dot1p substrate (Lys-79 ofhistone H3) is located in the conserved histone core, in a short turnconnecting the first and second helices, exposed on the nucleosome disksurface [, ]. In order for Lys-79 of H3 to be methylated by Dot1p, anotherlysine, Lys-123 of histone H2B, needs to be ubiquitinated. A possible reasonput forward for this requirement is that the ubiquitination may create a spacebetween adjacent nucleosomes, permitting access of Dot1p to its substrate[, ]. In yeast, different states of methylation on Lys-79 of histone H3(unmodified, mono-, di- and trimethylated) co-exist at the same time, but noclear function is associated with these different methylation states [].The strucure of the evolutionary conserved core of Dot1p, the DOT1 domain, hasfirst been described for the yeast Dot1p in complex withS-adenosyl-L-homocysteine (AdoHcy) and then for the humanDot1-like protein (Dot1Lp) in complex with SAM. The DOT1domain is about 300-350 amino acids long and is usually located at either ofthe extremities of the protein sequence: it stands at the C terminus of theyeast Dot1p and at the N terminus of the human Dot1Lp [, ]. DOT1 displays arather elongated structure and can be subdivided into two parts: the N- andthe C-terminal subdomains []. The N-terminal part is made up of five alphahelices and two pairs of short beta strand hairpins. The C-terminal partdisplays a Rossmann-like fold: it consists in aseven-stranded beta sheet tucked by five alpha helices (three helices on oneside of the sheet and two on the other), the sheet contains a centraltopological switchpoint resulting in a deep pocket where SAM is bound. The twosubdomains are linked covalently by a loop. Altogether the SAM binding pocketis formed by five segments of the DOT1 domain of which four are located in theC-terminal substructure of the DOT1 domain and one in the loop connecting bothparts; two of these segments are conserved across different Class ISAM-dependent MTs [].
This superfamily is composed of the Transcription factor IIS (TFIIS) and the Lens epithelium-derived growth factor (LEDGF) domains.Transcription factor IIS (TFIIS) is a transcription elongation factor that increases the overall transcription rate of RNA polymerase II by reactivating transcription elongation complexes that have arrested transcription. The three structural domains of TFIIS are conserved from yeast to human. The 80 or so N-terminal residues form a protein interaction domain containing a conserved motif, which has been called the LW motif because of the invariant leucine and tryptophan residues it contains. This N-terminal domain is not required for transcriptional activity, and while a similar sequence has been identified in other transcription factors, and proteins that are predominantly nuclear localized [, ], the domain is also found in proteins not directly involved in transcription. This domain is found in (amongst others):MED26 (also known as CRSP70 and ARC70), a subunit of the Mediator complex, which is required for the activity of the enhancer-binding protein Sp1. Elongin A, a subunit of a transcription elongation factor previously known as SIII. It increases the rate of transcription by suppressing transient pausing of the elongation complex. PPP1R10, a nuclear regulatory subunit of protein phosphatase 1 that was previously known as p99, FB19 or PNUTS. IWS1, which is thought to function in both transcription initiation and elongation. The TFIIS N-terminal domain is a compact four-helix bundle. The hydrophobic core residues of helices 2, 3, and 4 are well conserved among TFIIS domains, although helix 1 is less conserved []. Lens epithelium-derived growth factor (LEDGF), also known as transcriptional co-activator p75, is a chromatin-associated protein that protects cells from stress-induced apoptosis. It is the binding partner of HIV-1 integrase in human cells []. The integrase binding domain (IBD) of LEDGF is a compact right-handed bundle composed of five α-helices. The residues essential for the interaction with the integrase are present in the inter-helical loop regions of the bundle structure. The integrase binding domain is not unique to LEDGF, as a second human protein, hepatoma-derived growth factor-related protein 2 (HRP2), contains a homologous sequence [].
Major histocompatibility complex (MHC) class I molecules present antigenic peptides to CD8 T cells. The majority of peptides found associated with class I molecules are derived from nuclear and cytosolic proteins, and they are generated largely by the proteasome complex. These peptides are transported from cytosol into the lumen of the endoplasmic reticulum (ER) by a peptide transporter, which is known as the transporter associated with antigen processing (TAP). TAP is a trimeric complex consisting of TAP1, TAP2 and tapasin (TAP-A). TAP1 and TAP2 are required for peptide transport. Tapasin, which actually serves as a docking site on the TAP complex specific for interaction with class I MHC molecules, is essential for peptide loading (up to four MHC class I-tapasin complexes have beenfound to bind to each TAP molecule). However, since the exact mechanisms oftapasin functions are still unknown, it has also been speculated thattapasin may regulate the MHC class I release from the ER rather than directly loading peptides onto MHC class I molecules [, , , ].In studies of the interaction between MHC class I and TAP, it was found that TAP1, but not TAP2, is required for the association of TAP with class I molecules. Because tapasin is essential for the association of MHC class I to TAP, tapasin may directly interact with TAP1. Thus the predicted order of interaction between different molecules in the TAP complex is TAP2 to TAP1, TAP1 to tapasin, and tapasin to MHC class I molecules. Thus, by these linked events, the translocation and loading of peptides rapidly and efficientlyproceed in the same microenvironment [, ].Tapasin is a type I transmembrane (TM) glycoprotein with a double lysinemotif that is thought to be involved with mediating the retrieval of proteins back from the cis-Golgi, thus maintaining membrane proteins in theER []. It is encoded by an MHC-linked gene and is a member of theimmunoglobulin superfamily. Binding to TAP is mediated by the C-terminalregion, whereas its N-terminal 50 residues constitute the key element thatconverts the MHC class I molecules and TAP weak interactions into a stablecomplex [, ].
Zinc finger (Znf) domains are relatively small protein motifs which contain multiple finger-like protrusions that make tandem contacts with their target molecule. Some of these domains bind zinc, but many do not; instead binding other metals such as iron, or no metal at all. For example, some family members form salt bridges to stabilise the finger-like folds. They were first identified as a DNA-binding motif in transcription factor TFIIIA from Xenopus laevis (African clawed frog), however they are now recognised to bind DNA, RNA, protein and/or lipid substrates [, , , , ]. Their binding properties depend on the amino acid sequence of the finger domains and of the linker between fingers, as well as on the higher-order structures and the number of fingers. Znf domains are often found in clusters, where fingers can have different binding specificities. There are many superfamilies of Znf motifs, varying in both sequence and structure. They display considerable versatility in binding modes, even between members of the same class (e.g. some bind DNA, others protein), suggesting that Znf motifs are stable scaffolds that have evolved specialised functions. For example, Znf-containing proteins function in gene transcription, translation, mRNA trafficking, cytoskeleton organisation, epithelial development, cell adhesion, protein folding, chromatin remodelling and zinc sensing, to name but a few []. Zinc-binding motifs are stable structures, and they rarely undergo conformational changes upon binding their target. This entry represents the AN1-type zinc finger domain, which has a dimetal (zinc)-bound alpha/beta fold. This domain was first identified as a zinc finger at the C terminus of AN1 , a ubiquitin-likeprotein in Xenopus laevis []. The AN1-type zinc finger contains six conserved cysteines and two histidines that could potentially coordinate 2 zinc atoms.Certain stress-associated proteins (SAP) contain AN1 domain, often in combination with A20 zinc finger domains (SAP8) or C2H2 domains (SAP16) []. For example, the human protein Znf216 has an A20 zinc-finger at the N terminus and an AN1 zinc-finger at the C terminus, acting to negatively regulate the NFkappaB activation pathway and to interact with components of the immune response like RIP, IKKgamma and TRAF6. The interact of Znf216 with IKK-gamma and RIP is mediated by the A20 zinc-finger domain, while its interaction with TRAF6 is mediated by the AN1 zinc-finger domain; therefore, both zinc-finger domains are involved in regulating the immune response []. The AN1 zinc finger domain is also found in proteins containing a ubiquitin-like domain, which are involved in the ubiquitination pathway []. Proteins containing an AN1-type zinc finger include:Ascidian posterior end mark 6 (pem-6) protein [].Human AWP1 protein (associated with PRK1), which is expressed during early embryogenesis [].Human immunoglobulin mu binding protein 2 (SMUBP-2), mutations in which cause muscular atrophy with respiratory distress type 1 [].
Nucleosides are hydrophilic molecules and require specialised transport proteins for permeation of cell membranes. There are two types of nucleoside transport processes: equilibrative bidirectional processes driven by chemical gradients, and inwardly directed concentrative processes driven by an electrochemical gradient []. The two types of nucleoside transporters are classified into two families: the solute carrier (SLC) 29 and SLC28 families, corresponding to equilibrative and concentrative nucleoside transporters, respectively [].The microbial proteins include broad specificity transporters, such as the Escherichia coli NupC protein which transports all nucleosides (both ribo- and deoxyribonucleosides) except hypoxanthine and guanine nucleosides []. Bacillus subtilis NupC transporter has been shown to be involved in transport of the pyrimidine nucleoside uridine []. A recently characterised fungal protein, the first transporter of this type to be described in eukaryotes, exhibited transport activity for adenosine, uridine, inosine and guanosine but not cytidine, thymidine or the nucleobase hypoxanthine [].The characterised mammalian proteins can be divided into three subgroups; CNT1, CNT2 and CNT3 []. CNT1 preferentially transports pyrimidines and weakly transports adenosine. Several antiviral and anticancer nucleoside analogues, including AZT and dFdC are also substrates for CNT1. CNT2 selectively transports purines, and the human form has also been shown to facilitate the uptake of some antiviral compounds including ddI and ribavirin. CNT3 has a broader specificity, transporting both purines and pyrimidines. Several anticancer nucleoside analogues such as CdA, dFdC and FdU are also transported by CNT3. Substrate specificity appears to depend on a region containing transmembrane regions 7, 8 and 9. Mutation of just four residues in this region was sufficient to convert the activity of human CNT1 to that of CNT2. At least three other concentrative nucleoside transport activities have been described in mammalian cells, but the proteins responsible for these activities have not yet been identified.This entry represents a family of Concentrative Nucleoside Transporter (CNT) proteins found in bacteria and eukaryotes. Most of the bacteria and fungi homologues identified are H+ symporters, while the mammalian members (CNT1/2/3) are mostly Na+ symporters. However, mammalian CNT3 exhibits uniquely broad cation interactions with Na+, H+, and Li+ and can couple transport of uridine to uptake of H(+) [, ].
The bacterial CyaB like adenylyl cyclase and the mammalian thiamine triphosphatases (ThTPases) define a superfamily of catalytic domains called the CYTH (CyaB, thiamine triphosphatase) domain that is present in all three superkingdoms of life []. Proteins containing this domain act on triphosphorylated substrates and require at least one divalent metal cation for catalysis []. The catalytic core of the CYTH domain is predicted to contain an alpha beta scaffold with 6 conserved β-strands and 6 conserved α-helices. The CYTH domains contains several nearly universally conserved charged residues that are likely to form the active site. The most prominent of these are an EXEXK motif associated with strand-1 of the domain, two basic residues in helix-2, a K at the end of strand 3, an E in strand 4, a basic residue in helix-4, a D at the end of strand 5 and two acidic residues (typically glutamates) in strand 6. The presence of around 6 conserved acidic positions in the majority of the CYTH domains suggests that it coordinates two divalent metal ions. Both CyaB and ThTPase have been shown to require Mg(2) ions for their nucleotide cyclase and phosphatase activities. The four conserved basic residues in the CYTH domain are most probably involved in the binding of acidic phosphate moieties of their substrates. The conservation of these two sets of residues in the majority of CYTH domains suggests that most members of this group are likely to possess an activity dependent on two metal ions, with a preferencefor nucleotides or related phosphate-moiety -bearing substrates. The proposed biochemical activity, and the arrangement of predicted strands in the primary structure of the CYTH domain imply that they may adopt a barrel or sandwich-like configuration, with metal ions and the substrates bound in the central cavity [].Protein containing a CYTH-like domain include the yeast RNA triphosphatase Cet1, whose active site is located within a topologically closed hydrophilic β-barrel (composed of 8 antiparallel beta strands) known as the "triphosphate tunnel"[]. The superfamily including Cet-1-like RNA triphosphatases and all other CYTH proteins has been named Triphosphate Tunnel Metalloenzyme (TTM) []. Interestingly, bacterial CYTH-domain containing proteins, such as NeuTTM () from Nitrosomonas europaea []and CthTTM () from Clostridium thermocellum [], both have a high PPPase activity (though CthTM was less specific) but neither had any significant adenylyl cyclase activity. However, other bacterial CYTH-domain containing proteins, such as CyaB from A. hydrophyla and ygiF from E. coli, have been shown to have PPPase activity, but this activity is lower than their adenylate cyclase activity []. It has also been suggested that adenylyl cyclase and thiamine triphosphatase are secondary derivatives of proteins that performed an ancient role in polyphosphate and nucleotide metabolism [].
Ca2+ ions are unique in that they not only carry charge but they are also the most widely used of diffusible second messengers. Voltage-dependent Ca2+ channels (VDCC) are a family of molecules that allow cells to couple electrical activity to intracellular Ca2+ signalling. The opening and closing of these channels by depolarizing stimuli, such as action potentials, allows Ca2+ ions to enter neurons down a steep electrochemical gradient, producing transient intracellular Ca2+ signals. Many of the processes that occur in neurons, including transmitter release, gene transcription and metabolism are controlled by Ca2+ influx occurring simultaneously at different cellular locales. The pore is formed by the alpha-1 subunit which incorporates the conduction pore, the voltage sensor and gating apparatus, and the known sites of channel regulation by second messengers, drugs, and toxins []. The activity of this pore is modulated by four tightly-coupled subunits: an intracellular beta subunit; a transmembrane gamma subunit; and a disulphide-linked complex of alpha-2 and delta subunits, which are proteolytically cleaved from the same gene product. Properties of the protein including gating voltage-dependence, G protein modulation and kinase susceptibility can be influenced by these subunits.Voltage-gated calcium channels are classified as T, L, N, P, Q and R, and are distinguished by their sensitivity to pharmacological blocks, single-channel conductance kinetics, and voltage-dependence. On the basis of their voltage activation properties, the voltage-gated calcium classes can be further divided into two broad groups: the low (T-type) and high (L, N, P, Q and R-type) threshold-activated channels.The voltage-dependent calcium channel gamma (VDCCG) subunit family consistsof at least 8 members, which share a number of common structural features[]. Each member is predicted to possess 4 transmembrane domains, with intracellular N- and C-termini. The first extracellular loop contains a highly conserved N-glycosylation site and a pair of conserved cysteine residues. The C-terminal 7 residues of VDCCG-2, -3, -4 and -8 are also conserved andcontain a consensus site for phosphorylation by cAMP and cGMP-dependentprotein kinases, and a target site for binding by PDZ domain proteins [].The VDCCG-5 subunit was identified by genomic database searching, pursuingsequences similar to VDCCG-1 and -2. Mouse, human and rat isoforms havebeen cloned. VDCCG-5 is expressed in a range of tissues, including brain,kidney and testis [].
Ca2+ ions are unique in that they not only carry charge but they are also the most widely used of diffusible second messengers. Voltage-dependent Ca2+ channels (VDCC) are a family of molecules that allow cells to couple electrical activity to intracellular Ca2+ signalling. The opening and closing of these channels by depolarizing stimuli, such as action potentials, allows Ca2+ ions to enter neurons down a steep electrochemical gradient, producing transient intracellular Ca2+ signals. Many of the processes that occur in neurons, including transmitter release, gene transcription and metabolism are controlled by Ca2+ influx occurring simultaneously at different cellular locales. The pore is formed by the alpha-1 subunit which incorporates the conduction pore, the voltage sensor and gating apparatus, and the known sites of channel regulation by second messengers, drugs, and toxins []. The activity of this pore is modulated by four tightly-coupled subunits: an intracellular beta subunit; a transmembrane gamma subunit; and a disulphide-linked complex of alpha-2 and delta subunits, which are proteolytically cleaved from the same gene product. Properties of the protein including gating voltage-dependence, G protein modulation and kinase susceptibility can be influenced by these subunits.Voltage-gated calcium channels are classified as T, L, N, P, Q and R, and are distinguished by their sensitivity to pharmacological blocks, single-channel conductance kinetics, and voltage-dependence. On the basis of their voltage activation properties, the voltage-gated calcium classes can be further divided into two broad groups: the low (T-type) and high (L, N, P, Q and R-type) threshold-activated channels.The voltage-dependent calciumchannel gamma (VDCCG) subunit family consistsof at least 8 members, which share a number of common structural features[]. Each member is predicted to possess 4 transmembrane domains, with intracellular N- and C-termini. The first extracellular loop contains a highly conserved N-glycosylation site and a pair of conserved cysteine residues. The C-terminal 7 residues of VDCCG-2, -3, -4 and -8 are also conserved andcontain a consensus site for phosphorylation by cAMP and cGMP-dependentprotein kinases, and a target site for binding by PDZ domain proteins [].The VDCCG-6 subunit was identified by high throughput genomic sequencedatabase searching, pursuing sequences similar to VDCCG-1 to -5 [].Mouse, human and rat isoforms have been cloned. VDCCG-6 is expressed in arange of tissues including brain, kidney, lung, skeletal muscle, prostateand testis [].
Ca2+ ions are unique in that they not only carry charge but they are also the most widely used of diffusible second messengers. Voltage-dependent Ca2+ channels (VDCC) are a family of molecules that allow cells to couple electrical activity to intracellular Ca2+ signalling. The opening and closing of these channels by depolarizing stimuli, such as action potentials, allows Ca2+ ions to enter neurons down a steep electrochemical gradient, producing transient intracellular Ca2+ signals. Many of the processes that occur in neurons, including transmitter release, gene transcription and metabolism are controlled by Ca2+ influx occurring simultaneously at different cellular locales. The pore is formed by the alpha-1 subunit which incorporates the conduction pore, the voltage sensor and gating apparatus, and the known sites of channel regulation by second messengers, drugs, and toxins []. The activity of this pore is modulated by four tightly-coupled subunits: an intracellular beta subunit; a transmembrane gamma subunit; and a disulphide-linked complex of alpha-2 and delta subunits, which are proteolytically cleaved from the same gene product. Properties of the protein including gating voltage-dependence, G protein modulation and kinase susceptibility can be influenced by these subunits.Voltage-gated calcium channels are classified as T, L, N, P, Q and R, and are distinguished by their sensitivity to pharmacological blocks, single-channel conductance kinetics, and voltage-dependence. On the basis of their voltage activation properties, the voltage-gated calcium classes can be further divided into two broad groups: the low (T-type) and high (L, N, P, Q and R-type) threshold-activated channels.The voltage-dependent calcium channel gamma (VDCCG) subunit family consistsof at least 8 members, which share a number of common structural features[]. Each member is predicted to possess 4 transmembrane domains, with intracellular N- and C-termini. The first extracellular loop contains a highly conserved N-glycosylation site and a pair of conserved cysteine residues. The C-terminal 7 residues of VDCCG-2, -3, -4 and -8 are also conserved andcontain a consensus site for phosphorylation by cAMP and cGMP-dependentprotein kinases, and a target site for binding by PDZ domain proteins [].The VDCCG-7 subunit was identified by high throughput genomic sequencedatabase searching, pursuing sequences similar to VDCCG-1 to -5.Mouse and human isofroms have been cloned. VDCCG-7 is expressed in a rangeof tissues including brain, kidney, liver, small intestine and testis [].
Ca2+ ions are unique in that they not only carry charge but they are also the most widely used of diffusible second messengers. Voltage-dependent Ca2+ channels (VDCC) are a family of molecules that allow cells to couple electrical activity to intracellular Ca2+ signalling. The opening and closing of these channels by depolarizing stimuli, such as action potentials, allows Ca2+ ions to enter neurons down a steep electrochemical gradient, producing transient intracellular Ca2+ signals. Many of the processes that occur in neurons, including transmitter release, gene transcription and metabolism are controlled by Ca2+ influx occurring simultaneously at different cellular locales. The pore is formed by the alpha-1 subunit which incorporates the conduction pore, the voltage sensor and gating apparatus, and the known sites of channel regulation by second messengers, drugs, and toxins []. The activity of this pore is modulated by four tightly-coupled subunits: an intracellular beta subunit; a transmembrane gamma subunit; and a disulphide-linked complex of alpha-2 and delta subunits, which are proteolytically cleaved from the same gene product. Properties of the protein including gating voltage-dependence, G protein modulation and kinase susceptibility can be influenced by these subunits.Voltage-gated calcium channels are classifiedas T, L, N, P, Q and R, and are distinguished by their sensitivity to pharmacological blocks, single-channel conductance kinetics, and voltage-dependence. On the basis of their voltage activation properties, the voltage-gated calcium classes can be further divided into two broad groups: the low (T-type) and high (L, N, P, Q and R-type) threshold-activated channels.The voltage-dependent calcium channel gamma (VDCCG) subunit family consistsof at least 8 members, which share a number of common structural features[]. Each member is predicted to possess 4 transmembrane domains, with intracellular N- and C-termini. The first extracellular loop contains a highly conserved N-glycosylation site and a pair of conserved cysteine residues. The C-terminal 7 residues of VDCCG-2, -3, -4 and -8 are also conserved andcontain a consensus site for phosphorylation by cAMP and cGMP-dependentprotein kinases, and a target site for binding by PDZ domain proteins [].The VDCCG-8 subunit was identified by high throughput genomic sequencedatabase searching, pursuing sequences similar to VDCCG-1 to -5 [].Mouse and rat isoforms have been cloned. VDCCG-8 mRNA is expressed in thebrain and testis.
Transient receptor potential (TRP) channels can be described as tetramers formed by subunits with six transmembrane domains and containing cation-selective pores, which in several cases show high calcium permeability. The molecular architecture of TRP channels is reminiscent of voltage-gated channels and comprises six putative transmembrane segments (S1-S6), intracellular N- and C-termini, and a pore-forming reentrant loop between S5 and S6 [].TRP channels represent a superfamily conserved from worms to humans that comprise seven subfamilies []: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin or long TRPs), TRPA (ankyrin, whose only member is Transient receptor potential cation channel subfamily A member 1, TrpA1), TRPP (polycystin), TRPML (mucolipin) and TRPN (Nomp-C homologues), which has a single member that can be found in worms, flies, and zebrafish. TRPs are classified essentially according to their primary amino acid sequence rather than selectivity or ligand affinity, due to their heterogeneous properties and complex regulation.TRP channels are involved in many physiological functions, ranging from pure sensory functions, such as pheromone signalling, taste transduction, nociception, and temperature sensation, over homeostatic functions, such as Ca2+ and Mg2+ reabsorption and osmoregulation, to many other motile functions, such as muscle contraction and vaso-motor control [].The TRPV (vanilloid) subfamily can be divided into two distinct groups. The first, which comprises TrpV1, TrpV2, TrpV3, and TrpV4, with nonselective cation conducting pores, has memberswhich can be activated by temperature as well as chemical stimuli. They are involved in a range of functions including nociception, thermosensing and osmolarity sensing. The second group, which consists of TrpV5 and TrpV6, (also known as epithelial calcium channels 1 and 2), highly calcium selective, are involved in renal Ca2+ absorption/reabsorption [, ].TrpV6 was originally cloned from rabbit kidney cells, but has also been found in human. It is a calcium selective cation channel that mediates Ca2+ uptake in various tissues, including the intestine and epithelial tissues. [, , , , ]. TrpV6 has been related to a variety of diseases [, ]. Cryo-electron microscopy images of the open and closed states of this channel showed it adopts similar conformations in both states [].
Transient receptor potential (TRP) channels can be described as tetramers formed by subunits with six transmembrane domains and containing cation-selective pores, which in several cases show high calcium permeability. The molecular architecture of TRP channels is reminiscent of voltage-gated channels and comprises six putative transmembrane segments (S1-S6), intracellular N- and C-termini, and a pore-forming reentrant loop between S5 and S6 [].TRP channels represent a superfamily conserved from worms to humans that comprise seven subfamilies []: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin or long TRPs), TRPA (ankyrin, whose only member is Transient receptor potential cation channel subfamily A member 1, TrpA1), TRPP (polycystin), TRPML (mucolipin) and TRPN (Nomp-C homologues), which has a single member that can be found in worms, flies, and zebrafish. TRPs are classified essentially according to their primary amino acid sequence rather than selectivityor ligand affinity, due to their heterogeneous properties and complex regulation.TRP channels are involved in many physiological functions, ranging from pure sensory functions, such as pheromone signalling, taste transduction, nociception, and temperature sensation, over homeostatic functions, such as Ca2+ and Mg2+ reabsorption and osmoregulation, to many other motile functions, such as muscle contraction and vaso-motor control [].The TRPV (vanilloid) subfamily can be divided into two distinct groups. The first, which comprises TrpV1, TrpV2, TrpV3, and TrpV4, with nonselective cation conducting pores, has members which can be activated by temperature as well as chemical stimuli. They are involved in a range of functions including nociception, thermosensing and osmolarity sensing. The second group, which consists of TrpV5 and TrpV6, (also known as epithelial calcium channels 1 and 2), highly calcium selective, are involved in renal Ca2+ absorption/reabsorption [, ].TRPV5 was first isolated from rat duodenum using an expression cloning system and, subsequently, from human small intestine []. Its functionalrole is similar to TRPV6, being involved in response to a reduction incalcium. However, as its tissue distribution differs, the consequences ofmalfunction are likely to be different. TRPV5 appears to form the Icrac ionchannel, which has a pivotal role in maintenance and regulation of calcium.This means that it is implicated in processes as diverse as exocytosis,enzyme regulation, apoptosis and cell proliferation. More specifically,the classic descriptions of Icrac in T-cells predict that antagonists tothis channel should be useful in treating inflammatory diseases and inimmunomodulation. It is also potentially involved in cell proliferation,and may be linked to human cancer [].
Tubby, an autosomal recessive mutation, mapping to mouse chromosome 7, was recently found to be the result of a splicing defect in a novel gene with unknown function. This mutation maps to the tub gene [, ]. The mouse tubby mutation is the cause of maturity-onset obesity, insulin resistance and sensory deficits. By contrast with the rapid juvenile-onset weight gain seen in diabetes (db) and obese (ob) mice, obesity in tubby mice develops gradually, and strongly resembles the late-onset obesity observed in the human population. Excessive deposition of adipose tissue culminates in a two-fold increase of body weight. Tubby mice also suffer retinal degeneration and neurosensory hearing loss. The tripartite character of the tubby phenotype is highly similar to human obesity syndromes, such as Alstrom and Bardet-Biedl. Although these phenotypes indicate a vital role for tubby proteins, no biochemical function has yet been ascribed to any family member [], although it has been suggested that the phenotypic features of tubby mice may be the result of cellular apoptosis triggered by expression of the mutated tub gene. TUB is the founding-member of the tubby-like proteins, the TULPs. TULPs are found in multicellular organisms from both the plant and animal kingdoms. Ablation of members of this protein family cause disease phenotypes that are indicative of their importance in nervous-system function and development [].Mammalian TUB is a hydrophilic protein of ~500 residues. The N-terminal () portion of the protein is conserved neither in length nor sequence, but, in TUB, contains the nuclear localisation signal and may have transcriptional-activation activity. The C-terminal 250 residues are highly conserved. The C-terminal extremity contains a cysteine residue that might play an important role in the normal functioning of these proteins. The crystal structure of the C-terminal core domain from mouse tubby has been determined to 1.9A resolution. This domain is arranged as a 12-stranded, all anti-parallel, closed β-barrel that surrounds a central alpha helix, (which is at the extreme carboxyl terminus of the protein) that forms most of the hydrophobic core. Structural analyses suggest that TULPs constitute a unique family of bipartite transcription factors [].This entry represents conserved sites found in the C-terminal domain. The site closest to the C terminus contains a penultimate cysteine residue that could be critical to the normal functioning of these proteins.
Members of this group catalyze the first enzymatic reaction of the shikimate pathway. The common (shikimate) pathway links metabolism of carbohydrates to biosynthesis of aromatic amino acids phenylalanine, tyrosine, tryptophan, and derivatives in microorganisms and in plants. In a sequence of seven enzymatic reactions, D-erythrose 4-phosphate (E4P), an intermediate of the pentose phosphate pathway, and phosphoenol pyruvate (PEP), a glycolytic intermediate, are converted to chorismate. The pathway begins with the stereospecific condensation of E4P and PEP to yield 7-phospho 2-dehydro 3-deoxy-D-arabino-heptulosonate (DAHP), catalyzed by 3-deoxy-7-phosphoheptulonate synthase (DAHPS) (). The divalent metal cation requirement of this enzyme can be satisfied by a broad range of metals []. A Cys residue in a Cys-X-X-His motif has been identified as part of a metal binding site []. In Escherichia coli, the enzyme exists in three isoforms, each specifically inhibited by one of the three aromatic amino acids.DAHP synthetases fall into two classes, class I (represented by this entry) and class II. Class I was believed to be limited to microorganisms and class II to plants. However, a more recent study showed that class II also contains enzymes from a microbial eukaryote and several bacteria []. Brick and Woodard []proposed that the difference between the two classes lies in their metal ion requirement for activity. Whereas class I requires no metal cation, class II is dependent on a metal cation for activity. However, recently a class I DAHP synthase from Thermotoga maritima has been purified, characterised, and shown to be a metalloenzyme [].The three-dimentional structures of DAHP synthases have been determined [, , , , ]. The DAHPS(Phe) monomer is a (beta/alpha)8 barrel with an additional N-terminal beta strand and helices and an extra beta sheet near the C terminus []. The active site is located in a cleft at the carboxyl end of the barrel []. The allosteric feedback inhibition binding site of DAHPS(Phe) is composed of residues from two adjacent subunits of a tight dimer and is at least 20 angstroms away from the closest active site [].The absence of the shikimate pathway in animals makes it an attractive target for nontoxic herbicidal, antimicrobial, and antifungal agents. The nontoxic herbicide glyphosphate competitively inhibits 3-phosphoshikimate 1-carboxyvinyltransferase, the sixth enzymatic reaction of the pathway.This entry also includes phospho-2-dehydro-3-deoxyheptonate aldolase AMT16 from the Alternaria rot fungus. AMT16 is a component required for the non-ribosomal biosynthesis of the cyclic depsipeptides known as AM-toxins. The exact role of AMT16 is not known [].
The connexins are a family of integral membrane proteins that oligomerise to form intercellular channels that are clustered at gap junctions. These channels are specialised sites of cell-cell contact that allow the passage of ions, intracellular metabolites and messenger molecules (with molecular weight less than 1-2kDa) from the cytoplasm of one cell to its opposing neighbours. They are found in almost all vertebrate cell types, and somewhat similar proteins have been cloned from plant species. Invertebrates utilise a different family of molecules, innexins, that sharea similar predicted secondary structure to the vertebrate connexins, but have no sequence identity to them [].Vertebrate gap junction channels are thought to participate in diverse biological functions. For instance, in the heart they permit the rapid cell-cell transfer of action potentials, ensuring coordinated contraction of the cardiomyocytes. They are also responsible for neurotransmission at specialised 'electrical' synapses. In non-excitable tissues, such as the liver, they may allow metabolic cooperation between cells. In the brain, glial cells are extensively-coupled by gap junctions; this allows waves of intracellular Ca2+to propagate through nervous tissue, and may contribute to their ability to spatially-buffer local changes in extracellular K+concentration [].The connexin protein family is encoded by at least 13 genes in rodents, with many homologues cloned from other species. They show overlapping tissue expression patterns, most tissues expressing more than one connexin type. Their conductances, permeability to different molecules, phosphorylation and voltage-dependence of their gating, have been found to vary. Possible communication diversity is increased further by the fact that gap junctions may be formed by the association of different connexin isoforms from apposing cells. However, in vitro studies have shown that not all possible combinations of connexins produce active channels [, ].Hydropathy analysis predicts that all cloned connexins share a common transmembrane (TM) topology. Each connexin is thought to contain 4 TMdomains, with two extracellular and three cytoplasmic regions. This modelhas been validated for several of the family members by in vitrobiochemicalanalysis. Both N- and C-termini are thought to face the cytoplasm, and thethird TM domain has an amphipathic character, suggesting that it contributesto the lining of the formed-channel. Amino acid sequence identity betweenthe isoforms is ~50-80%, with the TM domains being well conserved. Bothextracellular loops contain characteristically conserved cysteine residues,which likely form intramolecular disulphide bonds. By contrast, the singleputative intracellular loop (between TM domains 2 and 3) and the cytoplasmicC terminus are highly variable among the family members.Six connexins arethought to associate to form a hemi-channel, or connexon. Two connexons theninteract (likely via the extracellular loops of their connexins) to form thecomplete gap junction channel.NH2-*** *** *************-COOH** ** ** **** ** ** ** Cytoplasmic---**----**-----**----**----------------** ** ** ** Membrane** ** ** **---**----**-----**----**----------------** ** ** ** Extracellular** ** ** **** **Two sets of nomenclature have been used to identify the connexins. Thefirst, and most commonly used, classifies the connexin molecules accordingto molecular weight, such as connexin43 (abbreviated to Cx43), indicatinga connexin of molecular weight close to 43kDa. However, studies haverevealed cases where clear functional homologues exist across speciesthat have quite different molecular masses; therefore, an alternativenomenclature was proposed based on evolutionary considerations, whichdivides the family into two major subclasses, alpha and beta, each with anumber of members []. Due to their ubiquity and overlapping tissue distributions, it has proved difficult to elucidate the functions of individual connexin isoforms. To circumvent this problem, particular connexin-encoding genes have been subjected to targeted-disruption in mice, and the phenotype of the resulting animals investigated. Around half the connexin isoforms have been investigated in this manner []. Further insight into the functional roles of connexins has come from the discovery that a number of human diseases are caused by mutations in connexin genes. For instance, mutations in Cx32 give rise to a form of inherited peripheral neuropathy called X-linked dominant Charcot-Marie-Tooth disease []. Similarly, mutations in Cx26 are responsible for both autosomal recessive and dominant forms of nonsyndromic deafness, a disorder characterised by hearing loss, with no apparent effects on other organ systems.Connexin36 (Cx36), which was recently cloned from mammalian brain, encodesa protein containing 321 amino acid residues and predicted molecular massof ~36kDa []. The rat and mouse forms of Cx36 are near-identical,differing by only a single residue. Studies of its distribution (by mRNAanalysis), have found that is is highly expressed in the adult retina ofthese species, and also (less-abundantly) in the brain. Within the latter,the highest expression levels are found in several discrete regions,including: the inferior olive, olfactory bulb, the CA3/CA4 sub-fields ofthe hippocampus, and several of the brainstem nuclei.
The connexins are a family of integral membrane proteins that oligomerise to form intercellular channels that are clustered at gap junctions. These channels are specialised sites of cell-cell contact that allow the passage of ions, intracellular metabolites and messenger molecules (with molecular weight less than 1-2kDa) from the cytoplasm of one cell to its opposing neighbours. They are found in almost all vertebrate cell types, and somewhat similar proteins have been cloned from plant species. Invertebrates utilise a different family of molecules, innexins, that share a similar predicted secondary structure to the vertebrate connexins, but have no sequence identity to them [].Vertebrate gap junction channels are thought to participate in diverse biological functions. For instance, in the heart they permit the rapid cell-cell transfer of action potentials, ensuring coordinated contraction of the cardiomyocytes. They are also responsible for neurotransmission at specialised 'electrical' synapses. In non-excitable tissues, such as the liver, they may allow metabolic cooperation between cells. In the brain, glial cells are extensively-coupled by gap junctions; this allows waves of intracellular Ca2+to propagate through nervous tissue, and may contribute to their ability to spatially-buffer local changes in extracellular K+concentration [].The connexin protein family is encoded by at least 13 genes in rodents, with many homologues cloned from other species. They show overlapping tissue expression patterns, most tissues expressing more than one connexin type. Their conductances, permeability to different molecules, phosphorylation and voltage-dependence of their gating, have been found to vary. Possible communication diversity is increased further by the fact that gap junctions may be formed by the association of different connexin isoforms from apposing cells. However, in vitro studies have shown that not all possible combinations of connexins produce active channels [, ].Hydropathy analysis predicts that all cloned connexins share a common transmembrane (TM) topology. Each connexin is thought to contain 4 TMdomains, with two extracellular and three cytoplasmic regions. This modelhas been validated for several of the family members by in vitrobiochemicalanalysis. Both N- and C-termini are thought to face the cytoplasm, and thethird TM domain has an amphipathic character, suggesting that it contributesto the lining of the formed-channel. Amino acid sequence identity betweenthe isoforms is ~50-80%, with the TM domains being well conserved. Bothextracellular loops contain characteristically conserved cysteine residues,which likely form intramolecular disulphide bonds. By contrast, the singleputative intracellular loop (between TM domains 2 and 3) and the cytoplasmicC terminus are highly variable among the family members.Six connexins arethought to associate to form a hemi-channel, or connexon. Two connexons theninteract (likely via the extracellular loops of their connexins) to form thecomplete gap junction channel.NH2-*** *** *************-COOH** ** ** **** ** ** ** Cytoplasmic---**----**-----**----**----------------** ** ** ** Membrane** ** ** **---**----**-----**----**----------------** ** ** ** Extracellular** ** ** **** **Two sets of nomenclature have been used to identify the connexins. Thefirst, and most commonly used, classifies the connexin molecules accordingto molecular weight, such as connexin43 (abbreviated to Cx43), indicatinga connexin of molecular weight close to 43kDa. However, studies haverevealed cases where clear functional homologues exist across speciesthat have quite different molecular masses; therefore, an alternativenomenclature was proposed based on evolutionary considerations, whichdivides the family into two major subclasses, alpha and beta, each with anumber of members []. Due to their ubiquity and overlapping tissue distributions, it has proved difficult to elucidate the functions of individual connexin isoforms. To circumvent this problem, particular connexin-encoding genes have been subjected to targeted-disruption in mice, and the phenotype of the resulting animals investigated. Around half the connexin isoforms have been investigated in this manner []. Further insight into the functional roles of connexins has come from the discovery that a number of human diseases are caused by mutations in connexin genes. For instance, mutations in Cx32 give rise to a form of inherited peripheral neuropathy called X-linked dominant Charcot-Marie-Tooth disease []. Similarly, mutations in Cx26 are responsible for both autosomal recessive and dominant forms of nonsyndromic deafness, a disorder characterised by hearing loss, with no apparent effects on other organ systems.Gap junction beta-4 protein (also called connexin30.3, or Cx30.3) is a structural component of gap junctions. It is closely related to Cx31.1, being ~70% identical. Together, these connexin molecules show a very restricted expression pattern, being preferentially expressed in the skin []. This protein was also found in the cochlea, being associated with normal auditory function [].
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.The Kv2 voltage-dependent potassium channels (also known as the Shab family) are responsible for much of the delayed rectifier current in Drosophila melanogaster (Fruit fly) nervous system and muscle. However, in vertebrates, Kv2 channels have been shwon to be involved in the delayed rectifier currents of the heart and skeletal muscles. They are also thought to be important in determining intrinsic neuronal excitability in both mammals and non-mammals []. Kv2 channels can be further divided into 2 subtypes, designated Kv2.1 and Kv2.2.The first Kv2.2 channel was cloned from rat and was originally referred to as the circumvillate papilla delayed rectifying K+channel or cDRK. Several mammalian channels have subsequently been found and, together with the rat Kv2.2 channel, form a small subfamily. They are predominantly expressed in the interneurones; however, their roles are largely undetermined.
Aconitase (aconitate hydratase; ) is an iron-sulphur protein that contains a [4Fe-4S]-cluster and catalyses the interconversion of isocitrate and citrate via a cis-aconitate intermediate. Aconitase functions in both the TCA and glyoxylate cycles, however unlike the majority of iron-sulphur proteins that function as electron carriers, the [4Fe-4S]-cluster of aconitase reacts directly with an enzyme substrate. In eukaryotes there is a cytosolic form (cAcn) and a mitochondrial form (mAcn) of the enzyme. In bacteria there are also 2 forms, aconitase A (AcnA) and B (AcnB). Several aconitases are known to be multi-functional enzymes with a second non-catalytic, but essential function that arises when the cellular environment changes, such as when iron levels drop [, ]. Eukaryotic cAcn and mAcn, and bacterial AcnA have the same domain organisation, consisting of three N-terminal alpha/beta/alpha domains, a linker region, followed by a C-terminal 'swivel' domain with a beta/beta/alpha structure (1-2-3-linker-4), although mAcn is smaller than cAcn. However, bacterial AcnB has a different organisation: it contains an N-terminal HEAT-like domain, followed by the 'swivel' domain, then the three alpha/beta/alpha domains (HEAT-4-1-2-3) [].Eukaryotic cAcn enzyme balances the amount of citrate and isocitrate in the cytoplasm, which in turn creates a balance between the amount of NADPH generated from isocitrate by isocitrate dehydrogenase with the amount of acetyl-CoA generated from citrate by citrate lyase. Fatty acid synthesis requires both NADPH and acetyl-CoA, as do other metabolic processes, including the need for NADPH to combat oxidative stress. The enzymatic form of cAcn predominates when iron levels are normal, but if they drop sufficiently to cause the disassembly of the [4Fe-4S]-cluster, then cAcn undergoes a conformational change from a compact enzyme to a more open L-shaped protein known as iron regulatory protein 1 (IRP1; or IRE-binding protein 1, IREBP1) [, ]. As IRP1, the catalytic site and the [4Fe-4S]-cluster are lost, and two new RNA-binding sites appear. IRP1 functions in the post-transcriptional regulation of genes involved in iron metabolism - it binds to mRNA iron-responsive elements (IRE), 30-nucleotide stem-loop structures at the 3' or 5' end of specific transcripts. Transcripts containing an IRE include ferritin L and H subunits (iron storage), transferrin (iron plasma chaperone), transferrin receptor (iron uptake into cells), ferroportin (iron exporter), mAcn, succinate dehydrogenase, erythroid aminolevulinic acid synthetase (tetrapyrrole biosynthesis), among others. If the IRE is in the 5'-UTR of the transcript (e.g. in ferritin mRNA), then IRP1-binding prevents its translation by blocking the transcript from binding to the ribosome. If the IRE is in the 3'-UTR of the transcript (e.g. transferrin receptor), then IRP1-binding protects it from endonuclease degradation, thereby prolonging the half-life of the transcript and enabling it to be translated [].IRP2 is another IRE-binding protein that binds to the same transcripts as IRP1. However, since IRP1 is predominantly in the enzymatic cAcn form, it is IRP2 that acts as the major metabolic regulator that maintains iron homeostasis []. Although IRP2 is homologous to IRP1, IRP2 lacks aconitase activity, and is known only to have a single function in the post-transcriptional regulation of iron metabolism genes []. In iron-replete cells, IRP2 activity is regulated primarily by iron-dependent degradation through the ubiquitin-proteasomal system.Bacterial AcnB is also known to be multi-functional. In addition to its role in the TCA cycle, AcnB was shown to be a post-transcriptional regulator of gene expression in Escherichia coli and Salmonella enterica [, ]. In S. enterica, AcnB initiates a regulatory cascade controlling flagella biosynthesis through an interaction with the ftsH transcript, an alternative RNA polymerase sigma factor. This binding lowers the intracellular concentration of FtsH protease, which in turn enhances the amount of RNA polymerase sigma32 factor (normally degraded by FtsH protease), and sigma32 then increases the synthesis of chaperone DnaK, which in turn promotes the synthesis of the flagellar protein FliC. AcnB regulates the synthesis of other proteins as well, such as superoxide dismutase (SodA) and other enzymes involved in oxidative stress.This entry represents the 'swivel' domain of bacterial aconitase B (AcnB) that is located in the N-terminal region following a HEAT-like domain. HEAT-like domains are usually implicated in protein-protein interactions, while the 'swivel' domain is usually a mobile unit in proteins that carry it. In AcnB, this N-terminal region was shown to be sufficient for dimerisation and for AcnB binding to mRNA. An iron-mediated dimerisation mechanism may be responsible for switching AcnB between its catalytic and regulatory roles, as dimerisation requires iron while mRNA binding is inhibited by iron.
The Macro or A1pp domain is a module of about 180 amino acids which can bind ADP-ribose (an NAD metabolite) or related ligands. Binding to ADP-ribose could be either covalent or non-covalent []: in certain cases it is believed to bind non-covalently []; while in other cases (such as Aprataxin) it appears to bind both non-covalently through a zinc finger motif, and covalently through a separate region of the protein []. The domain was described originally in association with ADP-ribose 1''-phosphate (Appr-1''-P) processing activity (A1pp) of the yeast YBR022W protein []. The domain is also called Macro domain as it is the C-terminal domain of mammalian core histone macro-H2A [, ]. Macro domain proteins can be found in eukaryotes, in (mostly pathogenic) bacteria, in archaea and in ssRNA viruses, such as coronaviruses [, ], Rubella and Hepatitis E viruses. In vertebrates the domain occurs e.g. in histone macroH2A, in predicted poly-ADP-ribose polymerases (PARPs) and in B aggressive lymphoma (BAL) protein. The macro domain can be associated with catalytic domains, such as PARP, or sirtuin. The Macro domain can recognise ADP-ribose or in some cases poly-ADP-ribose, which can be involved in ADP-ribosylation reactions that occur in important processes, such as chromatin biology, DNA repair and transcription regulation []. The human macroH2A1.1 Macro domain binds an NAD metabolite O-acetyl-ADP-ribose []. The Macro domain has been suggested to play a regulatory role in ADP-ribosylation, which is involved in inter- and intracellular signaling, transcriptional regulation, DNA repair pathways and maintenance of genomic stability, telomere dynamics, cell differentiation and proliferation, and necrosis and apoptosis. The 3D structure of the SARS-CoV Macro domain has a mixed α/β fold consisting of a central seven-stranded twisted mixed β-sheet sandwiched between two α-helices on one face, and three on the other. The final α-helix, located on the edge of the central β-sheet, forms the C terminus of the protein []. The crystal structure of AF1521 (a Macro domain-only protein from Archaeoglobus fulgidus) has also been reported and compared with other Macro domain containing proteins. Several Macro domain only proteins are shorter than AF1521, and appear to lack either the first strand of the β-sheet or the C-terminal helix 5. Well conserved residues form a hydrophobic cleft and cluster around the AF1521-ADP-ribose binding site [, , , ]. Aminopeptidases are exopeptidases involved in the processing and regular turnover of intracellular proteins, although their precise role in cellular metabolism is unclear [, ].Leucine aminopeptidases cleave leucine residues from the N-terminal of polypeptide chains; in general they are involved in the processing, catabolism and degradation of intracellular proteins [, , ]. Leucyl aminopeptidase forms a homohexamer containing two trimers stacked on top of one another []. Each monomer binds two zinc ions. The zinc-binding and catalytic sites are located within the C-terminal catalytic domain []. Leucine aminopeptidase has been shown to be identical with prolyl aminopeptidase () in mammals [].The N-terminal domain of these proteins has been shown in Escherichia coli PepA to function as a DNA-binding protein in Xer site-specific recombination and in transcriptional control of the carAB operon [, ].This superfamily represents the Macro domain as well as the N-terminal domain of Leucine aminopeptidase.
G protein-coupled receptors (GPCRs) constitute a vast protein family that encompasses a wide range of functions, including various autocrine, paracrine and endocrine processes. They show considerable diversity at the sequence level, on the basis of which they can be separated into distinct groups []. The term clan can be used to describe the GPCRs, as they embrace a group of families for which there are indications of evolutionary relationship, but between which there is no statistically significant similarity in sequence []. The currently known clan members include rhodopsin-like GPCRs (Class A, GPCRA), secretin-like GPCRs (Class B, GPCRB), metabotropic glutamate receptor family (Class C, GPCRC), fungal mating pheromone receptors (Class D, GPCRD), cAMP receptors (Class E, GPCRE) and frizzled/smoothened (Class F, GPCRF) [, , , , ]. GPCRs are major drug targets, and are consequently the subject of considerable research interest. It has been reported that the repertoire of GPCRs for endogenous ligands consists of approximately 400 receptors in humans and mice []. Most GPCRs are identified on the basis of their DNA sequences, rather than the ligand they bind, those that are unmatched to known natural ligands are designated by as orphan GPCRs, or unclassified GPCRs [].The rhodopsin-like GPCRs (GPCRA) represent a widespread protein family that includes hormone, neurotransmitter and light receptors, all of which transduce extracellular signals through interaction with guanine nucleotide-binding (G) proteins. Although their activating ligands vary widely in structure and character, the amino acid sequences of the receptors are very similar and are believed to adopt a common structural framework comprising 7 transmembrane (TM) helices [, , ].Lysophospholipids (LPs), such as lysophosphatidic acid (LPA), sphingosine1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), have long been known to act as signalling molecules in addition to their roles as intermediates in membrane biosynthesis []. They have roles in the regulation of cell growth, differentiation, apoptosis and development, and have been implicated in a wide range of pathophysiological conditions, including: blood clotting, corneal wounding, subarachinoid haemorrhage,inflammation and colitis []. A number of G protein-coupled receptors bind members of the lysophopholipid family - these include: the cannabinoid receptors; platelet activating factor receptor; OGR1, an SPC receptor identified in ovarian cancer cell lines; PSP24, an orphan receptor that has been proposed to bind LPA; and at least 8 closely related receptors, the EDG family, that bind LPA and S1P [].LPA is found in all cell types in small quantities (associated with membranebiosynthesis) but is produced in significant quantities by some cellularsources, accounting for the levels of LPA in serum. LPA is also found inelevated levels in ovarian cancer ascites, and acts to stimulate proliferation and promote survival of the cancer cells []. The effects of LPA on the proliferation and morphology of a number of other cell types have been well documented [, ]. However, identification of the mechanisms by which these effects are accomplished has been complicated by a number of factors, such as: a lack of antagonists, difficulty in ligand-binding experiments and the responsiveness of many cell types to LPA []. The G protein-coupled receptors EDG-2, EDG-4 and EDG-7 have now been identifiedas high affinity receptors for LPA. It has been suggested that these receptors should now be referred to as lpA1, lpA2 and lpA3 respectively [, ].EDG-2 was originally identified as a gene involved in neuron production fromembryonic cerebral cortex []. EDG-2 is widely distributed, with highest levels in the brain (in which expression correlates with development ofoligodendrocytes and Schwann cells) []. In the periphery, EDG-2 is found in many tissues, including the heart, kidney, testis, spleen and muscle in both humans and mouse []. The receptor is also expressed in a number of cancers []. Upon binding of LPA, EDG-2 couples to G proteins of the Gi, Gq and G12/13 classes, to mediate a range of effects including: inhibition of adenylyl cyclase; activation of phospholipase C, serum response element and MAP kinases; and actomyosin stimulation. These processes lead to cell rounding and proliferation [].
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.The Shal potassium channel was found in Drosophila melanogaster (Fruit fly). Several vertebrate potassium channels with similar amino acid sequences were subsequently found and, together with the D. melanogaster Shal channel, now constitute the Kv4 family. These channels are the primary subunits contributing to transient, voltage-dependent potassium currents in the nervous system (A currents) and the heart (transient outward current), and are inhibited by free fatty acids []. This family can be further divided into 3 subfamilies, designated Kv4.1(KCND1), Kv4.2(KCND2) and Kv4.3(KCND3).
Helicases have been classified in 5 superfamilies (SF1-SF5). All of the proteins bind ATP and, consequently, all of them carry the classical Walker A(phosphate-binding loop or P-loop) and Walker B(Mg2+-binding aspartic acid) motifs. For the two largest groups, commonlyreferred to as SF1 and SF2, a total of seven characteristic motifs has beenidentified []. These two superfamilies encompass a large number of DNA andRNA helicases from archaea, eubacteria, eukaryotes and viruses that seem to beactive as monomers or dimers. RNA and DNA helicases are considered to beenzymes that catalyze the separation of double-stranded nucleic acids in anenergy-dependent manner [].The various structures of SF1 and SF2 helicases present a common core with twoα-β RecA-like domains [, ]. Thestructural homology with the RecA recombination protein covers the fivecontiguous parallel beta strands and the tandem alpha helices. ATP binds tothe amino proximal α-β domain, where the Walker A (motif I) and WalkerB (motif II) are found. The N-terminal domain also contains motif III (S-A-T)which was proposed to participate in linking ATPase and helicase activities.The carboxy-terminal α-β domain is structurally very similar to theproximal one even though it is bereft of an ATP-binding site, suggesting thatit may have originally arisen through gene duplication of the first one.Some members of helicase superfamilies 1 and 2 are listed below:DEAD-box RNA helicases. The prototype of DEAD-boxproteins is the translation initiation factor eIF4A. The eIF4A protein isan RNA-dependent ATPase which functions together with eIF4B as an RNAhelicase [].DEAH-box RNA helicases. Mainly pre-mRNA-splicing factorATP-dependent RNA helicases [].Eukaryotic DNA repair helicase RAD3/ERCC-2, an ATP-dependent 5'-3' DNAhelicase involved in nucleotide excision repair of UV-damaged DNA.Eukaryotic TFIIH basal transcription factor complex helicase XPB subunit.An ATP-dependent 3'-5' DNA helicase which is a component of the core-TFIIHbasal transcription factor, involved in nucleotide excision repair (NER) ofDNA and, when complexed to CAK, in RNA transcription by RNA polymerase II.It acts by opening DNA either around the RNA transcription start site orthe DNA.Eukaryotic ATP-dependent DNA helicase Q. A DNA helicase that may play arole in the repair of DNA that is damaged by ultraviolet light or othermutagens.Bacterial and eukaryotic antiviral SKI2-like helicase. SKI2 has a role inthe 3'-mRNA degradation pathway, repressing dsRNA virus propagation byspecifically blocking translation of viral mRNAs, perhaps recognizing theabsence of CAP or poly(A).Bacterial DNA-damage-inducible protein G (DinG). A probable helicaseinvolved in DNA repair and perhaps also replication [].Bacterial primosomal protein N' (PriA). PriA protein is one of sevenproteins that make up the restart primosome, an apparatus that promotesassembly of replisomes at recombination intermediates and stalledreplication forks.Bacterial ATP-dependent DNA helicase recG. It has a critical role inrecombination and DNA repair, helping process Holliday junctionintermediates to mature products by catalyzing branch migration. It has aDNA unwinding activity characteristic of helicases with a 3' to 5'polarity.A variety of DNA and RNA virus helicases and transcription factorsThis entry represents the DNA-binding domain of classical SF1 and SF2 helicases. It does not recognize bacterial DinG and eukaryotic Rad3 which differ from other SF1-SF2 helicases by the presence of a large insert after the Walker A (see ).
RNA-directed RNA polymerase (RdRp) () is an essential protein encoded in the genomes of all RNA containing viruses with no DNA stage [, ]. It catalyses synthesis of the RNA strand complementary to a given RNA template, but the precise molecular mechanism remains unclear.The postulated RNA replication process is a two-step mechanism. First, the initiation step of RNA synthesis begins at or near the 3' end of the RNA template by means of a primer-independent (de novo) mechanism. The de novo initiation consists in the addition of a nucleotide tri-phosphate (NTP) to the 3'-OH of the first initiating NTP. During the following so-called elongation phase, this nucleotidyl transfer reaction is repeated with subsequent NTPs to generate the complementary RNA product []. All the RNA-directed RNA polymerases, and many DNA-directed polymerases, employ a fold whose organisation has been likened to the shape of a right hand with three subdomains termed fingers, palm and thumb []. Only the catalytic palm subdomain, composed of a four-stranded antiparallel β-sheet with two α-helices, is well conserved among all of these enzymes. In RdRp, the palm subdomain comprises three well conserved motifs (A, B and C). Motif A (D-x(4,5)-D) and motif C (GDD) are spatially juxtaposed; the Asp residues of these motifs are implied in the binding of Mg2+ and/or Mn2+. The Asn residue of motif B is involved in selection of ribonucleoside triphosphates over dNTPs and thus determines whether RNA is synthesised rather than DNA [].The domain organisation []and the 3D structure of the catalytic centre of a wide range of RdPp's, even those with a low overall sequence homology, are conserved. The catalytic centre is formed by several motifs containing a number of conserved amino acid residues.There are 4 superfamilies of viruses that cover all RNA containing viruses with no DNA stage:Viruses containing positive-strand RNA or double-strand RNA, except retroviruses and Birnaviridae: viral RNA-directed RNA polymerases including all positive-strand RNA viruses with no DNA stage, double-strand RNA viruses, and the Cystoviridae, Reoviridae, Hypoviridae, Partitiviridae, Totiviridae families.Mononegavirales (negative-strand RNA viruses with non-segmented genomes).Negative-strand RNA viruses with segmented genomes, i.e. Orthomyxoviruses (including influenza A, B, and C viruses, Thogotoviruses, and the infectious salmon anemia virus), Arenaviruses, Bunyaviruses, Hantaviruses, Nairoviruses, Phleboviruses, Tenuiviruses and Tospoviruses.Birnaviridae family of dsRNA viruses.The RNA-directed RNA polymerases in the first of the above superfamilies can be divided into the following three subgroups:All positive-strand RNA eukaryotic viruses with no DNA stage.All RNA-containing bacteriophages -there are two families of RNA-containing bacteriophages: Leviviridae (positive ssRNA phages) and Cystoviridae (dsRNA phages).Reoviridae family of dsRNA viruses.This entry represents RNA-directed RNA polymerase (also known as protein P2) from various Cystoviruses, such as Bacteriophage phi-6 and Bacteriophage phi-13 []. The RdRp protein of Cystoviruses is capable of primer-independent initiation, as are many RNA polymerases. The structure of this polymerase revealed an initiation platform, composed of a loop in the C-terminal domain that was essential for de novo initiation, a similar element having been identified in Hepatitis C virus RNA-dependent RNA polymerase [].
Helicases have been classified in 5 superfamilies (SF1-SF5). All of the proteins bind ATP and, consequently, all of them carry the classical Walker A(phosphate-binding loop or P-loop) and Walker B(Mg2+-binding aspartic acid) motifs. For the two largest groups, commonlyreferred to as SF1 and SF2, a total of seven characteristic motifs has beenidentified []. These two superfamilies encompass a large number of DNA andRNA helicases from archaea, eubacteria, eukaryotes and viruses that seem to beactive as monomers or dimers. RNA and DNA helicases are considered to beenzymes that catalyze the separation of double-stranded nucleic acids in anenergy-dependent manner [].The various structures of SF1 and SF2 helicases present a common core with twoα-β RecA-like domains [, ]. Thestructural homology with the RecA recombination protein covers the fivecontiguous parallel beta strands and the tandem alpha helices. ATP binds tothe amino proximal α-β domain, where the Walker A (motif I) and WalkerB (motif II) are found. The N-terminal domain also contains motif III (S-A-T)which was proposed to participate in linking ATPase and helicase activities.The carboxy-terminal α-β domain is structurally very similar to theproximal one even though it is bereft of an ATP-binding site, suggesting thatit may have originally arisen through gene duplication of the first one.Some members of helicase superfamilies 1 and 2 are listed below:DEAD-box RNA helicases. The prototype ofDEAD-boxproteins is the translation initiation factor eIF4A. The eIF4A protein isan RNA-dependent ATPase which functions together with eIF4B as an RNAhelicase [].DEAH-box RNA helicases. Mainly pre-mRNA-splicing factorATP-dependent RNA helicases [].Eukaryotic DNA repair helicase RAD3/ERCC-2, an ATP-dependent 5'-3' DNAhelicase involved in nucleotide excision repair of UV-damaged DNA.Eukaryotic TFIIH basal transcription factor complex helicase XPB subunit.An ATP-dependent 3'-5' DNA helicase which is a component of the core-TFIIHbasal transcription factor, involved in nucleotide excision repair (NER) ofDNA and, when complexed to CAK, in RNA transcription by RNA polymerase II.It acts by opening DNA either around the RNA transcription start site orthe DNA.Eukaryotic ATP-dependent DNA helicase Q. A DNA helicase that may play arole in the repair of DNA that is damaged by ultraviolet light or othermutagens.Bacterial and eukaryotic antiviral SKI2-like helicase. SKI2 has a role inthe 3'-mRNA degradation pathway, repressing dsRNA virus propagation byspecifically blocking translation of viral mRNAs, perhaps recognizing theabsence of CAP or poly(A).Bacterial DNA-damage-inducible protein G (DinG). A probable helicaseinvolved in DNA repair and perhaps also replication [].Bacterial primosomal protein N' (PriA). PriA protein is one of sevenproteins that make up the restart primosome, an apparatus that promotesassembly of replisomes at recombination intermediates and stalledreplication forks.Bacterial ATP-dependent DNA helicase recG. It has a critical role inrecombination and DNA repair, helping process Holliday junctionintermediates to mature products by catalyzing branch migration. It has aDNA unwinding activity characteristic of helicases with a 3' to 5'polarity.A variety of DNA and RNA virus helicases and transcription factorsThis entry represents the ATP-binding domain found within bacterial DinG and eukaryotic Rad3 proteins, differing from other SF1 and SF2 helicases by the presence of a large insert after the Walker A motif [].
The crystal structures of several lipocalins have been solved and show a novel 8-stranded anti-parallel β-barrel fold well conserved within thefamily. Sequence similarity within the family is at a much lower level andwould seem to be restricted to conserved disulphides and 3 motifs, whichform a juxtaposed cluster that may act as a common cell surface receptorsite []. By contrast, at the more variable end of the fold are found an internal ligand binding site and a putative surface for the formation of macromolecular complexes []. The anti-parallel β-barrel fold is alsoexploited by the fatty acid-binding proteins (which function similarly bybinding small hydrophobic molecules), by avidin and the closely relatedmetalloprotease inhibitors, and by triabin. Similarity at the sequence level, however, is less obvious, being confined to a single short N-terminal motif.The lipocalin family can be subdivided into kernal and outlier sets. Thekernal lipocalins form the largest self consistent group, comprising the subfamily of odour-binding proteins. The outlier lipocalins form several smaller distinct subgroups: the OBPs, the von Ebner's gland proteins, alpha-1-acid glycoproteins, tick histamine binding proteins and the nitrophorins.Odour Binding Proteins (OBPs) []are associated with olfactory tissue, and seem able to bind odorant molecules with high specificity. Rattus norvegicus (Rat) OBP is localised to the lateralnasal, or Sterno's, gland, the largest of the 20 discrete nasal glands of the rat. A similar protein from the olfactory tissue of Rana pipiens (Northern leopard frog), which was named protein BG (Bowman's gland), has been identified,cloned and sequenced. It is thought that the OBPsmay function by concentrating and delivering odorant molecules to their receptors.Aphrodisin []is the major macromolecular component of hamster vaginal discharge, and is secreted by vaginal tissue and the Bartholin's gland. These secretions, acting via the vomeronasal organ, are known to elicit a copulatory response in male hamsters. Aphrodisin is a mammalian proteinaceous pheromone.Probasin []is a lipocalin originally isolated from the nuclei of rat dorsolateral prostate epithelial cells. Probasin mRNA expression, which isregulated by androgens, gives rise to both a secreted and a nuclear form ofprobasin, the relative abundance of the two forms being correlated with celltype. Probasin concentration also seems to be closely linked with cell ageand state of differentiation.Bos taurus (Bovine) lipocalin allergen Bos d 2 is found in the secretory cells of skinapocrine sweat glands and the basement membranes of the epithelium and hairfollicles. Immunohistochemistry with a monoclonal anti-Bos d 2 antibody hasconfirmed that skin is the only tissue where mRNA encoding Bos d 2 is detected. This suggest that Bos d 2 is produced in sweat glands andtransported to the skin surface as a carrier of a pheromone. Because dander allergens of several mammalian species are lipocalins, the biological function of pheromone transport appears to be a common feature of animportant group of aeroallergens [].These lipocalins belong to the OBP group and may also act as odorant/pheromone carriers [].
5-hydroxytryptamine (5-HT) or serotonin, is a neurotransmitter that it is primarily found in the gastrointestinal (GI) tract, platelets, and in the central nervous system (CNS). It is implicated in a vast array of physiological and pathophysiological pathways. Receptors for 5-HT mediate both excitatory and inhibitory neurotransmission, and modulate the release of many neurotransmitters including glutamate, GABA, dopamine, epinephrine/norepinephrine, and acetylcholine, as well as many hormones, including oxytocin, prolactin, vasopressin and cortisol. In the CNS, 5-HT receptors can influence various neurological processes, such as aggression, anxiety and appetite and, as a, result are the target of a variety of pharmaceutical drugs, including many antidepressants, antipsychotics and anorectics []. The 5-HT receptors are grouped into a number of distinct subtypes, classified according to their antagonist susceptibilities and their affinities for 5-HT. With the exception of the 5-HT3 receptor, which is a ligand-gated ion channel [], all 5-HT receptors are members of the rhodopsin-like G protein-coupled receptor family [], and they activate an intracellular second messenger cascade to produce their responses. The 5-HT2 receptors mediate many of the central and peripheral physiologic functions of 5-hydroxytryptamine. The original 5HT2 receptor (now renamed as the 5-HT2A receptor) was initially classified according to its ability to display micromolar affinity for 5-HT, to be labelled with [3H]spiperone and by its susceptibility to 5-HT antagonists. At least 3 members of the 5HT2 receptor subfamily exist (5-HT2A, 5-HT2B, 5-HT2C), all of which share a high degree of sequence similarity and couple to Gq/G11 to stimulate the phosphoinositide pathway and elevate cytosolic calcium. Cardiovascular effects include contraction of blood vessels and shape changes in platelets; central nervous system effects include neuronal sensitisation to tactile stimuli and mediation of some of the effects of phenylisopropylamine hallucinogens. 5-HT2 receptors display functional selectivity in which the same agonist in different cell types or different agonists in the same cell type can differentially activate multiple, distinct signalling pathways [].This entry represents the 5-HT2A receptor (previously classified just as 5-HT2), which is one of the main excitatory serotonin receptors. It is expressed throughout the central nervous system [, ]with high concentrations found on the apical dendrites of pyramidal cells in layer V of the cortex [, , ], which are thought to modulate cognitive processes by enhancing glutamate release followed by a complex range of interactions with the 5-HT1A [], GABAA [], adenosine A1 [], AMPA [], mGluR2/3 [], mGlu5 []and OX2 receptors []. In the periphery, the 5-HT2A receptor is highly expressed in platelets and many cell types of the cardiovascular system, in fibroblasts, and in neurons of the peripheralnervous system. Additionally, 5-HT2A mRNA expression has been observed in human monocytes [], and platelet aggregation []with increased capillary permeability following exposure to 5-HT have been attributed to 5-HT2A receptor-mediated functions. 5-HT2A receptors also mediate contractile responses in a series of vascular smooth muscle preparations []and activation in hypothalamus causes increases in hormonal levels of oxytocin, prolactin, ACTH, corticosterone, and renin [].
The K+Cl- cotransporters (KCCs) constitute a branch of the electroneutral cation-coupled chloride cotransporter family SLC12 (Solute carrier family 12). The other branch of the SLC12 family is composed of the Na+-coupled chloride cotransporters, generally called N(K)CCs, that following the driving force imposed by the Na+:K+:ATPase translocate ions from the outside to the inside of the cell, thus having similar important roles as the KCCs in many physiological aspects, but in the opposite direction [].The Na-K-Cl co-transporters are a family of integral membrane proteins that are ubiquitously expressed in animal tissues, serving a variety offunctions. In cells of Cl-absorptive and Cl-secretory epithelia, Na-K-Cl co-transport serves as the major Cl-entry pathway, and functions in concert with other membrane ion channels and pumps to carry out net transepithelial movement of salt. This vectorial transport of Cl-across epithelia is involved in the reabsorption of salt in the vertebrate kidney (which is crucial for urinary concentration), and in the secretion of salt in such tissues as the mammalian intestine and trachea. In addition, Na-K-Cl co-transport is known to play a role in cell volume regulation in most mammalian cell types. The proteins mediate the coupled, electroneutral transport of sodium, potassium and chloride ions across the plasma membrane of cells (with a stoichiometry of 1:1:2, respectively). Co-transport of all three ions is obligatory, since absence of one is sufficient to prevent ion movement. Their transport activity does not alter the cell's membrane potential, thus the driving force for the transport is determined solely by the chemical gradients of the three transported ions; hence, under normal physiological conditions, the direction will be inward.Recent molecular studies have identified two distinct isoforms: one from Cl-secretory epithelia, NKCC1; and another, NKCC2, found specifically in the diluting segment of the vertebrate kidney, a Cl-absorptive epithelium []. They show lowish amino acid sequence identity (~58%); nevertheless, they have rather similar hydropathy profiles, with hydrophilic N- and C-termini, flanking a central hydrophobic domain. Their N-termini show considerable variation, unlike the central domain (containing the 12 putative transmembrane (TM) domains) and their C-termini, which are well conserved (~70%). Both isoforms are known to be glycosylated and, consistent with this, consensus sites for N-linked glycosylation are located within the large hydrophilic loop between presumed TM domains 7 and 8. Sequence comparisons with other cloned ion co-transporters reveals that Na-K-Cl co-transporters belong to a superfamily of electroneutral cation-chloride co-transporters, which includes the K-Cl co-transporter ) and the thiazide-sensitive Na-Cl co-transporter. All share a similar predicted membrane topology of 12 TM regions in a central hydrophobic domain, together with hydrophilic N- and C-termini that are likely cytoplasmic.Mutations in the gene encoding the renal-specific isoform of the Na-K-Cl co-transporter (NKCC2) give rise to Bartter's Syndrome Type 1, an inherited kidney disease characterised by hypokalemia, metabolic alkalosis, salt-wasting and hypotension [].NKCC1 (SLC12A2, BSC2) was first cloned from the shark rectal gland, a model secretory epithelium []. Subsequently, mammalian homologues were cloned from mouse and human tissues. NKCC1 has a wide distribution and is a basolateral secretory isoform, that plays fundamental roles in regulating trans-epithelial ion movement, cell volume, chloride homeostasis and neuronal excitability in a diverse range of tissues []. Its broad distribution also suggests that it may be involved in cell volume regulation and ionic homeostasis [, ]. The human isoform consists of 1212 amino acid residues and shares ~90 identity with the mouse homologue. It shows lower identity to other members of the cation-chloride co-transporter superfamily, being ~40% identical to the thiazide-sensitive Na-Cl co-transporter.
G protein-coupled receptors (GPCRs) constitute a vast protein family that encompasses a wide range of functions, including various autocrine, paracrine and endocrine processes. They show considerable diversity at the sequence level, on the basis of which they can be separated into distinct groups []. The term clan can be used to describe the GPCRs, as they embrace a group of families for which there are indications of evolutionary relationship, but between which there is no statistically significant similarity in sequence []. The currently known clan members include rhodopsin-like GPCRs (Class A, GPCRA), secretin-like GPCRs (Class B, GPCRB), metabotropic glutamate receptor family (Class C, GPCRC), fungal mating pheromone receptors (Class D, GPCRD), cAMP receptors (Class E, GPCRE) and frizzled/smoothened (Class F, GPCRF) [, , , , ]. GPCRs are major drug targets, and are consequently the subject of considerable research interest. It has been reported that the repertoire of GPCRs for endogenous ligands consists of approximately 400 receptors in humans and mice []. Most GPCRs are identified on the basis of their DNA sequences, rather than the ligand they bind, those that are unmatched to known natural ligands are designated by as orphan GPCRs, or unclassified GPCRs [].The rhodopsin-like GPCRs (GPCRA) represent a widespread protein family that includes hormone, neurotransmitter and light receptors, all of which transduce extracellular signals through interaction with guanine nucleotide-binding (G) proteins. Although their activating ligands vary widely in structure and character, the amino acid sequences of the receptors are very similar and are believed to adopt a common structural framework comprising 7 transmembrane (TM) helices [, , ].Adrenocorticotrophin (ACTH), melanocyte-stimulating hormones (MSH) andbeta-endorphin are peptide products of pituitary pro-opiomelanocortin.ACTH regulates synthesis and release of glucocorticoids and aldosteronein the adrenal cortex; it also has a trophic action on these cells.ACTH and beta-endorphin are synthesised and released in response tocorticotrophin-releasing factor at times of stress (heat, cold, infections,etc.) - their release leads to increased metabolism and analgesia.MSH has a trophic action on melanocytes, and regulates pigment productionin fish and amphibia. The ACTH receptor is found in high levels inthe adrenal cortex - binding sites are present in lower levels in theCNS. The MSH receptor is expressed in high levels in melanocytes,melanomas and their derived cell lines. Receptors are found in lowlevels in the CNS. MSH regulates temperature control in the septal regionof the brain and releases prolactin from the pituitary.A further gene, which encodes a melanocortin receptor that is functionallydistinct from the ACTH and MSH receptors, has also been characterised [, , , , ].The protein contains ~300 amino acids, withcalculated molecular mass of~36kDa, and potential N-linked glycosylation and phosphorylation sites[]. The melanocortin 4 receptor (MC4-R) is regulated by opiateadministration []. Rat MC4-R is 95% identical to human MC4-R, and thepotency of melanocortin peptides to stimulate cAMP production is similar inthese two species homologues []. Expression of MC4-R mRNA was found to beenriched in the striatum, nucleus accumbens, and periaque-ductal gray, allof which are regions implicated in the behavioral effects of opiates(and are regions in which MC1-, MC3- and MC5-R are expressed at low orundetectable levels) []. MC4-R mRNA has been found in multiple sites invirtually every brain region, including the cortex, thalamus, hypothalamus,brainstem, and spinal cord []. Unlike the MC3-R, MC4-R mRNA is found inboth parvicellular and magnocellular neurons of the paraventricular nucleusof the hypothalamus, suggesting a role in the central control of pituitaryfunction [].
Members of this group are signal transduction proteins that are direct oxygen sensors and are involved in regulation of cellular processes via the effector molecule cyclic diguanylate (c-di-GMP, bis(3',5')-cyclic diguanylic acid). They contain PAS/PAC, GGDEF, and EAL domains and have diguanylate cyclase and phosphodiesterase activities. Related groups with similar domain architectures contain different versions of PAS/PAC domain, and are thought to have different, often not yet determined biological functions.Escherichia coli Dos (YddU or DosP) and Komagataeibacter xylinus (Gluconacetobacter xylinus or Acetobacter xylinum) PdeA1 proteins have been shown to be direct, haem-based oxygen sensors [, , ]. Their N-terminal PAS domains are responsible for haem-binding [, ]. PAS/PAC is a ubiquitous intracellular sensory domain. It is located in the cytoplasm and sense changes in redox potential in the electron transport system or overall cellular redox status. PAS domains can monitor changes in light, oxygen or small ligands in a cell, and sense environmental factors that cross the cell membrane and/or affect cell metabolism [, , ]. In the haem-containing subgroup of PAS domains, the haem pocket acts as a ligand-specific trap []. The ligand binding to a haem-containing PAS domain leads to either activation or inhibition of a regulated (catalytic) domain (here, GGDEF and/or EAL domains). Phosphodiesterase activity with cAMP of E. coli Dos has been shown to be regulated by the haem redox state []. Similarly, Komagataeibacter xylinus PdeA1 is regulated by reversible binding of O2to the haem [].The catalytic function of the members of this group has also been experimentally determined.Cyclic di-GMP (c-di-GMP) is the specific nucleotide regulator of beta-1,4-glucan (cellulose) synthase in Komagataeibacter xylinus []. In a study of the regulation of biosynthesis of extracellular cellulose in Komagataeibacter xylinus [], the search for the enzymes that synthesise and hydrolyse cyclic di-GMP resulted in the identification of six proteins with identical domain architecture containing PAS, GGDEF and EAL domains. Three of them exhibited diguanylate cyclase activity (Dgc1-3), and three others - phosphodiesterase activity (PdeA1-3) [, ]. Likewise, E. coli Dos has been shown to have phosphodiesterase activity [].Genetic complementation using genes from three different bacteria encoding proteins with GGDEF domains as the only element in common indicate that the GGDEF domain is responsible for the diguanylate cyclase activity of these proteins []. Even prior to these results, the notion that the GGDEF domain is a diguanylate cyclase was supported by the detailed analysis of its sequence, which shows conservation of the proposed nucleotide-binding loop in alignment with eukaryotic adenylate cyclases []. By exclusion, the EAL domain emerged as the best candidate for the role of c-di-GMP phosphodiesterase. Indeed, the sequence of this domain contains several conserved aspartates, which could participate in metal binding and form a phosphodiesterase active site []. It is not clear what differences make one subgroup of these proteins to act as phosphodiesterases, and another - as diguanylate cyclases, while containing both domains.For additional information please see [, ].
G protein-coupled receptors (GPCRs) constitute a vast protein family that encompasses a wide range of functions, including various autocrine, paracrine and endocrine processes. They show considerable diversity at the sequence level, on the basis of which they can be separated into distinct groups []. The term clan can be used to describe the GPCRs, as they embrace a group of families for which there are indications of evolutionary relationship, but between which there is no statistically significant similarity in sequence []. The currently known clan members include rhodopsin-like GPCRs (Class A, GPCRA), secretin-like GPCRs (Class B, GPCRB), metabotropic glutamate receptor family (Class C, GPCRC), fungal mating pheromone receptors (Class D, GPCRD), cAMP receptors (Class E, GPCRE) and frizzled/smoothened (Class F, GPCRF) [, , , , ]. GPCRs are major drug targets, and are consequently the subject of considerable research interest. It has been reported that the repertoire of GPCRs for endogenous ligands consists of approximately 400 receptors in humans and mice []. Most GPCRs are identified on the basis of their DNA sequences, rather than the ligand they bind, those that are unmatched to known natural ligands are designated by as orphan GPCRs, or unclassified GPCRs [].The rhodopsin-like GPCRs (GPCRA) represent a widespread protein family that includes hormone, neurotransmitter and light receptors, all of which transduce extracellular signals through interaction with guanine nucleotide-binding (G) proteins. Although their activating ligands vary widely in structure and character, the amino acid sequences of the receptors are very similar and are believed to adopt a common structural framework comprising 7 transmembrane (TM) helices [, , ].Hypothalamic peptide hormones regulate secretion of anterior pituitary hormones, such as growth hormone, follicle stimulating hormone, luteinisinghormone and thyrotropin. A novel bioactive peptide has been identified from bovine hypothalamus and found to increase prolactin secretion from the anterior pituitary []. This peptide - prolactin-releasing peptide (PrRP) - is a member of the structurally related RF-amide family, which includes neuropeptide FF []. The peptide exists in two forms: a 31-amino acid form and a truncated 20-amino acid form []. PrRP has been found in the medulla oblongata, hypothalamus and pituitary, as well as in a number of other tissues. This distribution suggests the peptide may have other roles in addition to prolactin release [].The receptor for PrRP was identified to be an orphan receptor, previously known as GPR10 []. This receptor is expressed in the central nervous system with highest levels in the pituitary. Expression has also been detected in the cerebellum, brainstem, hypothalamus, thalamus and spinal cord in rat []. Binding of PrRP to the receptor results in activation of extracellular signal-related kinase (ERK) in a mainly pertussis toxin sensitive manner, suggesting coupling to Gi/o proteins []. PrRP can also cause increases in intracellular calcium and activation of c-Jun N-terminal protein kinase (JNK) in a pertussis toxin insensitive manner, indicating that the receptor can also couple to Gq proteins, depending on the cell type in which it is expressed [].
This aspartic peptidase domain is found in viral enzymatic polyproteins. It belongs to MEROPS peptidase family A3, subfamily A3A (cauliflower mosaic virus-type endopeptidase, clan AA). Cauliflower mosaic virus belongs to the Retro-transcribing viruses, which have a double-stranded DNA genome. The genome includes an open reading frame (ORF V) that shows similarities to the polgene of retroviruses. This ORF codes for a polyprotein that includes a reverse transcriptase, which, on the basisof a DTG triplet near the N terminus, was suggested to include an aspartic protease. The presence of an asparticprotease has been confirmed by mutational studies, implicating Asp-45 in catalysis. The protease releases itselffrom the polyprotein and is involved in reactions required to process the ORF IV polyprotein, which includes theviral coat protein []. The viral aspartic peptidase domain has also been found associated with a polyprotein encoded by integrated pararetrovirus-like sequences in the genome of Nicotiana tabacum (Common tobacco) []. Aspartic peptidases, also known as aspartyl proteases ([intenz:3.4.23.-]), are widely distributed proteolytic enzymes [, , ]known to exist in vertebrates, fungi, plants, protozoa, bacteria, archaea, retroviruses and some plant viruses. All known aspartic peptidases are endopeptidases. A water molecule, activated by two aspartic acid residues, acts as the nucleophile in catalysis. Aspartic peptidases can be grouped into five clans, each of which shows a unique structural fold [].Peptidases in clan AA are either bilobed (family A1 or the pepsin family) or are a homodimer (all other families in the clan, including retropepsin from HIV-1/AIDS) []. Each lobe consists of a single domain with a closed β-barrel and each lobe contributes one Asp to form the active site. Most peptidases in the clan are inhibited by the naturally occurring small-molecule inhibitor pepstatin [].Clan AC contains the single family A8: the signal peptidase 2 family. Members of the family are found in all bacteria. Signal peptidase 2 processes the premurein precursor, removing the signal peptide. The peptidase has four transmembrane domains and the active site is on the periplasmic side of the cell membrane. Cleavage occurs on the amino side of a cysteine where the thiol group has been substituted by a diacylglyceryl group. Site-directed mutagenesis has identified two essential aspartic acid residues which occur in the motifs GNXXDRX and FNXAD (where X is a hydrophobic residue) []. No tertiary structures have been solved for any member of the family, but because of the intramembrane location, the structure is assumed not to be pepsin-like.Clan AD contains two families of transmembrane endopeptidases: A22 and A24. These are also known as "GXGD peptidases"because of a common GXGD motif which includes one of the pair of catalytic aspartic acid residues. Structures are known for members of both families and show a unique, common fold with up to nine transmembrane regions []. The active site aspartic acids are located within a large cavity in the membrane into which water can gain access [].Clan AE contains two families, A25 and A31. Tertiary structures have been solved for members of both families and show a common fold consisting of an α-β-alpha sandwich, in which the beta sheet is five stranded [, ].Clan AF contains the single family A26. Members of the clan are membrane-proteins with a unique fold. Homologues are known only from bacteria. The structure of omptin (also known as OmpT) shows a cylindrical barrel containing ten beta strands inserted in the membrane with the active site residues on the outer surface [].There are two families of aspartic peptidases for which neither structure nor active site residues are known and these are not assigned to clans. Family A5 includes thermopsin, an endopeptidase found only in thermophilic archaea. Family A36 contains sporulation factor SpoIIGA, which is known to process and activate sigma factor E, one of the transcription factors that controls sporulation in bacteria [].
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.The Shal potassium channel was found in Drosophila melanogaster (Fruit fly). Several vertebrate potassium channels with similar amino acid sequences were subsequently found and, together with the D. melanogaster Shal channel, now constitute the Kv4 family. These channels are the primary subunits contributing to transient, voltage-dependent potassium currents in the nervous system (A currents) and the heart (transient outward current), and are inhibited by free fatty acids []. This family can be further divided into 3 subfamilies, designated Kv4.1(KCND1), Kv4.2(KCND2) and Kv4.3(KCND3).This uncharacterised C-terminal domain is associated with the Shal (Kv4) potassium channel.
Ribosomes are the particles that catalyse mRNA-directed protein synthesis in all organisms. The codons of the mRNA are exposed on the ribosome to allow tRNA binding. This leads to the incorporation of amino acids into the growing polypeptide chain in accordance with the genetic information. Incoming amino acid monomers enter the ribosomal A site in the form of aminoacyl-tRNAs complexed with elongation factor Tu (EF-Tu) and GTP. The growing polypeptide chain, situated in the P site as peptidyl-tRNA, is then transferred to aminoacyl-tRNA and the new peptidyl-tRNA, extended by one residue, is translocated to the P site with the aid the elongation factor G (EF-G) and GTP as the deacylated tRNA is released from the ribosome through one or more exit sites [, ]. About 2/3 of the mass of the ribosome consists of RNA and 1/3 of protein. The proteins are named in accordance with the subunit of the ribosome which they belong to - the small (S1 to S31) and the large (L1 to L44). Usually they decorate the rRNA cores of the subunits. Many ribosomal proteins, particularly those of the large subunit, are composed of a globular, surfaced-exposed domain with long finger-like projections that extend into the rRNA core to stabilise its structure. Most of the proteins interact with multiple RNA elements, often from different domains. In the large subunit, about 1/3 of the 23S rRNA nucleotides are at least in van der Waal's contact with protein, and L22 interacts with all six domains of the 23S rRNA. Proteins S4 and S7, which initiate assembly of the 16S rRNA, are located at junctions of five and four RNA helices, respectively. In this way proteins serve to organise and stabilise the rRNA tertiary structure. While the crucial activities of decoding and peptide transfer are RNA based, proteins play an active role in functions that may have evolved to streamline the process of protein synthesis. In addition to their function in the ribosome, many ribosomal proteins have some function 'outside' the ribosome [, ].L6 is a protein from the large (50S) subunit. In Escherichia coli, it is located in the aminoacyl-tRNA bindingsite of the peptidyltransferase centre, and is known to bind directly to 23S rRNA. It belongs to a family of ribosomal proteins, including L6 from bacteria, cyanelles (structures that perform similar functions to chloroplasts, but have structural and biochemical characteristics of Cyanobacteria) and mitochondria; and L9 from mammals, Drosophila, plants and yeast. L6 contains two domains with almost identical folds, suggesting that is was derived by the duplication of anancient RNA-binding protein gene. Analysis reveals several sites on the protein surface where interactions with other ribosome components may occur, the N terminus being involved in protein-protein interactions and the C terminus containing possible RNA-binding sites [].
Ribosomes are the particles that catalyse mRNA-directed protein synthesis in all organisms. The codons of the mRNA are exposed on the ribosome to allow tRNA binding. This leads to the incorporation of amino acids into the growing polypeptide chain in accordance with the genetic information. Incoming amino acid monomers enter the ribosomal A site in the form of aminoacyl-tRNAs complexed with elongation factor Tu (EF-Tu) and GTP. The growing polypeptide chain, situated in the P site as peptidyl-tRNA, is then transferred to aminoacyl-tRNA and the new peptidyl-tRNA, extended by one residue, is translocated to the P site with the aid the elongation factor G (EF-G) and GTP as the deacylated tRNA is released from the ribosome through one or more exit sites [, ]. About 2/3 of the mass of the ribosome consists of RNA and 1/3 of protein. The proteins are named in accordance with the subunit of the ribosome which they belong to - the small (S1 to S31) and the large (L1 to L44). Usually they decorate the rRNA cores of the subunits. Many ribosomal proteins, particularly those of the large subunit, are composed of a globular, surfaced-exposed domain with long finger-like projections that extend into the rRNA core to stabilise its structure. Most of the proteins interact with multiple RNA elements, often from different domains. In the large subunit, about 1/3 of the 23S rRNA nucleotides are at least in van der Waal's contact with protein, and L22 interacts with all six domains of the 23S rRNA. Proteins S4 and S7, which initiate assembly of the 16S rRNA, are located at junctions of five and four RNA helices, respectively. In this way proteins serve to organise and stabilise the rRNA tertiary structure. While the crucial activities of decoding and peptide transfer are RNA based, proteins play an active role in functions that may have evolved to streamline the process of protein synthesis. In addition to their function in the ribosome, many ribosomal proteins have some function 'outside' the ribosome [, ].L6 is a protein from the large (50S) subunit. In Escherichia coli, it is located in the aminoacyl-tRNA bindingsite of the peptidyltransferase centre, and is known to bind directly to 23S rRNA. It belongs to a family of ribosomal proteins, including L6 from bacteria, cyanelles (structures that perform similar functions to chloroplasts, but have structural and biochemical characteristics of Cyanobacteria) and mitochondria; and L9 from mammals, Drosophila, plants and yeast. L6 contains two domains with almost identical folds, suggesting that is was derived by the duplication of anancient RNA-binding protein gene. Analysis reveals several sites on the protein surface where interactions with other ribosome components may occur, the N terminus being involved in protein-protein interactions and the C terminus containing possible RNA-binding sites [].
Ribosomes are the particles that catalyse mRNA-directed protein synthesis in all organisms. The codons of the mRNA are exposed on the ribosome to allow tRNA binding. This leads to the incorporation of amino acids into the growing polypeptide chain in accordance with the genetic information. Incoming amino acid monomers enter the ribosomal A site in the form of aminoacyl-tRNAs complexed with elongation factor Tu (EF-Tu) and GTP. The growing polypeptide chain, situated in the P site as peptidyl-tRNA, is then transferred to aminoacyl-tRNA and the new peptidyl-tRNA, extended by one residue, is translocated to the P site with the aid the elongation factor G (EF-G) and GTP as the deacylated tRNA is released from the ribosome through one or more exit sites [, ]. About 2/3 of the mass of the ribosome consists of RNA and 1/3 of protein. The proteins are named in accordance with the subunit of the ribosome which they belong to - the small (S1 to S31) and the large (L1 to L44). Usually they decorate the rRNA cores of the subunits. Many ribosomal proteins, particularly those of the large subunit, are composed of a globular, surfaced-exposed domain with long finger-like projections that extend into the rRNA core to stabilise its structure. Most of the proteins interact with multiple RNA elements, often from different domains. In the large subunit, about 1/3 of the 23S rRNA nucleotides are at least in van der Waal's contact with protein, and L22 interacts with all six domains of the 23S rRNA. Proteins S4 and S7, which initiate assembly of the 16S rRNA, are located at junctions of five and four RNA helices, respectively. In this way proteins serve to organise and stabilise the rRNA tertiary structure. While the crucial activities of decoding and peptide transfer are RNA based, proteins play an active role in functions that may have evolved to streamline the process of protein synthesis. In addition to their function in the ribosome, many ribosomal proteins have some function 'outside' the ribosome [, ].L6 is a protein from the large (50S) subunit. In Escherichia coli, it is located in the aminoacyl-tRNA bindingsite of the peptidyltransferase centre, and is known to bind directly to 23S rRNA. It belongs to a family of ribosomal proteins, including L6 from bacteria, cyanelles (structures that perform similar functions to chloroplasts, but have structural and biochemical characteristics of Cyanobacteria) and mitochondria; and L9 from mammals, Drosophila, plants and yeast. L6 contains two domains with almost identical folds, suggesting that is was derived by the duplication of anancient RNA-binding protein gene. Analysis reveals several sites on the protein surface where interactions with other ribosome components may occur, the N terminus being involved in protein-protein interactions and the C terminus containing possible RNA-binding sites [].This α-β domain found duplicated in ribosomal L6 proteins consists of two β-sheets and one α-helix packed around single core [].
Zinc finger (Znf) domains are relatively small protein motifs which contain multiple finger-like protrusions that make tandem contacts with their target molecule. Some of these domains bind zinc, but many do not; instead binding other metals such as iron, or no metal at all. For example, some family members form salt bridges to stabilise the finger-like folds. They were first identified as a DNA-binding motif in transcription factor TFIIIA from Xenopus laevis (African clawed frog), however they are now recognised to bind DNA, RNA, protein and/or lipid substrates [, , , , ]. Their binding properties depend on the amino acid sequence of the finger domains and of the linker between fingers, as well as on the higher-order structures and the number of fingers. Znf domains are often found in clusters, where fingers can have different binding specificities. There are many superfamilies of Znf motifs, varying in both sequence and structure. They display considerable versatility in binding modes, even between members of the same class (e.g. some bind DNA, others protein), suggesting that Znf motifs are stable scaffolds that have evolved specialised functions. For example, Znf-containing proteins function in gene transcription, translation, mRNA trafficking, cytoskeleton organisation, epithelial development, cell adhesion, protein folding, chromatin remodelling and zinc sensing, to name but a few []. Zinc-binding motifs are stable structures, and they rarely undergo conformational changes upon binding their target. This entry represents a zinc finger motif found in transcription factor IIs (TFIIS). In eukaryotes the initiation of transcription of protein encoding genes by polymerase II (Pol II) is modulated by general and specific transcription factors. The general transcription factors operate through common promoters elements (such as the TATA box). At least eight different proteins associate to form the general transcription factors: TFIIA, -IIB, -IID, -IIE, -IIF, -IIG, -IIH and -IIS []. During mRNA elongation, Pol II can encounter DNA sequences that cause reverse movement of the enzyme. Such backtracking involves extrusion of the RNA 3'-end into the pore, and can lead to transcriptional arrest. Escape from arrest requires cleavage of the extruded RNA with the help of TFIIS, which induces mRNA cleavage by enhancing the intrinsic nuclease activity of RNA polymerase (Pol) II, past template-encoded pause sites []. TFIIS extends from the polymerase surface via a pore to the internal active site. Two essential and invariant acidic residues in a TFIIS loop complement the Pol II active site and could position a metal ion and a water molecule for hydrolytic RNA cleavage. TFIIS also induces extensive structural changes in Pol II that would realign nucleic acids in the active centre.TFIIS is a protein of about 300 amino acids. It contains three regions: a variable N-terminal domain not required for TFIIS activity; a conserved central domain required for Pol II binding; and a conserved C-terminal C4-type zinc finger essential for RNA cleavage. The zinc finger folds in a conformation termed a zinc ribbon []characterised by a three-stranded antiparallel β-sheet and two β-hairpins. A backbone model for Pol II-TFIIS complex was obtained from X-ray analysis. It shows that a beta hairpin protrudes from the zinc finger and complements the pol II active site []. Some viral proteins also contain the TFIIS zinc ribbon C-terminal domain. The Vaccinia virus protein, unlike its eukaryotic homologue, is an integral RNA polymerase subunit rather than a readily separable transcription factor [].
The antibiotic tetracycline has a broad spectrum of activity, acting to inhibit bacterial protein synthesis by binding to the 30S ribosomal subunit, which prevents the association of the aminoacyl-tRNA to the ribosomal acceptor A site. Tetracycline binding is reversible, therefore diluting out the antibiotic can reverse its effects. Tetracycline resistance genes are often located on mobile elements, such as plasmids, transposons and/or conjugative transposons, which can sometimes be transferred between bacterial species. In certain cases, tetracycline can enhance the transfer of these elements, thereby promoting resistance amongst a bacterial colony. There are three types of tetracycline resistance: tetracycline efflux, ribosomal protection, and tetracycline modification [, ]: Tetracycline efflux proteins belong to the major facilitator superfamily. Efflux proteins are membrane-associated proteins that recognise and export tetracycline from the cell. They are found in both Gram-positive and Gram-negative bacteria []. There are at least 22 different tetracycline efflux proteins, grouped according to sequence similarity: Group 1 are Tet(A), Tet(B), Tet(C), Tet(D), Tet(E), Tet(G), Tet(H), Tet(J), Tet(Z) and Tet(30); Group 2 are Tet(K) and Tet(L); Group 3 are Otr(B) and Tcr(3); Group 4 is TetA(P); Group 5 is Tet(V). In addition, there are the efflux proteins Tet(31), Tet(33), Tet(V), Tet(Y), Tet(34), and Tet(35).Ribosomal protection proteins are cytoplasmic proteins that display homology with the elongation factors EF-Tu and EF-G. Protection proteins bind the ribosome, causing an alteration in ribosomal conformation that prevents tetracycline from binding. There areat least ten ribosomal protection proteins: Tet(M), Tet(O), Tet(S), Tet(W), Tet(32), Tet(36), Tet(Q), Tet(T), Otr(A), and TetB(P). Both Tet(M) and Tet(O) have ribosome-dependent GTPase activity, the hydrolysis of GTP providing the energy for the ribosomal conformational changes. Tetracycline modification proteins include the enzymes Tet(37) and Tet(X), both of which inactivate tetracycline. In addition, there are the tetracycline resistance proteins Tet(U) and Otr(C).The expression of several of these tet genes is controlled by a family of tetracycline transcriptional regulators known as TetR. TetR family regulators are involved in the transcriptional control of multidrug efflux pumps, pathways for the biosynthesis of antibiotics, response to osmotic stress and toxic chemicals, control of catabolic pathways, differentiation processes, and pathogenicity []. The TetR proteins identified in over 115 genera of bacteria and archaea share a common helix-turn-helix (HTH) structure in their DNA-binding domain. However, TetR proteins can work in different ways: they can bind a target operator directly to exert their effect (e.g. TetR binds Tet(A) gene to repress it in the absence of tetracycline), or they can be involved in complex regulatory cascades in which the TetR protein can either be modulated by another regulator or TetR can trigger the cellular response. This entry represents the tetracycline resistance leader peptide, which can be found in Tet(M) ribosomal protection proteins. A short open reading frame corresponding to a 28 amino acid peptide, which contains a number of inverted repeat sequences was found immediately upstream of tet(M). Transcriptional analyses has found that expression of tet(M) resulted from an extension of a small transcript representing the upstream leader region into the resistance determinant. Therefore, this leader sequence is responsible for transcriptional attenuation and thus regulation of the transcription of tet(M) [].
Thioredoxins (Trxs) are ubiquitous enzymes with a CXXC active site that catalyses the reduction of disulfide bonds. This entry represents a group of plant thioredoxin-like proteins, including AtCDSP32 from Arabidopsis and OsCDSP32 from rice. AtCDSP32 includes two Trx modules with one potential active site (219)CGPC(222) and three extra Cys, this region is responsible for the insulin reduction activity of the protein []. It forms a heterodimeric complex with MSRB1 (methionine sulfoxide reductases B 1) via reduction of the sulfenic acid formed on MSRB1 catalytic Cys after methionine sulfoxide reduction [].Thioredoxins [, , , ]are small disulphide-containing redox proteins that have been found in all the kingdoms of living organisms. Thioredoxin serves as a general protein disulphide oxidoreductase. It interacts with a broad range of proteins by a redox mechanism based on reversible oxidation of two cysteine thiol groups to a disulphide, accompanied by the transfer of two electrons and two protons. The net result is the covalent interconversion of a disulphide and a dithiol. In the NADPH-dependent protein disulphide reduction, thioredoxin reductase (TR) catalyses the reduction of oxidised thioredoxin (trx) by NADPH using FAD and its redox-active disulphide; reduced thioredoxin then directly reduces the disulphide in the substrate protein [].Thioredoxin is present in prokaryotes and eukaryotes and the sequence around the redox-active disulphide bond is well conserved. All thioredoxins contain a cis-proline located in a loop preceding β-strand 4, which makes contact with the active site cysteines, and is important for stability and function []. Thioredoxin belongs to astructural family that includes glutaredoxin, glutathione peroxidase, bacterial protein disulphide isomerase DsbA, and the N-terminal domain of glutathione transferase []. Thioredoxins have a beta-alpha unit preceding the motif common to all these proteins.A number of eukaryotic proteins contain domains evolutionary related to thioredoxin, most of them are protein disulphide isomerases (PDI). PDI () [, , ]is an endoplasmic reticulum multi-functional enzyme that catalyses the formation and rearrangement of disulphide bonds during protein folding []. All PDI contains two or three (ERp72) copies of the thioredoxin domain, each of which contributes to disulphide isomerase activity, but which are functionally non-equivalent []. Moreover, PDI exhibits chaperone-like activity towards proteins that contain no disulphide bonds, i.e. behaving independently of its disulphide isomerase activity []. The various forms of PDI which are currently known are:PDI major isozyme; a multifunctional protein that also function as the beta subunit of prolyl 4-hydroxylase (), as a component of oligosaccharyl transferase (), as thyroxine deiodinase (), as glutathione-insulin transhydrogenase () and as a thyroid hormone-binding proteinERp60 (ER-60; 58 Kd microsomal protein). ERp60 was originally thought to be a phosphoinositide-specific phospholipase C isozyme and later to be a protease.ERp72.ERp5.Bacterial proteins that act as thiol:disulphide interchange proteins that allows disulphide bond formation in some periplasmic proteins also contain a thioredoxin domain. These proteins include:Escherichia coli DsbA (or PrfA) and its orthologs in Vibrio cholerae (TtcpG) and Haemophilus influenzae (Por).E. coli DsbC (or XpRA) and its orthologues in Erwinia chrysanthemi and H. influenzae.E. coli DsbD (or DipZ) and its H. influenzae orthologue.E. coli DsbE (or CcmG) and orthologues in H. influenzae.Rhodobacter capsulatus (Rhodopseudomonas capsulata) (HelX), Rhiziobiacae (CycY and TlpA).
Ribosomes are the particles that catalyse mRNA-directed protein synthesis in all organisms. The codons of the mRNA are exposed on the ribosome to allow tRNA binding. This leads to the incorporation of amino acids into the growing polypeptide chain in accordance with the genetic information. Incoming amino acid monomers enter the ribosomal A site in the form of aminoacyl-tRNAs complexed with elongation factor Tu (EF-Tu) and GTP. The growing polypeptide chain, situated in the P site as peptidyl-tRNA, is then transferred to aminoacyl-tRNA and the new peptidyl-tRNA, extended by one residue, is translocated to the P site with the aid the elongation factor G (EF-G) and GTP as the deacylated tRNA is released from the ribosome through one or more exit sites [, ]. About 2/3 of the mass of the ribosome consists of RNA and 1/3 of protein. The proteins are named in accordance with the subunit of the ribosome which they belong to - the small (S1 to S31) and the large (L1 to L44). Usually they decorate the rRNA cores of the subunits. Many ribosomal proteins, particularly those of the large subunit, are composed of a globular, surfaced-exposed domain with long finger-like projections that extend into the rRNA core to stabilise its structure. Most of the proteins interact with multiple RNA elements, often from different domains. In the large subunit, about 1/3 of the 23S rRNA nucleotides are at least in van der Waal's contact with protein, and L22 interacts with all six domains of the 23S rRNA. Proteins S4 and S7, which initiate assembly of the 16S rRNA, are located at junctions of five and four RNA helices, respectively. In this way proteins serve to organise and stabilise the rRNA tertiary structure. While the crucial activities of decoding and peptide transfer are RNA based, proteins play an active role in functions that may have evolved to streamline the process of protein synthesis. In addition to their function in the ribosome, many ribosomal proteins have some function 'outside' the ribosome [, ].L6 is a protein from the large (50S) subunit. In Escherichia coli, it is located in the aminoacyl-tRNA bindingsite of the peptidyltransferase centre, and is known to bind directly to 23S rRNA. It belongs to a family of ribosomal proteins, including L6 from bacteria, cyanelles (structures that perform similar functions to chloroplasts, but have structural and biochemical characteristics of Cyanobacteria) and mitochondria; and L9 from mammals, Drosophila, plants and yeast. L6 contains two domains with almost identical folds, suggesting that is was derived by the duplication of anancient RNA-binding protein gene. Analysis reveals several sites on the protein surface where interactions with other ribosome components may occur, the N terminus being involved in protein-protein interactions and the C terminus containing possible RNA-binding sites [].This entry represents the α-β domain found duplicated in ribosomal L6 proteins. This domain consists of two β-sheets and one α-helix packed around single core [].
Zinc finger (Znf) domains are relatively small protein motifs which contain multiple finger-like protrusions that make tandem contacts with their target molecule. Some of these domains bind zinc, but many do not; instead binding other metals such as iron, or no metal at all. For example, some family members form salt bridges to stabilise the finger-like folds. They were first identified as a DNA-binding motif in transcription factor TFIIIA from Xenopus laevis (African clawed frog), however they are now recognised to bind DNA, RNA, protein and/or lipid substrates [, , , , ]. Their binding properties depend on the amino acid sequence of the finger domains and of the linker between fingers, as well as on the higher-order structures and the number of fingers. Znf domains are often found in clusters, where fingers can have different binding specificities. There are many superfamilies of Znf motifs, varying in both sequence and structure. They display considerable versatility in binding modes, even between members of the same class (e.g. some bind DNA, others protein), suggesting that Znf motifs are stable scaffolds that have evolved specialised functions. For example, Znf-containing proteins function in gene transcription, translation, mRNA trafficking, cytoskeleton organisation, epithelial development, cell adhesion, protein folding, chromatin remodelling and zinc sensing, to name but a few []. Zinc-binding motifs are stable structures, and they rarely undergo conformational changes upon binding their target. C2H2-type (classical) zinc fingers (Znf) were the first class to be characterised. They contain a short β hairpin and an α helix (β/β/α structure), where a single zinc atom is held in place by Cys(2)His(2) (C2H2) residues in a tetrahedral array. C2H2 Znf's can be divided into three groups based on the number and pattern of fingers: triple-C2H2 (binds single ligand), multiple-adjacent-C2H2 (binds multiple ligands), and separated paired-C2H2 []. C2H2 Znf's are the most common DNA-binding motifs found in eukaryotic transcription factors, and have also been identified in prokaryotes []. Transcription factors usually contain several Znf's (each with a conserved β/β/α structure) capable of making multiple contacts along the DNA, where the C2H2 Znf motifs recognise DNA sequences by binding to the major groove of DNA via a short α-helix in the Znf, the Znf spanning 3-4 bases of the DNA []. C2H2 Znf's can also bind to RNA and protein targets [].This entry represents a potential FLYWCH Zn-finger domain found in a number of eukaryotic proteins. FLYWCH is a C2H2-type zinc finger characterised by five conserved hydrophobic residues, containing the conserved sequence motif:F/Y-X(n)-L-X(n)-F/Y-X(n)-WXCX(6-12)CX(17-22)HXHwhere X indicates any amino acid. This domain was first characterised in Drosophila Modifier of mdg4 proteins, Mod(mgd4), putative chromatin modulators involved in higher order chromatin domains. Mod(mdg4) proteins share a common N-terminal BTB/POZ domain, but differ in their C-terminal region, most containing C-terminal FLYWCH zinc finger motifs []. The FLYWCH domain in Mod(mdg4) proteins has a putative role in protein-protein interactions; for example, Mod(mdg4)-67.2 interacts with DNA-binding protein Su(Hw) via its FLYWCH domain.FLYWCH domains have been described in other proteins as well, including suppressor of killer of prune, Su(Kpn), which contains 4 terminal FLYWCH zinc finger motifs in a tandem array and a C-terminal glutathione SH-transferase (GST) domain [].
Ribosomes are the particles that catalyse mRNA-directed protein synthesis in all organisms. The codons of the mRNA are exposed on the ribosome to allow tRNA binding. This leads to the incorporation of amino acids into the growing polypeptide chain in accordance with the genetic information. Incoming amino acid monomers enter the ribosomal A site in the form of aminoacyl-tRNAs complexed with elongation factor Tu (EF-Tu) and GTP. The growing polypeptide chain, situated in the P site as peptidyl-tRNA, is then transferred to aminoacyl-tRNA and the new peptidyl-tRNA, extended by one residue, is translocated to the P site with the aid the elongation factor G (EF-G) and GTP as the deacylated tRNA is released from the ribosome through one or more exit sites [, ]. About 2/3 of the mass of the ribosome consists of RNA and 1/3 of protein. The proteins are named in accordance with the subunit of the ribosome which they belong to - the small (S1 to S31) and the large (L1 to L44). Usually they decorate the rRNA cores of the subunits. Many ribosomal proteins, particularly those of the large subunit, are composed of a globular, surfaced-exposed domain with long finger-like projections that extend into the rRNA core to stabilise its structure. Most of the proteins interact with multiple RNA elements, often from different domains. In the large subunit, about 1/3 of the 23S rRNA nucleotides are at least in van der Waal's contact with protein, and L22 interacts with all six domains of the 23S rRNA. Proteins S4 and S7, which initiate assembly of the 16S rRNA, are located at junctions of five and four RNA helices, respectively. In this way proteins serve to organise and stabilise the rRNA tertiary structure. While the crucial activities of decoding and peptide transfer are RNA based, proteins play an active role in functions that may have evolved to streamline the process of protein synthesis. In addition to their function in the ribosome, many ribosomal proteins have some function 'outside' the ribosome [, ].L6 is a protein from the large (50S) subunit. In Escherichia coli, it is located in the aminoacyl-tRNA bindingsite of the peptidyltransferase centre, and is known to bind directly to 23S rRNA. It belongs to a family of ribosomal proteins, including L6 from bacteria, cyanelles (structures that perform similar functions to chloroplasts, but have structural and biochemical characteristics of Cyanobacteria) and mitochondria; and L9 from mammals, Drosophila, plants and yeast. L6 contains two domains with almost identical folds, suggesting that is was derived by the duplication of anancient RNA-binding protein gene. Analysis reveals several sites on the protein surface where interactions with other ribosome components may occur, the N terminus being involved in protein-protein interactions and the C terminus containing possible RNA-binding sites [].The spinach plastid 50S subunit comprises 33 proteins, of which 31 are orthologues of Escherichia coli ribosomal proteins and two are plastid-specific ribosomal proteins (PSRP-5 and PSRP-6) having no homologues in other types of ribosomes [].
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and therapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The first EAG K+ channel was identified in Drosophila melanogaster (Fruit fly), following a screen for mutations giving rise to behavioural abnormalities. Disruption of the Eag gene caused an ether-induced, leg-shaking behaviour. Subsequent studies have revealed a conserved multi-gene family of EAG-like K+ channels, which are present in human and many other species. Based on the varying functional properties of the channels, the family has been divided into 3 subfamilies: EAG, ELK and ERG. Interestingly, Caenorhabditis elegans appears to lack the ELK type [].The human ether-a-go-go-related gene (HERG), cloned from hippocampus, shares 49% amino acid identity with EAG. It is also found in the heart, where it helps to control K+ efflux []. Mutations in HERG result in the disruption of the repolarising current and the disease LQT2 syndrome, an inheriteddisorder of cardiac repolarisation that predisposes affected individuals tolife-threatening arrhythmias [].
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The first EAG K+ channel was identified in Drosophila melanogaster (Fruit fly), following a screen for mutations giving rise to behavioural abnormalities. Disruption of the Eag gene caused an ether-induced, leg-shaking behaviour. Subsequent studies have revealed a conserved multi-gene family of EAG-like K+ channels, which are present in human and many other species. Based on the varying functional properties of the channels, the family has been divided into 3 subfamilies: EAG, ELK and ERG. Interestingly, Caenorhabditis elegans appears to lack the ELK type [].The EAG subfamily has been expressed in heterologous systems to reveal their biophysical and pharmacological functions and to determine their currents in native tissues. All mammalian EAG subfamily K+ channels that have been identified have properties typical of delayed rectifiers, with no measurable inactivation []. Only the Drosophila melanogaster Eag channel exhibits partial inactivation.
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The first EAG K+ channel was identified in Drosophila melanogaster (Fruit fly), following a screen for mutations giving rise to behavioural abnormalities. Disruption of the Eag gene caused an ether-induced, leg-shaking behaviour. Subsequent studies have revealed a conserved multi-gene family of EAG-like K+ channels, which are present in human and many other species. Based on the varying functional properties of the channels, the family has been divided into 3 subfamilies: EAG, ELK and ERG. Interestingly, Caenorhabditis elegans appears to lack the ELK type [].
Ca2+ ions are unique in that they not only carry charge but they are also the most widely used of diffusible second messengers. Voltage-dependent Ca2+ channels (VDCC) are a family of molecules that allow cells to couple electrical activity to intracellular Ca2+ signalling. The opening and closing of these channels by depolarizing stimuli, such as action potentials, allows Ca2+ ions to enter neurons down a steep electrochemical gradient, producing transient intracellular Ca2+ signals. Many of the processes that occur in neurons, including transmitter release, gene transcription and metabolism are controlled by Ca2+ influx occurring simultaneously at different cellular locales. The pore is formed by the alpha-1 subunit which incorporates the conduction pore, the voltage sensor and gating apparatus, and the known sites of channel regulation by second messengers, drugs, and toxins []. The activity of this pore is modulated by four tightly-coupled subunits: an intracellular beta subunit; a transmembrane gamma subunit; and a disulphide-linked complex of alpha-2 and delta subunits, which are proteolytically cleaved from the same gene product. Properties of the protein including gating voltage-dependence, G protein modulation and kinase susceptibility can be influenced by these subunits.Voltage-gated calcium channels are classified as T, L, N, P, Q and R, and are distinguished by their sensitivity to pharmacological blocks, single-channel conductance kinetics, and voltage-dependence. On the basis of their voltage activation properties, the voltage-gated calcium classes can be further divided into two broad groups: the low (T-type) and high (L, N, P, Q and R-type) threshold-activated channels.The voltage-dependent calcium channel gamma (VDCCG) subunit family consistsof at least 8 members, which share a number of common structural features[]. Each member is predicted to possess 4 transmembrane domains, with intracellular N- and C-termini. The first extracellular loop contains a highly conserved N-glycosylation site and a pair of conserved cysteine residues. The C-terminal 7 residues of VDCCG-2, -3, -4 and -8 are also conserved andcontain a consensus site for phosphorylation by cAMP and cGMP-dependentprotein kinases, and a target site for binding by PDZ domain proteins [].The VDCCG-2 subunit (also known as stargazin) was isolated by identifying the locus of the genetic disruption in the epileptic mouse mutant lineknown as stargazer []. VDCCG-2 subunits are brain specific and enriched in synaptic plasma membranes. In vitro studies using recombinant P/Q-type calcium channels show that VDCCG-2 subunit expression increases steady-statechannel inactivation, leading to the suggestion that, in stargazer mutants, inappropriate calcium entry may contribute to the seizure phenotype.VDCCG-2 subunits are also implicated in cellular trafficking. They interact with ionotropic glutamate AMPA receptor subunits, a process that has beenshown to be essential in delivering functional AMPA receptors to the surfacemembranes of cerebellar granule cells []. In addition, VDCCG-2 subunits are capable of associating with PDZ proteins, such as PSD-95, through their C-terminal PDZ binding domains. This interaction is required to target AMPAreceptors to cerebellar synapses.
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.A voltage-dependent potassium channel gene designated Shaw was initially isolated from Drosophila melanogaster (Fruit fly). Subsequently, several vetebrate potassium channels with similar amino acid sequences were found and, together with the D. melanogaster channel, now constitute the Kv3 family. These channels are thought to play a role in shortening of action potential durations and modulating pre-synaptic neurotransmitter release. In mammals, the family consists of 4 genes (Kv3.1, Kv3.2, Kv3.3 and Kv3.4). Each gene product has its own subcellular location and function.
ABC transporters belong to the ATP-Binding Cassette (ABC) superfamily, which uses the hydrolysis of ATP to energise diverse biological systems. ABC transporters minimally consist of two conserved regions: a highly conserved ATP binding cassette (ABC) and a less conserved transmembrane domain (TMD). These can be found on the same protein or on two different ones. Most ABC transporters function as a dimer and therefore are constituted of four domains, two ABC modules and two TMDs.ABC transporters are involved in the export or import of a wide variety of substrates ranging from small ions to macromolecules. The major function of ABC import systems is to provide essential nutrients to bacteria. They are found only in prokaryotes and their four constitutive domains are usually encoded by independent polypeptides (two ABC proteins and two TMD proteins). Prokaryotic importers require additional extracytoplasmic binding proteins (one or more per systems) for function. In contrast, export systems are involved in the extrusion of noxious substances, the export of extracellular toxins and the targeting of membrane components. They are found in all living organisms and in general the TMD is fused to the ABC module in a variety of combinations. Some eukaryotic exporters encode the four domains on the same polypeptide chain [].The ABC module (approximately two hundred amino acid residues) is known to bind and hydrolyse ATP, thereby coupling transport to ATP hydrolysis in a large number of biological processes. The cassette is duplicated in several subfamilies. Its primary sequence is highly conserved, displaying a typical phosphate-binding loop: Walker A, and a magnesium binding site: Walker B. Besides these two regions, three other conserved motifs are present in the ABC cassette: the switch region which contains a histidine loop, postulated to polarise the attaching water molecule for hydrolysis, the signature conserved motif (LSGGQ) specific to the ABC transporter, and the Q-motif (between Walker A and the signature), which interacts with the gamma phosphate through a water bond. The Walker A, Walker B, Q-loop and switch region form the nucleotide binding site [, , ].The 3D structure of a monomeric ABC module adopts a stubby L-shape with two distinct arms. ArmI (mainly β-strand) contains Walker A and Walker B. The important residues for ATP hydrolysis and/or binding are located in the P-loop. The ATP-binding pocket is located at the extremity of armI. The perpendicular armII contains mostly the alpha helical subdomain with the signature motif. It only seems to be required for structural integrity of the ABC module. ArmII is in direct contact with the TMD. The hinge between armI and armII contains both the histidine loop and the Q-loop, making contact with the gamma phosphate of the ATP molecule. ATP hydrolysis leads to a conformational change that could facilitate ADP release. In the dimer the two ABC cassettes contact each other through hydrophobic interactions at the antiparallel β-sheet of armI by a two-fold axis [, , , , , ].The ATP-Binding Cassette (ABC) superfamily forms one of the largest of all protein families with a diversity of physiological functions []. Several studies have shown that there is a correlation between the functional characterisation and the phylogenetic classification of the ABC cassette [, ]. More than 50 subfamilies have been described based on a phylogenetic and functional classification [, , ].This family consists of multi drug resistance-associated protein (MRP) in eukaryotes [, , ]. The multidrug resistance-associated protein is an integral membrane protein that causes multidrug resistance when overexpressed in mammalian cells. It belongs to the ABC transporter superfamily. The protein topology and function was experimentally demonstrated by epitope tagging and immunofluorescence. Insertion of tags in the critical regions associated with drug efflux reduced its function. The C-terminal domain seems to be highly conserved.
Helicases have been classified in 5 superfamilies (SF1-SF5). All of the proteins bind ATP and, consequently, all of them carry the classical Walker A(phosphate-binding loop or P-loop) and Walker B(Mg2+-binding aspartic acid) motifs. For the two largest groups, commonlyreferred to as SF1 and SF2, a total of seven characteristic motifs has beenidentified []. These two superfamilies encompass a large number of DNA andRNA helicases from archaea, eubacteria, eukaryotes and viruses that seem to beactive as monomers or dimers. RNA and DNA helicases are considered to beenzymes that catalyze the separation of double-stranded nucleic acids in anenergy-dependent manner [].The various structures of SF1 and SF2 helicases present a common core with twoα-β RecA-like domains [, ]. Thestructural homology with the RecA recombination protein covers the fivecontiguous parallel beta strands and the tandem alpha helices. ATP binds tothe amino proximal α-β domain, where the Walker A (motif I) and WalkerB (motif II) are found. The N-terminal domain also contains motif III (S-A-T)which was proposed to participate in linking ATPase and helicase activities.The carboxy-terminal α-β domain is structurally very similar to theproximal one even though it is bereft of an ATP-binding site, suggesting thatit may have originally arisen through gene duplication of the first one.Some members of helicase superfamilies 1 and 2 are listed below:DEAD-box RNA helicases. The prototype of DEAD-boxproteins is the translation initiation factor eIF4A. The eIF4A protein isan RNA-dependent ATPase which functions together with eIF4B as an RNAhelicase [].DEAH-box RNA helicases. Mainly pre-mRNA-splicing factorATP-dependent RNA helicases [].Eukaryotic DNA repair helicase RAD3/ERCC-2, an ATP-dependent 5'-3' DNAhelicase involved in nucleotide excision repair of UV-damaged DNA.Eukaryotic TFIIH basal transcription factor complex helicase XPB subunit.An ATP-dependent 3'-5' DNA helicase which is a component of the core-TFIIHbasal transcription factor, involved in nucleotide excision repair (NER) ofDNA and, when complexed to CAK, in RNA transcription by RNA polymerase II.It acts by opening DNA either around the RNA transcription start site orthe DNA.Eukaryotic ATP-dependent DNA helicase Q. A DNA helicase that may play arole in the repair of DNA that is damaged by ultraviolet light or othermutagens.Bacterial and eukaryotic antiviral SKI2-like helicase. SKI2 has a role inthe 3'-mRNA degradation pathway, repressing dsRNA virus propagation byspecifically blocking translation of viral mRNAs, perhaps recognizing theabsence of CAP or poly(A).Bacterial DNA-damage-inducible protein G (DinG). A probable helicaseinvolved in DNA repair and perhaps also replication [].Bacterial primosomal protein N' (PriA). PriA protein is one of sevenproteins that make up the restart primosome, an apparatus that promotesassembly of replisomes at recombination intermediates and stalledreplication forks.Bacterial ATP-dependent DNA helicase recG. It has a critical role inrecombination and DNA repair, helping process Holliday junctionintermediates to mature products by catalyzing branch migration. It has aDNA unwinding activity characteristic of helicases with a 3' to 5'polarity.A variety of DNA and RNA virus helicases and transcription factorsThis entry includes bacterial DinG and eukaryotic Rad3 proteins, differing from other SF1 and SF2 helicases by the presence of a large insert after the Walker A motif [].
Ribosomes are the particles that catalyse mRNA-directed protein synthesis in all organisms. The codons of the mRNA are exposed on the ribosome to allow tRNA binding. This leads to the incorporation of amino acids into the growing polypeptide chain in accordance with the genetic information. Incoming amino acid monomers enter the ribosomal A site in the form of aminoacyl-tRNAs complexed with elongation factor Tu (EF-Tu) and GTP. The growing polypeptide chain, situated in the P site as peptidyl-tRNA, is then transferred to aminoacyl-tRNA and the new peptidyl-tRNA, extended by one residue, is translocated to the P site with the aid the elongation factor G (EF-G) and GTP as the deacylated tRNA is released from the ribosome through one or more exit sites [, ]. About 2/3 of the mass of the ribosome consists of RNA and 1/3 of protein. The proteins are named in accordance with the subunit of the ribosome which they belong to - the small (S1 to S31) and the large (L1 to L44). Usually they decorate the rRNA cores of the subunits. Many ribosomal proteins, particularly those of the large subunit, are composed of a globular, surfaced-exposed domain with long finger-like projections that extend into the rRNA core to stabilise its structure. Most of the proteins interact with multiple RNA elements, often from different domains. In the large subunit, about 1/3 of the 23S rRNA nucleotides are at least in van der Waal's contact with protein, and L22 interacts with all six domains of the 23S rRNA. Proteins S4 and S7, which initiate assembly of the 16S rRNA, are located at junctions of five and four RNA helices, respectively. In this way proteins serve to organise and stabilise the rRNA tertiary structure. While the crucial activities of decoding and peptide transfer are RNA based, proteins play an active role in functions that may have evolved to streamline the process of protein synthesis. In addition to their function in the ribosome, many ribosomal proteins have some function 'outside' the ribosome [, ].L6 is a protein from the large (50S) subunit. In Escherichia coli, it is located in the aminoacyl-tRNA bindingsite of the peptidyltransferase centre, and is known to bind directly to 23S rRNA. It belongs to a family of ribosomal proteins, including L6 from bacteria, cyanelles (structures that perform similar functions to chloroplasts, but have structural and biochemical characteristics of Cyanobacteria) and mitochondria; and L9 from mammals, Drosophila, plants and yeast. L6 contains two domains with almost identical folds, suggesting that is was derived by the duplication of anancient RNA-binding protein gene. Analysis reveals several sites on the protein surface where interactions with other ribosome components may occur, the N terminus being involved in protein-protein interactions and the C terminus containing possible RNA-binding sites [].
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.KCNQ channels (also known as KQT-like channels) differ from other voltage-gated 6 TM helix channels, chiefly in that they possess no tetramerisation domain. Consequently, they rely on interaction with accessory subunits, or form heterotetramers with other members of the family []. Currently, 5 members of the KCNQ family are known. These have been found to be widely distributed within the body, having been shown to be expressed in the heart, brain, pancreas, lung, placenta and ear. They were initially cloned as a result of a search for proteins involved in cardiac arhythmia. Subsequently, mutations in other KCNQ family members have been shown to be responsible for some forms of hereditary deafness []and benign familial neonatal epilepsy [].KCNQ1 was the first member of the KCNQ channel family to be isolated, and has been found to be the most common cause of the disease `long QT syndrome' a cardiac arhythmia resulting in a prolonged QT interval. In exceptional cases, this can lead to sudden death, triggered by extreme stress [, ]. KCNQ1 is expressed in the stria vascularis of the inner ear, and may be the cause of hereditary deafness [].
Zinc finger (Znf) domains are relatively small protein motifs which contain multiple finger-like protrusions that make tandem contacts with their target molecule. Some of these domains bind zinc, but many do not; instead binding other metals such as iron, or no metal at all. For example, some family members form salt bridges to stabilise the finger-like folds. They were first identified as a DNA-binding motif in transcription factor TFIIIA from Xenopus laevis (African clawed frog), however they are now recognised to bind DNA, RNA, protein and/or lipid substrates [, , , , ]. Their binding properties depend on the amino acid sequence of the finger domains and of the linker between fingers, as well as on the higher-order structures and the number of fingers. Znf domains are often found in clusters, where fingers can have different binding specificities. There are many superfamilies of Znf motifs, varying in both sequence and structure. They display considerable versatility in binding modes, even between members of the same class (e.g. some bind DNA, others protein), suggesting that Znf motifs are stable scaffolds that have evolved specialised functions. For example, Znf-containing proteins function in gene transcription, translation, mRNA trafficking, cytoskeleton organisation, epithelial development, cell adhesion, protein folding, chromatin remodelling and zinc sensing, to name but a few []. Zinc-binding motifs are stable structures, and they rarely undergo conformational changes upon binding their target. This entry represents the zinc finger domain found in the large T-antigen (T-Ag) as the D1 domain. The T-Ag is found in a group of polyomaviruses consisting of the homonymous murine virus (Py) as well as other representative members such as the Simian virus 40 (SV40) and the human BK polyomavirus (BKPyV) and JC polyomavirus (JCPyV) []. Their large T antigen (T-Ag) protein binds to and activates DNA replication from the origin of DNA replication (ori). Insofar as is known, the T-Ag binds to the origin first as a monomer to its pentanucleotide recognition element. The monomers are then thought to assemble into hexamers and double hexamers, which constitute the form that is active in initiation of DNA replication. When bound to the ori, T-Ag double hexamers encircle DNA []. T-Ag is a multi-domain protein that contains an N-terminal J domain, a central origin-binding domain (OBD), and a C-terminal superfamily 3 helicase domain []. The helicase domain actually contains three distinct structural domains: D1 (domain 1), D2 and D3. D1 is the Zn domain at the N terminus and contains five α-helices (alpha1-alpha5). The Zn atom coordinated by a Zn motif is important in holding alpha3 (α-helix 3) and alpha4 together, which in turn provide an anchor for alpha1 and alpha2. The beginning of alpha5 packs with alpha1 and alpha2 of D1, but its C terminus extends to alpha6 of D3. The D2 domain contains three conserved helicase motifs related to SF3 helicases, namely the modified version of Walker A and B motifs and motif C. D2 folds into a core β-sheet consisting of five parallel β-strands sandwiched by α-helices. The third domain, D3, is all α-helical. Its seven α-helices originate from both the N-terminal region (alpha6-alpha8) and the C terminus (alpha13-alpha16), with D2 inserted between [].The Zn motif of T-Ag was proposed to form a canonical zinc-finger structure for DNA binding. However, the Zn domain (D1) has a globular fold stabilised by the coordination of a Zn atom through the Zn motif, and no classical zinc-finger structure specialised for DNA binding is present. The Zn motif is not directly involved in binding DNA but is instead important for stabilising the Zn-domain structure [].
Intercellular adhesion molecules (ICAMs) and vascular cell adhesion molecule-1 (VCAM-1) are part of the immunoglobulin superfamily. They are important in inflammation, immune responses and in intracellular signalling events []. The ICAM family consists of five members, designated ICAM-1 to ICAM-5. They are known to bind to leucocyte integrins CD11/CD18 during inflammation and in immune responses. In addition, ICAMs may exist in soluble forms in human plasma, due to activation and proteolysis mechanisms at cell surfaces.ICAM-1 (CD54) contains five Ig-like domains. It is expressed on leucocytes, endothelial and epithelial cells, and is upregulated in response to bacterial invasion. The protein is a ligand for lymphocyte-function associated (LFA) antigens and also a receptor for CD11a,b/CD18, fibrinogen, human rhinovirus and Plasmodium falciparum-infected erythrocytes. ICAM-1 binding sites for CD11a/CD18 and its other binding partners are located in the first domain and are overlapping. ICAM-1 domain 2 seems to play an important role in maintaining the conformation of domain 1 and particularly the structural integrity of the LFA-1 ligand-binding site [].The 3-dimensional atomic structure of the tandem N-terminal Ig-like domains (D1 and D2) of ICAM-1 has been determined to 2.2A resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus-ICAM-1 complex []. Extensive charge interactions between ICAM-1 and human rhinovirusesare largely conserved in major and minor receptor groups of rhinoviruses. The interaction of ICAMs with LFA-1 is mediated by a divalent cation bound to the insertion (I)-domain on the alpha chain of LFA-1 and the carboxyl group of a conserved glutamic acid residue on ICAMs.ICAM-2 (CD102) has two Ig-like domains. It is expressed on endothelial cells, leucocytes and platelets, and binds to CD11a'b/CD18. The protein is refractory to proinflammatory cytokines, and plays an important role in the adhesion of leucocytes to the uninduced endothelium [].ICAM-3 (CD50) contains five Ig-like domains and binds to leucocyte integrins CD11a'd/CD18. The protein plays an important role in the immune response and perhaps in signal transduction [].ICAM-4 (LW blood group Ag) is red blood cell (RBC) specific and binds to CD11a'b/CD18. It is associated with the RBC Rh antigens and could be important in retaining immature red cells in the bone marrow, or in the uptake of senescent cells into the spleen [].ICAM-5 (telencephalin) has nine Ig-like domains and is confined to the telencephalon of the brain. The role of this CD11a/CD18 binding molecule is not yet known [].VCAM-1 was first described as a cytokine-inducible endothelial adhesion molecule. It can bind to leucocyte integrin VL-4 (very late antigen-4) to recruit leucocytes to sites of inflammation []. The predominant form of VCAM-1 in vivo has an N-terminal extracellular region comprising seven Ig-like domains []. A conserved integrin-binding motif has been identified in domains 1 and 4, variants of which are present in the N-terminal domain of all members of the integrin-binding subgroup of the immunoglobulin superfamily. The structure of a VLA-4-binding fragment comprising the first two domains of VCAM-1 has been determined to 1.8A resolution. The integrin-binding motif is exposed and forms the N-terminal region of the loop between β-strands C and D of domain 1 []. VCAM-1 domains 1 and 2 are structurally similar to ICAM-1 and ICAM-2 [].Regulation of binding between integrins and their ICAM partners influences the ability of leukocytes interact with neurons [], to cross the blood-brain barrier [], to migrate through the vasculature [], and to interact with antigen-presenting cells []. This entry represents ICAM-2, a close relative of ICAM-1, which shares shares LFA-1, a leukocyte-expressed integrin, as its binding partner []. The two proteins have substantially overlapping functions.
Globins are haem-containing proteins involved in binding and/or transporting oxygen. They belong to a very large and well studied family that is widely distributed in many organisms []. Globins have evolved from a common ancestor and can be divided into three groups: single-domain globins, and two types of chimeric globins, flavohaemoglobins and globin-coupled sensors. Bacteria have all three types of globins, while archaea lack flavohaemoglobins, and eukaryotes lack globin-coupled sensors []. Several functionally different haemoglobins can coexist in the same species. The major types of globins include:Haemoglobin (Hb): tetramer of two alpha and two beta chains, although embryonic and foetal forms can substitute the alpha or beta chain for ones with higher oxygen affinity, such as gamma, delta, epsilon or zeta chains. Hb transports oxygen from lungs to other tissues in vertebrates []. Hb proteins are also present in unicellular organisms where they act as enzymes or sensors [].Myoglobin (Mb): monomeric protein responsible for oxygen storage in vertebrate muscle [].Neuroglobin: a myoglobin-like haemprotein expressed in vertebrate brain and retina, where it is involved in neuroprotection from damage due to hypoxia or ischemia []. Neuroglobin belongs to a branch of the globin family that diverged early in evolution. Cytoglobin: an oxygen sensor expressed in multiple tissues. Related to neuroglobin [].Erythrocruorin: highly cooperative extracellular respiratory proteins found in annelids and arthropods that are assembled from as many as 180 subunit into hexagonal bilayers [].Leghaemoglobin (legHb or symbiotic Hb): occurs in the root nodules of leguminous plants, where it facilitates the diffusion of oxygen to symbiotic bacteriods in order to promote nitrogen fixation.Non-symbiotic haemoglobin (NsHb): occurs in non-leguminous plants, and can be over-expressed in stressed plants [].Flavohaemoglobins (FHb): chimeric, with an N-terminal globin domain and a C-terminal ferredoxin reductase-like NAD/FAD-binding domain. FHb provides protection against nitric oxide via its C-terminal domain, which transfers electrons to haem in the globin [].Globin-coupled sensors: chimeric, with an N-terminal myoglobin-like domain and a C-terminal domain that resembles the cytoplasmic signalling domain of bacterial chemoreceptors. They bind oxygen, and act to initiate an aerotactic response or regulate gene expression [, ]. Protoglobin: a single domain globin found in archaea that is related to the N-terminal domain of globin-coupled sensors [].Truncated 2/2 globin: lack the first helix, giving them a 2-over-2 instead of the canonical 3-over-3 α-helical sandwich fold. Can be divided into three main groups (I, II and II) based on structural features [].Leghaemoglobins are haem-proteins, first identified in root nodules of leguminous plants, where they are crucial for supplying sufficient oxygen to root nodule bacteria for nitrogen fixation to occur [, ]. Although leghaemoglobin and myoglobin both share a common fold, and both regulate the facilitated diffusion of oxygen, leghemoglobins regulate oxygen affinity through a mechanism different from that of myoglobin using a novel combination of haem pocket amino acids that lower the oxygen affinity [, ]. The structure of leghaemoglobins is similar to that of haemoglobins and myoglobins, although there is little sequence conservation []. The protein is largely α-helical, eight helices providing the scaffold for a well-defined haem-binding pocket []. By contrast with the tetrameric mammalian globin assembly, the plant form is monomeric []. The structural similarity of leghaemoglobins and haemoglobins has suggested a common evolutionary origin. It was thought that haemoglobins may be found in plants other than legumes [], and indeed globins have now been identified in the roots of non-leguminous plants, where they have a role in respiratory metabolism in the root cells []. This entry also represents Non-symbiotic haemoglobins (NsHb) which play important roles in a variety of cellular processes. A class I NsHb from cotton plants can be induced in plant roots as a defence mechanism against pathogen invasions, possibly by modulating nitric oxide (NO) levels []. Several NsHbs appear to play a role NO scavenging in plants, indicating that the primordial function of haemoglobins may well be to protect against nitrosative stress and to modulate NO signalling functions [].
Zinc finger (Znf) domains are relatively small protein motifs which contain multiple finger-like protrusions that make tandem contacts with their target molecule. Some of these domains bind zinc, but many do not; instead binding other metals such as iron, or no metal at all. For example, some family members form salt bridges to stabilise the finger-like folds. They were first identified as a DNA-binding motif in transcription factor TFIIIA from Xenopus laevis (African clawed frog), however they are now recognised to bind DNA, RNA, protein and/or lipid substrates [, , , , ]. Their binding properties depend on the amino acid sequence of the finger domains and of the linker between fingers, as well as on the higher-order structures and the number of fingers. Znf domains are often found in clusters, where fingers can have different binding specificities. There are many superfamilies of Znf motifs, varying in both sequence and structure. They display considerable versatility in binding modes, even between members of the same class (e.g. some bind DNA, others protein), suggesting that Znf motifs are stable scaffolds that have evolved specialised functions. For example, Znf-containing proteins function in gene transcription, translation, mRNA trafficking, cytoskeleton organisation, epithelial development, cell adhesion, protein folding, chromatin remodelling and zinc sensing, to name but a few []. Zinc-binding motifs are stable structures, and they rarely undergo conformational changes upon binding their target. This entry represents the zinc finger domain superfamily found in the large T-antigen (T-Ag) as the D1 domain. The T-Ag is found in a group of polyomaviruses consisting of the homonymous murine virus (Py) as well as other representative members such as the Simian virus 40 (SV40) and the human BK polyomavirus (BKPyV) and JC polyomavirus (JCPyV) []. T-antigen and replication protein E1 share the same domain architecture and functionality despite low sequence similarity []. Their large T antigen (T-Ag) protein binds to and activates DNA replication from the origin of DNA replication (ori). Insofar as is known, the T-Ag binds to the origin first as a monomer to its pentanucleotide recognition element. The monomers are then thought to assemble into hexamers and double hexamers, which constitute the form that is active in initiation of DNA replication. When bound to the ori, T-Ag double hexamers encircle DNA []. T-Ag is a multi-domain protein that contains an N-terminal J domain, a central origin-binding domain (OBD), and a C-terminal superfamily 3 helicase domain []. The helicase domain actually contains three distinct structural domains: D1 (domain 1), D2 and D3. D1 is the Zn domain at the N terminus and contains five α-helices (α1-α5). The Zn atom coordinated by a Zn motif is important in holding α3 (α-helix 3) and α4 together, which in turn provide an anchor for α1 and α2. The beginning of α5 packs with α1 and α2 of D1, but its C terminus extends to α6 of D3. The D2 domain contains three conserved helicase motifs related to SF3 helicases, namely the modified version of Walker A and B motifs and motif C. D2 folds into a core β-sheet consisting of five parallel β-strands sandwiched by α-helices. The third domain, D3, is all α-helical. Its seven α-helices originate from both the N-terminal region (α6-α8) and the C terminus (α13-α16), with D2 inserted between [].The Zn motif of T-Ag was proposed to form a canonical zinc-finger structure for DNA binding. However, the Zn domain (D1) has a globular fold stabilised by the coordination of a Zn atom through the Zn motif, and no classical zinc-finger structure specialised for DNA binding is present. The Zn motif is not directly involved in binding DNA but is instead important for stabilising the Zn-domain structure [].
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.BK channels (also referred to as high-conductance, maxi-K channels or Slo family channels) []are widely expressed in the body, being found in glandular tissue, smooth and skeletal muscle, as well as in neural tissues. They have been demonstrated to regulate arteriolar and airway diameter, and also neurotransmitter release. Each channel complex is thought to be composed of 2 types of subunit; the pore-forming (alpha) subunits and smaller accessory (beta) subunits.The alpha subunit of the BK channel was initially thought to share the characteristic 6TM organisation of the voltage-gated K+ channels. However, the molecule is now thought to possess an additional TM domain, with an extracellular N terminus and intracellular C terminus. This C-terminal region contains 4 predominantly hydrophobic domains, which are also thought to lie intracellularly. The extracellular N terminus and the first TM region are required for modulation by the beta subunit. The precise location of the Ca2+-binding site that modulates channel activation remains unknown, but it is thought to lie within the C-terminal hydrophobic domains.The sodium-activated potassium channels Slick (Slo2.1, KCNT2) and Slack (Slo2.2, KCNT1) belong to the structurally related high-conductance potassium channels of the Slo family []. Slo3, also a member of the Slo family, is exclusively expressed in mammalian sperm [].
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The first EAG K+ channel was identified in Drosophila melanogaster (Fruit fly), following a screen for mutations giving rise to behavioural abnormalities. Disruption of the Eag gene caused an ether-induced, leg-shaking behaviour. Subsequent studies have revealed a conserved multi-gene family of EAG-like K+ channels, which are present in human and many other species. Based on the varying functional properties of the channels, the family has been divided into 3 subfamilies: EAG, ELK and ERG. Interestingly, Caenorhabditis elegans appears to lack the ELK type [].Little is known about the properties of channels of the ELK subfamily. However, when expressed in frog oocytes, they show properties between thoseof the EAG and ERG subtypes. Included in this family are Bec1 and Bec2,brain-specific genes found in the human telencephalon regions. It is thoughtthat they are involved in cellular excitability of restricted neurons in thehuman central nervous system. Phylogenetic analysis reveals that these genesconstitute a subfamily with Elk within the Eag family []. Recently, afurther Elk subfamily member has been identified in the mouse (Melk). On thebasis of sequence similarity, this indicates a distinct subclass within this family [].
Intercellular adhesion molecules (ICAMs) and vascular cell adhesion molecule-1 (VCAM-1) are part of the immunoglobulin superfamily. They are important in inflammation, immune responses and in intracellular signalling events []. The ICAM family consists of five members, designated ICAM-1 to ICAM-5. They are known to bind to leucocyte integrins CD11/CD18 during inflammation and in immune responses. In addition, ICAMs may exist in soluble forms in human plasma, due to activation and proteolysis mechanisms at cell surfaces.ICAM-1 (CD54) contains five Ig-like domains. It is expressed on leucocytes, endothelial and epithelial cells, and is upregulated in response to bacterial invasion. The protein is a ligand for lymphocyte-function associated (LFA) antigens and also a receptor for CD11a,b/CD18, fibrinogen, human rhinovirus and Plasmodium falciparum-infected erythrocytes. ICAM-1 binding sites for CD11a/CD18 and its other binding partners are located in the first domain and are overlapping. ICAM-1 domain 2 seems to play an important role in maintaining the conformation of domain 1 and particularly the structural integrity of the LFA-1 ligand-binding site [].The 3-dimensional atomic structure of the tandem N-terminal Ig-like domains (D1 and D2) of ICAM-1 has been determined to 2.2A resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus-ICAM-1 complex []. Extensive charge interactions between ICAM-1 and human rhinovirusesare largely conserved in major and minor receptor groups of rhinoviruses. The interaction of ICAMs with LFA-1 is mediated by a divalent cation bound to the insertion (I)-domain on the alpha chain of LFA-1 and the carboxyl group of a conserved glutamic acid residue on ICAMs.ICAM-2 (CD102) has two Ig-like domains. It is expressed on endothelial cells, leucocytes and platelets, and bindsto CD11a'b/CD18. The protein is refractory to proinflammatory cytokines, and plays an important role in the adhesion of leucocytes to the uninduced endothelium [].ICAM-3 (CD50) contains five Ig-like domains and binds to leucocyte integrins CD11a'd/CD18. The protein plays an important role in the immune response and perhaps in signal transduction [].ICAM-4 (LW blood group Ag) is red blood cell (RBC) specific and binds to CD11a'b/CD18. It is associated with the RBC Rh antigens and could be important in retaining immature red cells in the bone marrow, or in the uptake of senescent cells into the spleen [].ICAM-5 (telencephalin) has nine Ig-like domains and is confined to the telencephalon of the brain. The role of this CD11a/CD18 binding molecule is not yet known [].VCAM-1 was first described as a cytokine-inducible endothelial adhesion molecule. It can bind to leucocyte integrin VL-4 (very late antigen-4) to recruit leucocytes to sites of inflammation []. The predominant form of VCAM-1 in vivo has an N-terminal extracellular region comprising seven Ig-like domains []. A conserved integrin-binding motif has been identified in domains 1 and 4, variants of which are present in the N-terminal domain of all members of the integrin-binding subgroup of the immunoglobulin superfamily. The structure of a VLA-4-binding fragment comprising the first two domains of VCAM-1 has been determined to 1.8A resolution. The integrin-binding motif is exposed and forms the N-terminal region of the loop between β-strands C and D of domain 1 []. VCAM-1 domains 1 and 2 are structurally similar to ICAM-1 and ICAM-2 [].This entry is specific to members of the ICAM family.
Intercellular adhesion molecules (ICAMs) and vascular cell adhesion molecule-1 (VCAM-1) are part of the immunoglobulin superfamily. They are important in inflammation, immune responses and in intracellular signalling events []. The ICAM family consists of five members, designated ICAM-1 to ICAM-5. They are known to bind to leucocyte integrins CD11/CD18 during inflammation and in immune responses. In addition, ICAMs may exist in soluble forms in human plasma, due to activation and proteolysis mechanisms at cell surfaces.ICAM-1 (CD54) contains five Ig-like domains. It is expressed on leucocytes, endothelial and epithelial cells, and is upregulated in response to bacterial invasion. The protein is a ligand for lymphocyte-function associated (LFA) antigens and also a receptor for CD11a,b/CD18, fibrinogen, human rhinovirus and Plasmodium falciparum-infected erythrocytes. ICAM-1 binding sites for CD11a/CD18 and its other binding partners are located in the first domain and are overlapping. ICAM-1 domain 2 seems to play an important role in maintaining the conformation of domain 1 and particularly the structural integrity of the LFA-1 ligand-binding site [].The 3-dimensional atomic structure of the tandem N-terminal Ig-like domains (D1 and D2) of ICAM-1 has been determined to 2.2A resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus-ICAM-1 complex []. Extensive charge interactions between ICAM-1 and human rhinovirusesare largely conserved in major and minor receptor groups of rhinoviruses. The interaction of ICAMs with LFA-1 is mediated by a divalent cation bound to the insertion (I)-domain on the alpha chain of LFA-1 and the carboxyl group of a conserved glutamic acid residue on ICAMs.ICAM-2 (CD102) has two Ig-like domains. It is expressed on endothelial cells, leucocytes and platelets, and binds to CD11a'b/CD18. The protein is refractory to proinflammatory cytokines, and plays an important role in the adhesion of leucocytes to the uninduced endothelium [].ICAM-3 (CD50) contains five Ig-like domains and binds to leucocyte integrins CD11a'd/CD18. The protein plays an important role in the immune response and perhaps in signal transduction [].ICAM-4 (LW blood group Ag) is red blood cell (RBC) specific and binds to CD11a'b/CD18. It is associated with the RBC Rh antigens and could be important in retaining immature red cells in the bone marrow, or in the uptake of senescent cells into the spleen [].ICAM-5 (telencephalin) has nine Ig-like domains and is confined to the telencephalon of the brain. The role of this CD11a/CD18 binding molecule is not yet known [].VCAM-1 was first described as a cytokine-inducible endothelial adhesion molecule. It can bind to leucocyte integrin VL-4 (very late antigen-4) to recruit leucocytes to sites of inflammation []. The predominant form of VCAM-1 in vivo has an N-terminal extracellular region comprising seven Ig-like domains []. A conserved integrin-binding motif has been identified in domains 1 and 4, variants of which are present in the N-terminal domain of all members of the integrin-binding subgroup of the immunoglobulin superfamily. The structure of a VLA-4-binding fragment comprising the first two domains of VCAM-1 has been determined to 1.8A resolution. The integrin-binding motif is exposed and forms the N-terminal region of the loop between β-strands C and D of domain 1 []. VCAM-1 domains 1 and 2 are structurally similar to ICAM-1 and ICAM-2 [].This entry represents the N-terminal domain of both intercellular adhesion molecules (ICAM) and of vascular cell adhesion molecules (VCAM), which are structurally similar.
Intercellular adhesion molecules (ICAMs) and vascular cell adhesion molecule-1 (VCAM-1) are part of the immunoglobulin superfamily. They are important in inflammation, immune responses and in intracellular signalling events []. The ICAM family consists of five members, designated ICAM-1 to ICAM-5. They are known to bind to leucocyte integrins CD11/CD18 during inflammation and in immune responses. In addition, ICAMs may exist in soluble forms in human plasma, due to activation and proteolysis mechanisms at cell surfaces.ICAM-1 (CD54) contains five Ig-like domains. It is expressed on leucocytes, endothelial and epithelial cells, and is upregulated in response to bacterial invasion. The protein is a ligand for lymphocyte-function associated (LFA) antigens and also a receptor for CD11a,b/CD18, fibrinogen, human rhinovirus and Plasmodium falciparum-infected erythrocytes. ICAM-1 binding sites for CD11a/CD18 and its other binding partners are located in the first domain and are overlapping. ICAM-1 domain 2 seems to play an important role in maintaining the conformation of domain 1 and particularly the structural integrity of the LFA-1 ligand-binding site [].The 3-dimensional atomic structure of the tandem N-terminal Ig-like domains (D1 and D2) of ICAM-1 has been determined to 2.2A resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus-ICAM-1 complex []. Extensive charge interactions between ICAM-1 and human rhinovirusesare largely conserved inmajor and minor receptor groups of rhinoviruses. The interaction of ICAMs with LFA-1 is mediated by a divalent cation bound to the insertion (I)-domain on the alpha chain of LFA-1 and the carboxyl group of a conserved glutamic acid residue on ICAMs.ICAM-2 (CD102) has two Ig-like domains. It is expressed on endothelial cells, leucocytes and platelets, and binds to CD11a'b/CD18. The protein is refractory to proinflammatory cytokines, and plays an important role in the adhesion of leucocytes to the uninduced endothelium [].ICAM-3 (CD50) contains five Ig-like domains and binds to leucocyte integrins CD11a'd/CD18. The protein plays an important role in the immune response and perhaps in signal transduction [].ICAM-4 (LW blood group Ag) is red blood cell (RBC) specific and binds to CD11a'b/CD18. It is associated with the RBC Rh antigens and could be important in retaining immature red cells in the bone marrow, or in the uptake of senescent cells into the spleen [].ICAM-5 (telencephalin) has nine Ig-like domains and is confined to the telencephalon of the brain. The role of this CD11a/CD18 binding molecule is not yet known [].VCAM-1 was first described as a cytokine-inducible endothelial adhesion molecule. It can bind to leucocyte integrin VL-4 (very late antigen-4) to recruit leucocytes to sites of inflammation []. The predominant form of VCAM-1 in vivo has an N-terminal extracellular region comprising seven Ig-like domains []. A conserved integrin-binding motif has been identified in domains 1 and 4, variants of which are present in the N-terminal domain of all members of the integrin-binding subgroup of the immunoglobulin superfamily. The structure of a VLA-4-binding fragment comprising the first two domains of VCAM-1 has been determined to 1.8A resolution. The integrin-binding motif is exposed and forms the N-terminal region of the loop between β-strands C and D of domain 1 []. VCAM-1 domains 1 and 2 are structurally similar to ICAM-1 and ICAM-2 [].This entry is specific to VCAM-1 proteins.
The paired domain is an approximately 126 amino acid DNA-binding domain, which is found in eukaryotic transcription regulatory proteins involved in embryogenesis. The domain was originally described as the 'paired box' in the Drosophila protein paired (prd) [, ]. The paired domain is generally located in the N-terminal part. An octapeptide []and/or a homeodomain can occur C-terminal to the paired domain, as well as a Pro-Ser-Thr-rich C terminus.Paired domain proteins can function as transcription repressors or activators. The paired domain contains three subdomains, which show functional differences in DNA-binding. The crystal structures of prd and Pax proteins show that the DNA-bound paired domain is bipartite, consisting of an N-terminal subdomain (PAI or NTD) and a C-terminal subdomain (RED or CTD), connected by a linker. PAI and RED each form a three-helical fold, with the most C-terminal helices comprising a helix-turn-helix (HTH) motif that binds the DNA major groove. In addition, the PAI subdomain encompasses an N-terminal β-turn andβ-hairpin, also named 'wing', participating in DNA-binding. The linker canbind into the DNA minor groove. Different Pax proteins and their alternativelyspliced isoforms use different (sub)domains for DNA-binding to mediate thespecificity of sequence recognition [, ].Some proteins known to contain a paired domain:Drosophila paired (prd), a segmentation pair-rule class protein.Drosophila gooseberry proximal (gsb-p) and gooseberry distal (gsb-d),segmentation polarity class proteins.Drosophila Pox-meso and Pox-neuro proteins.The Pax proteins:Mammalian protein Pax1, which may play a role in the formation of segmented structures in the embryo. In mouse, mutations in Pax1 produce the undulated phenotype, characterised by vertebral malformations along the entire rostro-caudal axis.Mammalian protein Pax2, a probable transcription factor that may have arole in kidney cell differentiation.Mammalian protein Pax3. Pax3 is expressed during early neurogenesis. In humans, defects in Pax3 are the cause of Waardenburg's syndrome (WS), anautosomal dominant combination of deafness and pigmentary disturbance.Mammalian protein Pax4 pays an important role in the differentiation and development of pancreatic islet beta cells. It binds to a common element in the glucagon, insulin and somatostatin promoters. In humans, it has been related to the rare, familial, clinically and genetically heterogeneous form of diabetes MODY (maturity-onset diabetes of the young).Mammalian protein Pax5, also known as B-cell specific transcription factor(BSAP). Pax5 is involved in the regulation of the CD19 gene. It plays animportant role in B-cell differentiation as well as neural development andspermatogenesis.Mammalian protein Pax6 (oculorhombin). Pax6 is a transcription factor withimportant functions in eye and nasal development. In Man, defects in Pax6are the cause of aniridia type II (AN2), an autosomal dominant disordercharacterised by complete or partial absence of the iris.Mammalian protein Pax7 is involved in the regulation of muscle stem cells proliferation, playing a role in myogenesis and muscle regeneration.Mammalian protein Pax8, required in thyroid development.Mammalian protein Pax9, required for normal development of thymus, parathyroid glands, ultimobranchial bodies, teeth, skeletal elements of skull and larynx as well as distal limbs. In man, defects in Pax9 cause oligodontia.Zebrafish protein Paired box protein Pax-2a, involved in the development of the midbrain/hindbrain boundary organizer and specification of neuronal cell fates.Xenopus laevis protein Paired box protein Pax-3-A, which promotes both hatching gland and neural crest cell fates, two of the cell populations that arise from the neural plate border.
Intercellular adhesion molecules (ICAMs) and vascular cell adhesion molecule-1 (VCAM-1) are part of the immunoglobulin superfamily. They are important in inflammation, immune responses and in intracellular signalling events []. The ICAM family consists of five members, designated ICAM-1 to ICAM-5. They are known to bind to leucocyte integrins CD11/CD18 during inflammation and in immune responses. In addition, ICAMs may exist in soluble forms in human plasma, due to activation and proteolysis mechanisms at cell surfaces.ICAM-1 (CD54) contains five Ig-like domains. It is expressed on leucocytes, endothelial and epithelial cells, and is upregulated in response to bacterial invasion. The protein is a ligand for lymphocyte-function associated (LFA) antigens and also a receptor for CD11a,b/CD18, fibrinogen, human rhinovirus and Plasmodium falciparum-infected erythrocytes. ICAM-1 binding sites for CD11a/CD18 and its other binding partners are located in the first domain and are overlapping. ICAM-1 domain 2 seems to play an important role in maintaining the conformation of domain 1 and particularly the structural integrity of the LFA-1 ligand-binding site [].The 3-dimensional atomic structure of the tandem N-terminal Ig-like domains (D1 and D2) of ICAM-1 has been determined to 2.2A resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus-ICAM-1 complex []. Extensive charge interactions between ICAM-1 and human rhinovirusesare largely conserved in major and minor receptor groups of rhinoviruses. The interaction of ICAMs with LFA-1 is mediated by a divalent cation bound to the insertion (I)-domain on the alpha chain of LFA-1 and the carboxyl group of a conserved glutamic acid residue on ICAMs.ICAM-2 (CD102) has two Ig-like domains. It is expressed on endothelial cells, leucocytes and platelets, and binds to CD11a'b/CD18. The protein is refractory to proinflammatory cytokines, and plays an important role in the adhesion of leucocytes to the uninduced endothelium [].ICAM-3 (CD50) contains five Ig-like domains and binds to leucocyte integrins CD11a'd/CD18. The protein plays an important role in the immune response and perhaps in signal transduction [].ICAM-4 (LW blood group Ag) is red blood cell (RBC) specific and binds to CD11a'b/CD18. It is associated with the RBC Rh antigens and could be important in retaining immature red cells in the bone marrow, or in the uptake of senescent cells into the spleen [].ICAM-5 (telencephalin) has nine Ig-like domains and is confined to the telencephalon of the brain. The role of this CD11a/CD18 binding molecule is not yet known [].VCAM-1 was first described as a cytokine-inducible endothelial adhesion molecule. It can bind to leucocyte integrin VL-4 (very late antigen-4) to recruit leucocytes to sites of inflammation []. The predominant form of VCAM-1 in vivo has an N-terminal extracellular region comprising seven Ig-like domains []. A conserved integrin-binding motif has been identified in domains 1 and 4, variants of which are present in the N-terminal domain of all members of the integrin-binding subgroup of the immunoglobulin superfamily. The structure of a VLA-4-binding fragment comprising the first two domains of VCAM-1 has been determined to 1.8A resolution. The integrin-binding motif is exposed and forms the N-terminal region of the loop between β-strands C and D of domain 1 []. VCAM-1 domains 1 and 2 are structurally similar to ICAM-1 and ICAM-2 [].This entry represents the N-terminal domain of ICAM proteins such as ICAM-2, ICAM-3 and ICAM-4.
Members of this family trimethylate 'Lys-9' of histone H3 using monomethylated H3 'Lys-9' as substrate. It also weakly methylates histone H1 (in vitro). H3 'Lys-9' trimethylation represents a specific tag for epigenetic transcriptional repression by recruiting HP1 (CBX1, CBX3 and/or CBX5) proteins to methylated histones. This enzyme mainly functions in heterochromatin regions, thereby playing a central role in the establishment of constitutive heterochromatin at pericentric and telomere regions. H3 'Lys-9' trimethylation is also required to direct DNA methylation at pericentric repeats [, , ]. SUV39H1 (the human ortholog) is targeted to histone H3 via its interaction with RB1 and is involved in many processes, such as repression of MYOD1-stimulated differentiation[], regulation of the control switch for exiting the cell cycle and entering differentiation, repression by the PML-RARA fusion protein [], BMP-induced repression, repression of switch recombination to IgA []and regulation of telomere length [, ]. SUV39H1 is a component of the eNoSC (energy-dependent nucleolar silencing) complex, a complex that mediates silencing of rDNA in response to intracellular energy status and acts by recruiting histone-modifying enzymes. The eNoSC complex is able to sense the energy status of cell: upon glucose starvation, elevation of NAD+/NADP+ ratio activates SIRT1, leading to histone H3 deacetylation followed by dimethylation of H3 at 'Lys-9' (H3K9me2) by SUV39H1 and the formation of silent chromatin in the rDNA locus []. The activity of this enzyme has been mapped to the SET domain and the adjacent cysteine-rich regions []. The SET domain was originally identified in Su(var)3-9, E(z) and Trithorax genes in Drosophila melanogaster (Fruit fly) []. The sequence conservation pattern and structure analysis of the SET domain provides clues regarding the possible active site residues of the domain. There are three conserved sequence motifs in most of the SET domains. The N-terminal motif (I) has characteristic glycines. The central motif (II) has a distinct pattern of polar and charged residues (Asn, His). The C-terminal conserved motif (III) has a characteristic dyad of polar residues. It has been shown that deregulated SUV39H1 interferes at multiple levels with mammalian higher-order chromatin organisation []and these properties depend primarily on the SET domain [, ]. Methyltransferases (EC [intenz:2.1.1.-]) constitute an important class of enzymes present in every life form. They transfer a methyl group most frequently from S-adenosyl L-methionine (SAM or AdoMet) to a nucleophilic acceptor such as oxygen leading to S-adenosyl-L-homocysteine (AdoHcy) and a methylated molecule [, , ]. All these enzymes have in common a conserved region of about 130 amino acid residues that allow them to bind SAM []. The substrates that are methylated by these enzymes cover virtually every kind of biomolecules ranging from small molecules, to lipids, proteins and nucleic acids [, , ]. Methyltransferase are therefore involved in many essential cellular processes including biosynthesis, signal transduction, protein repair, chromatin regulation and gene silencing [, , ]. More than 230 families of methyltransferases have been described so far, of which more than 220 use SAM as the methyl donor.
Neurotransmitter ligand-gated ion channels are transmembrane receptor-ion channel complexes that open transiently upon binding of specific ligands, allowing rapid transmission of signals at chemical synapses [, ]. Five of these ion channel receptor families have been shown to form a sequence-related superfamily:Nicotinic acetylcholine receptor (AchR), an excitatory cation channel in vertebrates and invertebrates; in vertebrate motor endplates it is composed of alpha, beta, gamma and delta/epsilon subunits; in neurons it is composed of alpha and non-alpha (or beta) subunits [].Glycine receptor, an inhibitory chloride ion channel composed of alpha and beta subunits [].Gamma-aminobutyric acid (GABA) receptor, an inhibitory chloride ion channel; at least four types of subunits (alpha, beta, gamma and delta) are known [].Serotonin 5HT3 receptor, of which there are seven major types (5HT3-5HT7) [].Glutamate receptor, an excitatory cation channel of which at least three types have been described (kainate, N-methyl-D-aspartate (NMDA) and quisqualate) [].These receptors possess a pentameric structure (made up of varying subunits), surrounding a central pore. All known sequences of subunits from neurotransmitter-gated ion-channels are structurally related. They are composed of a large extracellular glycosylated N-terminal ligand-binding domain, followed by three hydrophobic transmembrane regions which form the ionic channel, followed by an intracellular region of variable length. A fourth hydrophobic region is found at the C-terminal of the sequence [, ].Gamma-aminobutyric acid type A (GABAA) receptors are members of the neurotransmitter ligand-gated ion channels: they mediate neuronal inhibition on binding GABA. The effects of GABA on GABAA receptors are modulated by a range of therapeutically important drugs, including barbiturates, anaesthetics and benzodiazepines (BZs) []. The BZs are a diverse range of compounds, including widely prescribed drugs, such as librium and valium, and their interaction with GABAA receptors provides the most potent pharmacological means of distinguishing different GABAA receptor subtypes.GABAA receptors are pentameric membrane proteins that operate GABA-gated chloride channels []. Eight types of receptor subunit have been cloned, with multiple subtypes within some classes: alpha 1-6, beta 1-4, gamma 1-4, delta, epsilon, pi, rho 1-3 and theta [, ]. Subunits are typically 50-60kDa in size and comprise a long N-terminal extracellular domain, containing a putative signal peptide and a disulphide-bonded beta structural loop; 4 putative transmembrane (TM) domains; and a large cytoplasmic loop connecting the third and fourth TM domains. Amongst family members, the large cytoplasmic loop displays the most divergence in terms of primary structure, the TM domains showing the highest level of sequence conservation [].Most GABAA receptors contain one type of alpha and beta subunit, and a single gamma polypeptide in a ratio of 2:2:1 [], though in some cases other subunits such as epsilon or delta may replace gamma. The BZ binding site is located at the interface of adjacent alpha and gamma subunits; therefore, the type of alpha and gamma subunits present is instrumental in determining BZ selectivity and sensitivity. Receptors can be categorised into 3 groups based on their alpha subunit content and, hence, sensitivity to BZs: alpha 1-containing receptors have greatest sensitivity towards BZs (type I); alpha 2, 3 and 5-containing receptors have similar but distinguishable properties (type II); and alpha 4- and 6-containing assemblies have very low BZ affinity []. A conserved histidine residue in the alpha subunit of type I and II receptors is believed to be responsible for BZ affinity []. The epsilon subunit was first identified in 1997. Northern blot analysis of several humanbrain tissues showed that epsilon transcripts were relatively enriched inamygdala and thalamus, compared to whole brain, and particularly abundant inthe subthalmic nucleus. Heteromeric recombinant receptors containing theepsilon subunit in place of the more usual gamma subunit were found to be insenstitive to the potentiating effects ofanaesthetic agents [].
Glycine is a major inhibitory neurotransmitter (NT) in the adult vertebratecentral nervous system (CNS). Glycinergic synapses have a well-establishedrole in the processing of motor and sensory information that controlsmovement, vision and audition []. This action of glycine is mediatedthrough its interaction with the glycine receptor (GlyR): an intrinsicchloride channel is opened in response to agonist binding. The subsequentinflux of anions prevents membrane depolarisation and neuronal firinginduced by excitatory NTs. Strychnine acts as a competitive antagonist ofglycine binding, thereby reducing the activity of inhibitory neurones.Poisoning with strychnine is characterised by over-excitation, muscle spasmsand convulsions. Whilst glycine is the principal physiological agonist atGlyRs, taurine and beta-alanine also behave as agonists []. Compounds thatmodulate GlyR activity include zinc, some alcohols and anaesthetics,picrotoxin, cocaine and some anticonvulsants. GlyRs were thought for sometime to be localised exclusively in the brain stem and spinal cord, but havesincebeen found to be expressed more widely, including the cochlear nuclei,cerebellar cortex and forebrain [].GlyRs belong to the ligand-gated ion channel family, which also includes theinhibitory gamma-aminobutyric acid type A (GABAA) and excitatory nicotinicacetylcholine (nACh) and serotonin type 3 (5-HT3) receptors [].Affinity-purified GlyR was found to contain two glycosylated membraneproteins of 48kDa and 56kDa, corresponding to alpha and beta subunits,respectively. Four genes encoding alpha subunits have been identified (GLRA1to 4), together with a single beta polypeptide (GLRB). The heterogeneity ofalpha subunits is further increased by alternative exon splicing, yieldingtwo isoforms of GLRA1 to 3 []. The characteristics of different GlyRsubtypes, therefore, can be largely explained by their GLRA content.GlyRs are generally believed to adopt a pentameric structure in vivo: fivesubunits assemble to form a ring structure with a central pore. Typically, astoichiometry of 3:2 (alpha:beta) is observed []. GlyR subunits share ahigh overall level of sequence similarity both with themselves and with thesubunits of the GABAA and nACh receptors. Four highly conserved segmentshave been proposed to correspond to transmembrane (TM) alpha helices (TM1-4), the second of which is thought to contribute to the pore wall []. A long extracellular N-terminal segment precedes TM1 and a long cytoplasmic loop links TM3 and 4. Short cytoplasmic and extracellular loops join TM1-2 andTM2-3, respectively, and a short C-terminal sequence follows TM4. Studiesusing radiolabelled strychnine have shown the alpha subunit to beresponsible for ligand binding, the critical residues for this interaction lying within the N-terminal domain. The beta subunit plays a structuralrole, contributing one of its TM domains to the pore wall as well as playinga putative role in postsynaptic clustering of the receptor.In several mammalian species, defects in glycinergic transmission areassociated with complex motor disorders. Mutations in the gene encodingGLRA1 give rise to hyperplexia, or startle disease []. This ischaracterised by muscular spasms in response to unexpected light or noisestimuli, similar to the symptoms of sublethal doses of strychnine. Themutations result in amino acid substitutions within the TM1-2 and TM3-4loops, suggesting that these regions are involved in the transduction ofligand binding into channel activation.
Neurotransmitter ligand-gated ion channels are transmembrane receptor-ion channel complexes that open transiently upon binding of specific ligands, allowing rapid transmission of signals at chemical synapses [, ]. Five of these ion channel receptor families have been shown to form a sequence-related superfamily:Nicotinic acetylcholine receptor (AchR), an excitatory cation channel in vertebrates and invertebrates; in vertebrate motor endplates it is composed of alpha, beta, gamma and delta/epsilon subunits; in neurons it is composed of alpha and non-alpha (or beta) subunits [].Glycine receptor, an inhibitory chloride ion channel composed of alpha and beta subunits [].Gamma-aminobutyric acid (GABA) receptor, an inhibitory chloride ion channel; at least four types of subunits (alpha, beta, gamma and delta) are known [].Serotonin 5HT3 receptor, of which there are seven major types (5HT3-5HT7) [].Glutamate receptor, an excitatory cation channel of which at least three types have been described (kainate, N-methyl-D-aspartate (NMDA) and quisqualate) [].These receptors possess a pentameric structure (made up of varying subunits), surrounding a central pore. All known sequences of subunits from neurotransmitter-gated ion-channels are structurally related. They are composed of a large extracellular glycosylated N-terminal ligand-binding domain, followed by three hydrophobic transmembrane regions which form the ionic channel, followed by an intracellular region of variable length. A fourth hydrophobic region is found at the C-terminal of the sequence [, ].Gamma-aminobutyric acid type A (GABAA) receptors are members of the neurotransmitter ligand-gated ion channels: they mediate neuronal inhibition on binding GABA. The effects of GABA on GABAA receptors are modulated by a range of therapeutically important drugs, including barbiturates, anaesthetics and benzodiazepines (BZs) []. The BZs are a diverse range of compounds, including widely prescribed drugs, such as librium and valium, and their interaction with GABAA receptors provides the most potent pharmacological means of distinguishing different GABAA receptor subtypes.GABAA receptors are pentameric membrane proteins that operate GABA-gated chloride channels []. Eight types of receptor subunit have been cloned, with multiple subtypes withinsome classes: alpha 1-6, beta 1-4, gamma 1-4, delta, epsilon, pi, rho 1-3 and theta [, ]. Subunits are typically 50-60kDa in size and comprise a long N-terminal extracellular domain, containing a putative signal peptide and a disulphide-bonded beta structural loop; 4 putative transmembrane (TM) domains; and a large cytoplasmic loop connecting the third and fourth TM domains. Amongst family members, the large cytoplasmic loop displays the most divergence in terms of primary structure, the TM domains showing the highest level of sequence conservation [].Most GABAA receptors contain one type of alpha and beta subunit, and a single gamma polypeptide in a ratio of 2:2:1 [], though in some cases other subunits such as epsilon or delta may replace gamma. The BZ binding site is located at the interface of adjacent alpha and gamma subunits; therefore, the type of alpha and gamma subunits present is instrumental in determining BZ selectivity and sensitivity. Receptors can be categorised into 3 groups based on their alpha subunit content and, hence, sensitivity to BZs: alpha 1-containing receptors have greatest sensitivity towards BZs (type I); alpha 2, 3 and 5-containing receptors have similar but distinguishable properties (type II); and alpha 4- and 6-containing assemblies have very low BZ affinity []. A conserved histidine residue in the alpha subunit of type I and II receptors is believed to be responsible for BZ affinity []. The existence of a pi subunit was first reported in 1997, where it wasdetected in a number of human and rat tissues. The subunit shares 30-40%amino acid identity with other members of the GABAA receptor subunit family.The polypeptide is found in several peripheral tissues, including theuterus, where its function appears to be related to tissue contractility: pisubunits can co-assemble with other GABAA receptor subunits to formrecombinant receptors with altered sensitivity to pregnenalone [].
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.The first Kv1 sequence (also known as Shaker) was found in Drosophila melanogaster (Fruit fly). Several vertebrate potassium channels with similar amino acid sequences were subsequently found and, together with the D. melanogaster Shaker channel, now constitute the Kv1 family. The family consists of at least 6 genes (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5 and Kv1.6) which each play distinct physiological roles. A conserved motif found towards the C terminus of these channels is required for efficient processing and surface expression []. Variations in this motif account for the differences in cell surface expression and localisation between family members. These channels are mostly expressed in the brain, but can also be found in non-excitable cells, such as lymphocytes []. Kv1.2 channels are uniformly distributed in the heart and brain. They play diverse functional roles in several neuronal compartments, especially in the regulation of pre- and post-synaptic membrane excitability. Kv1.2 subunits can co-localise with other Kv1 subunits. For example, Kv1.2 colocalises with Kv1.1 in the nodes of Ranvier in myelinated axons, and in the brain, in particular, the axons and nerve terminals; Kv1.2 coassembles with Kv1.4 subunits. In addition, Kv1.2 assembles with the Kv-beta2 subunit resulting in the promotion of Kv1.2 transport to the cell surface.
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.The first Kv1 sequence (also known as Shaker) was found in Drosophila melanogaster (Fruit fly). Several vertebrate potassium channels with similar amino acid sequences were subsequently found and, together with the D. melanogaster Shaker channel, now constitute the Kv1 family. The family consists of at least 6 genes (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5 and Kv1.6) which each play distinct physiological roles. A conserved motif found towards the C terminus of these channels is required for efficient processing and surface expression []. Variations in this motif account for the differences in cell surface expression and localisation between family members. These channels are mostly expressed in the brain, but can also be found in non-excitable cells, such as lymphocytes []. This entry represents the Kv1 group of voltage-dependent potassium channels.
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.The first Kv1 sequence (also known as Shaker) was found in Drosophila melanogaster (Fruit fly). Several vertebrate potassium channels with similar amino acid sequences were subsequently found and, together with the D. melanogaster Shaker channel, now constitute the Kv1 family. The family consists of at least 6 genes (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5 and Kv1.6) which each play distinct physiological roles. A conserved motif found towards the C terminus of these channels is required for efficient processing and surface expression []. Variations in this motif account for the differences in cell surface expression and localisation between family members. These channels are mostly expressed in the brain, but can also be found in non-excitable cells, such as lymphocytes []. The Kv1.1 subfamily is expressed in the embryonic nervous system, brain andlymphoid thymocyte precursors. The Kv1.1 subunits can associate with Kv1.2 and Kv1.4 subunits, especially in the cerebellum. Point mutations in Kv1.1 result in the disruption of this association and episodic ataxia type I, a rare autosomal dominant neurological disorder characterised by brief episodes of ataxia [].
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.The Shal potassium channel was found in Drosophila melanogaster (Fruit fly). Several vertebrate potassium channels with similar amino acid sequences were subsequently found and, together with the D. melanogaster Shal channel, now constitute the Kv4 family. These channels are the primary subunits contributing to transient, voltage-dependent potassium currents in the nervous system (A currents) and the heart (transient outward current), and are inhibited by free fatty acids []. This family can be further divided into 3 subfamilies, designated Kv4.1(KCND1), Kv4.2(KCND2) and Kv4.3(KCND3).Kv4.1 channels are expressed in the heart, brain, liver, kidney and pancreas. Association with specific subunits increases their surface expression. Essential to their function are the N and C termini, which work together to control the inactivation of these channels.
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form aselective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.The Shal potassium channel was found in Drosophila melanogaster (Fruit fly). Several vertebrate potassium channels with similar amino acid sequences were subsequently found and, together with the D. melanogaster Shal channel, now constitute the Kv4 family. These channels are the primary subunits contributing to transient, voltage-dependent potassium currents in the nervous system (A currents) and the heart (transient outward current), and are inhibited by free fatty acids []. This family can be further divided into 3 subfamilies, designated Kv4.1(KCND1), Kv4.2(KCND2) and Kv4.3(KCND3).Kv4.2 channels are the major contributors to somatodendritic A-type potassium channels in the basal ganglia and basal forebrain neurons []. They are also expressed in the heart and CNS. In rodents, they are an important constituent of the cardiac transient outward current, Ito. In addition, they have a predicted role in regulating neuronal transmission at post-synaptic loci in defined brain regions. Kv4.2 channels contain a number of conserved sites for mitogen-activated protein kinase ERK phosphorylation, suggesting that ERK may regulate potassium channel function by direct phosphorylation [].
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.The first Kv1 sequence (also known as Shaker) was found in Drosophila melanogaster (Fruit fly). Several vertebrate potassium channels with similar amino acid sequences were subsequently found and, together with the D. melanogaster Shaker channel, now constitute the Kv1 family. The family consists of at least 6 genes (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5 and Kv1.6) which each play distinct physiological roles. A conserved motif found towards the C terminus of these channels is required for efficient processing and surface expression []. Variations in this motif account for the differences in cell surface expression and localisation between family members. These channels are mostly expressed in the brain, but can also be found in non-excitable cells, such as lymphocytes []. Kv1.5 channels are expressed in the heart and pancreatic beta-cells. Themost studied is the human Kv1.5 channel (hKv1.5), which is thought to underlie the ultra-rapidly activating delayed rectifier K+current Ikur found in human atrial myocytes []. It is also expressed in the human ventricle where it is possible that it contributes to the K+current through formation of heteromultimeric K+channels with other Kv-alpha subunits []. Further experiments have shown that it is regulated by extracellular potassium and pH [].
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channelactivity, but at lower levels show more specific modulatory actions.The first Kv1 sequence (also known as Shaker) was found in Drosophila melanogaster (Fruit fly). Several vertebrate potassium channels with similar amino acid sequences were subsequently found and, together with the D. melanogaster Shaker channel, now constitute the Kv1 family. The family consists of at least 6 genes (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5 and Kv1.6) which each play distinct physiological roles. A conserved motif found towards the C terminus of these channels is required for efficient processing and surface expression []. Variations in this motif account for the differences in cell surface expression and localisation between family members. These channels are mostly expressed in the brain, but can also be found in non-excitable cells, such as lymphocytes []. KCNA6is expressed in the heart, smooth muscle cells and brain, especiallythe midbrain areas and brainstem. The KCNA6 alpha-subunits can also coassemble with other Shaker alpha subunits, for example, with Kv1.5 to form heteromultimeric K+channels.
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.The first Kv1 sequence (also known as Shaker) was found in Drosophila melanogaster (Fruit fly). Several vertebrate potassium channels with similar amino acid sequences were subsequently found and, together with the D. melanogaster Shaker channel, now constitute the Kv1 family. The family consists of at least 6 genes (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5 and Kv1.6) which each play distinct physiological roles. A conserved motif found towards the C terminus of these channels is required for efficient processing and surface expression []. Variations in this motif account for the differences in cell surface expression and localisation between family members. These channels are mostly expressed in the brain, but can also be found in non-excitable cells, such as lymphocytes []. Kv1.3 subunits are expressed in T-lymphocytes, microglia and osteoclasts. In activated T-lymphocytes, they maintain the secretion of the lymphokineIL-2. They are also believed to be responsible for the decrease in regulatory volume in response to hypotonic shock, and a Kv1.3 homologue has predicted roles in renal medullary K+transport.
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.The Shal potassium channel was found in Drosophila melanogaster (Fruit fly). Several vertebrate potassium channels with similar amino acid sequences were subsequently found and, together with the D. melanogaster Shal channel, now constitute the Kv4 family. These channels are the primary subunits contributing to transient, voltage-dependent potassium currents in the nervous system (A currents) and the heart (transient outward current), and are inhibited by free fatty acids []. This family can be further divided into 3 subfamilies, designated Kv4.1(KCND1), Kv4.2(KCND2) and Kv4.3(KCND3).Two isoforms of Kv4.3 have been cloned: one is full length and the other hasa small amino acid deletion. Both forms are expressed in the brain, whereasin the heart only the longer isoform is found []. Kv4.3 channels may have an important role in damping synaptic and excitatory membrane potentials. In the brain, they can also associate with Kv-beta2 subunits via the C terminus, resulting in increased channel density and protein expression [].
Protein phosphorylation, which plays a key role in most cellular activities, is a reversible process mediated by protein kinases and phosphoprotein phosphatases. Protein kinases catalyse the transfer of the gamma phosphate from nucleotide triphosphates (often ATP) to one or more amino acid residues in a protein substrate side chain, resulting in a conformational change affecting protein function. Phosphoprotein phosphatases catalyse the reverse process. Protein kinases fall into three broad classes, characterised with respect to substrate specificity []:Serine/threonine-protein kinasesTyrosine-protein kinasesDual specificity protein kinases (e.g. MEK - phosphorylates both Thr and Tyr on target proteins)Protein kinase function is evolutionarily conserved from Escherichia coli to human []. Protein kinases play a role in a multitude of cellular processes, including division, proliferation, apoptosis, and differentiation []. Phosphorylation usually results in a functional change of the target protein by changing enzyme activity, cellular location, or association with other proteins. The catalytic subunits of protein kinases are highly conserved, and several structures have been solved [], leading to large screens to develop kinase-specific inhibitors for the treatments of a number of diseases [].Tyrosine-protein kinases can transfer a phosphate group from ATP to a tyrosine residue in a protein. These enzymes can be divided into two main groups []:Receptor tyrosine kinases (RTK), which are transmembrane proteins involved in signal transduction; they play key roles in growth, differentiation, metabolism, adhesion, motility, death and oncogenesis []. RTKs are composed of 3 domains: an extracellular domain (binds ligand), a transmembrane (TM) domain, and an intracellular catalytic domain (phosphorylates substrate). The TM domain plays an important role in the dimerisation process necessary for signal transduction []. Cytoplasmic / non-receptor tyrosine kinases, which act as regulatory proteins, playing key roles in cell differentiation, motility, proliferation, and survival. For example, the Src-family of protein-tyrosine kinases [].Janus kinases (JAKs) are tyrosine kinases that function in membrane-proximal signalling events initiated by a variety of extracellular factors binding to cell surface receptors []. Many type I and II cytokine receptors lack a protein tyrosine kinase domain and rely on JAKs to initiate the cytoplasmic signal transduction cascade. Ligand binding induces oligomerisation of the receptors, which then activates the cytoplasmic receptor-associated JAKs. These subsequently phosphorylate tyrosine residues along the receptor chains with which they are associated. The phosphotyrosine residues are a target for a variety of SH2 domain-containing transducer proteins. Amongst these are the signal transducers and activators of transcription (STAT) proteins, which, after binding to the receptor chains, are phosphorylated by the JAK proteins. Phosphorylation enables the STAT proteins to dimerise and translocate into the nucleus, where they alter the expression of cytokine-regulated genes. This system is known as the JAK-STAT pathway.Four mammalian JAK family members have been identified: JAK1, JAK2, JAK3, and TYK2. They are relatively large kinases of approximately 1150 amino acids, with molecular weights of ~120-130kDa. Their amino acid sequences are characterised by the presence of 7 highly conserved domains, termed JAK homology (JH) domains. The C-terminal domain (JH1) is responsible for the tyrosine kinase function. The next domain in the sequence (JH2) is known as the tyrosine kinase-like domain, as its sequence shows high similarity to functional kinases but does not possess any catalytic activity. Although the function of this domain is not well established, there is some evidence for a regulatory role on the JH1 domain, thus modulating catalytic activity. The N-terminal portion of the JAKs (spanning JH7 to JH3) is important for receptor association and non-catalytic activity, and consists of JH3-JH4, which is homologous to the SH2 domain, and lastly JH5-JH7, which is a FERM domain.This represents the non-receptor tyrosine kinase JAK1, which is involved in the IFN-alpha/beta/gamma signal pathway. Jak1 acts as the kinase partner for the interleukin (IL)-2 receptor []and interleukin (IL)-10 receptor []. It directly phosphorylates STAT but also activates STAT signalling through the transactivation of other JAK kinases associated with signalling receptors [, ].JAK1 was initially cloned using a PCR-based strategy utilising degenerateprimers corresponding to conserved motifs within the catalytic domain of protein-tyrosine kinases []. In common with JAK2 and TYK2, and by contrastwith JAK3, JAK1 appears to be ubiquitously expressed.
Aconitase (aconitate hydratase; ) is an iron-sulphur protein that contains a [4Fe-4S]-cluster and catalyses the interconversion of isocitrate and citrate via a cis-aconitate intermediate. Aconitase functions in both the TCA and glyoxylate cycles, however unlike the majority of iron-sulphur proteins that function as electron carriers, the [4Fe-4S]-cluster of aconitase reacts directly with an enzyme substrate. In eukaryotes there is a cytosolic form (cAcn) and a mitochondrial form (mAcn) of the enzyme. In bacteria there are also 2 forms, aconitase A (AcnA) and B (AcnB). Several aconitases are known to be multi-functional enzymes with a second non-catalytic, but essential function thatarises when the cellular environment changes, such as when iron levels drop [, ]. Eukaryotic cAcn and mAcn, and bacterial AcnA have the same domain organisation, consisting of three N-terminal alpha/beta/alpha domains, a linker region, followed by a C-terminal 'swivel' domain with a beta/beta/alpha structure (1-2-3-linker-4), although mAcn is smaller than cAcn. However, bacterial AcnB has a different organisation: it contains an N-terminal HEAT-like domain, followed by the 'swivel' domain, then the three alpha/beta/alpha domains (HEAT-4-1-2-3) [].Eukaryotic cAcn enzyme balances the amount of citrate and isocitrate in the cytoplasm, which in turn creates a balance between the amount of NADPH generated from isocitrate by isocitrate dehydrogenase with the amount of acetyl-CoA generated from citrate by citrate lyase. Fatty acid synthesis requires both NADPH and acetyl-CoA, as do other metabolic processes, including the need for NADPH to combat oxidative stress. The enzymatic form of cAcn predominates when iron levels are normal, but if they drop sufficiently to cause the disassembly of the [4Fe-4S]-cluster, then cAcn undergoes a conformational change from a compact enzyme to a more open L-shaped protein known as iron regulatory protein 1 (IRP1; or IRE-binding protein 1, IREBP1) [, ]. As IRP1, the catalytic site and the [4Fe-4S]-cluster are lost, and two new RNA-binding sites appear. IRP1 functions in the post-transcriptional regulation of genes involved in iron metabolism - it binds to mRNA iron-responsive elements (IRE), 30-nucleotide stem-loop structures at the 3' or 5' end of specific transcripts. Transcripts containing an IRE include ferritin L and H subunits (iron storage), transferrin (iron plasma chaperone), transferrin receptor (iron uptake into cells), ferroportin (iron exporter), mAcn, succinate dehydrogenase, erythroid aminolevulinic acid synthetase (tetrapyrrole biosynthesis), among others. If the IRE is in the 5'-UTR of the transcript (e.g. in ferritin mRNA), then IRP1-binding prevents its translation by blocking the transcript from binding to the ribosome. If the IRE is in the 3'-UTR of the transcript (e.g. transferrin receptor), then IRP1-binding protects it from endonuclease degradation, thereby prolonging the half-life of the transcript and enabling it to be translated [].IRP2 is another IRE-binding protein that binds to the same transcripts as IRP1. However, since IRP1 is predominantly in the enzymatic cAcn form, it is IRP2 that acts as the major metabolic regulator that maintains iron homeostasis []. Although IRP2 is homologous to IRP1, IRP2 lacks aconitase activity, and is known only to have a single function in the post-transcriptional regulation of iron metabolism genes []. In iron-replete cells, IRP2 activity is regulated primarily by iron-dependent degradation through the ubiquitin-proteasomal system.Bacterial AcnB is also known to be multi-functional. In addition to its role in the TCA cycle, AcnB was shown to be a post-transcriptional regulator of gene expression in Escherichia coli and Salmonella enterica [, ]. In S. enterica, AcnB initiates a regulatory cascade controlling flagella biosynthesis through an interaction with the ftsH transcript, an alternative RNA polymerase sigma factor. This binding lowers the intracellular concentration of FtsH protease, which in turn enhances the amount of RNA polymerase sigma32 factor (normally degraded by FtsH protease), and sigma32 then increases the synthesis of chaperone DnaK, which in turn promotes thesynthesis of the flagellar protein FliC. AcnB regulates the synthesis of other proteins as well, such as superoxide dismutase (SodA) and other enzymes involved in oxidative stress.This entry represents bacterial aconitase B (AcnB) enzymes, which can switch between aconitase enzyme activity and post-translational gene regulation. An iron-mediated dimerisation mechanism may be responsible for switching AcnB between its catalytic and regulatory roles, as dimerisation requires iron while mRNA binding is inhibited by iron.
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activatedK+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.Inwardly-rectifying potassium channels (Kir) are the principal class of two-TM domain potassium channels. They are characterised by the property of inward-rectification, which is described as the ability to allow large inward currents and smaller outward currents. Inwardly rectifying potassium channels (Kir) are responsible for regulating diverse processes including: cellular excitability, vascular tone, heart rate, renal salt flow, and insulin release []. To date, around twenty members of this superfamily have been cloned, which can be grouped into six families by sequence similarity, and these are designated Kir1.x-6.x [, ].Cloned Kir channel cDNAs encode proteins of between ~370-500 residues, both N- and C-termini are thought to be cytoplasmic, and the N terminus lacks a signal sequence. Kir channel alpha subunits possess only 2TM domains linked with a P-domain. Thus, Kir channels share similarity with the fifth and sixth domains, and P-domain of the other families. It is thought that four Kir subunits assemble to form a tetrameric channel complex, which may be hetero- or homomeric [].Kir1.3 (also known as Kir4.2) was cloned from human kidney, where it appearsto be found most abundantly. At lower levels, it has also been detected inthe pancreas and lung. It is most closely related to Kir1.2, to which it is62% identical. Unlike the other Kir1.x family members, in the Kir1.3sequence, the Walker Type A consensus motif (for nucleotide binding) isnot conserved. Heterologous expression of Kir1.3 in Xenopus oocytes did notlead to detectable channel activity. Indeed, co-expression of Kir1.3 witheither Kir1.1 or Kir1.2 reduced currents resulting from expression of theseinward rectifier subunits alone, consistent with a dominant negativeinfluence of Kir1.3 on Kir1.1 and Kir1.2 expression [].
Zinc finger (Znf) domains are relatively small protein motifs which contain multiple finger-like protrusions that make tandem contacts with their target molecule. Some of these domains bind zinc, but many do not; instead binding other metals such as iron, or no metal at all. For example, some family members form salt bridges to stabilise the finger-like folds. They were first identified as a DNA-binding motif in transcription factor TFIIIA from Xenopus laevis (African clawed frog), however they are now recognised to bind DNA, RNA, protein and/or lipid substrates [, , , , ]. Their binding properties depend on the amino acid sequence of the finger domains and of the linker between fingers, as well as on the higher-order structures and the number of fingers. Znf domains are often found in clusters, where fingers can have different binding specificities. There are many superfamilies of Znf motifs, varying in both sequence and structure. They display considerable versatility in binding modes, even between members of the same class (e.g. some bind DNA, others protein), suggesting that Znf motifs are stable scaffolds that have evolved specialised functions. For example, Znf-containing proteins function in gene transcription, translation, mRNA trafficking, cytoskeleton organisation, epithelial development, cell adhesion, protein folding, chromatin remodelling and zinc sensing, to name but a few []. Zinc-binding motifs are stable structures, and they rarely undergo conformational changes upon binding their target. This entry represents ZZ-type zinc finger domains, named because of their ability to bind two zinc ions []. These domains contain 4-6 Cys residues that participate in zinc binding (plus additional Ser/His residues), including a Cys-X2-Cys motif found in other zinc finger domains. These zinc fingers are thought to be involved in protein-protein interactions. The structure of the ZZ domain shows that it belongs to the family of cross-brace zinc finger motifs that include the PHD, RING, and FYVE domains []. ZZ-type zinc finger domains are found in:Transcription factors P300 and CBP.Plant proteins involved in light responses, such as Hrb1.E3 ubiquitin ligases MEX and MIB2 ().Dystrophin and its homologues.Single copies of the ZZ zinc finger occur in the transcriptional adaptor/coactivator proteins P300, in cAMP response element-binding protein (CREB)-binding protein (CBP) and ADA2. CBP provides several binding sites for transcriptional coactivators. The site of interaction with the tumour suppressor protein p53 and the oncoprotein E1A with CBP/P300 is a Cys-rich region that incorporates two zinc-binding motifs: ZZ-type and TAZ2-type. The ZZ-type zinc finger of CBP contains two twisted anti-parallel β-sheets and a short α-helix, and binds two zinc ions []. One zinc ion is coordinated by four cysteine residues via 2 Cys-X2-Cys motifs, and the third zinc ion viaa third Cys-X-Cys motif and a His-X-His motif. The first zinc cluster is strictly conserved, whereas the second zinc cluster displays variability in the position of the two His residues.In Arabidopsis thaliana (Mouse-ear cress), the hypersensitive to red and blue 1 (Hrb1) protein, which regulating both red and blue light responses, contains a ZZ-type zinc finger domain [].ZZ-type zinc finger domains have also been identified in the testis-specific E3 ubiquitin ligase MEX that promotes death receptor-induced apoptosis []. MEX has four putative zinc finger domains: one ZZ-type, one SWIM-type and two RING-type. The region containing the ZZ-type and RING-type zinc fingers is required for interaction with UbcH5a and MEX self-association, whereas the SWIM domain was critical for MEX ubiquitination.In addition, the Cys-rich domains of dystrophin, utrophin and an 87kDa post-synaptic protein contain a ZZ-type zinc finger with high sequence identity to P300/CBP ZZ-type zinc fingers. In dystrophin and utrophin, the ZZ-type zinc finger lies between a WW domain (flanked by and EF hand) and the C-terminal coiled-coil domain. Dystrophin is thought to act as a link between the actin cytoskeleton and the extracellular matrix, and perturbations of the dystrophin-associated complex, for example, between dystrophin and the transmembrane glycoprotein beta-dystroglycan, may lead to muscular dystrophy. Dystrophin and its autosomal homologue utrophin interact with beta-dystroglycan via their C-terminal regions, which are comprised of a WW domain, an EF hand domain and a ZZ-type zinc finger domain []. The WW domain is the primary site of interaction between dystrophin or utrophin and dystroglycan, while the EF hand and ZZ-type zinc finger domains stabilise and strengthen this interaction.
The connexins are a family of integral membrane proteins that oligomerise to form intercellular channels that are clustered at gap junctions. These channels are specialised sites of cell-cell contact that allow the passage of ions, intracellular metabolites and messenger molecules (with molecular weight less than 1-2kDa) from the cytoplasm of one cell to its opposing neighbours. They are found in almost all vertebrate cell types, and somewhat similar proteins have been cloned from plant species. Invertebrates utilise a different family of molecules, innexins, that share a similar predicted secondary structure to the vertebrate connexins, but have no sequence identity to them [].Vertebrate gap junction channels are thought to participate in diverse biological functions. For instance, in the heart they permit the rapid cell-cell transfer of action potentials, ensuring coordinated contraction of the cardiomyocytes. They are also responsible for neurotransmission at specialised 'electrical' synapses. In non-excitable tissues, such as the liver, they may allow metabolic cooperation between cells. In the brain, glial cells are extensively-coupled by gap junctions; this allows waves of intracellular Ca2+to propagate through nervous tissue, and may contribute to their ability to spatially-buffer local changes in extracellular K+concentration [].The connexin protein family is encoded by at least 13 genes in rodents, with many homologues cloned from other species. They show overlapping tissue expression patterns, most tissues expressing more than one connexin type. Their conductances, permeability to different molecules, phosphorylation and voltage-dependence of their gating, have been found to vary. Possible communication diversity is increased further by the fact that gap junctions may be formed by the association of different connexin isoforms from apposing cells. However, in vitro studies have shown that not all possible combinations of connexins produce active channels [, ].Hydropathy analysis predicts that all cloned connexins share a common transmembrane (TM) topology. Each connexin is thought to contain 4 TMdomains, with two extracellular and three cytoplasmic regions. This modelhas been validated for several of the family members by in vitrobiochemicalanalysis. Both N- and C-termini are thought to face the cytoplasm, and thethird TM domain has an amphipathic character, suggesting that it contributesto the lining of the formed-channel. Amino acid sequence identity betweenthe isoforms is ~50-80%, with the TM domains being well conserved. Bothextracellular loops contain characteristically conserved cysteine residues,which likely form intramolecular disulphide bonds. By contrast, the singleputative intracellular loop (between TM domains 2 and 3) and the cytoplasmicC terminus are highly variable among the family members.Six connexins arethought to associate to form a hemi-channel, or connexon. Two connexons theninteract (likely via the extracellular loops of their connexins) to form thecomplete gap junction channel.NH2-*** *** *************-COOH** ** ** **** ** ** ** Cytoplasmic---**----**-----**----**----------------** ** ** ** Membrane** ** ** **---**----**-----**----**----------------** ** ** ** Extracellular** ** ** **** **Two sets of nomenclature have been used to identify the connexins. Thefirst, and most commonly used, classifies the connexin molecules accordingto molecular weight, such as connexin43 (abbreviated to Cx43), indicatinga connexin of molecular weight close to 43kDa. However, studies haverevealed cases where clear functional homologues exist across speciesthat have quite different molecular masses; therefore, an alternativenomenclature was proposed based on evolutionary considerations, whichdivides the family into two major subclasses, alpha and beta, each with anumber of members []. Due to their ubiquity and overlapping tissue distributions, it has proved difficult to elucidate the functions of individual connexin isoforms. To circumventthis problem, particular connexin-encoding genes have been subjected to targeted-disruption in mice, and the phenotype of the resulting animals investigated. Around half the connexin isoforms have been investigated in this manner []. Further insight into the functional roles of connexins has come from the discovery that a number of human diseases are caused by mutations in connexin genes. For instance, mutations in Cx32 give rise to a form of inherited peripheral neuropathy called X-linked dominant Charcot-Marie-Tooth disease []. Similarly, mutations in Cx26 are responsible for both autosomal recessive and dominant forms of nonsyndromic deafness, a disorder characterised by hearing loss, with no apparent effects on other organ systems.This superfamily represents the N-terminal domain of these proteins.
The connexins are a family of integral membrane proteins that oligomerise to form intercellular channels that are clustered at gap junctions. These channels are specialised sites of cell-cell contact that allow the passage of ions, intracellular metabolites and messenger molecules (with molecular weight less than 1-2kDa) from the cytoplasm of one cell to its opposing neighbours. They are found in almost all vertebrate cell types, and somewhat similar proteins have been cloned from plant species. Invertebrates utilise a different family of molecules, innexins, that share a similar predicted secondary structure to the vertebrate connexins, but have no sequence identity to them [].Vertebrate gap junction channels are thought to participate in diverse biological functions. For instance, in the heart they permit the rapid cell-cell transfer of action potentials, ensuring coordinated contraction of the cardiomyocytes. They are also responsible for neurotransmission at specialised 'electrical' synapses. In non-excitable tissues, such as the liver, they may allow metabolic cooperation between cells. In the brain, glial cells are extensively-coupled by gap junctions; this allows waves of intracellular Ca2+to propagate through nervous tissue, and may contribute to their ability to spatially-buffer local changes in extracellular K+concentration [].The connexin protein family is encoded by at least 13 genes in rodents, with many homologues cloned from other species. They show overlapping tissue expression patterns, most tissues expressing more than one connexin type. Their conductances, permeability to different molecules, phosphorylation and voltage-dependence of their gating, have been found to vary. Possible communication diversity is increased further by the fact that gap junctions may be formed by the association of different connexin isoforms from apposing cells. However, in vitro studies have shown that not all possible combinations of connexins produce active channels [, ].Hydropathy analysis predicts that all cloned connexins share a common transmembrane (TM) topology. Each connexin is thought to contain 4 TMdomains, with two extracellular and three cytoplasmic regions. This modelhas been validated for several of the family members by in vitrobiochemicalanalysis. Both N- and C-termini are thought to face the cytoplasm, and thethird TM domain has an amphipathic character, suggesting that it contributesto the lining of the formed-channel. Amino acid sequence identity betweenthe isoforms is ~50-80%, with the TM domains being well conserved. Bothextracellular loops contain characteristically conserved cysteine residues,which likely form intramolecular disulphide bonds. By contrast, the singleputative intracellular loop (between TM domains 2 and 3) and the cytoplasmicC terminus are highly variable among the family members.Six connexins arethought to associate to form a hemi-channel, or connexon. Two connexons theninteract (likely via the extracellular loops of their connexins) to form thecomplete gap junction channel.NH2-*** *** *************-COOH** ** ** **** ** ** ** Cytoplasmic---**----**-----**----**----------------** ** ** ** Membrane** ** ** **---**----**-----**----**----------------** ** ** ** Extracellular** ** ** **** **Two sets of nomenclature have been used to identify the connexins. Thefirst, and most commonly used, classifies the connexin molecules accordingto molecular weight, such as connexin43 (abbreviated to Cx43), indicatinga connexin of molecular weight close to 43kDa. However, studies haverevealed cases where clear functional homologues exist across speciesthat have quite different molecular masses; therefore, an alternativenomenclature was proposed based on evolutionary considerations, whichdivides the family into two major subclasses, alpha and beta, each with anumber of members []. Due to their ubiquity and overlapping tissue distributions, it has proved difficult to elucidate the functions of individual connexin isoforms. To circumvent this problem, particular connexin-encoding genes have been subjected to targeted-disruption in mice, and the phenotype of the resulting animals investigated. Around half the connexin isoforms have been investigated in this manner []. Further insight into the functional roles of connexins has come from the discovery that a number of human diseases are caused by mutations in connexin genes. For instance, mutations in Cx32 give rise to a form of inherited peripheral neuropathy called X-linked dominant Charcot-Marie-Tooth disease []. Similarly, mutations in Cx26 are responsible for both autosomal recessive and dominant forms of nonsyndromic deafness, a disorder characterised by hearing loss, with no apparent effects on other organ systems.This entry represents all types of connexins.
The connexins are a family of integral membrane proteins that oligomerise to form intercellular channels that are clustered at gap junctions. These channels are specialised sites of cell-cell contact that allow the passage of ions, intracellular metabolites and messenger molecules (with molecular weight less than 1-2kDa) from the cytoplasm of one cell to its opposing neighbours. They are found in almost all vertebrate cell types, and somewhat similar proteins have been cloned from plant species. Invertebrates utilise a different family of molecules, innexins, that share a similar predicted secondary structure to the vertebrate connexins, but have no sequence identity to them [].Vertebrate gap junction channels are thought to participate in diverse biological functions. For instance, in the heart they permit the rapid cell-cell transfer of action potentials, ensuring coordinated contraction of the cardiomyocytes. They are also responsible for neurotransmission at specialised 'electrical' synapses. In non-excitable tissues, such as the liver, they may allow metabolic cooperation between cells. In the brain, glial cells are extensively-coupled by gap junctions; this allows waves of intracellular Ca2+to propagate through nervous tissue, and may contribute to their ability to spatially-buffer local changes in extracellular K+concentration [].The connexin protein family is encoded by at least 13 genes in rodents, with many homologues cloned from other species. They show overlapping tissue expression patterns, most tissues expressing more than one connexin type. Their conductances, permeability to different molecules, phosphorylation and voltage-dependence of their gating, have been found to vary. Possible communication diversity is increased further by the fact that gap junctions may be formed by the association of different connexin isoforms from apposing cells. However, in vitro studies have shown that not all possible combinations of connexins produce active channels [, ].Hydropathy analysis predicts that all cloned connexins share a common transmembrane (TM) topology. Each connexin is thought to contain 4 TMdomains, with two extracellular and three cytoplasmic regions. This modelhas been validated for several of the family members by in vitrobiochemicalanalysis. Both N- and C-termini are thought to face the cytoplasm, and thethird TM domain has an amphipathic character, suggesting that it contributesto the lining of the formed-channel. Amino acid sequence identity betweenthe isoforms is ~50-80%, with the TM domains being well conserved. Bothextracellular loops contain characteristically conserved cysteine residues,which likely form intramolecular disulphide bonds. By contrast, the singleputative intracellular loop (between TM domains 2 and 3) and the cytoplasmicC terminus are highly variable among the family members.Six connexins arethought to associate to form a hemi-channel, or connexon. Two connexons theninteract (likely via the extracellular loops of their connexins) to form thecomplete gap junction channel.NH2-*** *** *************-COOH** ** ** **** ** ** ** Cytoplasmic---**----**-----**----**----------------** ** ** ** Membrane** ** ** **---**----**-----**----**----------------** ** ** ** Extracellular** ** ** **** **Two sets of nomenclature have been used to identify the connexins. Thefirst, and most commonly used, classifies the connexin molecules accordingto molecular weight, such as connexin43 (abbreviated to Cx43), indicatinga connexin of molecular weight close to 43kDa. However, studies haverevealed cases where clear functional homologues exist across speciesthat have quite different molecular masses; therefore, an alternativenomenclature was proposed based on evolutionary considerations, whichdivides the family into two major subclasses, alpha and beta, each with anumber of members []. Due to their ubiquity and overlapping tissue distributions, it has proved difficult to elucidate the functions of individual connexin isoforms. To circumvent this problem, particular connexin-encoding genes have been subjected to targeted-disruption in mice, and the phenotype of the resulting animals investigated. Around half the connexin isoforms have been investigated in this manner []. Further insight into the functional roles of connexins has come from the discovery that a number of human diseases are caused by mutations in connexin genes. For instance, mutations in Cx32 give rise to a form of inherited peripheral neuropathy called X-linked dominant Charcot-Marie-Tooth disease []. Similarly, mutations in Cx26 are responsible for both autosomal recessive and dominant forms of nonsyndromic deafness, a disorder characterised by hearing loss, with no apparent effects on other organ systems.Gap junction alpha-1 protein (also called connexin43, or Cx43) is a connexinof 381 amino acid residues (human isoform) that is widely expressed inseveral organs and cell types, and is the principal gap junction protein ofthe heart. Characterisation of genetically-engineered mice that lack Cx43,and also of human patients that have spontaneously-occurring mutations inthe gene encoding it (GJA1), suggest Cx43 is essential for the developmentof normal cardiac architecture and ventricular conduction. Mice lacking Cx43survive to term but die shortly after birth. They have cardiac malformationsthat lead to the obstruction of the pulmonary artery, leading to neonatalcyanosis, and subsequent death. This phenotype is reminiscent of some formsof stenosis of the pulmonary artery. Human subjects with visceroatrialheterotaxia (a heart disorder characterised by arterial defects), have beenfound to have points mutations in the Cx43-encoding gene, as a result of which a potential phosphorylation site within the C terminus is disrupted. Consequently, although these mutant Cx43 molecules still form functional gapjunction channels, their response to protein kinase activation is impaired.
The connexins are a family of integral membrane proteins that oligomerise to form intercellular channels that are clustered at gap junctions. These channels are specialised sites of cell-cell contact that allow the passage of ions, intracellular metabolites and messenger molecules (with molecular weight less than 1-2kDa) from the cytoplasm of one cell to its opposing neighbours. They are found in almost all vertebrate cell types, and somewhat similar proteins have been cloned from plant species. Invertebrates utilise a different family of molecules, innexins, that share a similar predicted secondary structure to the vertebrate connexins, but have no sequence identity to them [].Vertebrate gap junction channels are thought to participate in diverse biological functions. For instance, in the heart they permit the rapid cell-cell transfer of action potentials, ensuring coordinated contraction of the cardiomyocytes. They are also responsible for neurotransmission at specialised 'electrical' synapses. In non-excitable tissues, such as the liver, they may allow metabolic cooperation between cells. In the brain, glial cells are extensively-coupled by gap junctions; this allows waves of intracellular Ca2+to propagate through nervous tissue, and may contribute to their ability to spatially-buffer local changes in extracellular K+concentration [].The connexin protein family is encoded by at least 13 genes in rodents, with many homologues cloned from other species. They show overlapping tissue expression patterns, most tissues expressing more than one connexin type. Their conductances, permeability to different molecules, phosphorylation and voltage-dependence of their gating, have been found to vary. Possible communication diversity is increased further by the fact that gap junctions may be formed by the association of different connexin isoforms from apposing cells. However, in vitro studies have shown that not all possible combinations of connexins produce active channels [, ].Hydropathy analysis predicts that all cloned connexins share a common transmembrane (TM) topology. Each connexin is thought to contain 4 TMdomains, with two extracellular and three cytoplasmic regions. This modelhas been validated for several of the family members by in vitrobiochemicalanalysis. Both N- and C-termini are thought to face the cytoplasm, and thethird TM domain has an amphipathic character, suggesting that it contributesto the lining of the formed-channel. Amino acid sequence identity betweenthe isoforms is ~50-80%, with the TM domains being well conserved. Bothextracellular loops contain characteristically conserved cysteine residues,which likely form intramolecular disulphide bonds. By contrast, the singleputative intracellular loop (between TM domains 2 and 3) and the cytoplasmicC terminus are highly variable among the family members.Six connexins arethought to associate to form a hemi-channel, or connexon. Two connexons theninteract (likely via the extracellular loops of their connexins) to form thecomplete gap junction channel.NH2-*** *** *************-COOH** ** ** **** ** ** ** Cytoplasmic---**----**-----**----**----------------** ** ** ** Membrane** ** ** **---**----**-----**----**----------------** ** ** ** Extracellular** ** ** **** **Two sets of nomenclature have been used to identify the connexins. Thefirst, and most commonly used, classifies the connexin molecules accordingto molecular weight, such as connexin43 (abbreviated to Cx43), indicatinga connexin of molecular weight close to 43kDa. However, studies haverevealed cases where clear functional homologues exist across speciesthat have quite different molecular masses; therefore, an alternativenomenclature was proposed based on evolutionary considerations, whichdivides the family into two major subclasses, alpha andbeta, each with anumber of members []. Due to their ubiquity and overlapping tissue distributions, it has proved difficult to elucidate the functions of individual connexin isoforms. To circumvent this problem, particular connexin-encoding genes have been subjected to targeted-disruption in mice, and the phenotype of the resulting animals investigated. Around half the connexin isoforms have been investigated in this manner []. Further insight into the functional roles of connexins has come from the discovery that a number of human diseases are caused by mutations in connexin genes. For instance, mutations in Cx32 give rise to a form of inherited peripheral neuropathy called X-linked dominant Charcot-Marie-Tooth disease []. Similarly, mutations in Cx26 are responsible for both autosomal recessive and dominant forms of nonsyndromic deafness, a disorder characterised by hearing loss, with no apparent effects on other organ systems.Gap junction beta-2 protein (also called connexin26, or Cx26) is a connexinof 226 amino acid residues that is often found together with Cx32 inepithelial tissues. In rodents, it seems essential for normal embryonicdevelopment; mice lacking Cx26 die in utero at about embryonic day 11.Absence of Cx26 impairs transplacental movement of glucose, thus impairingembryonic development. In humans, spontaneously-occurring mutations in thegene encoding Cx26 have been found to be associated with a quite differentdisorder, non-syndromic neurosensory autosomal recessive deafness, the mostcommon form of inherited hearing loss. Consistent with a role in auditorytransduction, Cx26 is the predominant connexin isoform expressed in theorgan of Corti of the cochlea. Here, it may be involved in maintaining theionic balance of the endolymph, by providing a pathway for ions necessaryto maintain this potassium-rich fluid.
The connexins are a family of integral membrane proteins that oligomerise to form intercellular channels that are clustered at gap junctions. These channels are specialised sites of cell-cell contact that allow the passage of ions, intracellular metabolites and messenger molecules (with molecular weight less than 1-2kDa) from the cytoplasm of one cell to its opposing neighbours. They are found in almost all vertebrate cell types, and somewhat similar proteins have been cloned from plant species. Invertebrates utilise a different family of molecules, innexins, that share a similar predicted secondary structure to the vertebrate connexins, but have no sequence identity to them [].Vertebrate gap junction channels are thought to participate in diverse biological functions. For instance, in the heart they permit the rapid cell-cell transfer of action potentials, ensuring coordinated contraction of the cardiomyocytes. They are also responsible for neurotransmission at specialised 'electrical' synapses. In non-excitable tissues, such as the liver, they may allow metabolic cooperation between cells. In the brain, glial cells are extensively-coupled by gap junctions; this allows waves of intracellular Ca2+to propagate through nervous tissue, and may contribute to their ability to spatially-buffer local changes in extracellular K+concentration [].Theconnexin protein family is encoded by at least 13 genes in rodents, with many homologues cloned from other species. They show overlapping tissue expression patterns, most tissues expressing more than one connexin type. Their conductances, permeability to different molecules, phosphorylation and voltage-dependence of their gating, have been found to vary. Possible communication diversity is increased further by the fact that gap junctions may be formed by the association of different connexin isoforms from apposing cells. However, in vitro studies have shown that not all possible combinations of connexins produce active channels [, ].Hydropathy analysis predicts that all cloned connexins share a common transmembrane (TM) topology. Each connexin is thought to contain 4 TMdomains, with two extracellular and three cytoplasmic regions. This modelhas been validated for several of the family members by in vitrobiochemicalanalysis. Both N- and C-termini are thought to face the cytoplasm, and thethird TM domain has an amphipathic character, suggesting that it contributesto the lining of the formed-channel. Amino acid sequence identity betweenthe isoforms is ~50-80%, with the TM domains being well conserved. Bothextracellular loops contain characteristically conserved cysteine residues,which likely form intramolecular disulphide bonds. By contrast, the singleputative intracellular loop (between TM domains 2 and 3) and the cytoplasmicC terminus are highly variable among the family members.Six connexins arethought to associate to form a hemi-channel, or connexon. Two connexons theninteract (likely via the extracellular loops of their connexins) to form thecomplete gap junction channel.NH2-*** *** *************-COOH** ** ** **** ** ** ** Cytoplasmic---**----**-----**----**----------------** ** ** ** Membrane** ** ** **---**----**-----**----**----------------** ** ** ** Extracellular** ** ** **** **Two sets of nomenclature have been used to identify the connexins. Thefirst, and most commonly used, classifies the connexin molecules accordingto molecular weight, such as connexin43 (abbreviated to Cx43), indicatinga connexin of molecular weight close to 43kDa. However, studies haverevealed cases where clear functional homologues exist across speciesthat have quite different molecular masses; therefore, an alternativenomenclature was proposed based on evolutionary considerations, whichdivides the family into two major subclasses, alpha and beta, each with anumber of members []. Due to their ubiquity and overlapping tissue distributions, it has proved difficult to elucidate the functions of individual connexin isoforms. To circumvent this problem, particular connexin-encoding genes have been subjected to targeted-disruption in mice, and the phenotype of the resulting animals investigated. Around half the connexin isoforms have been investigated in this manner []. Further insight into the functional roles of connexins has come from the discovery that a number of human diseases are caused by mutations in connexin genes. For instance, mutations in Cx32 give rise to a form of inherited peripheral neuropathy called X-linked dominant Charcot-Marie-Tooth disease []. Similarly, mutations in Cx26 are responsible for both autosomal recessive and dominant forms of nonsyndromic deafness, a disorder characterised by hearing loss, with no apparent effects on other organ systems.Gap junction beta-1 protein (also called connexin32, or Cx32) is a connexinof 283 amino acid residues (human isoform) that is widely expressed in manytissues, including the liver, exocrine pancreas, central nervous system andepithelium of the gastrointestinal tract. The amphibian isoform from theXenopus laevis (African clawed frog), is slightly shorter, containing 264amino acid residues. In the adult frog, the protein is present in the lungs,alimentary tract and ovaries [].In humans, Cx32 appears to be critical to the functioning of Schwann cells,which are responsible for the myelination of nerves in the peripheralnervous system. Mutations in the gene encoding Cx32 give rise to a form ofinherited neuropathy called X-linked Charcot-Marie-Tooth disease, whichaffects nervous conduction in both motor and sensory axons. To date, >40different mutations have been identified, and these are spread throughoutmost of the Cx32 molecule. The effects of some of these mutations have beendetermined, and several of them lead to a complete loss of gap junctionfunction. Targeted-gene disruption of Cx32 in mice has confirmed its rolein Schwann cell function; such Cx32-null mice also develop a form ofperipheral neuropathy similar to Charcot-Marie-Tooth disease.
The connexins are a family of integral membrane proteins that oligomerise to form intercellular channels that are clustered at gap junctions. These channels are specialised sites of cell-cell contact that allow the passage of ions, intracellular metabolites and messenger molecules (with molecular weight less than 1-2kDa) from the cytoplasm of one cell to its opposing neighbours. They are found in almost all vertebrate cell types, and somewhat similar proteins have been cloned from plant species. Invertebrates utilise a different family of molecules, innexins, that share a similar predicted secondary structure to the vertebrate connexins, but have no sequence identity to them [].Vertebrate gap junction channels are thought to participate in diverse biological functions. For instance, in the heart they permit the rapid cell-cell transfer of action potentials, ensuring coordinated contraction of the cardiomyocytes. They are also responsible for neurotransmission at specialised 'electrical' synapses. In non-excitable tissues, such as the liver, they may allow metabolic cooperation between cells. In the brain, glial cells are extensively-coupled by gap junctions; this allows waves of intracellular Ca2+to propagate through nervous tissue, and may contribute to their ability to spatially-buffer local changes in extracellular K+concentration [].The connexin protein family is encoded by at least 13 genes in rodents, with many homologues cloned from other species. They show overlapping tissue expression patterns, most tissues expressing more than one connexin type. Their conductances, permeability to different molecules, phosphorylation and voltage-dependence of their gating, have been found to vary. Possible communication diversity is increased further by the fact that gap junctions may be formed by the association of different connexin isoforms from apposing cells. However, in vitro studies have shown that not all possible combinations of connexins produce active channels [, ].Hydropathy analysis predicts that all cloned connexins share a common transmembrane (TM) topology. Each connexin is thought to contain 4 TMdomains, with two extracellular and three cytoplasmic regions. This modelhas been validated for several of the family members by in vitrobiochemicalanalysis. Both N- and C-termini are thought to face the cytoplasm, and thethird TM domain has an amphipathic character, suggesting that it contributesto the lining of the formed-channel. Amino acid sequence identity betweenthe isoforms is ~50-80%, with the TM domains being well conserved. Bothextracellular loops contain characteristically conserved cysteine residues,which likely form intramolecular disulphide bonds. By contrast, the singleputative intracellular loop (between TM domains 2 and 3) and the cytoplasmicC terminus are highly variable among the family members.Six connexins arethought to associate to form a hemi-channel, or connexon. Two connexons theninteract (likely via the extracellular loops of their connexins) to form thecomplete gap junction channel.NH2-*** *** *************-COOH** ** ** **** ** ** ** Cytoplasmic---**----**-----**----**----------------** ** ** ** Membrane** ** ** **---**----**-----**----**----------------** ** ** ** Extracellular** ** ** **** **Two sets of nomenclature have been used to identify the connexins. Thefirst, and most commonly used, classifies the connexin molecules accordingto molecular weight, such as connexin43 (abbreviated to Cx43), indicatinga connexin of molecular weight close to 43kDa. However, studies haverevealed cases where clear functional homologues exist across speciesthat have quite different molecular masses; therefore, an alternativenomenclature was proposed based on evolutionary considerations, whichdivides the family into two major subclasses, alpha and beta, each with anumber of members []. Due to their ubiquity and overlapping tissue distributions, it has proved difficult to elucidate the functions of individual connexin isoforms. To circumvent this problem, particular connexin-encoding genes have been subjected to targeted-disruption in mice, and the phenotype of the resulting animals investigated. Around half the connexin isoforms have been investigated in this manner []. Further insight into the functional roles of connexins has come from the discovery that a number of human diseases are caused by mutations in connexin genes. For instance, mutations in Cx32 give rise to a form of inherited peripheral neuropathy called X-linked dominant Charcot-Marie-Tooth disease []. Similarly, mutations in Cx26 are responsible for both autosomal recessive and dominant forms of nonsyndromic deafness, a disorder characterised by hearing loss, with no apparent effects on other organ systems.Gap junction beta-3 protein (also called connexin31, or Cx31) is a connexinof 270 amino acid residues, and belongs to a family that also includes Cx26,Cx31.1 and Cx32. At the mRNA level, it has been found to be expressed in theskin, ear, placenta and eye. Mutations in Cx31 have been found to beresponsible for two quite different inherited human diseases: erythro-keratomdermia variablis and autosomal dominant hearing impairment. Theformer is a hereditary skin disease characterised by transient figurate redpatches of skin and hyperkeratosis. In the Cx31 molecule of these patients,either a conserved glycine has been substituted by a charged residue, or acysteine has been changed to a to serine residue []. In the latter,mutations in Cx31 result in high-frequency hearing impairment, making itthe second connexin molecule (together with Cx26) in which mutations havebeen found to be responsible for an inherited hearing disorder.
The connexins are a family of integral membrane proteins that oligomerise to form intercellular channels that are clustered at gap junctions. These channels are specialised sites of cell-cell contact that allow the passage of ions, intracellular metabolites and messenger molecules (with molecular weight less than 1-2kDa) from the cytoplasm of one cell to its opposing neighbours. They are found in almost all vertebrate cell types, and somewhat similar proteins have been cloned from plant species. Invertebrates utilise a different family of molecules, innexins, that share a similar predicted secondary structure to the vertebrate connexins, but have no sequence identity to them [].Vertebrate gap junction channels are thought to participatein diverse biological functions. For instance, in the heart they permit the rapid cell-cell transfer of action potentials, ensuring coordinated contraction of the cardiomyocytes. They are also responsible for neurotransmission at specialised 'electrical' synapses. In non-excitable tissues, such as the liver, they may allow metabolic cooperation between cells. In the brain, glial cells are extensively-coupled by gap junctions; this allows waves of intracellular Ca2+to propagate through nervous tissue, and may contribute to their ability to spatially-buffer local changes in extracellular K+concentration [].The connexin protein family is encoded by at least 13 genes in rodents, with many homologues cloned from other species. They show overlapping tissue expression patterns, most tissues expressing more than one connexin type. Their conductances, permeability to different molecules, phosphorylation and voltage-dependence of their gating, have been found to vary. Possible communication diversity is increased further by the fact that gap junctions may be formed by the association of different connexin isoforms from apposing cells. However, in vitro studies have shown that not all possible combinations of connexins produce active channels [, ].Hydropathy analysis predicts that all cloned connexins share a common transmembrane (TM) topology. Each connexin is thought to contain 4 TMdomains, with two extracellular and three cytoplasmic regions. This modelhas been validated for several of the family members by in vitrobiochemicalanalysis. Both N- and C-termini are thought to face the cytoplasm, and thethird TM domain has an amphipathic character, suggesting that it contributesto the lining of the formed-channel. Amino acid sequence identity betweenthe isoforms is~50-80%, with the TM domains being well conserved. Bothextracellular loops contain characteristically conserved cysteine residues,which likely form intramolecular disulphide bonds. By contrast, the singleputative intracellular loop (between TM domains 2 and 3) and the cytoplasmicC terminus are highly variable among the family members.Six connexins arethought to associate to form a hemi-channel, or connexon. Two connexons theninteract (likely via the extracellular loops of their connexins) to form thecomplete gap junction channel.NH2-*** *** *************-COOH** ** ** **** ** ** ** Cytoplasmic---**----**-----**----**----------------** ** ** ** Membrane** ** ** **---**----**-----**----**----------------** ** ** ** Extracellular** ** ** **** **Two sets of nomenclature have been used to identify the connexins. Thefirst, and most commonly used, classifies the connexin molecules accordingto molecular weight, such as connexin43 (abbreviated to Cx43), indicatinga connexin of molecular weight close to 43kDa. However, studies haverevealed cases where clear functional homologues exist across speciesthat have quite different molecular masses; therefore, an alternativenomenclature was proposed based on evolutionary considerations, whichdivides the family into two major subclasses, alpha and beta, each with anumber of members []. Due to their ubiquity and overlapping tissue distributions, it has proved difficult to elucidate the functions of individual connexin isoforms. To circumvent this problem, particular connexin-encoding genes have been subjected to targeted-disruption in mice, and the phenotype of the resulting animals investigated. Around half the connexin isoforms have been investigated inthis manner []. Further insight into the functional roles of connexins has come from the discovery that a number of human diseases are caused by mutations in connexin genes. For instance, mutations in Cx32 give rise to a form of inherited peripheral neuropathy called X-linked dominant Charcot-Marie-Tooth disease []. Similarly, mutations in Cx26 are responsible for both autosomal recessive and dominant forms of nonsyndromic deafness, a disorder characterised by hearing loss, with no apparent effects on other organ systems.Gap junction alpha-5 protein (also called connexin40, or Cx40) is a connexinof ~357 amino acid residues. The chicken isoform is about ten residueslonger, and is hence known as connexin42 (Cx42), as it has a molecular massof ~42 kD. Targeted disruption of the gene encoding Cx40 in mice suggeststhat Cx40-containing gap junctions are involved in the rapid conduction ofimpulses in the His-Purkinje system of the heart, which is responsible forthe coordinated spread of excitation from the atrioventricular (A-V) nodeto the ventricular myocardium. Mice lacking Cx40 are viable and fertile;however, they have subtle electrocardio-graphic abnormalities, such aspartial A-V block. Studies of the distribution of Cx40 support thesefindings, since Cx40 has been reported to be prominently expressed in thePurkinje fibres of the heart.
The connexins are a family of integral membrane proteins that oligomerise to form intercellular channels that are clustered at gap junctions. These channels are specialised sites of cell-cell contact that allow the passage of ions, intracellular metabolites and messenger molecules (with molecular weight less than 1-2kDa) from the cytoplasm of one cell to its opposing neighbours. They are found in almost all vertebrate cell types, and somewhat similar proteins have been cloned from plant species. Invertebrates utilise a different family of molecules, innexins, that share a similar predicted secondary structure to the vertebrate connexins, but have no sequence identity to them [].Vertebrate gap junction channels are thought to participate in diverse biological functions. For instance, in the heart they permit the rapid cell-cell transfer of action potentials, ensuring coordinated contraction of the cardiomyocytes. They are also responsible for neurotransmission at specialised 'electrical' synapses.In non-excitable tissues, such as the liver, they may allow metabolic cooperation between cells. In the brain, glial cells are extensively-coupled by gap junctions; this allows waves of intracellular Ca2+to propagate through nervous tissue, and may contribute to their ability to spatially-buffer local changes in extracellular K+concentration [].The connexin protein family is encoded by at least 13 genes in rodents, with many homologues cloned from other species. They show overlapping tissue expression patterns, most tissues expressing more than one connexin type. Their conductances, permeability to different molecules, phosphorylation and voltage-dependence of their gating, have been found to vary. Possible communication diversity is increased further by the fact that gap junctions may be formed by the association of different connexin isoforms from apposing cells. However, in vitro studies have shown that not all possible combinations of connexins produce active channels [, ].Hydropathy analysis predicts that all cloned connexins share a common transmembrane (TM) topology. Each connexin is thought to contain 4 TMdomains, with two extracellular and three cytoplasmic regions. This modelhas been validated for several of the family members by in vitrobiochemicalanalysis. Both N- and C-termini are thought to face the cytoplasm, and thethird TM domain has an amphipathic character, suggesting that it contributesto the lining of the formed-channel. Amino acid sequence identity betweenthe isoforms is ~50-80%, with the TM domains being well conserved. Bothextracellular loops contain characteristically conserved cysteine residues,which likely form intramolecular disulphide bonds. By contrast, the singleputative intracellular loop (between TM domains 2 and 3) and the cytoplasmicC terminus are highly variable among the family members.Six connexins arethought to associate to form a hemi-channel, or connexon. Two connexons theninteract (likely via the extracellular loops of their connexins) to form thecomplete gap junction channel.NH2-*** *** *************-COOH** ** ** **** ** ** ** Cytoplasmic---**----**-----**----**----------------** ** ** ** Membrane** ** ** **---**----**-----**----**----------------** ** ** ** Extracellular** ** ** **** **Two sets of nomenclature have been used to identify the connexins. Thefirst, and most commonly used, classifies the connexin molecules accordingto molecular weight, such as connexin43 (abbreviated to Cx43), indicatinga connexin of molecular weight close to 43kDa. However, studies haverevealed cases where clear functional homologues exist across speciesthat have quite different molecular masses; therefore, an alternativenomenclature was proposed based on evolutionary considerations, whichdivides the family into two major subclasses, alpha and beta, each with anumber of members []. Due to their ubiquity and overlapping tissue distributions, it has proved difficult to elucidate the functions of individual connexin isoforms. To circumvent this problem, particular connexin-encoding genes have been subjected to targeted-disruption in mice, and the phenotype of the resulting animals investigated. Around half the connexin isoforms have been investigated in this manner []. Further insight into the functional roles of connexins has come from the discovery that a number of human diseases are caused by mutations in connexin genes. For instance, mutations in Cx32 give rise to a form of inherited peripheral neuropathy called X-linked dominant Charcot-Marie-Tooth disease []. Similarly, mutations in Cx26 are responsible for both autosomal recessive and dominant forms of nonsyndromic deafness, a disorder characterised by hearing loss, with no apparent effects on other organ systems.Gap junction alpha-4 protein (also called connexin37, or Cx37) is a connexinof 333 amino acids (human isoform) with a predicted molecular mass of ~37kD. It is expressed in many organs and tissues, including: brain, heart,uterus, ovary, and endothelial cells of blood vessels. When heterologouslyexpressed, Cx37 forms intercellular channels that are more sensitive tovoltage and show faster voltage-gating kinetics than most other previously-characterised gap junction channels. The recent generation of mice lackingthe gene encoding Cx37 (GJA4 or CXN-37) has shed some light on its functionin vivo. Female mice lacking Cx37 are viable and apparently in good health,but are rendered infertile, as a result of a failure to ovulate. It appearsthat in the ovarian follicle, functional gap junctions (formed by Cx37) arecritical for communication between the oocyte and the surrounding granulosacells. Without Cx37, follicular development is arrested, and subsequentlyovulation does not occur.
The connexins are a family of integral membrane proteins that oligomerise to form intercellular channels that are clustered at gap junctions. These channels are specialised sites of cell-cell contact that allow the passage of ions, intracellular metabolites and messenger molecules (with molecular weight less than 1-2kDa) from the cytoplasm of one cell to its opposing neighbours. They are found in almost all vertebrate cell types, and somewhat similar proteins have been cloned from plant species. Invertebrates utilise a different family of molecules, innexins, that share a similar predicted secondary structure to the vertebrate connexins, but have no sequence identity to them [].Vertebrate gap junction channels are thought to participate in diverse biological functions. For instance, in the heart they permit the rapid cell-cell transfer of action potentials, ensuring coordinated contraction of the cardiomyocytes. They are also responsible for neurotransmission at specialised 'electrical' synapses. In non-excitable tissues, such as the liver, they may allow metabolic cooperation between cells. In the brain, glial cells are extensively-coupled by gap junctions; this allows waves of intracellular Ca2+to propagate through nervous tissue, and may contribute to their ability to spatially-buffer local changes in extracellular K+concentration [].The connexin protein family is encoded by at least 13 genes in rodents, with many homologues cloned from other species. They show overlapping tissue expression patterns, most tissues expressing more than one connexin type. Their conductances, permeability to different molecules, phosphorylation and voltage-dependence of their gating, have been found to vary. Possible communication diversity is increased further by the fact that gap junctions may be formed by the association of different connexin isoforms from apposing cells. However, in vitro studies have shown that not all possible combinations of connexins produce active channels [, ].Hydropathy analysis predicts that all cloned connexins share a common transmembrane (TM) topology. Each connexin is thought to contain 4 TMdomains, with two extracellular and three cytoplasmic regions. This modelhas been validated for several of the family members by in vitrobiochemicalanalysis. Both N- and C-termini are thought to face the cytoplasm, and thethird TM domain has an amphipathic character, suggesting that it contributesto the lining of the formed-channel. Amino acid sequence identity betweenthe isoforms is ~50-80%, with the TM domains being well conserved. Bothextracellular loops contain characteristically conserved cysteine residues,which likely form intramolecular disulphide bonds. By contrast, the singleputative intracellular loop (between TM domains 2 and 3) and the cytoplasmicC terminus are highly variable among the family members.Six connexins arethought to associate to form a hemi-channel, or connexon. Two connexons theninteract (likely via the extracellular loops of their connexins) to form thecomplete gap junction channel.NH2-*** *** *************-COOH** ** ** **** ** ** ** Cytoplasmic---**----**-----**----**----------------** ** ** ** Membrane** ** ** **---**----**-----**----**----------------** ** ** ** Extracellular** ** ** **** **Two sets of nomenclature have been used to identify the connexins. Thefirst, and most commonly used, classifies the connexin molecules accordingto molecular weight, such as connexin43 (abbreviated to Cx43), indicatinga connexin of molecular weight close to 43kDa. However, studies haverevealed cases where clear functional homologues exist across speciesthat have quite different molecular masses; therefore, an alternativenomenclature was proposed based on evolutionary considerations, whichdivides the family into two major subclasses, alpha and beta, each with anumber of members []. Due to their ubiquity and overlapping tissue distributions, it has proved difficult to elucidate the functions of individual connexin isoforms. To circumvent this problem, particular connexin-encoding genes have been subjected to targeted-disruption in mice, and the phenotype of the resulting animals investigated. Around half the connexin isoforms have been investigated in this manner []. Further insight into the functional roles of connexins has come from the discovery that a number of human diseases are caused by mutations in connexin genes. For instance, mutations in Cx32 give rise to a form of inherited peripheral neuropathy called X-linked dominant Charcot-Marie-Tooth disease []. Similarly, mutations in Cx26 are responsible for both autosomal recessive and dominant forms of nonsyndromic deafness, a disorder characterised by hearing loss, with no apparent effects on other organ systems.Gap junction alpha-8 protein (also called connexin50, Cx50, or lens fibreprotein MP70) is a connexin of ~431 amino acid residues. The chicken isoformis shorter (399 residues) and is hence known as Cx45.6. Cx50 and Cx46 arethe two gap junction proteins normally found in lens fibre cells of the eye.Evidence from both genetically-engineered mice, and from the identificationof mutations in the human Cx50-encoding gene, highlight the importance ofthis connexin in maintaining lens transparency. Deletion of mice Cx50produces a viable phenotype, but these animals start to develop cataracts(of the zonular pulverant type) at about one week old. They also haveabnormally small eyes and lenses. Similarly, mutations in the human geneencoding Cx50 have been associated with the occurrence of congenitalcataracts. Affected individuals develop cataracts (with zonular pulverentopacities), and analysis shows they have a single point mutation in the Cx50coding region, resulting in a non-conservative substitution in the secondputative TM domain of a serine residue for a proline.
The connexins are a family of integral membrane proteins that oligomerise to form intercellular channels that are clustered at gap junctions. These channels are specialised sites of cell-cell contact that allow the passage of ions, intracellular metabolites and messenger molecules (with molecular weight less than 1-2kDa) from the cytoplasm of one cell to its opposing neighbours. They are found in almost all vertebrate cell types, and somewhat similar proteins have been cloned from plant species. Invertebrates utilise a different family of molecules, innexins, that share a similar predicted secondary structure to the vertebrate connexins, but have no sequence identity to them [].Vertebrate gap junction channels are thought to participate in diverse biological functions. For instance, in the heart they permit the rapid cell-cell transfer of action potentials, ensuring coordinated contraction of the cardiomyocytes. They are also responsible for neurotransmission at specialised 'electrical' synapses. In non-excitable tissues, such as the liver, they may allow metabolic cooperation between cells. In the brain, glial cells are extensively-coupled by gap junctions; this allows waves of intracellular Ca2+to propagate through nervous tissue, and may contribute to their ability to spatially-buffer local changes in extracellular K+concentration [].The connexin protein family is encoded by at least 13 genes in rodents, with many homologues cloned from other species. They show overlapping tissue expression patterns, most tissues expressing more than one connexin type. Their conductances, permeability to different molecules, phosphorylation and voltage-dependence of their gating, have been found to vary. Possible communication diversity is increased further by the fact that gap junctions may be formed by the association of different connexin isoforms from apposing cells. However, in vitro studies have shown that not all possible combinations of connexins produce active channels [, ].Hydropathy analysis predicts that all cloned connexins share a common transmembrane (TM) topology. Each connexin is thought to contain 4 TMdomains, with two extracellular and three cytoplasmic regions. This modelhas been validated for several of the family members by in vitrobiochemicalanalysis. Both N- and C-termini are thought to face the cytoplasm, and thethird TM domain has an amphipathic character, suggesting that it contributesto the lining of the formed-channel. Amino acid sequence identity betweenthe isoforms is ~50-80%, with the TM domains being well conserved. Bothextracellular loops contain characteristically conserved cysteine residues,which likely form intramolecular disulphide bonds. By contrast, the singleputative intracellular loop (between TM domains 2 and 3) and the cytoplasmicC terminus are highly variable among the family members.Six connexins arethought to associate to form a hemi-channel, or connexon. Two connexons theninteract (likely via the extracellular loops of their connexins) to form thecomplete gap junction channel.NH2-*** *** *************-COOH** ** ** **** ** ** ** Cytoplasmic---**----**-----**----**----------------** ** ** ** Membrane** ** ** **---**----**-----**----**----------------** ** ** ** Extracellular** ** ** **** **Two sets of nomenclature have been used to identify the connexins. Thefirst, and most commonly used, classifies the connexin molecules accordingto molecular weight, such as connexin43 (abbreviated to Cx43), indicatinga connexin of molecular weight close to 43kDa. However, studies haverevealed cases where clear functional homologues exist across speciesthat have quite different molecular masses; therefore, an alternativenomenclature was proposed based on evolutionary considerations, whichdivides the family into two major subclasses, alpha and beta, each with anumber of members []. Due to their ubiquityand overlapping tissue distributions, it has proved difficult to elucidate the functions of individual connexin isoforms. To circumvent this problem, particular connexin-encoding genes have been subjected to targeted-disruption in mice, and the phenotype of the resulting animals investigated. Around half the connexin isoforms have been investigated in this manner []. Further insight into the functional roles of connexins has come from the discovery that a number of human diseases are caused by mutations in connexin genes. For instance, mutations in Cx32 give rise to a form of inherited peripheral neuropathy called X-linked dominant Charcot-Marie-Tooth disease []. Similarly, mutations in Cx26 are responsible for both autosomal recessive and dominant forms of nonsyndromic deafness, a disorder characterised by hearing loss, with no apparent effects on other organ systems.Gap junction alpha-6 protein (also called connexin45, or Cx45) is a connexinof 396 amino acid residues. It is one of four isoforms expressed in theheart (together with Cx43, Cx40 and Cx37). All four isoforms show differingdistribution patterns within the human heart: Cx45 tends to be detectableonly at rather low levels, with a trend toward higher levels in the atriathan the ventricles. Cx45 is also thought to be involved in the formation ofgap junctions between the bone-forming cells, osteoblasts; the extent oftheir cell-cell coupling may act to modulate their gene expression.
The connexins are a family of integral membrane proteins that oligomerise to form intercellular channels that are clustered at gap junctions. These channels are specialised sites of cell-cell contact that allow the passage of ions, intracellular metabolites and messenger molecules (with molecular weight less than 1-2kDa) from the cytoplasm of one cell to its opposing neighbours. They are found in almost all vertebrate cell types, and somewhat similar proteins have been cloned from plant species. Invertebrates utilise a different family of molecules, innexins, that share a similar predicted secondary structure to the vertebrate connexins, but have no sequence identity to them [].Vertebrate gap junction channels are thought to participate in diverse biological functions. For instance, in the heart they permit the rapid cell-cell transfer of action potentials, ensuring coordinated contraction of the cardiomyocytes. They are also responsible for neurotransmission at specialised 'electrical' synapses. In non-excitable tissues, such as the liver, they may allow metabolic cooperation between cells. In the brain, glial cells are extensively-coupled by gap junctions; this allows waves of intracellular Ca2+to propagate through nervous tissue, and may contribute to their ability to spatially-buffer local changes in extracellular K+concentration [].The connexin protein family is encoded by at least 13 genes in rodents, with many homologues cloned from other species. They show overlapping tissue expression patterns, most tissues expressing more than one connexin type. Their conductances, permeability to different molecules, phosphorylation and voltage-dependence of their gating, have been found to vary. Possible communication diversity is increased further by the fact that gap junctions may be formed by the association of different connexin isoforms from apposing cells. However, in vitro studies have shown that not all possible combinations of connexins produce active channels [, ].Hydropathy analysis predicts that all cloned connexins share a common transmembrane (TM) topology. Each connexin is thought to contain 4 TMdomains, with two extracellular and three cytoplasmic regions. This modelhas been validated for several of the family members by in vitrobiochemicalanalysis. Both N- and C-termini are thought to face the cytoplasm, and thethird TM domain has an amphipathic character, suggesting that it contributesto the lining of the formed-channel. Amino acid sequence identity betweenthe isoforms is ~50-80%, with the TM domains being well conserved. Bothextracellular loops contain characteristically conserved cysteine residues,which likely form intramolecular disulphide bonds. By contrast, the singleputative intracellular loop (between TM domains 2 and 3) and the cytoplasmicC terminus are highly variable among the family members.Six connexins arethought to associate to form a hemi-channel, or connexon. Two connexons theninteract (likely via the extracellular loops of their connexins) to form thecomplete gap junction channel.NH2-*** *** *************-COOH** ** ** **** ** ** ** Cytoplasmic---**----**-----**----**----------------** ** ** ** Membrane** ** ** **---**----**-----**----**----------------** ** ** ** Extracellular** ** ** **** **Two sets of nomenclature have been used to identify the connexins. Thefirst, and most commonly used, classifies the connexin molecules accordingto molecular weight, such as connexin43 (abbreviated to Cx43), indicatinga connexin of molecular weight close to 43kDa. However, studies haverevealed cases where clear functional homologues exist across speciesthat have quite different molecular masses; therefore, an alternativenomenclature was proposed based on evolutionary considerations, whichdivides the family into two major subclasses, alpha and beta, each with anumber of members []. Due to their ubiquity and overlapping tissue distributions, it has proved difficult to elucidate the functions of individual connexin isoforms. To circumvent this problem, particular connexin-encoding genes have been subjected to targeted-disruption in mice, and the phenotype of the resulting animals investigated. Around half the connexin isoforms have been investigated in this manner []. Further insight into the functional roles of connexins has come from the discovery that a number of human diseases are caused by mutations in connexin genes. For instance, mutations in Cx32 give rise to a form of inherited peripheral neuropathy called X-linked dominant Charcot-Marie-Tooth disease []. Similarly, mutations in Cx26 are responsible for both autosomal recessive and dominant forms of nonsyndromic deafness, a disorder characterised by hearing loss, with no apparent effects on other organ systems.Gap junction beta-5 protein (also called connexin 31.1, or Cx31.1) is aconnexin of 271 amino acid residues that shares ~70% amino acid identitywith both Cx30.3 and Cx31 [, ]. It is most closely related to the former;together these molecules share arather restricted expression pattern,being preferentially expressed in the skin, and undetectable in most othertissues. Additionally, the genes encoding them have been found to berather closely-linked on mouse chromosome 4 [].
This group of aspartic peptidases belongs to the MEROPS family A26 (clan AF). Members of the family are transmembrane proteins. The type example for the family is omptin (also known as protease VII) from Escherichia coli, the product of the ompTgene. Omptin preferentially cleaves polypeptides between two basically-charged amino acids []. The tertiary structure has been solved and shows a ten-stranded beta barrel, and because the strands are amphipathic, hydrolphilic rsidue point into the barrel with the hydrophobic residue on the outside. The active site residues, two pairs of aspartic acids and a histidine, are on opposite sides of the active site groove and at the periplasmic surface []. Because the enzyme is sensitive to the serine protease inhibitor diisopropylfluoro-phosphate [], omptin was incorrectly identified as a serine peptidase.The family also includes the surface protease Pla from the plague organism Yersinia pestis, which is an important virulence factor. Pla can activate plasminogen and inactivate plasmin inhibitor, which may lead to uncontrolled proteolysis and aid entry of the bacteriuminto the circulation []. Pla is temperature sensitive, with proteolytic activity changing with temperature. At temperatures below 30 C, Pla acts as a coagulase, but at temperatures above 30 C it is fibrolytic [].Aspartic peptidases, also known as aspartyl proteases ([intenz:3.4.23.-]), are widely distributed proteolytic enzymes [, , ]known to exist in vertebrates, fungi, plants, protozoa, bacteria, archaea, retroviruses and some plant viruses. All known aspartic peptidases are endopeptidases. A water molecule, activated by two aspartic acid residues, acts as the nucleophile in catalysis. Aspartic peptidases can be grouped into five clans, each of which shows a unique structural fold [].Peptidases in clan AA are either bilobed (family A1 or the pepsin family) or are a homodimer (all other families in the clan, including retropepsin from HIV-1/AIDS) []. Each lobe consists of a single domain with a closed β-barrel and each lobe contributes one Asp to form the active site. Most peptidases in the clan are inhibited by the naturally occurring small-molecule inhibitor pepstatin [].Clan AC contains the single family A8: the signal peptidase 2 family. Members of the family are found in all bacteria. Signal peptidase 2 processes the premurein precursor, removing the signal peptide. The peptidase has four transmembrane domains and the active site is on the periplasmic side of the cell membrane. Cleavage occurs on the amino side of a cysteine where the thiol group has been substituted by a diacylglyceryl group. Site-directed mutagenesis has identified two essential aspartic acid residues which occur in the motifs GNXXDRX and FNXAD (where X is a hydrophobic residue) []. No tertiary structures have been solved for any member of the family, but because of the intramembrane location, the structure is assumed not to be pepsin-like.Clan AD contains two families of transmembrane endopeptidases: A22 and A24. These are also known as "GXGD peptidases"because of a common GXGD motif which includes one of the pair of catalytic aspartic acid residues. Structures are known for members of both families and show a unique, common fold with up to nine transmembrane regions []. The active site aspartic acids are located within a large cavity in the membrane into which water can gain access [].Clan AE contains two families, A25 and A31. Tertiary structures have been solved for members of both families and show a common fold consisting of an α-β-alpha sandwich, in which the beta sheet is five stranded [, ].Clan AF contains the single family A26. Members of the clan are membrane-proteins with a unique fold. Homologues are known only from bacteria. The structure of omptin (also known as OmpT) shows a cylindrical barrel containing ten beta strands inserted in the membrane with the active site residues on the outer surface [].There are two families of aspartic peptidases for which neither structure nor active site residues are known and these are not assigned to clans. Family A5 includes thermopsin, an endopeptidase found only in thermophilic archaea. Family A36 contains sporulation factor SpoIIGA, which is known to process and activate sigma factor E, one of the transcription factors that controls sporulation in bacteria [].
Aconitase (aconitate hydratase; ) is an iron-sulphur protein that contains a [4Fe-4S]-cluster and catalyses the interconversion of isocitrate and citrate via a cis-aconitate intermediate. Aconitase functions in both the TCA and glyoxylate cycles, however unlike the majority of iron-sulphur proteins that function as electron carriers, the [4Fe-4S]-cluster of aconitase reacts directly with an enzyme substrate. In eukaryotes there is a cytosolic form (cAcn) and a mitochondrial form (mAcn) of the enzyme. In bacteria there are also 2 forms, aconitase A (AcnA) and B (AcnB). Several aconitases are known to be multi-functional enzymes with a second non-catalytic, but essential function that arises when the cellular environment changes, such as when iron levels drop [, ]. Eukaryotic cAcn and mAcn, and bacterial AcnA have the same domain organisation, consisting of three N-terminal alpha/beta/alpha domains, a linker region, followed by a C-terminal 'swivel' domain with a beta/beta/alpha structure (1-2-3-linker-4), although mAcn is smaller than cAcn. However, bacterial AcnB has a different organisation: it contains an N-terminal HEAT-like domain, followed by the 'swivel' domain, then the three alpha/beta/alpha domains (HEAT-4-1-2-3) [].Eukaryotic cAcn enzyme balances the amount of citrate and isocitrate in the cytoplasm, which in turn creates a balance between the amount of NADPH generated from isocitrate by isocitrate dehydrogenase with the amount of acetyl-CoA generated from citrate by citrate lyase. Fatty acid synthesis requires both NADPH and acetyl-CoA, as do other metabolic processes, including the need for NADPH to combat oxidative stress. The enzymatic form of cAcn predominates when iron levels are normal, but if they drop sufficiently to cause the disassembly of the [4Fe-4S]-cluster, then cAcn undergoes a conformational change from a compact enzyme to a more open L-shaped protein known as iron regulatory protein 1 (IRP1; or IRE-binding protein 1, IREBP1) [, ]. As IRP1, the catalytic site and the [4Fe-4S]-cluster are lost, and two new RNA-binding sites appear. IRP1 functions in the post-transcriptional regulation of genes involved in iron metabolism - it binds to mRNA iron-responsive elements (IRE), 30-nucleotide stem-loop structures at the 3' or 5' end of specific transcripts. Transcripts containing an IRE include ferritin L and H subunits (iron storage), transferrin (iron plasma chaperone), transferrin receptor (iron uptake into cells), ferroportin (iron exporter), mAcn, succinate dehydrogenase, erythroid aminolevulinic acid synthetase (tetrapyrrole biosynthesis), among others. If the IRE is in the 5'-UTR of the transcript (e.g. in ferritin mRNA), then IRP1-binding prevents its translation by blocking the transcript from binding to the ribosome. If the IRE is in the 3'-UTR of the transcript (e.g. transferrin receptor), then IRP1-binding protects it from endonuclease degradation, thereby prolonging the half-life of the transcript and enabling it to be translated [].IRP2 is another IRE-binding protein that binds to the same transcripts as IRP1. However, since IRP1 is predominantly in the enzymatic cAcn form, it is IRP2 that acts as the major metabolic regulator that maintains iron homeostasis []. Although IRP2 is homologous to IRP1, IRP2 lacks aconitase activity, and is known only to have a single function in the post-transcriptional regulation of iron metabolism genes []. In iron-replete cells, IRP2 activity is regulated primarily by iron-dependent degradation through the ubiquitin-proteasomal system.Bacterial AcnB is also known to be multi-functional. In addition to its role in the TCA cycle, AcnB was shown to be a post-transcriptional regulator of gene expression in Escherichia coli and Salmonella enterica [, ]. In S. enterica, AcnB initiates a regulatory cascade controlling flagella biosynthesis through an interaction with the ftsH transcript, an alternative RNA polymerase sigma factor. This binding lowers the intracellular concentration of FtsH protease, which in turn enhances the amount of RNA polymerase sigma32 factor (normally degraded by FtsH protease), and sigma32 then increases the synthesis of chaperone DnaK, which in turn promotes the synthesis of the flagellar protein FliC. AcnB regulates the synthesis of other proteins as well, such as superoxide dismutase (SodA) and other enzymes involved in oxidative stress.3-isopropylmalate dehydratase (or isopropylmalate isomerase; ) catalyses the stereo-specific isomerisation of 2-isopropylmalate and 3-isopropylmalate, via the formation of 2-isopropylmaleate. This enzyme performs the second step in the biosynthesis of leucine, and is present in most prokaryotes and many fungal species. The prokaryotic enzyme is a heterodimer composed of a large (LeuC) and small (LeuD) subunit, while the fungal form is a monomeric enzyme. Both forms of isopropylmalate are related and are part of the larger aconitase family []. Aconitases are mostly monomeric proteins which share four domains in common and contain a single, labile [4Fe-4S]cluster. Three structural domains (1, 2 and 3) are tightly packed around the iron-sulphur cluster, while a fourth domain (4) forms a deep active-site cleft. The prokaryotic enzyme is encoded by two adjacent genes, leuC and leuD, corresponding to aconitase domains 1-3 and 4 respectively [, ]. LeuC does not bind an iron-sulphur cluster. It is thought that some prokaryotic isopropylamalate dehydrogenases can also function as homoaconitase , converting cis-homoaconitate to homoisocitric acid in lysine biosynthesis []. Homoaconitase has been identified in higher fungi (mitochondria) and several archaea and one thermophilic species of bacteria, Thermus thermophilus []. It is also found in the higher plant Arabidopsis thaliana, where it is targeted to the chloroplast [].This entry represents a region containing 3 domains, each with a 3-layer alpha/beta/alpha topology. This region represents the [4Fe-4S]cluster-binding region found at the N-terminal of eukaryotic mAcn, cAcn/IPR1 and IRP2, and bacterial AcnA, but in the C-terminal of bacterial AcnB. This domain is also found in the large subunit of isopropylmalate dehydratase (LeuC).
