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Träfflista för sökning "hsv:(MEDICIN OCH HÄLSOVETENSKAP) hsv:(Medicinska och farmaceutiska grundvetenskaper) hsv:(Läkemedelskemi) ;pers:(Villoutreix Bruno O.)"

Sökning: hsv:(MEDICIN OCH HÄLSOVETENSKAP) hsv:(Medicinska och farmaceutiska grundvetenskaper) hsv:(Läkemedelskemi) > Villoutreix Bruno O.

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1.
  • Friedrich, Ute, et al. (författare)
  • Structural and energetic characteristics of the heparin-binding site in antithrombotic protein C
  • 2001
  • Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 276:26, s. 24122-24128
  • Tidskriftsartikel (refereegranskat)abstract
    • Human activated protein C (APC) is a key component of a natural anticoagulant system that regulates blood coagulation. In vivo, the catalytic activity of APC is regulated by two serpins, alpha1-antitrypsin and the protein C inhibitor (PCI), the inhibition by the latter being stimulated by heparin. We have identified a heparin-binding site in the serine protease domain of APC and characterized the energetic basis of the interaction with heparin. According to the counter-ion condensation theory, the binding of heparin to APC is 66% ionic in nature and comprises four to six net ionic interactions. To localize the heparin-binding site, five recombinant APC variants containing amino acid exchanges in loops 37, 60, and 70 (chymotrypsinogen numbering) were created. As demonstrated by surface plasmon resonance, reduction of the electropositive character of loops 37 and 60 resulted in complete loss of heparin binding. The functional consequence was loss in heparin-induced stimulation of APC inhibition by PCI, whereas the PCI-induced APC inhibition in the absence of heparin was enhanced. Presumably, the former observations were due to the inability of heparin to bridge some APC mutants to PCI, whereas the increased inhibition of certain APC variants by PCI in the absence of heparin was due to reduced repulsion between the enzymes and the serpin. The heparin-binding site of APC was also shown to interact with heparan sulfate, albeit with lower affinity. In conclusion, we have characterized and spatially localized the functionally important heparin/heparan sulfate-binding site of APC.
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2.
  • Nilsson, Sara, et al. (författare)
  • Mutations in complement factor I as found in atypical hemolytic uremic syndrome lead to either altered secretion or altered function of factor I
  • 2010
  • Ingår i: European Journal of Immunology. - : Wiley. - 1521-4141 .- 0014-2980. ; 40:1, s. 172-185
  • Tidskriftsartikel (refereegranskat)abstract
    • The complement system is regulated by inhibitors such as factor I (FI), a serine protease that degrades activated complement factors C4b and C3b in the presence of specific cofactors. Mutations and polymorphisms in FI and its cofactors are associated with atypical hemolytic uremic syndrome (aHUS). All 14 complement factor I mutations associated with aHUS analyzed in this study were heterozygous and generated premature stop codons (six) or amino acid substitutions (eight). Almost all of the mutants were expressed by human embryonic kidney 293 cells but only six mutants were secreted into the medium, three of which were at lower levels than WT. The remaining eight mutants were not secreted but sensitive to deglycosylation with endoglycosidase H, indicating that they were retained early in the secretory pathway. Six secreted mutants were purified and five of them were functionally altered in degradation of C4b/C3b in the fluid-phase in the presence of various cofactors and on endothelial cells. Three mutants cleaved surface-bound C3b less efficiently than WT. The D501N mutant was severely impaired both in solution and on surface irrespective of the cofactor used. in conclusion, mutations in complement factor I affect both secretion and function of FI, which leads to impaired regulation of the complement system in aHUS.
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3.
  • Villoutreix, Bruno O., et al. (författare)
  • Screening the molecular surface of human anticoagulant protein C: a search for interaction sites
  • 2001
  • Ingår i: Journal of Computer-Aided Molecular Design. - 1573-4951. ; 15:1, s. 13-27
  • Tidskriftsartikel (refereegranskat)abstract
    • Protein C (PC), a 62 kDa multi-modular zymogen, is activated to an anticoagulant serine protease (activated PC or APC) by thrombin bound to thrombomodulin on the surface of endothelial cells. PC/APC interacts with many proteins and the characterisation of these interactions is not trivial. However, molecular modelling methods help to study these complex biological processes and provide basis for rational experimental design and interpretation of the results. PC/APC consists of a Gla domain followed by two EGF modules and a serine protease domain. In this report, we present two structural models for full-length APC and two equivalent models for full-length PC, based on the X-ray structures of Gla-domainless APC and of known serine protease zymogens. The overall elongated shape of the models is further cross-validated using size exclusion chromatography which allows evaluation of the Stokes radius (rs for PC = 33.15 A; rs for APC = 34.19 A), frictional ratio and axial ratio. We then propose potential binding sites at the surface of PC/APC using surface hydrophobicity as a determinant of the preferred sites of intermolecular recognition. Most of the predicted binding sites are consistent with previously reported experimental data, while some clusters highlight new regions that should be involved in protein-protein interactions.
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4.
  • Webb, Joanna H., et al. (författare)
  • Localization of a hydrophobic binding site for anticoagulant protein S on the beta -chain of complement regulator C4b-binding protein
  • 2001
  • Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 276:6, s. 4330-4337
  • Tidskriftsartikel (refereegranskat)abstract
    • C4b-binding protein (C4BP) is a plasma glycoprotein involved in regulation of the complement system. C4BP consists of seven alpha-chains and one unique beta-chain, all constructed of repeating complement control protein (CCP) modules. The beta-chain, made up of three CCPs, binds tightly to vitamin K-dependent protein S, a cofactor to anticoagulant activated protein C. When bound to C4BP, protein S loses its activated protein C cofactor function. In this study, we have mutated potentially important amino acids located at the surface of CCP1 of the beta-chain to probe the protein S-C4BP interaction. The substitutions were designed after analysis of a homology-based three-dimensional structure of the beta-chain and were L27T/F45Q, I16S/V18S, V31T/I33N, I16S/V18S/V31T/I33N, L38S/V39S, and K41E/K42E. The mutants were expressed in a prokaryotic system, purified using an N-terminal His-tag, refolded using an oxido-shuffling system, and tested in several assays for their ability to bind protein S. Our data define Ile(16), Val(18), Val(31), and Ile(33) as crucial for protein S binding, with secondary effects from Leu(38) and Val(39). In addition, Lys(41) and Lys(42) contribute slightly to the interaction. Our results further confirm that surface hydrophobicity analysis may be used to identify ligand recognition sites.
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7.
  • Knobe, Karin, et al. (författare)
  • Functional Analysis of the Factor IX Epidermal Growth Factor-Like Domain Mutation Ile66Thr Associated with Mild Hemophilia B.
  • 2006
  • Ingår i: Pathophysiology of Haemostasis and Thrombosis. - : S. Karger AG. - 1424-8832 .- 1424-8840. ; 35:5, s. 370-375
  • Tidskriftsartikel (refereegranskat)abstract
    • he present study focused on the functional role of the mutation Ile66Thr located in the N-terminal epidermal growth factor-like domain of coagulation factor IX (FIX). This mutation causes mild hemophilia B with approximately 25% FIX coagulant activity and FIX antigen levels of around 90% of normal. In the 3-dimensional structure of porcine FIXa and in the subsequent 3-dimensional model of human FIXa that we have previously developed, residue 66 is exposed to the solvent and can be replaced by many amino acids, including Thr, without affecting the major folding/stability of the molecule. This is consistent with the basically normal antigen levels observed. We found that the FIX Ile66Thr mutant was activated to a normal extent by FVIIa/TF and FXIa. However, the ability of FIX Ile66Thr to activate FX was impaired in both the presence and absence of FVIIIa, indicating that Ile66 is not directly involved in the binding of FIX to FVIIIa.
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8.
  • Knobe, Karin, et al. (författare)
  • Probing the activation of protein C by the thrombin-thrombomodulin complex using structural analysis, site-directed mutagenesis, and computer modeling
  • 1999
  • Ingår i: Proteins. - 0887-3585. ; 35:2, s. 218-234
  • Tidskriftsartikel (refereegranskat)abstract
    • Protein C (PC) is activated to an essential anticoagulant enzyme (activated PC or APC) by thrombin (T) bound to thrombomodulin (TM), a membrane receptor present on the surface of endothelial cells. The understanding of this complex biological system is in part limited due to the lack of integration of experimental and structural data. In the work presented here, we analyze the PC-T-TM pathway in the context of both types of information. First, structural analysis of the serine protease domain of PC suggests that a positively charged cluster of amino acids could be involved in the activation process. To investigate the importance of these basic amino acids, two recombinant PC mutants were constructed using computer-guided site-directed mutagenesis. The double mutant had the K62[217]N/K63[218]D substitution and in the single mutant, K86[241] was changed to S. Both mutants were activated by free thrombin at rates equivalent to that of wild-type PC (wt-PC) and they demonstrated similar calcium-dependent inhibition of their activation. The K86[241]S mutant and wt-PC were activated by thrombin bound to soluble TM at a similar rate. In contrast, the K62[217]N/ K63[218]D mutant was activated by the T-TM complex at a 10-fold lower catalytic efficiency due to a lowering in k(cat) and increase in Km. Molecular models for PC and thrombin bound to a segment of TM were developed. The experimental results and the modeling data both indicate that electrostatic interactions are of crucial importance to orient PC onto the T-TM complex. A key electropositive region centered around loops 37[191] and 60[214] of PC is defined. PC loop 37[191] is located 7-8 A from the TM epidermal growth factor (EGF) 4 while the loop 60[214] is about 10 A away from TM EGF4. Both loops are far from thrombin. A key function of TM could be to create an additional binding site for PC. The Gla domain of PC points toward the membrane and away from thrombin or the EGF modules of TM during the activation process.
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10.
  • Mark, Linda, et al. (författare)
  • KSHV complement control protein mimics human molecular mechanisms for inhibition of the complement system.
  • 2004
  • Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 279:43, s. 45093-45101
  • Tidskriftsartikel (refereegranskat)abstract
    • Kaposi's sarcoma-associated human herpesvirus (KSHV) is thought to cause Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Previously, we reported that the KSHV complement control protein (KCP) encoded within the viral genome is a potent regulator of the complement system; it acts both as a cofactor for factor I and accelerates decay of the C3 convertases (Spiller, O.B., Blackbourn, D.J., Mark, L., Proctor, D. G., and Blom, A. M. (2003) J. Biol. Chem. 278, 9283-9289). KCP is a homologue to human complement regulators, being comprised of four complement control protein (CCP) domains. In this, the first study to identify the functional sites of a viral homologue at the amino acid level, we created a three-dimensional homology-based model followed by site-directed mutagenesis to locate complement regulatory sites. Classical pathway regulation, both through decay acceleration and factor I cleavage of C4b, required a cluster of positively charged amino acids in CCP1 stretching into CCP2 (Arg-20, Arg-33, Arg-35, Lys-64, Lys-65, and Lys-88) as well as positively (Lys-131, Lys-133, and His-135) and negatively (Glu-99, Glu-152, and Asp-155) charged areas at opposing faces of the border region between CCPs 2 and 3. The regulation of the alternative pathway (via factor I-mediated C3b cleavage) was found to both overlap with classical pathway regulatory sites (Lys-64, Lys-65, Lys-88 and Lys-131, Lys-133, His-135) as well as require unique, more C-terminal residues in CCPs 3 and 4 (His-158, His-171, and His-213) and CCP 4 (Phe-195, Phe-207, and Leu-209). We show here that KCP has evolved to maintain the spatial structure of its functional sites, especially the positively charged patches, compared with host complement regulators.
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