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Repeat Domain (repeat + domain)
Kinds of Repeat Domain Selected AbstractsActin-binding domain of mouse plectinFEBS JOURNAL, Issue 10 2004Crystal structure, binding to vimentin Plectin, a large and widely expressed cytolinker protein, is composed of several subdomains that harbor binding sites for a variety of different interaction partners. A canonical actin-binding domain (ABD) comprising two calponin homology domains (CH1 and CH2) is located in proximity to its amino terminus. However, the ABD of plectin is unique among actin-binding proteins as it is expressed in the form of distinct, plectin isoform-specific versions. We have determined the three-dimensional structure of two distinct crystalline forms of one of its ABD versions (pleABD/2,) from mouse, to a resolution of 1.95 and 2.0 Å. Comparison of pleABD/2, with the ABDs of fimbrin and utrophin revealed structural similarity between plectin and fimbrin, although the proteins share only low sequence identity. In fact, pleABD/2, has been found to have the same compact fold as the human plectin ABD and the fimbrin ABD, differing from the open conformation described for the ABDs of utrophin and dystrophin. Plectin harbors a specific binding site for intermediate filaments of various types within its carboxy-terminal R5 repeat domain. Our experiments revealed an additional vimentin-binding site of plectin, residing within the CH1 subdomain of its ABD. We show that vimentin binds to this site via the amino-terminal part of its rod domain. This additional amino-terminal intermediate filament protein binding site of plectin may have a function in intermediate filament dynamics and assembly, rather than in linking and stabilizing intermediate filament networks. [source] Cleavage and conformational changes of tau protein follow phosphorylation during Alzheimer's diseaseINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 2 2008Siddhartha Mondragón-Rodríguez Summary Phosphorylation, cleavage and conformational changes in tau protein all play pivotal roles during Alzheimer's disease (AD). In an effort to determine the chronological sequence of these changes, in this study, using confocal microscopy, we compared phosphorylation at several sites (Ser199/202/396/404/422 -Thr205 and the second repeat domain), cleavage of tau (D421) and the canonical conformational Alz-50 epitope. While all of these posttranslational modifications are found in neurofibrillary tangles (NFTs) at all stages of the disease, we found significantly higher numbers of phospho-tau positive NFTs when compared with cleaved tau (P = 0.006 in Braak III; P = 0.002 in Braak IV; P = 0.012 in Braak V) or compared with the Alz-50 epitope (P < 0.05). Consistent with these findings, in a double transgenic mice model (Tet/GSK-3,/VLW) overexpressing the enzyme glycogen synthase kinase-3, (GSK-3,) and tau with a triple FTDP-17 mutation (VLW) with AD-like neurodegeneration, phosphorylation at sites Ser199/202 -Thr205 was greater than truncated tau. Taken together, these data strongly support the notion that the conformational changes and truncation of tau occur after the phosphorylation of tau. We propose two probable pathways for the pathological processing of tau protein during AD, either phosphorylation and cleavage of tau followed by the Alz-50 conformational change or phosphorylation followed by the conformational change and cleavage as the last step. [source] Tubulin and CRMP-2 complex is transported via Kinesin-1JOURNAL OF NEUROCHEMISTRY, Issue 6 2005Nariko, Toshihide, Yuko Kimura Arimura Fukata Abstract The transport of tubulin and microtubules in a growing axon is essential for axonal growth and maintenance. However, the molecular mechanism underlying the linkage of tubulin and microtubules to motor proteins is not yet clear. Collapsin response mediator protein-2 (CRMP-2) is enriched at the distal part of growing axons in primary hippocampal neurons and plays a critical role in axon differentiation through its interaction with tubulin dimer and Numb. In this study, we show that CRMP-2 regulates tubulin transport by linking tubulin and Kinesin-1. The C-terminal region of CRMP-2 directly binds to the tetratricopeptide repeat domain of kinesin light chain 1 (KLC1). Soluble tubulin binds to the middle of CRMP-2 and forms a trimeric complex with CRMP-2/KLC1. Furthermore, the movement of GFP,tubulin in the photobleached area is weakened by knockdown of KLCs or CRMP-2. These results indicate that the CRMP-2/Kinesin-1 complex regulates soluble tubulin transport to the distal part of the growing axon. [source] Chemical synthesis and biotinylation of the thrombospondin domain TSR2PROTEIN SCIENCE, Issue 5 2009Theresa K. Tiefenbrunn Abstract The type 1 repeat domain from thrombospondin has potent antiangiogenic activity and a structurally interesting fold, making it an attractive target for protein engineering. Chemical synthesis is an attractive approach for studying protein domains because it enables the use of unnatural amino acids for site-specific labeling and detailed structure-function analysis. Here, we demonstrate the first total chemical synthesis of the thrombospondin type 1 repeat domain by native chemical ligation. In addition to the natural domain, five sites for side chain modification were evaluated and two were found to be compatible with oxidative folding. Several challenges were encountered during peptide synthesis due to the functional complexity of the domain. These challenges were overcome by the use of new solid supports, scavengers, and the testing of multiple ligation sites. We also describe an unusual sequence-specific protecting group migration observed during cleavage resulting in +90 Da and +194 Da adducts. Synthetic access to this domain enables the synthesis of a number of variants that can be used to further our understanding of the biochemical interaction network of thrombospondin and provide insight into the structure and function of this important antitumorogenic protein domain. [source] Structure and stability of the ankyrin domain of the Drosophila Notch receptorPROTEIN SCIENCE, Issue 11 2003Mark E. Zweifel RMSD, root mean square deviation; CD, circular dichroism spectroscopy Abstract The Notch receptor contains a conserved ankyrin repeat domain that is required for Notch-mediated signal transduction. The ankyrin domain of Drosophila Notch contains six ankyrin sequence repeats previously identified as closely matching the ankyrin repeat consensus sequence, and a putative seventh C-terminal sequence repeat that exhibits lower similarity to the consensus sequence. To better understand the role of the Notch ankyrin domain in Notch-mediated signaling and to examine how structure is distributed among the seven ankyrin sequence repeats, we have determined the crystal structure of this domain to 2.0 Å resolution. The seventh, C-terminal, ankyrin sequence repeat adopts a regular ankyrin fold, but the first, N-terminal ankyrin repeat, which contains a 15-residue insertion, appears to be largely disordered. The structure reveals a substantial interface between ankyrin polypeptides, showing a high degree of shape and charge complementarity, which may be related to homotypic interactions suggested from indirect studies. However, the Notch ankyrin domain remains largely monomeric in solution, demonstrating that this interface alone is not sufficient to promote tight association. Using the structure, we have classified reported mutations within the Notch ankyrin domain that are known to disrupt signaling into those that affect buried residues and those restricted to surface residues. We show that the buried substitutions greatly decrease protein stability, whereas the surface substitutions have only a marginal affect on stability. The surface substitutions are thus likely to interfere with Notch signaling by disrupting specific Notch-effector interactions and map the sites of these interactions. [source] High levels of intra-specific variation in the NG repeat region of the Pinctada maxima N66 organic matrix proteinAQUACULTURE RESEARCH, Issue 9 2009Carolyn Smith-Keune Abstract There has been wide speculation about the functional importance, and potential contribution to the varying nacre characteristics of pearl oysters, of variation in the length of an asparagine,glycine-rich repeat domain (the NG region) within the key organic matrix proteins nacrein and N66. This speculation has centred on large inter-species differences in the length of the NG protein domain. We report for the first time the presence of significant intra-specific (allelic) polymorphism in both the length and the amino acid sequence of the NG repeat domain of N66 within both wild and hatchery populations of Pinctada maxima. We also detected the presence of a second putative N66-like locus within the P. maxima genome. Confirmation of gene expression of the various alleles at the two loci identified in P. maxima is now required, together with experimental testing of functional differences in the carbonic anhydrase activity of the alleles identified. [source] Role of the leucine-rich repeat domain of cryopyrin/NALP3 in monosodium urate crystal,induced inflammation in miceARTHRITIS & RHEUMATISM, Issue 7 2010Hal M. Hoffman Objective The mechanism by which monosodium urate monohydrate (MSU) crystals intracellularly activate the cryopyrin inflammasome is unknown. The aim of this study was to use a mouse molecular genetics,based approach to test whether the leucine-rich repeat (LRR) domain of cryopyrin is required for MSU crystal,induced inflammation. Methods Cryopyrin-knockout lacZ (Cryo,Z/,Z) mice and mice with the cryopyrin LRR domain deleted and fused to the lacZ reporter (Cryo,LRR Z/,LRR Z) were generated using bacterial artificial chromosome,based targeting vectors, which allow for large genomic deletions. Bone marrow,derived macrophages from Cryo,LRR Z/,LRR Z mice, Cryo,Z/,Z mice, and congenic wild-type (WT) mice were challenged with endotoxin-free MSU crystals under serum-free conditions. Phagocytosis and cytokine expression were assessed by flow cytometry and enzyme-linked immunosorbent assay. MSU crystals also were injected into mouse synovial-like subcutaneous air pouches. The in vivo inflammatory responses were examined. Results Release of interleukin-1, (IL-1,), but not CXCL1 and tumor necrosis factor ,, was impaired in Cryo,LRR Z/,LRR Z and Cryo,Z/,Z mouse bone marrow,derived macrophages compared with WT mouse bone marrow,derived macrophages in response to not only MSU crystals but also other known stimuli that activate the cryopyrin inflammasome. In addition, a comparable percentage of MSU crystals taken up by each type of bone marrow,derived macrophage was observed. Moreover, total leukocyte infiltration in the air pouch and IL-1, production were attenuated in Cryo,Z/,Z and Cryo,LRR Z/,LRR Z mice at 6 hours postinjection of MSU crystals compared with WT mice. Conclusion MSU crystal,induced inflammatory responses were comparably attenuated both in vitro and in vivo in Cryo,LRR Z/,LRR Z and Cryo,Z/,Z mice. Hence, the LRR domain of cryopyrin plays a role in mediating MSU crystal,induced inflammation in this model. [source] Functional consequences of a germline mutation in the leucine-rich repeat domain of NLRP3 identified in an atypical autoinflammatory disorderARTHRITIS & RHEUMATISM, Issue 4 2010Isabelle Jéru Objective To gain insight into the pathophysiology of an atypical familial form of an autoinflammatory disorder, characterized by autosomal-dominant sensorineural hearing loss, systemic inflammation, increased secretion of interleukin-1, (IL-1,), and the absence of any cutaneous manifestations, and to assess the functional consequences of a missense mutation identified in the leucine-rich repeat (LRR) domain of NLRP3. Methods Microsatellite markers were used to test the familial segregation of the NLRP3 locus with the disease phenotype. All NLRP3 exons were screened for mutations by sequencing. Functional assays were performed in HEK 293T cells to determine the effects of mutated (versus normal) NLRP3 proteins on NF-,B activation, caspase 1 signaling, and speck formation. Results A heterozygous NLRP3 missense mutation (p.Tyr859Cys) was identified in exon 6, which encodes the LRR domain of the protein. This mutation was found to segregate with the disease phenotype within the family, and had a moderate activating effect on speck formation and procaspase 1 processing and did not alter the inhibitory properties of NLRP3 on NF-,B signaling. Conclusion This report is the first to describe a familial form of a cryopyrinopathy associated with a mutation outside of exon 3 of NLRP3. This finding, together with the known efficacy of anti,IL-1 treatments in these disorders, underlines the importance of screening all exons of NLRP3 in patients who present with atypical manifestations. In addition, the gain of function associated with this mutation in terms of activation of caspase 1 signaling was consistent with the observed inflammatory phenotype. Therefore, this study of the functional consequences of an LRR mutation sheds new light on the clinical relevance of in vitro assays. [source] Ligand-induced activation of the insulin receptor: a multi-step process involving structural changes in both the ligand and the receptorBIOESSAYS, Issue 4 2009Colin W. Ward Abstract Current models of insulin binding to the insulin receptor (IR) propose (i) that there are two binding sites on the surface of insulin which engage with two binding sites on the receptor and (ii) that ligand binding involves structural changes in both the ligand and the receptor. Many of the features of insulin binding to its receptor, namely B-chain helix interactions with the leucine-rich repeat domain and A-chain residue interactions with peptide loops from another part of the receptor, are also seen in models of relaxin and insulin-like peptide 3 binding to their receptors. We show that these principles can likely be extended to the group of mimetic peptides described by Schäffer and coworkers, which are reported to have no sequence identity with insulin. This review summarizes our current understanding of ligand-induced activation of the IR and highlights the key issues that remain to be addressed. [source] Molecular and functional characterization of a novel splice variant of ANKHD1 that lacks the KH domain and its role in cell survival and apoptosiscFEBS JOURNAL, Issue 16 2005Melissa C. Miles Multiple ankyrin repeat motif-containing proteins play an important role in protein,protein interactions. ANKHD1 proteins are known to possess multiple ankyrin repeat domains and a single KH domain with no known function. Using yeast two-hybrid system analysis, we identified a novel splice variant of ANKHD1. This splice variant of ANKHD1, which we designated as HIV-1 Vpr-binding ankyrin repeat protein (VBARP), does not contain the signature KH domain, and codes for only a single ankyrin repeat motif. We characterized VBARP by molecular and functional analysis, revealing that VBARP is ubiquitously expressed in different tissues as well as cell lines of different lineage. In addition, blast searches indicated that orthologs and homologs to VBARP exist in different phyla, suggesting that VBARP might be evolutionarily conserved, and thus may be involved in basic cellular function(s). Furthermore, biochemical analysis revealed the presence of two VBARP isoforms coding for 69 and 49 kDa polypeptides, respectively, that are primarily localized in the cytoplasm. Functional analysis using short interfering RNA approaches indicate that this gene product is essential for cell survival through its regulation of caspases. Taken together, these results indicate that VBARP is a novel splice variant of ANKHD1 and may play a role in cellular apoptosis (antiapoptotic) and cell survival pathway(s). [source] Characterization of epitopes recognized by anti- Streptococcus mutans P1 monoclonal antibodiesFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2007William P. McArthur Abstract Sequences contributing to epitopes recognized by a panel of monoclonal antibodies (mAbs) against the Streptococcus mutans surface protein P1 were delineated by Western blot and enzyme-linked immunosorbent assay using a battery of deletion constructs and recombinant polypeptides. mAbs that recognize complex discontinuous epitopes reconstituted by combining the alanine-rich and proline-rich repeat domains and varying degrees of flanking sequence were identified as well as mAbs that bound epitopes contained within contiguous segments of P1. Cross-reactivity with SspA and SspB from Streptococcus gordonii is also reported. This information enables insight into the structure and function of a streptococcal adhesin and its correlates of protection and furthers our understanding of the immunomodulatory and bacterial-adherence inhibition activities of anti-P1 mAbs. [source] Biological activity, membrane-targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coliPROTEIN SCIENCE, Issue 9 2004Jennifer White CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; GPI, glycophosphatidyl inositol; PpDAF, human DAF1,4 expressed in Pichia pastoris, N glycosylated and with an oligohistidine tag; EcDAF, nonglycosylated human DAF 1,4 expressed in Escherichia coli; nDAF, human native glycosylated (GPI-anchored) DAF from erythrocytes; EcDAF-MP, soluble E. coli human DAF linked through a C-terminal cysteine to the myristoylated peptide APT542; PCR, polymerase chain reaction; SCR, short consensus repeat; TCEP, Tris- (2-carboxyethyl) phosphine Abstract Decay-accelerating factor (DAF, CD55) is a glycophosphatidyl inositol-anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane-targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C-terminal cysteine residue to permit site-specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane-localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF-derived therapeutic. Crystals of the E. coli -derived protein were obtained and diffracted to 2.2 Å, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential. [source] The ankyrin repeat as molecular architecture for protein recognitionPROTEIN SCIENCE, Issue 6 2004Leila K. Mosavi Abstract The ankyrin repeat is one of the most frequently observed amino acid motifs in protein databases. This protein,protein interaction module is involved in a diverse set of cellular functions, and consequently, defects in ankyrin repeat proteins have been found in a number of human diseases. Recent biophysical, crystallographic, and NMR studies have been used to measure the stability and define the various topological features of this motif in an effort to understand the structural basis of ankyrin repeat-mediated protein,protein interactions. Characterization of the folding and assembly pathways suggests that ankyrin repeat domains generally undergo a two-state folding transition despite their modular structure. Also, the large number of available sequences has allowed the ankyrin repeat to be used as a template for consensus-based protein design. Such projects have been successful in revealing positions responsible for structure and function in the ankyrin repeat as well as creating a potential universal scaffold for molecular recognition. [source] Binding of platelet glycoprotein Ib, through the convex surface of leucine-rich repeats domain of glycoprotein IXJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 9 2009X. MO Summary.,Background: The mechanism of assembly of the platelet glycoprotein (GP) Ib-IX complex from GPIb,, GPIb, and GPIX subunits is not entirely clear. In this complex, ectodomains of both GPIb, and GPIX subunits contain two leucine-rich repeats (LRR) and share high sequence similarity. However, they differ noticeably in stability, hampering further analysis of their interaction. Objectives and methods: Guided by analysis of the LRR structure, we report a well-folded Ib,/IX chimera and its usage in dissecting GPIX function. Results: In this chimera, three non-contiguous sequences that may constitute the putative convex surface of the GPIb, ectodomain are replaced by their GPIX counterparts. Like GPIb, but unlike GPIX ectodomain, it can secrete from transfected Chinese hamster ovary cells and fold into a stable conformation. Furthermore, replacing the ectodomain in GPIX with the Ib,/IX chimera, but not the GPIb, ectodomain, preserved its interaction with GPIb, as demonstrated by its native-like GPIb,-induced increase in surface expression and coimmunoprecipitation. Conclusions: The putative convex surface of the LRR domain in GPIX is sufficient, in the context of full-length subunit, to mediate its association with GPIb,. [source] The Flavobacterium psychrophilum OmpA, an outer membrane glycoprotein, induces a humoral response in rainbow troutJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2007F. Dumetz Abstract Aims:, The purpose of this study was to characterize OmpA, a major glycoprotein isolated from the membrane fraction of Flavobacterium psychrophilum, and to evaluate its potential as antigenic unit in a possible vaccine. Methods and Results:, The expression product of ompA is a 465-amino-acid protein precursor that contains a 21-amino acid signal peptide and has overall homology (up to 60% identity) with similarly sized proteins of some bacteria belonging to the Flavobacteriaceae family. The carboxy-terminal region contains the ,OmpA/MotB' domain/signature and five putative ,Thrombospondin type 3 repeats' domains have been identified in the central region. OmpA was clearly detected in the outer membrane fraction and its surface exposure was demonstrated. OmpA is one of the immunodominant antigens and binding of specific anti-OmpA antibodies lead to cell lysis in the presence of complement. Fish immunized with OmpA emulsified with Freund's adjuvant developed a high antibody titter. Conclusions:, Collectively, the data obtained here indicate that OmpA may be involved in Fl. psychrophilum/host cell interactions and appears to be a potential immunogen for a vaccine. Significance and Impact of the Study:, This study is one step in the direction of understanding pathogenesis of Fl. psychrophilum and development of future vaccine. [source] | ||||||||||||||||