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Robert Ménard

Publications and source records attributed to Robert Ménard.

At least 19 recordsLinked to original sources
PubMed2006

Quantitative imaging of Plasmodium sporozoites in the mammalian host.

Malaria, the disease caused by Plasmodium, kills more than 1 million people annually. Little is known of the pre-erythrocytic phase of the parasite life cycle, i.e., after the sporozoite stage is inoculated in the dermis by a mosquito and before the erythrocyte-infecting stage is released from hepatocytes. We present here a quantitative, real-time analysis of the fate of parasites transmitted in a rodent system. We describe previously unrecognized steps in the parasite's journey to the liver of the host, which are likely to play an important role in the host immune response.

Animals↗
PubMed2006

Binding site-based classification of coronaviral papain-like proteases.

The coronavirus replicase gene encodes one or two papain-like proteases (termed PL1pro and PL2pro) implicated in the N-terminal processing of the replicase polyprotein and thus contributing to the formation of the viral replicase complex that mediates genome replication. Using consensus fold recognition with the 3D-JURY meta-predictor followed by model building and refinement, we developed a structural model for the single PLpro present in the severe acute respiratory syndrome coronavirus (SCoV) genome, based on significant structural relationships to the catalytic core domain of HAUSP, a ubiquitin-specific protease (USP). By combining the SCoV PLpro model with comparative sequence analyses we show that all currently known coronaviral PLpros can be classified into two groups according to their binding site architectures. One group includes all PL2pros and some of the PL1pros, which are characterized by a restricted USP-like binding site. This group is designated the R-group. The remaining PL1pros from some of the coronaviruses form the other group, featuring a more open papain-like binding site, and is referred to as the O-group. This two-group, binding site-based classification is consistent with experimental data accumulated to date for the specificity of PLpro-mediated polyprotein processing and PLpro inhibition. It also provides an independent evaluation of the similarity-based annotation of PLpro-mediated cleavage sites, as well as a basis for comparison with previous groupings based on phylogenetic analyses.

Amino Acid Sequence↗
PubMed2006

Quantitative imaging of Plasmodium transmission from mosquito to mammal.

Plasmodium, the parasite that causes malaria, is transmitted by a mosquito into the dermis and must reach the liver before infecting erythrocytes and causing disease. We present here a quantitative, real-time analysis of the fate of parasites transmitted in a rodent system. We show that only a proportion of the parasites enter blood capillaries, whereas others are drained by lymphatics. Lymph sporozoites stop at the proximal lymph node, where most are degraded inside dendritic leucocytes, but some can partially differentiate into exoerythrocytic stages. This previously unrecognized step of the parasite life cycle could influence the immune response of the host, and may have implications for vaccination strategies against the preerythrocytic stages of the parasite.

Animals↗
PubMed2006

Structural aspects of recently discovered viral deubiquitinating activities.

Protein ubiquitination has been identified as a regulatory mechanism in key cellular activities, and deubiquitination is recognized as an important step in processes governed by ubiquitin and ubiquitin-like modifiers. Viruses are known to target ubiquitin and ubiquitin-like modifier pathways using various strategies, including the recruitment of host deubiquitinating enzymes. Deubiquitinating activities have recently been described for proteins from three different virus families (adenovirus, coronavirus and herpesvirus), and predicted for others. This review centers on structural-functional aspects that characterize the confirmed viral deubiquitinating enzymes, and their relationships to established families of cellular deubiquitinating enzymes.

Amino Acid Sequence↗
PubMed2005

Roles of dimerization domain residues in binding and catalysis by aminoacylase-1.

The aminoacylase-1/metallopeptidase 20 (Acy1/M20) family is the largest metallopeptidase family. Several crystal structures feature a metal-binding and a dimerization-mediating domain, both arranged in an extended open conformation. We have recently shown [Lindner et al. (2003) J. Biol. Chem. 278, 44496-44504] that in human Acy1 the invariant residues Glu147 and His206 from the metal-binding and the dimerization domain, respectively, are recruited to the active site from opposite dimer subunits. We hypothesized that, to facilitate this, formation of the binary complex is associated with domain closure, which would also position additional residues in the functional active site of Acy1. These would include two partially conserved dimerization domain residues: an asparagine (Asn263) and an arginine (Arg276) from the same subunit as His206 and Glu147, respectively. In this paper, we investigate the significance of the three dimerization domain residues of human Acy1 His206, Asn263, and Arg276 and, additionally, the nearby Asp274 for catalysis using site-directed mutagenesis. Enzyme complementation assays confirm the putative subunit allocations of these residues, and steady-state kinetics support roles for all of them in catalysis but only involve the Arg276 in substrate-binding. The results are consistent with a model of the closed conformation for the structure of the related enzyme carboxypeptidase G2. This study demonstrates experimentally for the first time for a member of the Acy1/M20 family that several residues outside of the metal-binding domain are involved in binding and catalysis.

Amidohydrolases↗
PubMed2005

Towards systematic identification of Plasmodium essential genes by transposon shuttle mutagenesis.

After the deciphering of the genome sequences of several Plasmodium species, efforts must turn to elucidating gene function and identifying essential gene products. However, random approaches are lacking and gene targeting is inefficient in Plasmodium. Here, we established shuttle transposon mutagenesis in Plasmodium berghei. We constructed a mini-Tn5 derivative that can transpose into parasite genes cloned in Escherichia coli, providing an efficient means of generating knockout fragments. A 10(4)-fold increase in frequencies of double-crossover homologous recombination in the parasite using a new electroporation technology permits to reproducibly generate pools of distinct mutants after transfection with mini-Tn5-interrupted sequences. The procedure opens the way to the systematic identification of essential genes in Plasmodium.

Animals↗
PubMed2005

Carboxy-monopeptidase substrate specificity of human cathepsin X.

Cathepsin X is a papain-like cysteine protease with restricted positional specificity, acting primarily as a carboxy-monopeptidase. We mapped the specificities at the S2, S1, and S1' subsites of human cathepsin X by systematically and independently substituting the P2, P1, and P1' positions of the carboxy-monopeptidase substrate Abz-FRF(4NO(2)) with natural amino acids. Human cathepsin X has broad S2, S1, and S1' specificities within two orders of magnitude in k(cat)/K(M), excluding proline that is not tolerated at these subsites. Glycine is not favored in S2, but is among the preferred residues in S1 and S1', which highlights S2 as the affinity-determinant subsite. The presence of peculiar residues at several binding site positions (Asp76, His234, Asn75, and Glu72) does not translate into a markedly different sequence specificity profile relative to other human cathepsins. These findings suggest that a specific function of human cathepsin X is unlikely to result from sequence specificity, but rather from a combination of its unique positional specificity and the co-localization of enzyme and substrate in a specific cellular environment.

Amino Acid Sequence↗
PubMed2005

In vivo imaging of malaria parasites--recent advances and future directions.

A new view into the life of malaria parasites is now possible owing to recent advances in imaging techniques and to the generation of tagged parasites. Insights into how parasites interact with their insect vectors and mammalian hosts have been gained by the study of various parasitic forms in their natural environment. Quantitative analysis of Plasmodium ookinete motility has revealed different modes of motility in parasite invasion of the mosquito gut and the extrusion of invaded gut cells from the epithelium. Similar analysis with Plasmodium sporozoites has revealed the importance of parasite motility in transmission from the mosquito vector to the mammalian host.

Animals↗
PubMed2005

The papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme.

The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) is involved in the processing of the viral polyprotein and, thereby, contributes to the biogenesis of the virus replication complex. Structural bioinformatics has revealed a relationship for the SARS-CoV PLpro to herpesvirus-associated ubiquitin-specific protease (HAUSP), a ubiquitin-specific protease, indicating potential deubiquitinating activity in addition to its function in polyprotein processing (T. Sulea, H. A. Lindner, E. O. Purisima, and R. Menard, J. Virol. 79:4550-4551, 2005). In order to confirm this prediction, we overexpressed and purified SARS-CoV PLpro (amino acids [aa]1507 to 1858) from Escherichia coli. The purified enzyme hydrolyzed ubiquitin-7-amino-4-methylcoumarin (Ub-AMC), a general deubiquitinating enzyme substrate, with a catalytic efficiency of 13,100 M(-1)s(-1), 220-fold more efficiently than the small synthetic peptide substrate Z-LRGG-AMC, which incorporates the C-terminal four residues of ubiquitin. In addition, SARS-CoV PLpro was inhibited by the specific deubiquitinating enzyme inhibitor ubiquitin aldehyde, with an inhibition constant of 210 nM. The purified SARS-CoV PLpro disassembles branched polyubiquitin chains with lengths of two to seven (Ub2-7) or four (Ub4) units, which involves isopeptide bond cleavage. SARS-CoV PLpro processing activity was also detected against a protein fused to the C terminus of the ubiquitin-like modifier ISG15, both in vitro using the purified enzyme and in HeLa cells by coexpression with SARS-CoV PLpro (aa 1198 to 2009). These results clearly establish that SARS-CoV PLpro is a deubiquitinating enzyme, thereby confirming our earlier prediction. This unexpected activity for a coronavirus papain-like protease suggests a novel viral strategy to modulate the host cell ubiquitination machinery to its advantage.

Amino Acid Sequence↗
PubMed2005

Manipulating the Plasmodium genome.

Genome manipulation, the primary tool for assigning function to sequence, will be essential for understanding Plasmodium biology and malaria pathogenesis in molecular terms. The first success in transfecting Plasmodium was reported almost ten years ago. Gene-targeting studies have since flourished, as Plasmodium is haploid and integrates DNA only by homologous recombination. These studies have shed new light on the function of many proteins, including vaccine candidates and drug resistance factors. However, many essential proteins, including those involved in parasite invasion of erythrocytes, cannot be characterized in the absence of conditional mutagenesis. Proteins also cannot be identified on a functional basis as random DNA integration has not been achieved. We overview here the ways in which the Plasmodium genome can be manipulated. We also point to the tools that should be established if our goal is to address parasite infectivity in a systematic way and to conduct refined structure-function analysis of selected products.

Animals↗
PubMed2004

Site-directed mutagenesis of the active site region in the quinate/shikimate 5-dehydrogenase YdiB of Escherichia coli.

YdiB and its paralog AroE are members of the quinate/shikimate 5-dehdrogenase family. Enzymes from this family function in the shikimate pathway that is essential for survival of microorganisms and plants and represent potential drug targets. Recent YdiB and AroE crystal structures revealed the presence of a NAD(P)-binding and a catalytic domain. We carried out site-directed mutagenesis of 8 putative active site residues in YdiB from Escherichia coli and analyzed structural and kinetic properties of the mutant enzymes. Our data indicate critical roles for an invariant lysine and aspartate residue in substrate binding and allowed us to differentiate between two previously proposed models for the binding of the substrate in the active site. Comparison of several YdiB and AroE structures led us to conclude that, upon cofactor binding and domain closure, the 2 identified binding residues are repositioned to bind to the substrate. Although the lysine residue contributes to some extent to the stabilization of the transition state, we did not identify any residue as catalytically essential. This indicates that catalysis does not operate through a general acid-base mechanism, as thought originally. Our improved understanding of the medically and agriculturally important quinate/shikimate 5-dehydrogenase family at the molecular level may prove useful in the development of novel herbicides and antimicrobial agents.

Alcohol Oxidoreductases↗
PubMed2004

Conditional mutagenesis using site-specific recombination in Plasmodium berghei.

Reverse genetics in Plasmodium, the genus of parasites that cause malaria, still faces major limitations. Only red blood cell stages of this haploid parasite can be transfected. Consequently, the function of many essential genes in these and subsequent stages, including those encoding vaccine candidates, cannot be addressed genetically. Here, we establish conditional mutagenesis in Plasmodium by using site-specific recombination and the Flp/FRT system of yeast. Site-specific recombination is induced after cross-fertilization in the mosquito vector of two clones containing either the target sequence flanked by two FRT sites or the Flp recombinase. Parasites that have undergone recombination are recognized in the cross progeny through the expression of a fluorescence marker. This approach should permit to dissect the function of any essential gene of Plasmodium during the haploid phase of its life, i.e., during infection of salivary glands in the mosquito and infection of both the liver and red blood cells in the mammal.

Animals↗
PubMed2004

The elusive malaria sporozoite in the mammalian host.

Malaria infection is initiated when Plasmodium sporozoites are injected into a host during the bite of an infected mosquito. In the mammal, the sporozoite must rapidly reach an intravacuolar niche within a hepatocyte, where it will generate the parasite stage that invades red blood cells and causes the symptoms of the disease. Herein, we describe our understanding of the way in which sporozoites travel from the site of the mosquito bite to the liver, arrest in the liver, cross the sinusoidal barrier and eventually gain access to hepatocytes. We also highlight some of the recent advances in our understanding of these processes at the molecular level.

Animals↗
PubMed2004

Imaging movement of malaria parasites during transmission by Anopheles mosquitoes.

Malaria is contracted when Plasmodium sporozoites are inoculated into the vertebrate host during the blood meal of a mosquito. In infected mosquitoes, sporozoites are present in large numbers in the secretory cavities of the salivary glands at the most distal site of the salivary system. However, how sporozoites move through the salivary system of the mosquito, both in resting and feeding mosquitoes, is unknown. Here, we observed fluorescent Plasmodium berghei sporozoites within live Anopheles stephensi mosquitoes and their salivary glands and ducts. We show that sporozoites move in the mosquito by gliding, a type of motility associated with their capacity to invade host cells. Unlike in vitro, sporozoite gliding inside salivary cavities and ducts is modulated in speed and motion pattern. Imaging of sporozoite discharge through the proboscis of salivating mosquitoes indicates that sporozoites need to locomote from cavities into ducts to be ejected and that their progression inside ducts favours their early ejection. These observations suggest that sporozoite gliding allows not only for cell invasion but also for parasite locomotion in host tissues, and that it may control parasite transmission.

Animals↗
PubMed2003

Essential roles of zinc ligation and enzyme dimerization for catalysis in the aminoacylase-1/M20 family.

Members of the aminoacylase-1 (Acy1)/M20 family of aminoacylases and exopeptidases exist as either monomers or homodimers. They contain a zinc-binding domain and a second domain mediating dimerization in the latter case. The roles that both domains play in catalysis have been investigated for human Acy1 (hAcy1) by x-ray crystallography and by site-directed mutagenesis. Structure comparison of the dinuclear zinc center in a mutant of hAcy1 reported here with dizinc centers in related enzymes points to a difference in zinc ligation in the Acy1/M20 family. Mutational analysis supports catalytic roles of zinc ions, a vicinal glutamate, and a histidine from the dimerization domain. By complementing different active site mutants of hAcy1, we show that catalysis occurs at the dimer interface. Reinterpretation of the structure of a monomeric homolog, peptidase V, reveals that a domain insertion mimics dimerization. We conclude that monomeric and dimeric Acy1/M20 family members share a unique active site architecture involving both enzyme domains. The study may provide means to improve homologous carboxypeptidase G2 toward application in antibody-directed enzyme prodrug therapy.

Amidohydrolases↗