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Robert Ménard

Publications and source records attributed to Robert Ménard.

26 records · Page 2Linked to original sources
PubMed2003

Specificity determinants of human cathepsin s revealed by crystal structures of complexes.

Cathepsin S, a lysosomal cysteine protease of the papain superfamily, has been implicated in the preparation of MHC class II alphabeta-heterodimers for antigen presentation to CD4+ T lymphocytes and is considered a potential target for autoimmune-disease therapy. Selective inhibition of this enzyme may be therapeutically useful for attenuating the hyperimmune responses in a number of disorders. We determined the three-dimensional crystal structures of human cathepsin S in complex with potent covalent inhibitors, the aldehyde inhibitor 4-morpholinecarbonyl-Phe-(S-benzyl)Cys-Psi(CH=O), and the vinyl sulfone irreversible inhibitor 4-morpholinecarbonyl-Leu-Hph-Psi(CH=CH-SO(2)-phenyl) at resolutions of 1.8 and 2.0 A, respectively. In the structure of the cathepsin S-aldehyde complex, the tetrahedral thiohemiacetal adduct favors the S-configuration, in which the oxygen atom interacts with the imidazole group of the active site His164 rather than with the oxyanion hole. The present structures provide a detailed map of noncovalent intermolecular interactions established in the substrate-binding subsites S3 to S1' of cathepsin S. In the S2 pocket, which is the binding affinity hot spot of cathepsin S, the Phe211 side chain can assume two stable conformations that accommodate either the P2-Leu or a bulkier P2-Phe side chain. This structural plasticity of the S2 pocket in cathepsin S explains the selective inhibition of cathepsin S over cathepsin K afforded by inhibitors with the P2-Phe side chain. Comparison with the structures of cathepsins K, V, and L allows delineation of local intermolecular contacts that are unique to cathepsin S.

Base Sequence↗
PubMed2003

Family C1 cysteine proteases: biological diversity or redundancy?

Recent progress in the identification and partial characterization of novel genes encoding cysteine proteases of the papain family has considerably increased our knowledge of this family of enzymes. Kinetic data available to date for this large family indicate relatively broad, overlapping specificities for most enzymes, thus inspiring a growing conviction that they may exhibit functional redundancy. This is also supported in part by phenotypes of cathepsin knockout mice and suggests that several proteases can substitute for each other to degrade or process a given substrate. On the other hand, specific functions of one particular protease have also been documented. In addition, differences in cellular distribution and intracellular localization may contribute to defining specific functional roles for some of these proteases.

Animals↗
PubMed2002

Design of noncovalent inhibitors of human cathepsin L. From the 96-residue proregion to optimized tripeptides.

A novel series of noncovalent inhibitors of cathepsin L have been designed to mimic the mode of autoinhibition of procathepsin L. Just like the propeptide, these peptide-based inhibitors have a reverse-binding mode relative to a substrate and span both the S' and S subsites of the enzyme active site. In contrast to previous studies in which even moderate truncation of the full-length propeptide led to rapid reduction in potency, these blocked tripeptide-sized inhibitors maintain nanomolar potency. Moreover, these short peptides show higher selectivity (up to 310-fold) for inhibiting cathepsin L over K versus only 2-fold selectivity of the 96-residue propeptide of cathepsin L. A 1.9 A X-ray crystallographic structure of the complex of cathepsin L with one of the inhibitors confirms the designed reverse-binding mode of the inhibitor as well as its noncovalent nature. Enzymatic analysis also shows the inhibitors to be resistant to hydrolysis at elevated concentrations of the enzyme. The mode of inhibition of these molecules provides a general strategy for inhibiting other cathepsins as well as other proteases.

Binding Sites↗
PubMed2002

A noninvasive cell-based assay for monitoring proteolytic activity within a specific subcellular compartment.

A noninvasive cell-based assay has been developed to monitor the proteolytic activity of cathepsin L within a specific subcellular compartment, the lysosome. The green fluorescent protein (GFP) of Aequorea victoria was selected as a substrate. Targeting to lysosomes was achieved by fusing GFP to preprocathepsin L, which also ensures colocalization of the enzyme and the substrate. Stably transfected HeLa-rtTA (reverse tetracycline-controlled transactivator) cells were induced with doxycycline and cultured in the presence of various concentrations of cysteine protease inhibitors for 48 h. In the absence of inhibitor, proteolytic degradation of GFP leads to loss of fluorescence, which is due almost exclusively to the action of recombinant cathepsin L. However, a dose-dependent increase of GFP fluorescence is observed for cells treated with the potent cathepsin L inhibitor benzyloxycarbonyl-LeuLeuTyr-CHN(2). Fluorescence is also observed when GFP is fused to an inactive preprocathepsin L (C25A mutant). Targeting of GFP to an acidic cellular compartment can destabilize the protein and render it susceptible to proteolytic degradation. The approach should be generally applicable for proteases localized in acidic environments. Such an assay can be of great value in validating the participation of a specific enzyme in a given process or in testing the ability of putative inhibitors to reach their intracellular target.

Amino Acid Sequence↗
PubMed2002

Levels of circumsporozoite protein in the Plasmodium oocyst determine sporozoite morphology.

The sporozoite stage of the Plasmodium parasite is formed by budding from a multinucleate oocyst in the mosquito midgut. During their life, sporozoites must infect the salivary glands of the mosquito vector and the liver of the mammalian host; both events depend on the major sporozoite surface protein, the circumsporozoite protein (CS). We previously reported that Plasmodium berghei oocysts in which the CS gene is inactivated do not form sporozoites. Here, we analyzed the ultrastructure of P.berghei oocyst differentiation in the wild type, recombinants that do not produce or produce reduced amounts of CS, and corresponding complemented clones. The results indicate that CS is essential for establishing polarity in the oocyst. The amounts of CS protein correlate with the extent of development of the inner membranes and associated microtubules underneath the oocyst outer membrane, which normally demarcate focal budding sites. This is a first example of a protein controlling both morphogenesis and infectivity of a parasite stage.

Animals↗
PubMed2002

Plasmodium sporozoite invasion into insect and mammalian cells is directed by the same dual binding system.

Plasmodium sporozoites, the transmission form of the malaria parasite, successively invade salivary glands in the mosquito vector and the liver in the mammalian host. Sporozoite capacity to invade host cells is mechanistically related to their ability to glide on solid substrates, both activities depending on the transmembrane protein TRAP. Here, we show that loss-of- function mutations in two adhesive modules of the TRAP ectodomain, an integrin-like A-domain and a thrombospondin type I repeat, specifically decrease sporozoite invasion of host cells but do not affect sporozoite gliding and adhesion to cells. Irrespective of the target cell, i.e. in mosquitoes, rodents and cultured human or hamster cells, sporozoites bearing mutations in one module are less invasive, while those bearing mutations in both modules are non-invasive. In Chinese hamster ovary cells, the TRAP modules interact with distinct cell receptors during sporozoite invasion, and thus act as independently active pass keys. As these modules are also present in other members of the TRAP family of proteins in Apicomplexa, they may account for the capacity of these parasites to enter many cell types of phylogenetically distant origins.

Amino Acid Sequence↗
PubMed2002

S2' substrate specificity and the role of His110 and His111 in the exopeptidase activity of human cathepsin B.

The ability of the lysosomal cysteine protease cathepsin B to function as a peptidyldipeptidase (removing C-terminal dipeptides) has been attributed to the presence of two histidine residues (His(110) and His(111)) present in the occluding loop, an extra peptide segment located in the primed side of the active-site cleft. Whereas His(111) is unpaired, His(110) is present as an ion pair with Asp(22) on the main body of the protease. This ion pair appears to act as a latch to hold the loop in a closed position. The exopeptidase activity of cathepsin B, examined using quenched fluorescence substrates, was shown to have a 20-fold preference for aromatic side chains in the P2' position relative to glutamic acid as the least favourable residue. Site-directed mutagenesis demonstrated that His(111) makes a positive 10-fold contribution to the exopeptidase activity, whereas His(110) is critical for this action with the Asp(22)-His(110) ion pair stabilizing the electrostatic interaction by a maximum of 13.9 kJ/mol (3.3 kcal/mol). These studies showed that cathepsin B is optimized to act as an exopeptidase, cleaving dipeptides from protein substrates in a successive manner, because of its relaxed specificity in P2' and its other subsites.

Amidohydrolases↗