The Experts below are selected from a list of 3558 Experts worldwide ranked by ideXlab platform
Margaret Kielian - One of the best experts on this subject based on the ideXlab platform.
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E1 mutants identify a critical region in the trimer interface of the Semliki Forest virus fusion protein
2016Co-Authors: Catherine Y. Liu, Margaret KielianAbstract:The alphavirus Semliki Forest virus (SFV) uses a membrane fusion reaction to infect host cells. Fusion of the virus and cell membranes is triggered by low pH in the endosome and is mediated by the viral membrane protein E1. During fusion, E1 inserts into the target membrane, trimerizes, and refolds into a hairpin conformation. Formation of the E1 Homotrimer is critical to membrane fusion, but the mechanism of tri-merization is not understood. The crystal structure of the postfusion E1 trimer shows that an aspartate residue, D188, is positioned in the central core trimer interface. D188 is conserved in all reported alphavirus E1 sequences. We tested the contribution of this amino acid to trimerization and fusion by replacing D188 with alanine (D188A) or lysine (D188K) in an SFV infectious clone. These mutations were predicted to disrupt specific interactions at this position and/or change their pH dependence. Our results indicated that the D188K mutation blocked SFV fusion and infection. At low pH, D188K E1 inserted into target membranes but was trapped as a target membrane-inserted monomer that did not efficiently form the stable core trimer. In contrast, the D188A mutant was infectious, although trimerization and fusion required a lower pH. While there are extensive contacts between E1 subunits in the Homotrimer, the D188K mutant identifies an important “hot spot ” for protein-protein interactions within the core trimer. In an aqueous environment, phospholipid bilayers are stabl
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Second-Site Revertants of a Semliki Forest Virus Fusion-Block Mutation Reveal the Dynamics of a Class II Membrane Fusion Protein
Journal of virology, 2006Co-Authors: Chantal Chanel-vos, Margaret KielianAbstract:The alphavirus Semliki Forest virus (SFV) infects cells through low-pH-induced membrane fusion mediated by the E1 protein, a class II virus membrane fusion protein. During fusion, E1 inserts into target membranes via its hydrophobic fusion loop and refolds to form a stable E1 Homotrimer. Mutation of a highly conserved histidine (the H230A mutation) within a loop adjacent to the fusion loop was previously shown to block SFV fusion and infection, although the mutant E1 protein still inserts into target membranes and forms a Homotrimer. Here we report on second-site mutations in E1 that rescue the H230A mutant. These mutations were located in a cluster within the hinge region, at the membrane-interacting tip, and within the groove where the E1 stem is believed to pack. Together the revertants reveal specific and interconnected aspects of the fusion protein refolding reaction.
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a conserved histidine in the ij loop of the semliki forest virus e1 protein plays an important role in membrane fusion
Journal of Virology, 2004Co-Authors: Chantal Chanelvos, Margaret KielianAbstract:The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a low pH-triggered membrane fusion reaction mediated by the E1 protein. E1 is a class II fusion protein that contains the hydrophobic fusion peptide loop and converts to a stable Homotrimer during the fusion reaction. Intriguingly, the fusion loop is closely associated with a loop connecting the i and j β-strands. This ij loop plays a role in the cholesterol dependence of membrane fusion and is specifically susceptible to proteolysis in the protease-resistant E1 Homotrimer. The SFV ij loop contains a histidine residue at position 230. Sequence comparisons revealed that an analogous histidine is completely conserved in all alphavirus and flavivirus fusion proteins. An E1 H230A mutant was constructed using the SFV infectious clone. Although cells infected with H230A RNA produced virus particles, these virions were completely noninfectious and were blocked in both cell-cell fusion and lipid mixing assays. The H230A virions efficiently bound to cell surface receptors and responded to low pH by undergoing acid-dependent conformational changes including dissociation of the E1/E2 dimer, exposure of the fusion loop, association with target liposomes, exposure of acid-conformation-specific epitopes, and formation of the stable E1 Homotrimer. Studies with a soluble fragment of E1 showed that the mutant protein was defective in lipid-dependent conformational changes. Our results indicate that the E1 ij loop and the conserved H230 residue play a critical role in alphavirus-membrane fusion and suggest the presence of a previously undescribed late intermediate in the fusion reaction.
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Purification and crystallization reveal two types of interactions of the fusion protein Homotrimer of Semliki Forest Virus
Journal of Virology, 2004Co-Authors: Don L Gibbons, Brigid Reilly, Anna Ahn, Marie-christine Vaney, Armelle Vigouroux, Felix A. Rey, Margaret KielianAbstract:The fusion proteins of the alphaviruses and flaviviruses have a similar native structure and convert to a highly stable Homotrimer conformation during the fusion of the viral and target membranes. The properties of the alpha- and flavivirus fusion proteins distinguish them from the class I viral fusion proteins, such as influenza virus hemagglutinin, and establish them as the first members of the class II fusion proteins. Understanding how this new class carries out membrane fusion will require analysis of the structural basis for both the interaction of the protein subunits within the Homotrimer and their interaction with the viral and target membranes. To this end we report a purification method for the El ectodomain Homotrimer from the alphavirus Semliki Forest virus. The purified protein is trimeric, detergent soluble, retains the characteristic stability of the starting Homotrimer, and is free of lipid and other contaminants. In contrast to the postfusion structures that have been determined for the class I proteins, the El Homotrimer contains the fusion peptide region responsible for interaction with target membranes. This E1 trimer preparation is an excellent candidate for structural studies of the class II viral fusion proteins, and we report conditions that generate three-dimensional crystals suitable for analysis by X-ray diffraction. Determination of the structure will provide our first high-resolution views of both the low-pH-induced trimeric conformation and the target membraneinteracting region of the alphavirus fusion protein.
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visualization of the target membrane inserted fusion protein of semliki forest virus by combined electron microscopy and crystallography
Cell, 2003Co-Authors: Don L Gibbons, Margaret Kielian, Brigid Reilly, Jorge Navaza, Jean LepaultAbstract:Abstract Semliki Forest virus enters cells by receptor-mediated endocytosis. The acidic environment of the endosome triggers a membrane fusion reaction that is mediated by the E1 glycoprotein. During fusion, E1 rearranges from an E1/E2 heterodimer to a highly stable, membrane-inserted E1 Homotrimer (E1HT). In this study, we analyzed E1HT by a combination of electron cryomicroscopy, electron crystallography of negatively stained 2D crystals, and fitting of the available X-ray structure of the monomeric E1 ectodomain into the resulting 3D reconstruction. The visualized E1HT reveals that the ectodomain has reoriented vertically and inserted the distal tip of domain II into the lipid bilayer. Our data allow the visualization of a viral fusion protein inserted in its target membrane and demonstrate that insertion is a cooperative process, resulting in rings composed of five to six Homotrimers.
Sergey Leikin - One of the best experts on this subject based on the ideXlab platform.
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molecular mechanism of type i collagen Homotrimer resistance to mammalian collagenases
Journal of Biological Chemistry, 2010Co-Authors: Elena Makareeva, Charlotte L. Phillips, Robert Visse, Hideaki Nagase, Wolfgang Losert, Natalia V Kuznetsova, Angela M Deridder, Mary Beth Sutter, Sergey LeikinAbstract:Abstract Type I collagen cleavage is crucial for tissue remodeling, but its Homotrimeric isoform is resistant to all collagenases. The Homotrimers occur in fetal tissues, fibrosis, and cancer, where their collagenase resistance may play an important physiological role. To understand the mechanism of this resistance, we studied interactions of α1(I)3 Homotrimers and normal α1(I)2α2(I) heterotrimers with fibroblast collagenase (MMP-1). Similar MMP-1 binding to the two isoforms and similar cleavage efficiency of unwound α1(I) and α2(I) chains suggested increased stability and less efficient unwinding of the Homotrimer triple helix at the collagenase cleavage site. The unwinding, necessary for placing individual chains inside the catalytic cleft of the enzyme, was the rate-limiting cleavage step for both collagen isoforms. Comparative analysis of the homo- and heterotrimer cleavage kinetics revealed that MMP-1 binding promotes stochastic helix unwinding, resolving the controversy between different models of collagenase action.
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Carcinomas Contain a Matrix Metalloproteinase–Resistant Isoform of Type I Collagen Exerting Selective Support to Invasion
Cancer research, 2010Co-Authors: Elena Makareeva, Sejin Han, Charlotte L. Phillips, Robert Visse, Hideaki Nagase, Juan Carlos Vera, Dan L. Sackett, Kenn Holmbeck, Sergey LeikinAbstract:Collagen fibers affect metastasis in two opposing ways, by supporting invasive cells but also by generating a barrier to invasion. We hypothesized that these functions might be performed by different isoforms of type I collagen. Carcinomas are reported to contain α1(I)3 Homotrimers, a type I collagen isoform normally not present in healthy tissues, but the role of the Homotrimers in cancer pathophysiology is unclear. In this study, we found that these Homotrimers were resistant to all collagenolytic matrix metalloproteinases (MMP). MMPs are massively produced and used by cancer cells and cancer-associated fibroblasts for degrading stromal collagen at the leading edge of tumor invasion. The MMP-resistant Homotrimers were produced by all invasive cancer cell lines tested, both in culture and in tumor xenografts, but they were not produced by cancer-associated fibroblasts, thereby comprising a specialized fraction of tumor collagen. We observed the Homotrimer fibers to be resistant to pericellular degradation, even upon stimulation of the cells with proinflammatory cytokines. Furthermore, we confirmed an enhanced proliferation and migration of invasive cancer cells on the surface of Homotrimeric versus normal (heterotrimeric) type I collagen fibers. In summary, our findings suggest that invasive cancer cells may use Homotrimers for building MMP-resistant invasion paths, supporting local proliferation and directed migration of the cells whereas surrounding normal stromal collagens are cleaved. Because the Homotrimers are universally secreted by cancer cells and deposited as insoluble, MMP-resistant fibers, they offer an appealing target for cancer diagnostics and therapy. Cancer Res; 70(11); 4366–74. ©2010 AACR.
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Bending rigidity of type I collagen Homotrimer fibrils
Biophysical Journal, 2009Co-Authors: Sejin Han, Charlotte L. Phillips, Daniel J. Mcbride, Robert Visse, Hideaki Nagase, Wolfgang Losert, Sergey LeikinAbstract:Normal type I collagen is an α1(I)2α2(I) heterotrimeric triple helix, but α1(I)3 Homotrimers are also found in fetal tissues and various pathological conditions, e.g., causing bone fragility and reducing tendon tensile strength. It remains unclear whether α1(I)3 Homotrimers alter mechanical properties of individual fibrils or affect tissues by altering their organization at a higher level. To address this question, we investigated how Homotrimers affect fibril bending rigidity. Homotrimer fibrils have been shown to be more loosely packed so that we expected them to be more susceptible to bending. However, confocal imaging of in vitro fibrillogenesis revealed straight, spear-like Homotrimer fibrils and curved heterotrimer fibrils. Surprisingly, Homotrimer fibrils were more rigid despite being thinner and more hydrated. To quantify fibril rigidity, we analyzed their shape by Fourier decomposition, determined the correlation function for the direction along each fibril, and calculated the distribution of local fibril curvature. The fibril persistence length of Homotrimers was 3 ∼ 10 times longer than for Homotrimers. These persistence length values indicated much higher bending rigidity of Homotrimer helices. We conjectured that the increased rigidity might be related to stabilization of the region surrounding the mammalian collagenase cleavage site. In heterotrimers, this region is known to be the most flexible along the helix. We corroborated this hypothesis by probing the susceptibility of the collagenase cleavage site to MMP-1. Dissection of the observed effects revealed an increased stability of the Homotrimer helix at this site. We argue that the loss of the α2(I) chain reduces type I collagen flexibility within the region most vulnerable to bending, thereby increasing the overall bending rigidity of the helix and fibrils. Higher fibril rigidity may alter tissue mechanics not only directly but also by changing the tissue scaffold architecture.
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Segregation of type I collagen homo- and heterotrimers in fibrils.
Journal of molecular biology, 2008Co-Authors: Sejin Han, Daniel J. Mcbride, Wolfgang Losert, Sergey LeikinAbstract:Abstract Normal type I collagen is a heterotrimer of two α1(I) and one α2(I) chains, but various genetic and environmental factors result in synthesis of Homotrimers that consist of three α1(I) chains. The Homotrimers completely replace the heterotrimers only in rare recessive disorders. In the general population, they may compose just a small fraction of type I collagen. Nevertheless, they may play a significant role in pathology; for example, synthesis of 10–15% Homotrimers due to a polymorphism in the α1(I) gene may contribute to osteoporosis. Homotrimer triple helices have different stability and less efficient fibrillogenesis than heterotrimers. Their fibrils have different mechanical properties. However, very little is known about their molecular interactions and fibrillogenesis in mixtures with normal heterotrimers. Here we studied the kinetics and thermodynamics of fibril formation in such mixtures by combining traditional approaches with 3D confocal imaging of fibrils, in which homo- and heterotrimers were labeled with different fluorescent colors. In a mixture, following a temperature jump from 4 to 32 °C, we observed a rapid increase in turbidity most likely caused by formation of Homotrimer aggregates. The aggregates promoted nucleation of Homotrimer fibrils that served as seeds for mixed and heterotrimer fibrils. The separation of colors in confocal images indicated segregation of homo- and heterotrimers at a subfibrillar level throughout the process. The fibril color patterns continued to change slowly after the fibrillogenesis appeared to be complete, due to dissociation and reassociation of the pepsin-treated homo- and heterotrimers, but this remixing did not significantly reduce the segregation even after several days. Independent homo- and heterotrimer solubility measurements in mixtures confirmed that the subfibrillar segregation was an equilibrium property of intermolecular interactions and not just a kinetic phenomenon. We argue that the subfibrillar segregation may exacerbate effects of a small fraction of α1(I) Homotrimers on formation, properties, and remodeling of collagen fibers.
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Changes in Thermal Stability and Microunfolding Pattern of Collagen Helix Resulting from the Loss of α2(I) Chain in Osteogenesis Imperfecta Murine
Journal of molecular biology, 2003Co-Authors: Natalia V Kuznetsova, Daniel J. Mcbride, Sergey LeikinAbstract:Abstract Homozygous mutations resulting in formation of α1(I) 3 Homotrimers instead of normal type I collagen cause mild to severe osteogenesis imperfecta (OI) in humans and mice. Limited studies of changes in thermal stability of type I Homotrimers were reported previously, but the results were not fully consistent. We revisited this question in more detail using purified tendon collagen from wild-type (α1(I) 2 α2(I) heterotrimers) and oim (α1(I) 3 ) mice as well as artificial α1(I) 3 Homotrimers obtained by refolding of rat-tail-tendon collagen. We found that at the same heating rate oim Homotrimers completely denature at ∼2.5 deg.C higher temperature than wild-type heterotrimers, as determined by differential scanning calorimetry. At the same, constant temperature, Homotrimers denature ∼100 times slower than heterotrimers, as determined by circular dichroism. Detailed analysis of proteolytic cleavage at different temperatures revealed that microunfolding of oim Homotrimers and wild-type heterotrimers occurs at similar rate but within a number of different sites. In particular, the weakest spot on the oim triple helix is located ∼100 amino acid residues from the C-terminal end within the cyanogen bromide peptide CB6. The same microunfolding site is also present in wild-type collagen, but the weakest spot of the latter is located close to the N-terminal end of CB8. Amino acid analysis and differential gel electrophoresis showed virtually no posttranslational overmodification of oim mouse tendon collagen. Moreover, thermal stability and microunfolding of artificial rat-tail-tendon Homotrimers were similar to oim Homotrimers. Thus, the observed changes are associated with difference in the amino acid composition of α1(I) and α2(I) chains rather than posttranslational overmodification.
Florence Ruggiero - One of the best experts on this subject based on the ideXlab platform.
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In vivo evidence for a bridging role of a collagen V subtype at the epidermis-dermis interface.
The Journal of investigative dermatology, 2012Co-Authors: Christelle Bonod-bidaud, M. Malbouyres, Muriel Roulet, Uwe Hansen, Ahmed Elsheikh, Sylvie Ricard-blum, Clément Faye, Elisabeth Vaganay, Patricia Rousselle, Florence RuggieroAbstract:Collagen V is the defective product in most cases of classical Ehlers–Danlos syndrome (EDS), a connective tissue disorder typically characterized by skin fragility and abnormal wound healing. Collagen V assembles into diverse molecular forms. The predominant α1(V) 2 α2(V) heterotrimer controls fibrillogenesis in skin and other tissues. The α1(V) 3 minor form is thought to occur in skin, but its function is unknown. To elucidate its role, we generated transgenic mice that overexpress the human α1(V) 3 Homotrimer in the epidermis. The transgene-derived product is deposited as thin unstriated fibrillar material in the basement membrane zone of embryonic and perinatal epidermis and hair follicles. Accumulation of α1(V) 3 -containing fibrils leads to ultrastructural modifications at the epidermis–dermis interface and provokes changes in biomechanical properties, although not statistically significant. Using superparamagnetic immunobeads to isolate authentic suprastructures and protein-binding assays, we demonstrate that the Homotrimer is part of a protein network containing collagen IV, laminin-111, and the dermal collagen VI. Our data show that the Homotrimer serves as a bridging molecule that contributes to the stabilization of the epidermal–dermal interface. This finding strongly suggests that collagen V may be expressed in skin as different subtypes with important but distinct roles in matrix organization and stability.
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Development of a functional skin matrix requires deposition of collagen V heterotrimers.
Molecular and cellular biology, 2004Co-Authors: Hélène Chanut-delalande, Agnes Fichard, Christelle Bonod-bidaud, S. Cogne, M. Malbouyres, F. Ramirez, Florence RuggieroAbstract:Collagen V is a minor component of the heterotypic I/III/V collagen fibrils and the defective product in most cases of classical Ehlers Danlos syndrome (EDS). The present study was undertaken to elucidate the impact of collagen V mutations on skin development, the most severely affected EDS tissues, using mice harboring a targeted deletion of the α2(V) collagen gene (Col5a2). Contrary to the original report, our studies indicate that the Col5a2 deletion (a.k.a. the pN allele) represents a functionally null mutation that affects matrix assembly through a complex sequence of events. First the mutation impairs assembly and/or secretion of the α1(V)2α2(V) heterotrimer with the result that the α1(V) Homotrimer is the predominant species deposited into the matrix. Second, the α1(V) Homotrimer is excluded from incorporation into the heterotypic collagen fibrils and this in turn severely impairs matrix organization. Third, the mutant matrix stimulates a compensatory loop by the α1(V) collagen gene that leads to additional deposition of α1(V) Homotrimers. These data therefore underscore the importance of the collagen V heterotrimer in dermal fibrillogenesis. Furthermore, reduced thickness of the basement membranes underlying the epidermis and increased apoptosis of the stromal fibroblasts in pN/pN skin strongly indicate additional roles of collagen V in the development of a functional skin matrix.
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Development of a functional skin matrix requires deposition of collagen V heterotrimers.
Molecular and Cellular Biology, 2004Co-Authors: Hélène Chanut-delalande, Agnes Fichard, Christelle Bonod-bidaud, S. Cogne, M. Malbouyres, F. Ramirez, Florence RuggieroAbstract:Collagen V is a minor component of the heterotypic I/III/V collagen fibrils and the defective product in most cases of classical Ehlers Danlos syndrome (EDS). The present study was undertaken to elucidate the impact of collagen V mutations on skin development, the most severely affected EDS tissues, using mice harboring a targeted deletion of the alpha2(V) collagen gene (Col5a2). Contrary to the original report, our studies indicate that the Col5a2 deletion (a.k.a. the pN allele) represents a functionally null mutation that affects matrix assembly through a complex sequence of events. First the mutation impairs assembly and/or secretion of the alpha1(V)(2)alpha2(V) heterotrimer with the result that the alpha1(V) Homotrimer is the predominant species deposited into the matrix. Second, the alpha1(V) Homotrimer is excluded from incorporation into the heterotypic collagen fibrils and this in turn severely impairs matrix organization. Third, the mutant matrix stimulates a compensatory loop by the alpha1(V) collagen gene that leads to additional deposition of alpha1(V) Homotrimers. These data therefore underscore the importance of the collagen V heterotrimer in dermal fibrillogenesis. Furthermore, reduced thickness of the basement membranes underlying the epidermis and increased apoptosis of the stromal fibroblasts in pN/pN skin strongly indicate additional roles of collagen V in the development of a functional skin matrix.Collagen V is a minor component of the heterotypic I/III/V collagen fibrils and the defective product in most cases of classical Ehlers Danlos syndrome (EDS). The present study was undertaken to elucidate the impact of collagen V mutations on skin development, the most severely affected EDS tissues, using mice harboring a targeted deletion of the alpha2(V) collagen gene (Col5a2). Contrary to the original report, our studies indicate that the Col5a2 deletion (a.k.a. the pN allele) represents a functionally null mutation that affects matrix assembly through a complex sequence of events. First the mutation impairs assembly and/or secretion of the alpha1(V)(2)alpha2(V) heterotrimer with the result that the alpha1(V) Homotrimer is the predominant species deposited into the matrix. Second, the alpha1(V) Homotrimer is excluded from incorporation into the heterotypic collagen fibrils and this in turn severely impairs matrix organization. Third, the mutant matrix stimulates a compensatory loop by the alpha1(V) collagen gene that leads to additional deposition of alpha1(V) Homotrimers. These data therefore underscore the importance of the collagen V heterotrimer in dermal fibrillogenesis. Furthermore, reduced thickness of the basement membranes underlying the epidermis and increased apoptosis of the stromal fibroblasts in pN/pN skin strongly indicate additional roles of collagen V in the development of a functional skin matrix.
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Control of Heterotypic Fibril Formation by Collagen V Is Determined by Chain Stoichiometry
Journal of Biological Chemistry, 2001Co-Authors: Hélène Chanut-delalande, Agnes Fichard, Robert Garrone, Simonetta Bernocco, David J S Hulmes, Florence RuggieroAbstract:Abstract Although the collagen V heterotrimer is known to be involved in the control of fibril assembly, the role of the Homotrimer in fibrillar organization has not yet been examined. Here, the production of substantial amounts of recombinant collagen V Homotrimer has allowed a detailed study of its role in homotypic and heterotypic fibril formation. After removal of terminal regions by pepsin digestion, both the collagen V heterotrimer and Homotrimer formed thin homotypic fibrils, thus showing that diameter limitation is at least in part an intrinsic property of the collagen V triple helix. When mixed with collagen I, however, various complementary approaches indicated that the collagen V heterotrimer and Homotrimer exerted different effects in heterotypic fibril formation. Unlike the heterotrimer, which was buried in the fibril interior, the Homotrimer was localized as thin filamentous structures at the surface of wide collagen I fibrils and did not regulate fibril assembly. Its localization at the fibril surface suggests that the Homotrimer can act as a molecular linker between collagen fibrils or macromolecules in the extracellular matrix or both. Thus, depending on their respective distribution in tissues, the different collagen V isoforms might fulfill specific biological functions.
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Bone morphogenetic protein-1 (BMP-1) mediates C-terminal processing of procollagen V Homotrimer.
The Journal of biological chemistry, 2001Co-Authors: E. Kessler, Hélène Chanut-delalande, Agnes Fichard, M. Brusel, Florence RuggieroAbstract:Abstract The processing of the fibrillar procollagen precursors to mature collagens is an essential requirement for fibril formation. The enzymes involved in these events are known as the procollagen N and C proteinases. The latter, which cleaves the C-propeptides of the fibrillar procollagens I-III, is identical to the previously described bone morphogenetic protein-1 (BMP-1). Surprisingly, unlike the other fibrillar collagens, the processing of the C-propeptide domain of the procollagen V Homotrimer was found to be mediated by furin rather than BMP-1. However, the presence of putative BMP-1 cleavage sites in the α1(V) C-propeptide sequence prompted us to reconsider the procollagen V C-propeptide cleavage by BMP-1. Using a recombinant system to produce substantial amounts of the proα1(V) Homotrimer, we have previously shown that the C-propeptide is spontaneously released in the culture medium. The trimeric C-propeptide fragment, resulting from the furin cleavage, still encompassed the predicted BMP-1 cleavage sites. It was purified and tested as a substrate for BMP-1. In parallel, the release of the C-propeptide in the culture medium was inhibited by the addition of a specific furin inhibitor, allowing the re-examination of BMP-1 activity on the intact molecule. We showed that BMP-1 does cleave both substrates at one of the two predicted C-proteinase cleavage sites. Our results favor a role for PCP/BMP-1 in physiological C-terminal processing of procollagen V and imply a general mechanism for fibrillar collagen C-terminal processing.
Shigeru Utsumi - One of the best experts on this subject based on the ideXlab platform.
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Structure-physicochemical function relationships of soybean beta-conglycinin heterotrimers.
Journal of agricultural and food chemistry, 2002Co-Authors: Nobuyuki Maruyama, Mohamad Ramlan Mohamed Salleh, Koji Takahashi, Kazuhiro Yagasaki, Hideyuki Goto, Naho Hontani, Shuko Nakagawa, Shigeru UtsumiAbstract:We purified four single molecular species of beta-conglycinin heterotrimers consisting of the alpha and beta subunits or the alpha' and beta subunits from mutant soybean cultivars lacking the alpha or alpha' subunit, respectively, and examined their structural features and physicochemical functions. The extent of the hydrophobicities of the heterotrimers was related to the number of the alpha or alpha' subunit. The thermal stabilities of the heterotrimers were mainly conferred by the subunit which had lower thermal stability. Solubilities at low ionic strength (mu = 0.08) of the heterotrimers containing the alpha or alpha' subunit were very similar to those of the alpha and alpha' Homotrimers, respectively. Emulsifying abilities and heat-induced associations of the heterotrimers containing one beta subunit were similar to those of the alpha or alpha' Homotrimer, whereas those of the heterotrimers containing two beta subunits were similar to those of the beta Homotrimer.
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The effect of the N-linked glycans on structural features and physicochemical functions of soybean β-conglycinin Homotrimers
Journal of the American Oil Chemists' Society, 2002Co-Authors: Nobuyuki Maruyama, Mohamad Ramlan Mohamed Salleh, Koji Takahashi, Kazuhiro Yagasaki, Hideyuki Goto, Naho Hontani, Shuko Nakagawa, Shigeru UtsumiAbstract:β-Conglycinin is a trimeric protein consisting of three subunits, α,α′,and β, which are N-glycosylated. The α and α′ subunits contain extension regions in addition to core regions common to all subunits. We purified homogeneous trimers consisting of only α, α′, or β from mutant soybean cultivars containing β-conglycinin lacking one or two subunits: α Homotrimers from an α′-lacking mutant, α′ Homotrimers from an α-lacking mutant, and β Homotrimers from an α-and α′-lacking mutant. Structural features and physicochemical functions of the three Homotrimers were examined and compared with those of recombinant Homotrimers having no N-linked glycans. The native Homotrimers have secondary structures very similar to those of the recombinant ones. In analogy with the recombinant Homotrimers, the native ones exhibit different thermal stabilities from one another (β>α′>α), and the native α and α′ Homotrimers exhibit better solubility, emulsifying ability, and heat-induced association than the native β Homotrimer. Further, the N-linked glycans contribute to solubilities of the three subunits at low ionic strength (μ=0.08) and to the emulsifying ability of the native β Homotrimer. N-Linked glycans also prevent heat-induced associations of the native α and α′ Homotrimers but do not contribute to the secondary structure and the thermal stability of β-conglycinin.
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Crystal structures of recombinant and native soybean beta-conglycinin beta Homotrimers.
European journal of biochemistry, 2001Co-Authors: Nobuyuki Maruyama, Koji Takahashi, Kazuhiro Yagasaki, Shuko Nakagawa, Motoyasu Adachi, Mitsutaka Kohno, Yasuyuki Takenaka, Eiko Okuda, Bunzo Mikami, Shigeru UtsumiAbstract:The crystal structures of recombinant and native beta Homotrimers of soybean beta-conglycinin were determined by X-ray crystallography at 2.7 and 2.8 A resolutions, respectively. The crystals of the recombinant and native beta Homotrimers belong to space group P21 with cell parameters a = 80.51 A, b = 63.48 A, c = 131.43 A, and beta = 90.01 degrees and with cell parameters a = 82.78 A, b = 69.47 A, c = 125.33 A and beta = 97.22 degrees, respectively. The beta monomers consist of amino-terminal and carboxyl-terminal modules that are very similar to each other and are related by a pseudo-dyad axis. Each module of the beta monomer is subdivided into a core and a loop domain. These structural features of both beta Homotrimers are consistent with those of canavalin and phaseolin, which are similar vicilin class proteins. The superposition of the models of the native and recombinant beta monomers shows a root mean square deviation of 0.43-0.51 A for 343 common Calpha atoms within 2.0 A. This result indicates that the N-linked glycans do not influence the final structure of the beta Homotrimer. Comparison of the models of beta-conglycinin, phaseolin and canavalin indicates that beta-conglycinin resembles canavalin rather than phaseolin, and that canavalin and phaseolin differ the most among them. The evolutional relationships are discussed.
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Crystal structure of soybean proglycinin A1aB1b Homotrimer.
Journal of molecular biology, 2001Co-Authors: Motoyasu Adachi, Yasuyuki Takenaka, Bunzo Mikami, Andrew B. Gidamis, Shigeru UtsumiAbstract:Soybean glycinin is a member of the 11 S globulin family. The crystal structure of proglycinin was determined by X-ray crystallography at 2.8 A resolution with an R-factor of 0.199 and a free R-factor of 0.250. A trimer molecule was found in an asymmetric unit of crystals. The trimer model contains three A1aB1b subunits and comprises 1128 amino acid residues and 34 water molecules. The constituent protomers of the homo-trimeric protein are arranged around a 3-fold symmetry axis with dimensions of 95 Ax95 Ax40 A. The protomer model is composed of five fragments which correspond roughly to conserved regions based on the sequence alignment of various 11 S globulins. The core of the protomer consists of two jelly-roll beta-barrels and two extended helix domains. This structure of proglycinin is similar to those of canavalin and phaseolin belonging to the 7 S globulin family, strongly supporting the hypothesis that both 7 S and 11 S globulins are derived from a common ancestor. The inter and intra-chain disulfide bonds conserved in the 11 S globulin family are clearly observed. It is found that the face with the inter-chain disulfide bond (IE face) contains more hydrophobic residues than that with the intra-chain disulfide bond. This suggests that a mature hexamer is formed by the interaction between the IE faces after processing.
Hélène Chanut-delalande - One of the best experts on this subject based on the ideXlab platform.
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Development of a functional skin matrix requires deposition of collagen V heterotrimers.
Molecular and cellular biology, 2004Co-Authors: Hélène Chanut-delalande, Agnes Fichard, Christelle Bonod-bidaud, S. Cogne, M. Malbouyres, F. Ramirez, Florence RuggieroAbstract:Collagen V is a minor component of the heterotypic I/III/V collagen fibrils and the defective product in most cases of classical Ehlers Danlos syndrome (EDS). The present study was undertaken to elucidate the impact of collagen V mutations on skin development, the most severely affected EDS tissues, using mice harboring a targeted deletion of the α2(V) collagen gene (Col5a2). Contrary to the original report, our studies indicate that the Col5a2 deletion (a.k.a. the pN allele) represents a functionally null mutation that affects matrix assembly through a complex sequence of events. First the mutation impairs assembly and/or secretion of the α1(V)2α2(V) heterotrimer with the result that the α1(V) Homotrimer is the predominant species deposited into the matrix. Second, the α1(V) Homotrimer is excluded from incorporation into the heterotypic collagen fibrils and this in turn severely impairs matrix organization. Third, the mutant matrix stimulates a compensatory loop by the α1(V) collagen gene that leads to additional deposition of α1(V) Homotrimers. These data therefore underscore the importance of the collagen V heterotrimer in dermal fibrillogenesis. Furthermore, reduced thickness of the basement membranes underlying the epidermis and increased apoptosis of the stromal fibroblasts in pN/pN skin strongly indicate additional roles of collagen V in the development of a functional skin matrix.
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Development of a functional skin matrix requires deposition of collagen V heterotrimers.
Molecular and Cellular Biology, 2004Co-Authors: Hélène Chanut-delalande, Agnes Fichard, Christelle Bonod-bidaud, S. Cogne, M. Malbouyres, F. Ramirez, Florence RuggieroAbstract:Collagen V is a minor component of the heterotypic I/III/V collagen fibrils and the defective product in most cases of classical Ehlers Danlos syndrome (EDS). The present study was undertaken to elucidate the impact of collagen V mutations on skin development, the most severely affected EDS tissues, using mice harboring a targeted deletion of the alpha2(V) collagen gene (Col5a2). Contrary to the original report, our studies indicate that the Col5a2 deletion (a.k.a. the pN allele) represents a functionally null mutation that affects matrix assembly through a complex sequence of events. First the mutation impairs assembly and/or secretion of the alpha1(V)(2)alpha2(V) heterotrimer with the result that the alpha1(V) Homotrimer is the predominant species deposited into the matrix. Second, the alpha1(V) Homotrimer is excluded from incorporation into the heterotypic collagen fibrils and this in turn severely impairs matrix organization. Third, the mutant matrix stimulates a compensatory loop by the alpha1(V) collagen gene that leads to additional deposition of alpha1(V) Homotrimers. These data therefore underscore the importance of the collagen V heterotrimer in dermal fibrillogenesis. Furthermore, reduced thickness of the basement membranes underlying the epidermis and increased apoptosis of the stromal fibroblasts in pN/pN skin strongly indicate additional roles of collagen V in the development of a functional skin matrix.Collagen V is a minor component of the heterotypic I/III/V collagen fibrils and the defective product in most cases of classical Ehlers Danlos syndrome (EDS). The present study was undertaken to elucidate the impact of collagen V mutations on skin development, the most severely affected EDS tissues, using mice harboring a targeted deletion of the alpha2(V) collagen gene (Col5a2). Contrary to the original report, our studies indicate that the Col5a2 deletion (a.k.a. the pN allele) represents a functionally null mutation that affects matrix assembly through a complex sequence of events. First the mutation impairs assembly and/or secretion of the alpha1(V)(2)alpha2(V) heterotrimer with the result that the alpha1(V) Homotrimer is the predominant species deposited into the matrix. Second, the alpha1(V) Homotrimer is excluded from incorporation into the heterotypic collagen fibrils and this in turn severely impairs matrix organization. Third, the mutant matrix stimulates a compensatory loop by the alpha1(V) collagen gene that leads to additional deposition of alpha1(V) Homotrimers. These data therefore underscore the importance of the collagen V heterotrimer in dermal fibrillogenesis. Furthermore, reduced thickness of the basement membranes underlying the epidermis and increased apoptosis of the stromal fibroblasts in pN/pN skin strongly indicate additional roles of collagen V in the development of a functional skin matrix.
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Control of Heterotypic Fibril Formation by Collagen V Is Determined by Chain Stoichiometry
Journal of Biological Chemistry, 2001Co-Authors: Hélène Chanut-delalande, Agnes Fichard, Robert Garrone, Simonetta Bernocco, David J S Hulmes, Florence RuggieroAbstract:Abstract Although the collagen V heterotrimer is known to be involved in the control of fibril assembly, the role of the Homotrimer in fibrillar organization has not yet been examined. Here, the production of substantial amounts of recombinant collagen V Homotrimer has allowed a detailed study of its role in homotypic and heterotypic fibril formation. After removal of terminal regions by pepsin digestion, both the collagen V heterotrimer and Homotrimer formed thin homotypic fibrils, thus showing that diameter limitation is at least in part an intrinsic property of the collagen V triple helix. When mixed with collagen I, however, various complementary approaches indicated that the collagen V heterotrimer and Homotrimer exerted different effects in heterotypic fibril formation. Unlike the heterotrimer, which was buried in the fibril interior, the Homotrimer was localized as thin filamentous structures at the surface of wide collagen I fibrils and did not regulate fibril assembly. Its localization at the fibril surface suggests that the Homotrimer can act as a molecular linker between collagen fibrils or macromolecules in the extracellular matrix or both. Thus, depending on their respective distribution in tissues, the different collagen V isoforms might fulfill specific biological functions.
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Bone morphogenetic protein-1 (BMP-1) mediates C-terminal processing of procollagen V Homotrimer.
The Journal of biological chemistry, 2001Co-Authors: E. Kessler, Hélène Chanut-delalande, Agnes Fichard, M. Brusel, Florence RuggieroAbstract:Abstract The processing of the fibrillar procollagen precursors to mature collagens is an essential requirement for fibril formation. The enzymes involved in these events are known as the procollagen N and C proteinases. The latter, which cleaves the C-propeptides of the fibrillar procollagens I-III, is identical to the previously described bone morphogenetic protein-1 (BMP-1). Surprisingly, unlike the other fibrillar collagens, the processing of the C-propeptide domain of the procollagen V Homotrimer was found to be mediated by furin rather than BMP-1. However, the presence of putative BMP-1 cleavage sites in the α1(V) C-propeptide sequence prompted us to reconsider the procollagen V C-propeptide cleavage by BMP-1. Using a recombinant system to produce substantial amounts of the proα1(V) Homotrimer, we have previously shown that the C-propeptide is spontaneously released in the culture medium. The trimeric C-propeptide fragment, resulting from the furin cleavage, still encompassed the predicted BMP-1 cleavage sites. It was purified and tested as a substrate for BMP-1. In parallel, the release of the C-propeptide in the culture medium was inhibited by the addition of a specific furin inhibitor, allowing the re-examination of BMP-1 activity on the intact molecule. We showed that BMP-1 does cleave both substrates at one of the two predicted C-proteinase cleavage sites. Our results favor a role for PCP/BMP-1 in physiological C-terminal processing of procollagen V and imply a general mechanism for fibrillar collagen C-terminal processing.
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Bone morphogenetic protein-1 (BMP-1) mediates C-terminal processing of procollagen V Homotrimer.
Journal of Biological Chemistry, 2001Co-Authors: E. Kessler, Hélène Chanut-delalande, Agnes Fichard, M. Brusel, Florence RuggieroAbstract:The processing of the fibrillar procollagen precursors to mature collagens is an essential requirement for fibril formation. The enzymes involved in these events are known as the procollagen N and C proteinases. The latter, which cleaves the C-propeptides of the fibrillar procollagens I-III, is identical to the previously described bone morphogenetic protein-1 (BMP-1). Surprisingly, unlike the other fibrillar collagens, the processing of the C-propeptide domain of the procollagen V Homotrimer was found to be mediated by furin rather than BMP-1. However, the presence of putative BMP-1 cleavage sites in the alpha1(V) C-propeptide sequence prompted us to reconsider the procollagen V C-propeptide cleavage by BMP-1. Using a recombinant system to produce substantial amounts of the proalpha1(V) Homotrimer, we have previously shown that the C-propeptide is spontaneously released in the culture medium. The trimeric C-propeptide fragment, resulting from the furin cleavage, still encompassed the predicted BMP-1 cleavage sites. It was purified and tested as a substrate for BMP-1. In parallel, the release of the C-propeptide in the culture medium was inhibited by the addition of a specific furin inhibitor, allowing the re-examination of BMP-1 activity on the intact molecule. We showed that BMP-1 does cleave both substrates at one of the two predicted C-proteinase cleavage sites. Our results favor a role for PCP/BMP-1 in physiological C-terminal processing of procollagen V and imply a general mechanism for fibrillar collagen C-terminal processing.The processing of the fibrillar procollagen precursors to mature collagens is an essential requirement for fibril formation. The enzymes involved in these events are known as the procollagen N and C proteinases. The latter, which cleaves the C-propeptides of the fibrillar procollagens I-III, is identical to the previously described bone morphogenetic protein-1 (BMP-1). Surprisingly, unlike the other fibrillar collagens, the processing of the C-propeptide domain of the procollagen V Homotrimer was found to be mediated by furin rather than BMP-1. However, the presence of putative BMP-1 cleavage sites in the alpha1(V) C-propeptide sequence prompted us to reconsider the procollagen V C-propeptide cleavage by BMP-1. Using a recombinant system to produce substantial amounts of the proalpha1(V) Homotrimer, we have previously shown that the C-propeptide is spontaneously released in the culture medium. The trimeric C-propeptide fragment, resulting from the furin cleavage, still encompassed the predicted BMP-1 cleavage sites. It was purified and tested as a substrate for BMP-1. In parallel, the release of the C-propeptide in the culture medium was inhibited by the addition of a specific furin inhibitor, allowing the re-examination of BMP-1 activity on the intact molecule. We showed that BMP-1 does cleave both substrates at one of the two predicted C-proteinase cleavage sites. Our results favor a role for PCP/BMP-1 in physiological C-terminal processing of procollagen V and imply a general mechanism for fibrillar collagen C-terminal processing.