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Bruno O Villoutreix

Publications and source records attributed to Bruno O Villoutreix.

At least 19 recordsLinked to original sources
PubMed2006

Kaposi's sarcoma-associated herpes virus complement control protein: KCP--complement inhibition and more.

The complement system is an important part of innate immunity providing immediate protection against pathogens without a need for previous exposure, as well as priming the adaptive immune response through opsonisation, leukocyte recruitment and enhancing humoral immune responses. Its importance is not only shown through recurring fulminant infections in individuals with complement component deficiencies, but also through the many complement evasion strategies discovered for a wide range of infectious microbes (including acquisition of endogenous host complement inhibitors and expression of own homologues). Knowledge of these mechanisms at a molecular level may aid development of vaccines and novel therapeutic strategies. Here, we review the structure-function studies of the membrane-bound complement inhibitor KCP that is expressed on the surface of Kaposi's sarcoma-associated herpesvirus (KSHV) virions and infected cells. KCP accelerates the decay of classical C3 convertase and induces the degradation of activated complement factors C4b and C3b by a serine proteinase, factor I. Molecular modeling and site-directed mutagenesis have identified sites on the surface of KCP required for complement inhibition and support the hypothesis that KCP has evolved to mimic the structure and function of endogenous human inhibitors. KCP additionally enhances virion binding to permissive cells through a heparin/heparan sulfate-binding site located at the N-terminus of the protein.

Amino Acids↗
PubMed2006

FAF-Drugs: free ADME/tox filtering of compound collections.

In silico screening based on the structures of the ligands or of the receptors has become an essential tool to facilitate the drug discovery process but compound collections are needed to carry out such in silico experiments. It has been recognized that absorption, distribution, metabolism, excretion and toxicity (ADME/tox) are key properties that need to be considered early on, even during the database preparation stage. FAF-Drugs is an online service based on Frowns (a chemoinformatics toolkit) that allows users to process their own compound collections via simple ADME/Tox filtering rules such as molecular weight, polar surface area, logP or number of rotatable bonds. SMILES (Simplified Molecular Input Line Entry System), CANSMILES (canonical smiles) or SDF (structure data file) files are required as input and molecules that pass or do not pass the filters are sent back in CANSMILES format. This service should thus help scientists engaging in drug discovery campaigns. Other utilities and several compound collections suitable for in silico screening are available at our site. FAF-Drugs can be accessed at http://bioserv.rpbs.jussieu.fr/FAFDrugs.html.

Drug Design↗
PubMed2006

Proposed structural models of the prothrombinase (FXa-FVa) complex.

Activated coagulation factor V (FVa) functions as a cofactor to factor Xa (FXa) in the conversion of prothrombin (PT) to thrombin. This essential procoagulant reaction, despite being the subject of extensive investigation, is not fully understood structurally and functionally. To elucidate the structure of the FXa-FVa complex, we have performed protein:protein (Pr:Pr) docking simulation with the pseudo-Brownian Pr:Pr docking ICM package and with the shape-complementarity Pr:Pr docking program PPD. The docking runs were carried out using a new model of full-length human FVa and the X-ray structure of human FXa. Five representative models of the FXa-FVa complex were in overall agreement with some of the available experimental data, but only one model was found to be consistent with almost all of the reported experimental results. The use of hybrid docking approach (theoretical plus experimental) is definitively important to study such large macromolecular complexes. The FXa-FVa model we have created will be instrumental for further investigation of this macromolecular system and will guide future site directed mutagenesis experiments.

Factor Va↗
PubMed2006

The Kaposi's sarcoma-associated herpesvirus complement control protein (KCP) binds to heparin and cell surfaces via positively charged amino acids in CCP1-2.

The Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP) inhibits the human complement system, and is similar in structure and function to endogenous complement inhibitors. Other inhibitors such as C4b-binding protein and factor H, as well as the viral homologue vaccinia virus complement control protein are known to bind heparin and, for the two latter, also to glycosaminoglycans at the surface of cells. We report here that KCP also binds to heparin at physiological ionic strength. With help of site directed mutagenesis, positively charged amino acids in the two N-terminal complement control protein (CCP) domains 1-2 were found to be necessary for heparin binding. In silico molecular docking of heparin to KCP confirmed the experimental data, and further explored the heparin binding site, enabling us to present a model of the KCP-heparin interaction. Furthermore, the docking analysis also yielded insights of the KCP structure, by indicating that the angle between CCP domains 1-2 during the initial binding of heparin is more extended than in the model we have previously presented. We also found that KCP binds to heparan sulfate and weakly to glycosaminoglycans at the surface of cells. This might indicate that KCP at the surface of viral particles aids in the primary attachment to the target cells, which is known to involve binding to heparan sulfate. Therefore, the present study contributes to the knowledge of heparin-protein interactions in general as well as to the understanding of the biology of KSHV.

Amino Acids, Basic↗
PubMed2006

Functional analysis of the factor IX epidermal growth factor-like domain mutation Ile66Thr associated with mild hemophilia B.

The 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.

Epidermal Growth Factor↗
PubMed2006

Receptor-based computational screening of compound databases: the main docking-scoring engines.

The processes used by academic and industrial scientists to discover new drugs have recently experienced a true renaissance with many new and exciting techniques. The number of protein structures and/or chemical ligands is constantly growing, through the use of parallel chemistry, X-ray crystallography, NMR or homology modeling methods and so is the theoretical understanding of protein-ligand interactions. As such, structure-based approaches to drug-design and in silico screening are becoming routine part of most modern lead discovery programs. Prioritization of compound libraries is an extremely important task that aims at the rapid identification of tight-binding ligands and ultimately new therapeutic compounds. These in silico approaches combined with other experimental methods facilitate the design of new medicines to treat cardiovascular, degenerative, infectious, and neoplastic diseases, among others. Here, we review key concepts and specific features of several selected ligand-receptor docking/scoring methods while several other topics pertaining to the field of in silico screening are reviewed in the following articles of this special issue of Current Protein and Peptide Science.

Animals↗
PubMed2005

A formylated hexapeptide ligand mimics the ability of Wnt-5a to impair migration of human breast epithelial cells.

Loss of Wnt-5a protein expression is associated with shorter recurrence-free survival in breast carcinoma patients and increased motility in mammary cell lines. Based on sequence analysis of Wnt-5a, we identified 14 peptide fragments and investigated their ability to mimic the effects of Wnt-5a on mammary cell adhesion and migration. Two of these peptides significantly increased adhesion and impaired migration in the non-tumorigenic HB2 breast epithelial cell line and in the MDA-MB-468 breast cancer cell line, both of which show little endogenous expression of the Wnt-5a protein. We removed two amino acids at a time from the N terminus of the shorter of these two peptides to identify the shortest peptide that still inhibited migration. The influence on tumor cell adhesion was gradually lost and was no longer detectable when only six amino acids remained. However, formylation of the N-terminal methionine of this hexapeptide restored its effect on adhesion and reduced tumor cell motility via a Frizzled-5 receptor-dependent mechanism, even at a low pH such as encountered in breast tumor tissue. This formylated hexapeptide ligand induced a rapid cytosolic calcium signal, whereas it did not affect the cellular levels of unphosphorylated beta-catenin or active JNK. The novel formyl-Met-Asp-Gly-Cys-Glu-Leu peptide ligand is not only a valuable experimental tool but has also a potential role in antimetastatic treatment of the 50% of human breast cancer patients that have reduced endogenous Wnt-5a protein expression.

Amino Acid Sequence↗
PubMed2005

Fast structure-based virtual ligand screening combining FRED, DOCK, and Surflex.

A protocol was devised in which FRED, DOCK, and Surflex were combined in a multistep virtual ligand screening (VLS) procedure to screen the pocket of four different proteins. One goal was to evaluate the impact of chaining "freely available packages to academic users" on docking/scoring accuracy and CPU time consumption. A bank of 65 660 compounds including 49 known actives was generated. Our procedure is successful because docking/scoring parameters are tuned according to the nature of the binding pocket and because a shape-based filtering tool is applied prior to flexible docking. The obtained enrichment factors are in line with those reported in recent studies. We suggest that consensus docking/scoring could be valuable to some drug discovery projects. The present protocol could process the entire bank for one receptor in less than a week on one processor, suggesting that VLS experiments could be performed even without large computer resources.

Binding Sites↗
PubMed2005

PCE: web tools to compute protein continuum electrostatics.

PCE (protein continuum electrostatics) is an online service for protein electrostatic computations presently based on the MEAD (macroscopic electrostatics with atomic detail) package initially developed by D. Bashford [(2004) Front Biosci., 9, 1082-1099]. This computer method uses a macroscopic electrostatic model for the calculation of protein electrostatic properties, such as pK(a) values of titratable groups and electrostatic potentials. The MEAD package generates electrostatic energies via finite difference solution to the Poisson-Boltzmann equation. Users submit a PDB file and PCE returns potentials and pK(a) values as well as color (static or animated) figures displaying electrostatic potentials mapped on the molecular surface. This service is intended to facilitate electrostatics analyses of proteins and thereby broaden the accessibility to continuum electrostatics to the biological community. PCE can be accessed at http://bioserv.rpbs.jussieu.fr/PCE.

Hydrogen-Ion Concentration↗
PubMed2005

Regulation of blood coagulation by the protein C anticoagulant pathway: novel insights into structure-function relationships and molecular recognition.

The protein C system provides important control of blood coagulation by regulating the activities of factor VIIIa (FVIIIa) and factor Va (FVa), cofactors in the activation of factor X and prothrombin, respectively. The system comprises membrane-bound and circulating proteins that assemble into multi-molecular complexes on cell surfaces. Vitamin K-dependent protein C, the key component of the system, circulates in blood as zymogen to an anticoagulant serine protease. It is efficiently activated on the surface of endothelial cells by thrombin bound to the membrane protein thrombomodulin. The endothelial protein C receptor (EPCR) further stimulates the protein C activation. Activated protein C (APC) together with its cofactor protein S inhibits coagulation by degrading FVIIIa and FVa on the surface of negatively charged phospholipid membranes. Efficient FVIIIa degradation by APC requires not only protein S but also intact FV, which like thrombin is a Janus-faced protein with both procoagulant and anticoagulant potential. In addition to its anticoagulant properties, APC has antiinflammatory and antiapoptotic functions, which are exerted when APC binds to EPCR and proteolytic cleaves protease-activated receptor 1 (PAR-1). The protein C system is physiologically important, and genetic defects affecting the system are the most common risk factors of venous thrombosis. The proteins of the protein C system are composed of multiple domains and the 3-dimensional structures of several of the proteins are known. The molecular recognition of the protein C system is progressively being unraveled, giving us new insights into this fascinating and intricate molecular scenario at the atomic level.

Blood Coagulation↗
PubMed2005

The anticoagulant protein C pathway.

The anticoagulant protein C system regulates the activity of coagulation factors VIIIa and Va, cofactors in the activation of factor X and prothrombin, respectively. Protein C is activated on endothelium by the thrombin-thrombomodulin-EPCR (endothelial protein C receptor) complex. Activated protein C (APC)-mediated cleavages of factors VIIIa and Va occur on negatively charged phospholipid membranes and involve protein cofactors, protein S and factor V. APC also has anti-inflammatory and anti-apoptotic activities that involve binding of APC to EPCR and cleavage of PAR-1 (protease-activated receptor-1). Genetic defects affecting the protein C system are the most common risk factors of venous thrombosis. The protein C system contains multi-domain proteins, the molecular recognition of which will be reviewed.

Animals↗
PubMed2005

Noonan syndrome type I with PTPN11 3 bp deletion: structure-function implications.

Noonan syndrome was recently reported to be caused by mutations in the PTPN11 gene in 40% of the cases. This gene encodes the nonreceptor-type protein tyrosine phosphatase SHP-2 and has been shown to be self down-regulated with the concurrency of two SH2 domains. Insertion of a specific loop (D'EF) from N-terminal SH2 domain into the SHP-2 active-site is responsible for the reversible inhibition of the phosphatase activity. Here we report the first in frame trinucleotide deletion resulting in the removal of Aspartate 61 (D61del), a key residue of the N-terminal SH2 D'EF loop. Energetic-based structural analysis and electrostatic calculations carried out on the wild-type and mutant proteins predict lower stability of the D'EF loop for the D61del variant as compared to the wild type indicating better access to the active site and most likely an enzyme activated for longer extent. Similar computations were performed on the previously functionally characterized gain-of-function D61Y mutant and similar behaviors were observed. The simulation data for the D61del and D61Y mutants suggest that both variants could yield more catalytic cycles than the wild-type molecule in the same timespan because of the opening of the active site. It also supports the notion that D61 plays a major role for proper down-regulation of the protein tyrosine phosphatase activity of SHP-2.

Adult↗
PubMed2004

The gamma-carboxyglutamic acid domain of anticoagulant protein S is involved in activated protein C cofactor activity, independently of phospholipid binding.

We expressed 2 chimeras between human protein S (PS) and human prothrombin (FII) in which the prothrombin gamma-carboxyglutamic acid (Gla) domain replaced the PS Gla domain in native PS (Gla(FII)-PS) or in PS deleted of the thrombin-sensitive region (TSR) (Gla(FII)-DeltaTSR-PS). Neither PS/FII chimera had activated protein C (APC) cofactor activity in plasma clotting assays or purified systems, but both bound efficiently to phospholipids. This pointed to a direct involvement of the PS Gla domain in APC cofactor activity through molecular interaction with APC. Using computational methods, we identified 2 opposite faces of solvent-exposed residues on the PS Gla domain (designated faces 1 and 2) as potentially involved in this interaction. Their importance was supported by functional characterization of a PS mutant in which the face 1 and face 2 PS residues were reintroduced into Gla(FII)-PS, leading to significant APC cofactor activity, likely through restored interaction with APC. Furthermore, by characterizing PS mutants in which PS face 1 and PS face 2 were individually replaced by the corresponding prothrombin faces, we found that face 1 was necessary for efficient phospholipid binding but that face 2 residues were not strictly required for phospholipid binding and were involved in the interaction with APC.

1-Carboxyglutamic Acid↗
PubMed2004

The Kaposi's sarcoma-associated herpesvirus complement control protein mimics human molecular mechanisms for inhibition of the complement system.

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.

Amino Acid Motifs↗
PubMed2004

Functional properties of recombinant factor V mutated in a potential calcium-binding site.

Activated coagulation factor V (FVa) is a cofactor of activated factor X (FXa) in prothrombin activation. FVa is composed of a light chain (LC) and a heavy chain (HC) that are noncovalently associated in a calcium-dependent manner. We constructed a recombinant FV Asp111Asn/Asp112Asn mutant (rFV-NN) to abolish calcium binding to a potential calcium-binding site in FVa in order to study the specific role of these residues in the expression of FVa activity. Whereas thrombin-activated recombinant FV wild type (rFV-wt) presented with stable FVa activity, incubation of rFV-NN with thrombin resulted in a temporary increase in FVa activity, which was rapidly lost upon prolonged incubation. Loss of FVa activity was most likely due to dissociation of HC and LC since, upon chromatography of rFVa-NN on a SP-Sepharose column, the HC did not bind significantly to the resin whereas the LC bound and could be eluted at high ionic strength. In contrast, rFVa-wt adhered to the column, and both the HC and LC coeluted at high ionic strength. In the presence of phospholipid vesicles, the loss of rFVa-NN activity was partially prevented by FXa, active site inhibited FXa, and prothombin in a dose-dependent manner. We conclude that the introduced amino acid substitutions result in a loss of the high-affinity (calcium-dependent) interaction of the HC and LC of FVa. We propose that the introduced substitutions disrupt the calcium-binding site in FV, thereby yielding a FV molecule that rapidly loses activity following thrombin-catalyzed activation most likely via dissociation of the HC and LC.

Amino Acid Sequence↗
PubMed2004

Structural stability and heat-induced conformational change of two complement inhibitors: C4b-binding protein and factor H.

The complement inhibitors C4b-binding protein (C4BP) and factor H (FH) both consist of complement control protein (CCP) domains. Here we examined the secondary structure of both proteins by circular dichroism and Fourier-transform infrared technique at temperatures ranging from 30 degrees C-90 degrees C. We found that predominantly beta-sheet structure of both proteins was stable up to 70 degrees C, and that a reversible conformational change toward alpha-helix was apparent at temperatures ranging from 70 degrees C to 90 degrees C. The ability of both proteins to inhibit complement was not impaired after incubation at 95 degrees C, exposure to extreme pH conditions, and storage at room temperature for several months. Similar remarkable stability was previously observed for vaccinia virus control protein (VCP), which is also composed of CCP domains; it therefore seems to be a general property of CCP-containing proteins. A typical CCP domain has a hydrophobic core, which is wrapped in beta-sheets and stabilized by two disulphide bridges. How the CCP domains tolerate harsh conditions is unclear, but it could be due to a combination of high content of prolines, hydrophobic residues, and the presence of two disulphide bridges within each domain. These findings are of interest because CCP-containing complement inhibitors have been proposed as clinical agents to be used to control unwanted complement activation that contributes to many diseases.

Circular Dichroism↗
PubMed2004

Complement inhibitor C4b-binding protein-friend or foe in the innate immune system?

The complement system constitutes an important component of the defence against foreign organisms, functioning both in innate and adaptive immune systems. It is potentially harmful also to the own organism and is therefore tightly regulated by a number of membrane-bound and soluble factors. C4b-binding protein (C4BP) is a potent circulating soluble inhibitor of the classical and lectin pathways of complement. In recent years, the relationships between the structure of C4BP and its functions have been elucidated using a combination of computer-based molecular analysis and recombinant DNA technologies. Moreover, two novel functions have recently been ascribed to C4BP. One is the ability of C4BP to localize complement regulatory activity to the surface of apoptotic cells via its interaction with the membrane-binding vitamin K-dependent protein S. The other is the ability of C4BP to act as a survival factor for B cells due to an interaction with CD40. The complement regulatory activity of C4BP is not only beneficial because it is also explored by pathogens such as Neisseria gonorrhoeae, Bordetella pertussis, Streptococcus pyogenes, Escherichia coli K1, and Candida albicans, that bind C4BP to their surfaces. This contributes to the serum resistance and the pathogenicity of these bacteria. In this review, the structural requirements and functional importance of the interactions between C4BP and its various ligands are discussed.

Animals↗