<p>We present a computational structure-based redesign of the phenylalanine adenylation domain of the non-ribosomal peptide synthetase (NRPS) enzyme gramicidin S synthetase A (GrsA-PheA) for a set of non-cognate substrates for which the wild-type enzyme has little or virtually no specificity. Experimental validation of a set of top-ranked computationally-predicted enzyme mutants shows significant improvement in the specificity for the target substrates. We further present enhancements to the methodology for computational enzyme redesign that are experimentally shown to result in significant additional improvements in the target substrate specificity. The mutant with the highest activity for a non-cognate substrate exhibits 1/6 of the wild-type enzyme/wild-type substrate activity, further confirming the feasibility of our computational approach. Our results suggest that structure-based protein design can identify active mutants different from those selected by evolution.</p><p>Knowledge about the structures of individual domains and domain interactions can further our redesign of the NRPS enzymes for new bioactive nature product. So far, little structure information has been available for the auxiliary domains such as the epimerization domains and how they interact with the NRPS modules. Solution structure studies by nuclear magnetic resonance (NMR) provide advantages for understanding the dynamics of the domains and reveal active conformations that sometimes are not represented by the crystal structures. However, the large size of the NRPS proteins present challenges for structure studies in solution. In chapter 3, we study the solution structure of the 56 kDa epimerization domain of GrsA (GrsA-PheE) by NMR. We use multidimensional backbone resonance experiments as well as specific labeling strategy to assign the backbone resonances of GrsA-PheE. Secondary structures are determined by sets of residual dipolar couplings (RDCs) measured in multiple alignment media. To determine the global fold of the protein, we obtain long-range distance restraints by measuring the paramagnetic relaxation enhancements (PREs) from 15 site-directed spin labeling samples. </p><p>In chapter 4, we investigate the redox regulation of phosphatases. The activity levels of protein tyrosine phosphatases (PTPs) in cells are highly regulated in various ways including by phosphorylation, localization and protein-protein interaction. Additionally, redox-dependent modification has emerged as a critical part in attenuating PTPs activity in response to cellular stimuli. The tandem Src homology 2 domain-containing PTPs (SHPs) belong to the family of nonreceptor PTPs. The activity level of SHPs is highly regulated by interaction of SH2 domain, phosphorylation level of C-terminal tail and by reversible oxidation. In vivo evidence has shown the reversible oxidation of catalytic cysteine inhibits SHPs activity transiently as a result, affecting the phosphorylation level of its target proteins. In this chapter, we investigate in vitro the reversible oxidation of full-length and catalytic domain of SHP-1 and SHP-2 by using kinetic measurements and mass spectrometry. We have confirmed the susceptibility of the active site cysteines of SHPs to oxidative inactivation, with rate constants for oxidation similar to other PTPs (2-10 M-1s-1). Both SHP-1 and SHP-2 can be reduced and reactivated with the reductants DTT and gluthathione, whereas only the catalytic domain of SHP-2 is subject to reactivation by thioredoxin. Unlike PTPs whose oxidation contains a catalytic cysteine disulfide bonding to a backdoor cysteine or forms a sulfenylamide bonding to nearby backbone nitrogen, we have found that in the reversibly oxidized SHPs, the catalytic cysteines is re-reduced while two conserved backdoor cysteines form a disulfide linkage. Knocking out either of the backdoor cysteine preserves the reversibility of the oxidized SHPs with a disulfide formation between the catalytic cysteine and the remaining backdoor cysteine. However, removal of both backdoor cysteines leads to irreversible oxidative inactivation, demonstrating that these two cysteines are necessary and sufficient for ensuring reversible oxidation of the SHPs. Our results extend the mechanisms by which redox regulation of PTPs is used to modulate intracellular signaling pathways.</p> / Dissertation

Au cours des dernières années, une nouvelle classe de composés anticancéreux à base de ruthénium, appelés RDCs (Ruthenium-Derived Compounds), a été développé pour dépasser les limitations des agents chimiothérapiques contenants du platine. Contrairement à ces derniers, l’activité anticancéreuse des RDCs est en partie indépendante de l'interaction avec l'ADN. L’objectif principal de ma thèse a été ainsi de comprendre les mécanismes moléculaires quiinter viennent dans l’action anticancéreuse et antimétastatique des RDCs au-delà du dommage à l’ADN.J’ai démontré que le RDC11, contrairement au cisplatine, affecte les voies de signalisation de HIF-1 et mTOR, deux voies qui jouent un rôle clé dans le métabolisme cellulaire et qui sont souvent altérées dans les cellules cancéreuses.En parallèle, j’ai effectué un analyse structure/activité pour sélectionner des nouveaux RDCs ayant meilleures propriétés chimiques et pharmacologiques que le RDC11. Cette étude a permis d’identifier deux nouveaux RDCs qui réduisent la croissance tumorale in vivo avec un dosage beaucoup plus faible que le RDC11 et qui induisent la mort des cellules cancéreuses par une surproduction d'espèces réactives de l'oxygène et par l'activation de la caspase8. En conclusion, mes travaux ont conduit à l’identification de nouveaux mécanismes à la base de l’activité anticancéreuse du RDC11 qui pourraient expliquer certaines différences entre le mode d’action du RDC11 et du cisplatine. De plus, ils ont permis de sélectionner deux nouveaux RDCs plus efficaces que le RDC11. Ces résultat sont un impact important pour le développement de nouvelles thérapies anticancéreuses ou antimétastatiques. / In recent years, a new class of anticancer ruthenium-based drugs, called RDCs (Ruthenium-Derived Compounds), has been developed to overcome the limitations of classic platinum chemotherapeutics. Unlike the latter, the anticancer activity of RDCs is in part independent of DNA interaction. Therefore, the main objective of my thesis work was to elucidate the molecular mechanisms involved in RDCs anticancer and antimetastatic activity beyond DNA damage. I demonstrated that RDC11, unlike cisplatin, affects the HIF-1 and mTOR signaling pathways, two pathways that play a key role in cellular metabolism and that are frequently altered in cancer cells. In parallel, I performed a structure/activity analysis to select new RDCs endowed with better chemical and pharmacological properties than RDC11. This study allowed to identify two novel RDCs that reduce tumor growth in vivo at much lower doses than RDC11 and that induce cancer cell death by an overproduction of reactive oxygen species and activation of caspase 8. In conclusion, my work led to the identification of new mechanisms underlying the anticancer activity of RDC11 that could explain some of the differences between the mode of action of RDC11 and cisplatin. In addition, it allowed to select two novel RDCs which are more effective than RDC11. These results have a significant impact on the development of new anticancer or antimetastatic therapies.