To improve the existing methods based on phenotype and/or genotype a new genotyping method was investigated to identify Lactobacillus species. / The method used the polymerase chain reaction (PCR) to amplify specific sequences of aminopeptidase (pepN) genes. The primers for the PCR reactions derived from a pepN sequence of Lactobacillus rhamnosus S93. PepN amplification products of 387 bp were obtained from forty three Lactobacillus strains and from some strains of Lactococcus (3), Streptococcus (2) and Bifidobactertium (5). / Restriction fragment length polymorphisms (RFLPs) and single-strand conformation polymorphisms (SSCP) methods were used to detect polymorphisms among amplified aminopeptidase DNA fragments from the different Lactobacillus strains. / The results of RFLPs after digestions with Sau3A I, Rsa I and Tru9 I confirmed that the PCR products were specific. According to the fingerprints generated, Lactobacillus species tested could be grouped in four. / SSCP allowed a good discrimination between different pepN PCR products of the same size. Some Lactobacillus strains, Lb. plantarum and Lb. rhamnosus showed the different ssDNA patterns. Though for many strains of Lactobacillus the SSCP patterns were similar, no general comparison can be made because all the samples were not loaded on the same SSCP polyacrylamide gel. The SSCP, PCR-based method can be easily modified to increase the rate of polymorphism detection. / This new genetic identification method is different from others because it uses specific pepN DNA sequences for each strain tested and it uses SSCP to detect the presence of polymorphisms. The method is also applicable to other genera of lactic acid bacteria.

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La protéolyse est une fonction clé de la cellule pour le maintien de l'intégrité du protéome, pour le métabolisme et pour la régulation de nombreux processus physiologiques. Le travail présenté dans cette thèse porte sur une famille de complexes peptidases cytosoliques auto-compartimentés et énergie indépendants découverts chez les Archées, les aminopeptidases TET. Chez l'Archée hyperthermophile Pyrococcus horikoshii, organisme modèle de cette étude, il existe 3 peptidases TET présentant chacune des spécificités de substrats différentes. Les caractérisations structurales des différents membres connus de cette famille de peptidases ont révélé un assemblage dodécamériques creux en forme de tétraèdre d'environ 450 kDa. Des études récentes ont montré l'existence de complexes adoptant la même conformation que les TET dans les 3 domaines du vivant. La première partie du travail présenté a permis d'identifier des marqueurs structuraux caractéristiques de l'assemblage tétraédrique afin de déterminer sans ambiguïté l'appartenance de ces complexes à la famille des TET. La seconde partie de l'étude a conduit à élucider la question de la multiplicité des TET chez les Archées hyperthermophile mise en évidence grâce à une étude phylogénétique initiée pendant la thèse. L'étude en co-expression de PhTET2 et PhTET3 révèle que ces aminopeptidases sont capable de former un hétéro-oligomère présentant une activité enzymatique accrue vis-à-vis des homo-oligomères. La dernière partie du travail porte sur les relations oligomérisation-fonction chez les peptidases TET. L'étude d'un mutant de l'oligomérisation de PhTET2 via une stratégie intégrative alliant biochimie, enzymologie, biophysique (SAXS et AUC) et des études in vivo a permis de mettre en évidence un processus d'assemblage contrôlé permettant d'augmenter l'efficacité de la peptidase. Enfin, la méthode de variation de contraste en diffusion de neutrons aux petits angles (SANS) appliqué à l'étude de l'hétéro-oligomère a permis de révéler une topologie rationalise du complexe hétéro-oligomérique favorisant la formations de poches multi-catalytique. L'ensemble de ce travail contribue à mieux comprendre l'importance et le rôle physiologique des machines TETs dans les cellules. / Proteolysis is a key function in the cell for the maintenance of the proteome integrity, the metabolism and for the regulation of many physiological processes. The thesis work is focused on a family of self-compartmentalized energy-independent cytosolic peptidases discovered in Archaea, the TET aminopeptidases. Three different TET showing contrasted enzymatic specificities co-exist in the cytosol of the hyperthermophilic archaeon Pyrococcus horikoshii, which is the model organism for this study. The structural characterization of the known members of this family shows that they self-assemble in a unique 450 kDa hollow tetrahedral structure . Recent studies have revealed the existence of peptidases complexes that adopt the same conformation in the three domains of life. The first part of this work allowed identifying structural markers to assign without any ambiguity uncharacterized peptidases to the TET family. The second objective of the work was to understand the multiplicity of TET peptidases in hyperthermophilic archaeon that was highlighted by a phylogenomic study presented in this work . The co-expression of PhTET2 and PhTET3 in E. coli revealed that the two proteins form a hetero-oligomeric complex with enhanced enzymatic activity compared to the homo-oligomers. The last part of the work addressed the question of oligomerization-function relationship in TET particles. A mutagenesis strategy was used to slow down the oligomerization process of PhTET2, and, using an integrative strategy combining biochemistry, enzymology, biophysics (SAXS and AUC) and in vivo studies we were able to dissect the oligomerization pathway of the TET particles and to demonstrate that it is a highly controlled process aim to enhance the activity of the peptidases. Finally, the contrast variation technique in small angle neutron scattering studies (SANS) allowed us to unravel the rational topology of the TET hetero-oligomers that favored the formation of multi-catalytic enzymatic pockets in the complex. All theses studies contributed to specify the biological importance of the TET molecular machines in the cells.

Les aminopeptidases sont des cibles thérapeutiques importantes pour plusieurs maladies, car elles sont impliquées dans divers processus physiologiques et pathologiques comme la progression tumorale, l'angiogenèse, et certaines infections (virales, bactériennes, et parasitaires). Il en existe deux classes : les aminopeptidases avec un ion métallique (Aminopeptidase N [APN ou CD13] et leukotrien A4 hydrolase [LTA4H]) et les aminopeptidases avec deux ions métalliques (Aminopeptidase de l'Aeromonas proteolytica [APaero], Leucine Aminopeptidase cytosolique [LAPc] et Méthionine aminopeptidase 1 ou 2 [MetAP]). Deux types de composés dérivés des amino-benzosubérones ont été envisagés pour inhiber sélectivement chacune de ces classes d'aminopeptidases. L'étude des relations structures-activités (RSA) nous a permis de découvrir une molécule très puissante et sélective de l'APN (Ki 60 pM). L'APN est une enzyme monométallique considérée aujourd'hui comme une nouvelle cible pour la lutte contre le cancer car son inhibition bloque le processus de l'angiogenèse et donc la progression tumorale. L'étude d'une nouvelle classe de molécules trisubstituées dérivées des amino­benzosubérones a abouti à la découverte d'une seconde molécule active et sélective des enzymes bimétalliques notamment l'APaero (Ki 10 nM).

Coronary artery disease (CAD) is the most prevalent disorder and the leading cause of death worldwide. There are a number of CAD medications, which are effective and safe in most patients, but have been associated with adverse reactions such as angioedema induced by angiotensin I-converting enzyme inhibitors (AE-ACEi). In this study, we identified aminopeptidase P (APP) activity as an endophenotype for AE-ACEi, which is a heritable quantitative trait (heritability =0.336 +/- 0.251 SD) and is significantly reduced in a majority of our cases. Although initial mutation screening did not reveal any coding variants in XPNPEP2, which encodes membrane-bound APP, subsequent linkage analysis of APP activity in eight families provided a maximum LOD score (3.75) for this locus. Sequencing of additional cases identified a splice variant (314_431del) and a non-coding polymorphism (rs3788853) in this locus, which cosegregate with low plasma APP activity. The latter accounts for the linkage signal and is associated with AE-ACEi (P = 0.036). In addition, we identified other potential loci for APP activity and demonstrated that certain ACEi (Captopril and Enalapril) non-specifically inhibit APP activity. Furthermore, we detected polymorphisms associated with reduced APP and ACE activities among females with estrogen-dependent inherited angioedema. / We also conducted a genetic investigation of depression among CAD patients to identify common susceptibility loci which might explain the correlation between these diseases. Our candidate gene association study identified a polymorphism (rs216873) in the von Willebrand factor gene that was significantly associated (P = 7.4 x 10-5) with elevated depressive symptoms in our CAD cohort. These results suggest that risk factors for atherosclerosis also underlie susceptibility to depression among CAD patients. / This dissertation contributes to the field of genetics and pharmacogenomics of CAD. A better understanding of the toxic effects of CAD drugs will assist in the development of safer and more effective treatments. In addition, our results may facilitate clinical assays to identify individuals who are susceptible to angioedema prior to ACEi or estrogen therapy. Finally, our genetic investigation of depression in CAD patients reveals a novel drug target (VWF) for treatment of depression in cardiac cases.

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Les aminopeptidases sont des cibles thérapeutiques importantes pour plusieurs maladies, car elles sont impliquées dans divers processus physiologiques et pathologiques comme la progression tumorale, l'angiogenèse, et certaines infections (virales, bactériennes, et parasitaires). Il en existe deux classes : les aminopeptidases avec un ion métallique (Aminopeptidase N [APN ou CD13] et leukotrien A4 hydrolase [LTA4H]) et les aminopeptidases avec deux ions métalliques (Aminopeptidase de l'Aeromonas proteolytica [APaero], Leucine Aminopeptidase cytosolique [LAPc] et Méthionine aminopeptidase 1 ou 2 [MetAP]). Deux types de composés dérivés des amino-benzosubérones ont été envisagés pour inhiber sélectivement chacune de ces classes d'aminopeptidases. L'étude des relations structures-activités (RSA) nous a permis de découvrir une molécule très puissante et sélective de l'APN (Ki 60 pM). L'APN est une enzyme monométallique considérée aujourd'hui comme une nouvelle cible pour la lutte contre le cancer car son inhibition bloque le processus de l'angiogenèse et donc la progression tumorale. L'étude d'une nouvelle classe de molécules trisubstituées dérivées des amino­benzosubérones a abouti à la découverte d'une seconde molécule active et sélective des enzymes bimétalliques notamment l'APaero (Ki 10 nM). / The aminopeptidases are important therapeutic targets for several diseases, because they are implied in various physiological and pathological processes like the tumoral progression, the angiogenesis, and certain infections (viral, bacterial, and parasitic). There are two classes: aminopeptidases with one metal ion (Aminopeptidase N [APN or CD13] and leukotriene A4 hydrolase [LTA4H]) and aminopeptidases with two metal ions (Aminopeptidase of Aeromonas proteolytica [APaero], cytosolic leucine aminopeptidase [LAPc] and Methionine aminopeptidase 1 or 2 [MetAP]). Two types of compounds of amino-benzosuberone derivatives were envisaged to inhibit selectively each one of these classes of aminopeptidases. The study of the structure-activity relationship (SAR) enabled us to discover a very powerful and selective molecule of the APN (Ki 60 pM). The APN is a monometallic enzyme considered today as a new target for the fight against cancer because its inhibition blocks the angiogenesis process and thus the tumoral progression. The study of a new class of trisubstituted molecules derived from the amino-benzosuberone led us to discover another molecule which is active and selective of the bimetallic enzymes in particular APaero (Ki 10 nM).