|
| ||
|
|
||
C-terminal Domain (c-terminal + domain)
Kinds of C-terminal Domain Selected AbstractsStructural Models and Binding Site Prediction of the C-terminal Domain of Human Hsp90: A New Target for Anticancer DrugsCHEMICAL BIOLOGY & DRUG DESIGN, Issue 5 2008Miriam Sgobba Heat shock protein 90 is a valuable target for anticancer drugs because of its role in the activation and stabilization of multiple oncogenic signalling proteins. While several compounds inhibit heat shock protein 90 by binding the N-terminal domain, recent studies have proved that the C-terminal domain is important for dimerization of the chaperone and contains an additional binding site for inhibitors. Heat shock protein 90 inhibition achieved with molecules binding to the C-terminal domain provides an additional and novel opportunity to design and develop drugs. Therefore, for the first time, we have investigated the structure and the dynamic behaviour of the C-terminal domain of human heat shock protein 90 with and without the small-middle domain, using homology modelling and molecular dynamics simulations. In addition, secondary structure predictions and peptide folding simulations proved useful to investigate a putative additional ,-helix located between H18 and ,20 of the C-terminal domain. Finally, we used the structural information to infer the location of the binding site located in the C-terminal domain by using a number of computational tools. The predicted pocket is formed by two grooves located between helix H18, the loop downstream of H18 and the loop connecting helices H20 and H21 of each monomer of the C-terminal domain, with only two amino acids contributing from each middle domain. [source] The Photoreaction of the Photoactive Yellow Protein Domain in the Light Sensor Histidine Kinase Ppr is Influenced by the C-terminal Domains,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Hironari Kamikubo To study the role of the C-terminal domains in the photocycle of a light sensor histidine kinase (Ppr) having a photoactive yellow protein (PYP) domain as the photosensor domain, we analyzed the photocycles of the PYP domain of Ppr (Ppr-PYP) and full-length Ppr. The gene fragment for Ppr-PYP was expressed in Escherichia coli, and it was chemically reconstituted with p- coumaric acid; the full-length gene of Ppr was coexpressed with tyrosine ammonia-lyase and p -coumaric acid ligase for biosynthesis in cells. The light/dark difference spectra of Ppr-PYP were pH sensitive. They were represented as a linear combination of two independent difference spectra analogous to the PYPL/dark and PYPM/dark difference spectra of PYP from Halorhodospira halophila, suggesting that the pH dependence of the difference spectra is explained by the equilibrium shift between the PYPL - and PYPM -like intermediates. The light/dark difference spectrum of Ppr showed the equilibrium shift toward PYPL compared with that of Ppr-PYP. Kinetic measurements of the photocycles of Ppr and Ppr-PYP revealed that the C-terminal domains accelerate the recovery of the dark state. These observations suggest an interaction between the C-terminal domains and the PYP domain during the photocycle, by which light signals captured by the PYP domain are transferred to the C-terminal domains. [source] Two different unique cardiac isoforms of protein 4.1R in zebrafish, Danio rerio, and insights into their cardiac functions as related to their unique structuresDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 7 2010Kenji Murata Protein 4.1R (4.1R) has been identified as the major component of the human erythrocyte membrane skeleton. The members of the protein 4.1 gene family are expressed in a tissue-specific alternative splicing manner that increases their functions in each tissue; however, the exact roles of cardiac 4.1R in the developing myocardium are poorly understood. In zebrafish (ZF), we identified two heart-specific 4.1R isoforms, ZF4.1RH2 and ZF4.1RH3, encoding N-terminal 30 kDa (FERM) domain and spectrin-actin binding domain (SABD) and C-terminal domain (CTD), separately. Applying immunohistochemistry using specific antibodies for 30 kDa domain and CTD separately, the gene product of ZF4.1RH2 and ZF4.1RH3 appeared only in the ventricle and in the atrium, respectively, in mature hearts. During embryogenesis, both gene expressions are expressed starting 24 h post-fertilization (hpf). Following whole-mount in situ hybridization, ZF4.1RH3 gene expression was detected in the atrium of 37 hpf embryos. These results indicate that the gene product of ZF4.1RH3 is essential for normal morphological shape of the developing heart and to support the repetitive cycles of its muscle contraction and relaxation. [source] The capsid protein of human immunodeficiency virus: intersubunit interactions during virus assemblyFEBS JOURNAL, Issue 21 2009Mauricio G. Mateu The capsid protein (CA) of HIV-1 is composed of two domains, the N-terminal domain (NTD) and the C-terminal domain (CTD). During the assembly of the immature HIV-1 particle, both CA domains constitute a part of the Gag polyprotein, which forms a spherical capsid comprising up to 5000 radially arranged, extended subunits. Gag,Gag interactions in the immature capsid are mediated in large part by interactions between CA domains, which are involved in the formation of a lattice of connected Gag hexamers. After Gag proteolysis during virus maturation, the CA protein is released, and approximately 1000,1500 free CA subunits self-assemble into a truncated cone-shaped capsid. In the mature capsid, NTD,NTD and NTD,CTD interfaces are involved in the formation of CA hexamers, and CTD,CTD interfaces connect neighboring hexamers through homodimerization. The CA,CA interfaces involved in the assembly of the immature capsid and those forming the mature capsid are different, at least in part. CA appears to have evolved an extraordinary conformational plasticity, which allows the creation of multiple CA,CA interfaces and the occurrence of CA conformational switches. This minireview focuses on recent structure,function studies of the diverse CA,CA interactions and interfaces involved in HIV-1 assembly. Those studies are leading to a better understanding of molecular recognition events during virus morphogenesis, and are also relevant for the development of anti-HIV drugs that are able to interfere with capsid assembly or disassembly. [source] Definition of the residues required for the interaction between glycine-extended gastrin and transferrin in vitroFEBS JOURNAL, Issue 17 2009Suzana Kovac Transferrin is the main iron transport protein found in the circulation, and the level of transferrin saturation in the blood is an important indicator of iron status. The peptides amidated gastrin(17) (Gamide) and glycine-extended gastrin(17) (Ggly) are well known for their roles in controlling acid secretion and as growth factors in the gastrointestinal tract. Several lines of evidence, including the facts that transferrin binds gastrin, that gastrins bind ferric ions, and that the level of expression of gastrins positively correlates with transferrin saturation, suggest the possible involvement of the transferrin,gastrin interaction in iron homeostasis. In the present work, the interaction between gastrins and transferrin has been characterized by surface plasmon resonance and covalent crosslinking. First, an interaction between iron-free apo-transferrin and Gamide or Ggly was observed. The fact that no interaction was observed in the presence of the chelator EDTA suggested that the gastrin,ferric ion complex was the interacting species. Moreover, removal of ferric ions with EDTA reduced the stability of the complex between apo-transferrin and gastrins, and no interaction was observed between Gamide or Ggly and diferric transferrin. Second, some or all of glutamates at positions 8,10 of the Ggly molecule, together with the C-terminal domain, were necessary for the interaction with apo-transferrin. Third, monoferric transferrin mutants incapable of binding iron in either the N-terminal or C-terminal lobe still bound Ggly. These findings are consistent with the hypothesis that gastrin peptides bind to nonligand residues within the open cleft in each lobe of transferrin and are involved in iron loading of transferrin in vivo. Structured digital abstract ,,MINT-7212832, MINT-7212849: Apo-transferrin (uniprotkb:P02787) and Gamide (uniprotkb:P01350) bind (MI:0407) by surface plasmon resonance (MI:0107) ,,MINT-7212881, MINT-7212909: Ggly (uniprotkb:P01350) and Apo-transferrin (uniprotkb:P02787) bind (MI:0407) by cross-linking studies (MI:0030) ,,MINT-7212864: Apo-transferrin (uniprotkb:P02787) and Ggly (uniprotkb:P01350) bind (MI:0407) by competition binding (MI:0405) [source] Site-directed mutagenesis of the active site loop of the rhodanese-like domain of the human molybdopterin synthase sulfurase MOCS3FEBS JOURNAL, Issue 11 2007Major differences in substrate specificity between eukaryotic, bacterial homologs Sequence alignments of human molybdopterin synthase sulfurase, MOCS3, showed that the N-terminal domain is homologous to Escherichia coli MoeB, whereas the C-terminal domain is homologous to rhodanese-like proteins. Previous studies showed that the activity of the separately purified rhodanese-like domain of MOCS3 displayed 1000-fold lower activity in comparison to bovine rhodanese with thiosulfate as sulfur source. When the six amino acid active site loop of MOCS3 rhodanese-like domain was exchanged with the loop found in bovine rhodanese, thiosulfate:cyanide sulfurtransferase activity was increased 165-fold. Site-directed mutagenesis of each individual residue of the active site loop of the MOCS3 rhodanese-like domain showed that the charge of the last amino acid determines thiosulfate sulfurtransferase activity. Replacing Asp417 by threonine resulted in 90-fold increased activity, whereas replacing it by arginine increased the activity 470-fold. Using a fully defined in vitro system containing precursor Z, MOCS2A, E. coli MoaE, E. coli MoeB, Mg-ATP, MOCS3 rhodanese-like domain, and thiosulfate, it was shown that sulfur transfer to MOCS2A was also affected by the alterations, but not as drastically. Our studies revealed that in humans and most eukaryotes thiosulfate is not the physiologic sulfur donor for MOCS3, whereas in bacterial homologs, which have an arginine at the last position of the active site loop, thiosulfate can be used as a sulfur source for molybdenum cofactor biosynthesis. The phylogenetic analysis of MoeB homologs showed that eukaryotic homologs are of bacterial origin. Furthermore, it could be shown that an MoeB homolog named MoeZ, where the dual CXXC zinc-binding motif of the MoeB domain is not present, arose independently several times during evolution. [source] The domains carrying the opposing activities in adenylyltransferase are separated by a central regulatory domainFEBS JOURNAL, Issue 11 2007Paula Clancy Adenylyltransferase is a bifunctional enzyme that controls the enzymatic activity of dodecameric glutamine synthetase in Escherichia coli by reversible adenylylation and deadenylylation. Previous studies showed that the two similar but chemically distinct reactions are carried out by separate domains within adenylyltransferase. The N-terminal domain carries the deadenylylation activity, and the C-terminal domain carries the adenylylation activity [Jaggi R, van Heeswijk WC, Westerhoff HV, Ollis DL & Vasudevan SG (1997) EMBO J16, 5562,5571]. In this study, we further map the domain junctions of adenylyltransferase on the basis of solubility and enzymatic analysis of truncation constructs, and show for the first time that adenylyltransferase has three domains: the two activity domains and a central, probably regulatory (R), domain connected by interdomain Q-linkers (N-Q1-R-Q2-C). The various constructs, which have the opposing domain and or central domain removed, all retain their activity in the absence of their respective nitrogen status indicator, i.e. PII or PII-UMP. A panel of mAbs to adenylyltransferase was used to demonstrate that the cellular nitrogen status indicators, PII and PII-UMP, probably bind in the central regulatory domain to stimulate the adenylylation and deadenylylation reactions, respectively. In the light of these results, intramolecular signaling within adenylyltransferase is discussed. [source] Monomeric solution structure of the helicase-binding domain of Escherichia coli DnaG primaseFEBS JOURNAL, Issue 21 2006Xun-Cheng Su DnaG is the primase that lays down RNA primers on single-stranded DNA during bacterial DNA replication. The solution structure of the DnaB-helicase-binding C-terminal domain of Escherichia coli DnaG was determined by NMR spectroscopy at near-neutral pH. The structure is a rare fold that, besides occurring in DnaG C-terminal domains, has been described only for the N-terminal domain of DnaB. The C-terminal helix hairpin present in the DnaG C-terminal domain, however, is either less stable or absent in DnaB, as evidenced by high mobility of the C-terminal 35 residues in a construct comprising residues 1,171. The present structure identifies the previous crystal structure of the E. coli DnaG C-terminal domain as a domain-swapped dimer. It is also significantly different from the NMR structure reported for the corresponding domain of DnaG from the thermophile Bacillus stearothermophilus. NMR experiments showed that the DnaG C-terminal domain does not bind to residues 1,171 of the E. coli DnaB helicase with significant affinity. [source] Isolation, characterization, sequencing and crystal structure of charybdin, a type 1 ribosome-inactivating protein from Charybdis maritima agg.FEBS JOURNAL, Issue 12 2006Eleftherios Touloupakis A novel, type 1 ribosome-inactivating protein designated charybdin was isolated from bulbs of Charybdis maritima agg. The protein, consisting of a single polypeptide chain with a molecular mass of 29 kDa, inhibited translation in rabbit reticulocytes with an IC50 of 27.2 nm. Plant genomic DNA extracted from the bulb was amplified by PCR between primers based on the N-terminal and C-terminal sequence of the protein from dissolved crystals. The complete mature protein sequence was derived by partial DNA sequencing and terminal protein sequencing, and was confirmed by high-resolution crystal structure analysis. The protein contains Val at position 79 instead of the conserved Tyr residue of the ribosome-inactivating proteins known to date. To our knowledge, this is the first observation of a natural substitution of a catalytic residue at the active site of a natural ribosome-inactivating protein. This substitution in the active site may be responsible for the relatively low in vitro translation inhibitory effect compared with other ribosome-inactivating proteins. Single crystals were grown in the cold room from PEG6000 solutions. Diffraction data collected to 1.6 Å resolution were used to determine the protein structure by the molecular replacement method. The fold of the protein comprises two structural domains: an ,,+ , N-terminal domain (residues 4,190) and a mainly ,-helical C-terminal domain (residues 191,257). The active site is located in the interface between the two domains and comprises residues Val79, Tyr117, Glu167 and Arg170. [source] Inhibition of human ether à go-go potassium channels by Ca2+/calmodulin binding to the cytosolic N- and C-terminiFEBS JOURNAL, Issue 5 2006Ulrike Ziechner Human ether à go-go potassium channels (hEAG1) open in response to membrane depolarization and they are inhibited by Ca2+/calmodulin (CaM), presumably binding to the C-terminal domain of the channel subunits. Deletion of the cytosolic N-terminal domain resulted in complete abolition of Ca2+/CaM sensitivity suggesting the existence of further CaM binding sites. A peptide array-based screen of the entire cytosolic protein of hEAG1 identified three putative CaM-binding domains, two in the C-terminus (BD-C1: 674,683, BD-C2: 711,721) and one in the N-terminus (BD-N: 151,165). Binding of GST-fusion proteins to Ca2+/CaM was assayed with fluorescence correlation spectroscopy, surface plasmon resonance spectroscopy and precipitation assays. In the presence of Ca2+, BD-N and BD-C2 provided dissociation constants in the nanomolar range, BD-C1 bound with lower affinity. Mutations in the binding domains reduced inhibition of the functional channels by Ca2+/CaM. Employment of CaM-EF-hand mutants showed that CaM binding to the N- and C-terminus are primarily dependent on EF-hand motifs 3 and 4. Hence, closure of EAG channels presumably requires the binding of multiple CaM molecules in a manner more complex than previously assumed. [source] The calpain 1,,-actinin interactionFEBS JOURNAL, Issue 23 2003Resting complex between the calcium-dependant protease, its target in cytoskeleton Calpain 1 behaviour toward cytoskeletal targets was investigated using two ,-actinin isoforms from smooth and skeletal muscles. These two isoforms which are, respectively, sensitive and resistant to calpain cleavage, interact with the protease when using in vitro binding assays. The stability of the complexes in EGTA [Kd(,Ca2+) = 0.5 ± 0.1 µm] was improved in the presence of 1 mm calcium ions [Kd(+Ca2+) = 0.05 ± 0.01 µm]. Location of the binding structures shows that the C-terminal domain of ,-actinin and each calpain subunit, 28 and 80 kDa, participates in the interaction. In particular, the autolysed calpain form (76/18) affords a similar binding compared to the 80/28 intact enzyme, with an identified binding site in the catalytic subunit, located in the C-terminal region of the chain (domain III,IV). The in vivo colocalization of calpain 1 and ,-actinin was shown to be likely in the presence of calcium, when permeabilized muscle fibres were supplemented by exogenous calpain 1 and the presence of calpain 1 in Z-line cores was shown by gold-labelled antibodies. The demonstration of such a colocalization was brought by coimmunoprecipitation experiments of calpain 1 and ,-actinin from C2.7 myogenic cells. We propose that calpain 1 interacts in a resting state with cytoskeletal targets, and that this binding is strengthened in pathological conditions, such as ischaemia and dystrophies, associated with high calcium concentrations. [source] The N -acetylglutamate synthase/N -acetylglutamate kinase metabolon of Saccharomyces cerevisiae allows co-ordinated feedback regulation of the first two steps in arginine biosynthesisFEBS JOURNAL, Issue 5 2003Katia Pauwels In Saccharomyces cerevisiae, which uses the nonlinear pathway of arginine biosynthesis, the first two enzymes, N -acetylglutamate synthase (NAGS) and N -acetylglutamate kinase (NAGK), are controlled by feedback inhibition. We have previously shown that NAGS and NAGK associate in a complex, essential to synthase activity and protein level [Abadjieva, A., Pauwels, K., Hilven, P. & Crabeel, M. (2001) J. Biol. Chem.276, 42869,42880]. The NAGKs of ascomycetes possess, in addition to the catalytic domain that is shared by all other NAGKs and whose structure has been determined, a C-terminal domain of unknown function and structure. Exploring the role of these two domains in the synthase/kinase interaction, we demonstrate that the ascomycete-specific domain is required to maintain synthase activity and protein level. Previous results had suggested a participation of the third enzyme of the pathway, N -acetylglutamylphosphate reductase, in the metabolon. Here, genetic analyses conducted in yeast at physiological level, or in a heterologous background, clearly demonstrate that the reductase is dispensable for synthase activity and protein level. Most importantly, we show that the arginine feedback regulation of the NAGS and NAGK enzymes is mutually interdependent. First, the kinase becomes less sensitive to arginine feedback inhibition in the absence of the synthase. Second, and as in Neurospora crassa, in a yeast kinase mutant resistant to arginine feedback inhibition, the synthase becomes feedback resistant concomitantly. We conclude that the NAGS/NAGK metabolon promotes the co-ordination of the catalytic activities and feedback regulation of the first two, flux controlling, enzymes of the arginine pathway. [source] A hydrophobic segment within the C-terminal domain is essential for both client-binding and dimer formation of the HSP90-family molecular chaperoneFEBS JOURNAL, Issue 1 2003Shin-ichi Yamada The , isoform of human 90-kDa heat shock protein (HSP90,) is composed of three domains: the N-terminal (residues 1,400); middle (residues 401,615) and C-terminal (residues 621,732). The middle domain is simultaneously associated with the N- and C-terminal domains, and the interaction with the latter mediates the dimeric configuration of HSP90. Besides one in the N-terminal domain, an additional client-binding site exists in the C-terminal domain of HSP90. The aim of the present study is to elucidate the regions within the C-terminal domain responsible for the bindings to the middle domain and to a client protein, and to define the relationship between the two functions. A bacterial two-hybrid system revealed that residues 650,697 of HSP90, were essential for the binding to the middle domain. An almost identical region (residues 657,720) was required for the suppression of heat-induced aggregation of citrate synthase, a model client protein. Replacement of either Leu665-Leu666 or Leu671-Leu672 to Ser-Ser within the hydrophobic segment (residues 662,678) of the C-terminal domain caused the loss of bindings to both the middle domain and the client protein. The interaction between the middle and C-terminal domains was also found in human 94-kDa glucose-regulated protein. Moreover, Escherichia coli HtpG, a bacterial HSP90 homologue, formed heterodimeric complexes with HSP90, and the 94-kDa glucose-regulated protein through their middle-C-terminal domains. Taken together, it is concluded that the identical region including the hydrophobic segment of the C-terminal domain is essential for both the client binding and dimer formation of the HSP90-family molecular chaperone and that the dimeric configuration appears to be similar in the HSP90-family proteins. [source] Structural consequences of site-directed mutagenesis in flexible protein domainsFEBS JOURNAL, Issue 8 200156)S mutant of RhoGDI, NMR characterization of the L(5 The guanine dissociation inhibitor RhoGDI consists of a folded C-terminal domain and a highly flexible N-terminal region, both of which are essential for biological activity, that is, inhibition of GDP dissociation from Rho GTPases, and regulation of their partitioning between membrane and cytosol. It was shown previously that the double mutation L55S/L56S in the flexible region of RhoGDI drastically decreases its affinity for Rac1. In the present work we study the effect of this double mutation on the conformational and dynamic properties of RhoGDI, and describe the weak interaction of the mutant with Rac1 using chemical shift mapping. We show that the helical content of the region 45,56 of RhoGDI is greatly reduced upon mutation, thus increasing the entropic penalty for the immobilization of the helix, and contributing to the loss of binding. In contrast to wild-type RhoGDI, no interaction with Rac1 could be identified for amino-acid residues of the flexible domain of the mutant RhoGDI and only very weak binding was observed for the folded domain of the mutant. The origins of the effect of the L55S/L56S mutation on the binding constant (decreased by at least three orders of magnitude relative to wild-type) are discussed with particular reference to the flexibility of this part of the protein. [source] Identification of residues critical for toxicity in Clostridium perfringens phospholipase C, the key toxin in gas gangreneFEBS JOURNAL, Issue 16 2000Alberto Alape-Girón Clostridium perfringens phospholipase C (PLC), also called ,-toxin, is the major virulence factor in the pathogenesis of gas gangrene. The toxic activities of genetically engineered ,-toxin variants harboring single amino-acid substitutions in three loops of its C-terminal domain were studied. The substitutions were made in aspartic acid residues which bind calcium, and tyrosine residues of the putative membrane-interacting region. The variants D269N and D336N had less than 20% of the hemolytic activity and displayed a cytotoxic potency 103 -fold lower than that of the wild-type toxin. The variants in which Tyr275, Tyr307, and Tyr331 were substituted by Asn, Phe, or Leu had 11,73% of the hemolytic activity and exhibited a cytotoxic potency 102 - to 105 -fold lower than that of the wild-type toxin. The results demonstrated that the sphingomyelinase activity and the C-terminal domain are required for myotoxicity in vivo and that the variants D269N, D336N, Y275N, Y307F, and Y331L had less than 12% of the myotoxic activity displayed by the wild-type toxin. This work therefore identifies residues critical for the toxic activities of C. perfringens PLC and provides new insights toward understanding the mechanism of action of this toxin at a molecular level. [source] Identification of tudor repeat associator with PCTAIRE 2 (Trap)FEBS JOURNAL, Issue 7 2000A novel protein that interacts with the N-terminal domain of PCTAIRE 2 in rat brain PCTAIRE 2 is a Cdc2-related kinase that is predominantly expressed in the terminally differentiated neuron. To elucidate the function of PCTAIRE 2, proteins that associate with PCTAIRE 2 were screened by the yeast two-hybrid system. A positive clone was found to encode a novel protein that could bind to PCTAIRE 2 in vitro as well as in vivo, and was designated as Trap (tudor repeat associator with PCTAIRE 2). The overall structure of Trap shows no significant homology to any proteins, but contains five repeated domains (the tudor-like domain), conserved in Drosophila tudor protein. Trap associates with the N-terminal domain of PCTAIRE 2 through its C-terminal domain, which contains two tudor-like domains. PCTAIRE 1, but not PCTAIRE 3, can also associate with Trap. Trap is predominantly expressed in brain and testis, and gradually increases during brain development throughout life, consistent with the expression pattern of PCTAIRE 2. Immunoreactivities for PCTAIRE 2 and Trap were colocalized to the mitochondria in COS 7 cells. Immunohistochemical analyses showed that PCTAIRE 2 and Trap were distributed in the same cell layer of the cerebral cortex and cerebellum. These findings suggest that Trap is a physiological partner of PCTAIRE 2 in terminally differentiated neurons. [source] Regulation of anaerobic arginine catabolism in Bacillus licheniformis by a protein of the Crp/Fnr familyFEMS MICROBIOLOGY LETTERS, Issue 2 2000Abdelouahid Maghnouj Abstract Arginine anaerobic catabolism occurs in Bacillus licheniformis through the arginine deiminase pathway, encoded by the gene cluster arcABDC. We report here the involvement of a new protein, ArcR, in the regulation of the pathway. ArcR is a protein of the Crp/Fnr family encoded by a gene located 109 bp downstream from arcC. It binds to a palindromic sequence, very similar to an Escherichia coli Crp binding site, located upstream from arcA. Residues in the C-terminal domain of Crp that form the DNA binding motif, in particular residues Arg-180 and Glu-181 that make specific bonds with DNA, are conserved in ArcR, suggesting that the complexes formed with DNA by Crp and ArcR are similar. Moreover, the pattern of DNase I hypersensitivity sites induced by the binding of ArcR suggests that ArcR bends the DNA in the same way as Crp. From the absence of anaerobic induction following inactivation of arcR and from the existence of a binding site upstream of the arcA transcription start point, it can be inferred that ArcR is an activator of the arginine deiminase pathway. [source] Members of the IclR family of bacterial transcriptional regulators function as activators and/or repressorsFEMS MICROBIOLOGY REVIEWS, Issue 2 2006Antonio J. Molina-Henares Abstract Members of the IclR family of regulators are proteins with around 250 residues. The IclR family is best defined by a profile covering the effector binding domain. This is supported by structural data and by a number of mutants showing that effector specificity lies within a pocket in the C-terminal domain. These regulators have a helix-turn-helix DNA binding motif in the N-terminal domain and bind target promoters as dimers or as a dimer of dimers. This family comprises regulators acting as repressors, activators and proteins with a dual role. Members of the IclR family control genes whose products are involved in the glyoxylate shunt in Enterobacteriaceae, multidrug resistance, degradation of aromatics, inactivation of quorum-sensing signals, determinants of plant pathogenicity and sporulation. No clear consensus exists on the architecture of DNA binding sites for IclR activators: the MhpR binding site is formed by a 15-bp palindrome, but the binding sites of PcaU and PobR are three perfect 10-bp sequence repetitions forming an inverted and a direct repeat. IclR-type positive regulators bind their promoter DNA in the absence of effector. The mechanism of repression differs among IclR-type regulators. In most of them the binding sites of RNA polymerase and the repressor overlap, so that the repressor occludes RNA polymerase binding. In other cases the repressor binding site is distal to the RNA polymerase, so that the repressor destabilizes the open complex. [source] N-terminal extension of Saccharomyces cerevisiae translation termination factor eRF3 influences the suppression efficiency of sup35 mutationsFEMS YEAST RESEARCH, Issue 3 2007Kirill Volkov Abstract The eukaryotic translation termination factor eRF3 stimulates release of nascent polypeptides from the ribosome in a GTP-dependent manner. In most eukaryotes studied, eRF3 consists of an essential, conserved C-terminal domain and a nonessential, nonconserved N-terminal extension. However, in some species, this extension is required for efficient termination. Our data show that the N-terminal extension of Saccharomyces cerevisiae eRF3 also participates in regulation of termination efficiency, but acts as a negative factor, increasing nonsense suppression efficiency in sup35 mutants containing amino acid substitutions in the C-terminal domain of the protein. [source] Central forkhead domain of human TFIIE, plays a primary role in binding double-stranded DNA at transcription initiationGENES TO CELLS, Issue 3 2009Aki Tanaka The human general transcription factor, TFIIE, consists of two subunits, , and ,. Structural analyses indicated the presence of a forkhead motif within the central region of TFIIE,. This motif was essential for transcription and possessed a double-stranded DNA-binding activity. Protein-DNA photo-cross-linking studies indicated that TFIIE, binds within the promoter region, adjacent to the transcription initiation site where promoter melting begins at transcription initiation. Furthermore, neither TFIIE nor the other general transcription factor TFIIH, were required for basal transcription of adenovirus major late promoter artificially pre-melted at the initiation site. These data suggest a model in which TFIIE binds to a position adjacent to the initiation site via the forkhead domain, enabling TFIIH to begin opening the promoter. Here, we used systematic point mutations to further investigate the functional roles of this domain. The mutant proteins were expressed in bacteria, purified and used to examine transcription of two different forms of template, phosphorylation of the C-terminal domain of RNA polymerase II, as well as dsDNA-binding. Taken together, our results strongly demonstrated that the primary function of the forkhead region is dsDNA-binding in transcription. In addition, we identified three positively charged lysine residues which play a key role in this function. [source] Mammalian CLASPs are required for mitotic spindle organization and kinetochore alignmentGENES TO CELLS, Issue 8 2006Yuko Mimori-Kiyosue CLASP1 and CLASP2 are homologous mammalian proteins, which associate with the ends of growing microtubules, as well as the cell cortex and the kinetochores of mitotic chromosomes. Previous studies have shown that in interphase cells CLASPs can attach microtubule plus ends to the cortex and stabilize them by repeatedly rescuing them from depolymerization. Here we show that CLASP1 and 2 play similar and redundant roles in organizing the mitotic apparatus in HeLa cells. Simultaneous depletion of both CLASPs causes mitotic spindle defects and a significant metaphase delay, which often results in abnormal exit from mitosis. Metaphase delay is associated with decreased kinetochore tension, increased kinetochore oscillations and more rapid microtubule growth. We show that the association of CLASP2 with the kinetochores relies on its C-terminal domain, but is independent of microtubules or association with CLIP-170. We propose that CLASPs exhibit at the kinetochores an activity similar to that at the cortex, providing apparent stabilization of microtubules by locally reducing the amplitude of growth/shortening episodes at the microtubule ends. This local stabilization of microtubules is essential for the formation of normal metaphase spindle, completion of anaphase and cytokinesis. [source] Meu10 is required for spore wall maturation in Schizosaccharomyces pombeGENES TO CELLS, Issue 2 2002Takahiro Tougan Background: Many genes are meiosis and/or sporulation-specifically transcribed during this process. Isolation and analysis of these genes might help us to understand how meiosis and sporulation are regulated. For this purpose, we have isolated a large number of cDNA clones from Schizosaccharomyces pombe whose expression is up-regulated during meiosis. Results: We have isolated meu10+ gene, which encodes 416 amino acids and bears homology to SPS2 of Saccharomyces cerevisiae. A strain whose meu10+ gene has been deleted forms no viable spores. Thin-section electron micrographs showed that the meu10, strain has abnormally formed spore walls, and then they disrupt, allowing cytoplasmic material to escape. The Meu10-GFP fusion protein is localized to the spore periphery, thereafter returned to the cytoplasm after sporulation. Meu10-GFP localization to the spore wall was almost normal in the bgs2, or chs1, mutants that lack 1,3-,-glucan or chitin, respectively. In contrast, 1,3-,-glucan is abnormally localized in meu10, cells. Meu10 has an N-terminal domain with homology to the mammalian insulin receptor and a C-terminal domain with a transmembrane motif. Mutants whose N-terminal or C-terminal domain was truncated were severely defective for sporulation. Conclusions: Meu10 is a spore wall component and plays a pivotal role in the formation of the mature spore wall structure. [source] The use of membrane translocating peptides to identify sites of interaction between the C5a receptor and downstream effector proteinsIMMUNOLOGY, Issue 4 2004Graham A. Auger Summary The complement fragment C5a is a potent leucocyte chemoattractant and activator, mediating its effects through a G-protein-coupled receptor. Whilst the C-terminal domain of this receptor has been shown to be essential for receptor desensitization and internalization, it is not known which domains couple to the receptor's heterotrimeric G proteins. In this report we have used a membrane translocating sequence (MTS) to examine the effects of the four intracellular domains of the human C5a receptor (C5aR) on the receptor's signalling via G,i family heterotrimeric G proteins in intact RBL-2H3 cells. The results indicate that all of the intracellular domains couple to downstream signalling, with the proximal region of the C terminus being a major binding site and intracellular loop 3 playing a role in G protein activation or receptor desensitization. [source] From collagen chemistry towards cell therapy , a personal journeyINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2007Michael E. Grant Summary The Fell,Muir Award requires the recipient to deliver a lecture and a review manuscript which provides a personal overview of significant scientific developments in the field of matrix biology over the period of the recipient's career. In this context, this review considers the collagen family of structural proteins and the advances in biochemical, molecular biological and genetic techniques which led to the elucidation of the structure, synthesis and function of this important group of extracellular matrix constituents. Particular attention is focussed on early research on the identification and assembly of the soluble precursors of collagen types I and II, and the identification of the precursor of basement membrane collagen type IV. In subsequent studies investigating the maintenance of the chick chondrocyte phenotype in culture, the influence of the extracellular milieu was found to influence markedly both cell morphology and collagen gene expression. These studies led to the discovery of collagen type X whose expression is restricted to hypertrophic chondrocytes at sites of endochondral ossification. Such research provided a prelude to investigations of mammalian endochondral ossification which is known to be aberrant in a variety of human chondrodysplasias and is reactivated in bone fracture repair and in osteoarthritis. The cloning of bovine and then human collagen type X genes facilitated studies in relevant human diseases and contributed to the discovery of mutations in the COL10A1 gene in families with metaphyseal chondrodysplasia type Schmid. Clustering of mutations in the C-terminal domain of the type X collagen molecule has now been widely documented and investigations of the pathogenic mechanisms in animal models are beginning to suggest the prospect of novel treatment strategies. [source] STAT-1: a novel regulator of apoptosisINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 6 2003Anastasis Stephanou Summary., Extracellular signalling molecules binding to their specific receptors are able to modulate gene expression, leading to changes in development, cell growth and homeostasis. The signal transducers and activators of transcription (STAT) protein family members are among the best studied of the latent cytoplasmic signal-dependent transcription factors. The STAT factors are activated via phosphorylation on the C-terminal domain following cytokine signalling or by various stress-induced stimuli. Recently, STAT-1 has been implicated in modulating pro- and anti-apoptotic genes following several stress-induced responses. These effects are dependent on STAT-1 phosphorylation on serine-727 and require the C-terminal transactivation domain of STAT-1 to enhance its pro-apoptotic effect or inhibit its anti-apoptotic effects. The STAT-1 C-terminal domain has been demonstrated to be important for protein,protein interaction with other transcriptional activators. The reports that STAT-1-deficient mice develop spontaneous and chemically induced tumours more rapidly compared to wild-type mice and that STAT-1-deficient cells are more resistant to agents that induce apoptosis strongly support the argument that STAT-1 acts as a tumour suppressor. [source] Two potent transactivation domains in the C-terminal region of human NANOG mediate transcriptional activation in human embryonic carcinoma cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2009Hyun-Jin Do Abstract The core embryonic stem cell transcription factors Oct4, Sox2, and Nanog are expressed in germ cell tumors (GCTs) and have been proposed to play a regulatory role in tumorigenesis. However, little is known about the mechanism of regulation of tumorigenesis by the complicated network of these proteins. Nanog is a novel homeobox-containing transcription factor that is expressed in pluripotent cells as well as GCTs. To understand the molecular and functional role of human NANOG (hNANOG) in germ cells, mutagenesis of the C-terminal domain (CD) of hNANOG and transient transfection assays in NCCIT human embryonic carcinoma cells were carried out to identify critical transactivation motifs. We divided the CD into three putative functional subdomains, CD1, tryptophan-repeat (WR) subdomain, and CD2. WR subdomain and CD2 independently contained transcriptional potential and, in combination, had a synergistic effect on transcriptional activity, while CD1 was transcriptionally inactive. The glutamine (Q) motif in WR subdomain, and multiple acidic residues in CD2 were required for maximal and synergistic transcriptional activation by the hNANOG CD. The results of the current study contribute to a better understanding of the complicated molecular machinery of stem cell transcription factors and their role in unregulated proliferation in germ cell tumorigenesis. J. Cell. Biochem. 106: 1079,1089, 2009. © 2009 Wiley-Liss, Inc. [source] The C-terminal C1 cassette of the N -methyl- d -aspartate receptor 1 subunit contains a bi-partite nuclear localization sequenceJOURNAL OF NEUROCHEMISTRY, Issue 6 2002K. D. Holmes Abstract The N -methyl- d -aspartate receptor (NMDAR) is a multimeric transmembrane protein composed of at least two subunits. One subunit, NR1, is derived from a single gene and can be subdivided into three regions: the N-terminal extracellular domain, the transmembrane regions, and the C-terminal intracellular domain. The N-terminal domain is responsible for Mg2+ metal ion binding and channel activity, while the transmembrane domains are important for ion channel formation. The intracellular C-terminal domain is involved in regulating receptor activity and subcellular localization. Our recent experiments indicated that the intracellular C-terminal domain, when expressed independently, localizes almost exclusively in the nucleus. An examination of the amino acid sequence reveals the presence of a putative nuclear localization sequence (NLS) in the C1 cassette of the NR1 intracellular C-terminus. Using an expression vector designed to test whether a putative NLS sequence is a valid, functional NLS, we have demonstrated that a bi-partite NLS does in fact exist within the NR1-1 C-terminus. Computer algorithms identified a putative helix,loop,helix motif that spanned the C0C1 cassettes of the C-terminus. These data suggest that the NR1 subunit may represent another member of a family of transmembrane proteins that undergo intramembrane proteolysis, releasing a cytosolic peptide that is actively translocated to the nucleus leading to alterations in gene regulation. [source] Identification of a cellular protein specifically interacting with the precursor of the hepatitis B e antigenJOURNAL OF VIRAL HEPATITIS, Issue 3 2001S. Salhi In hepatitis B virus (HBV) the precore gene encodes a protein from which derives P22, the precursor of the mature secreted hepatitis B virus e antigen (HBeAg). Circumstantial evidences suggest that HBeAg and/or its precursor P22 are important for establishing persistent infection. Although P22 is essentially present in the secretory pathway, a substantial fraction has been found in the cytosol. In order to get new insights into the biological function of P22, we looked for cellular proteins which could strongly associate with this protein. Using immunoprecipitation studies on human cell extracts, we found that a non-secreted cellular protein of about 32 kDa (P32) bound with a high specificity to P22. P32 associated neither with HBeAg nor with the viral core protein P21 which exhibits the same amino acids sequence as P22 but is N-terminally shorter by 10 residues. We also demonstrated that this interaction depended on the presence of the P22 C-terminal domain. Our data argues for a potential biological function of P22. [source] Current trends in the structure,activity relationship studies of the endogenous agouti-related protein (AGRP) melanocortin receptor antagonistMEDICINAL RESEARCH REVIEWS, Issue 5 2005Andrzej M. Wilczynski Abstract Agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin-3 and -4 (MC3R and MC4) G-protein coupled receptors. The 87,132 amino acid C-terminal domain of hAGRP possesses five disulfide bridges and a well-defined three-dimensional structure that displays full biological activity as compared to the full-length protein. Based on the NMR structure of the C-terminal AGRP(87,132), a novel mini-protein, referred to as "Mini-AGRP" was designed that exhibited receptor binding affinity and antagonism similar to that of the parent hAGRP(87,132) protein. It was demonstrated that this new-engineered protein autonomously folds to the inhibitor cystine knot (ICK) motif. As this AGRP is a novel mammalian protein involved in energy homeostasis and possibly other physiological functions remaining to be identified, structure-function studies are starting to emerge toward the understanding of how this unique protein putatively interacts with the melanocortin receptors with the objective of designing potential therapeutic agents for in vivo physiological studies. This article summarizes the progress to date of AGRP-based structure,activity relationships and putative ligand,receptor interactions. © 2005 Wiley Periodicals, Inc. [source] The RNA degradosome in Bacillus subtilis: identification of CshA as the major RNA helicase in the multiprotein complexMOLECULAR MICROBIOLOGY, Issue 4 2010Martin Lehnik-Habrink Summary In most organisms, dedicated multiprotein complexes, called exosome or RNA degradosome, carry out RNA degradation and processing. In addition to varying exoribonucleases or endoribonucleases, most of these complexes contain a RNA helicase. In the Gram-positive bacterium Bacillus subtilis, a RNA degradosome has recently been described; however, no RNA helicase was identified. In this work, we tested the interaction of the four DEAD box RNA helicases encoded in the B. subtilis genome with the RNA degradosome components. One of these helicases, CshA, is able to interact with several of the degradosome proteins, i.e. RNase Y, the polynucleotide phosphorylase, and the glycolytic enzymes enolase and phosphofructokinase. The determination of in vivo protein,protein interactions revealed that CshA is indeed present in a complex with polynucleotide phosphorylase. CshA is composed of two RecA-like domains that are found in all DEAD box RNA helicases and a C-terminal domain that is present in some members of this protein family. An analysis of the contribution of the individual domains of CshA revealed that the C-terminal domain is crucial both for dimerization of CshA and for all interactions with components of the RNA degradosome, including RNase Y. A transfer of this domain to CshB allowed the resulting chimeric protein to interact with RNase Y suggesting that this domain confers interaction specificity. As a degradosome component, CshA is present in the cell in similar amounts under all conditions. Taken together, our results suggest that CshA is the functional equivalent of the RhlB helicase of the Escherichia coli RNA degradosome. [source] | ||||||||||||||||||||||||||||||||||