Analyse biochimique et inhibition de complexes macromoléculaires dans des cellules humaines et bactériennes

All cellular processes depend on macromolecular complexes. Studying their composition, structure and the interactions between their components is a crucial step toward understanding of their functions. Moreover, their components can be targeted in applied research aimed at drug design. During my Ph.D., we set up a multidisciplinary approach to study two macromolecular complexes: the eukaryotic selenocysteine (Sec) biosynthesis and incorporation machinery and the H. pylori type 4 secretion system. Sec is the 21st amino acid of the genetic code and is encoded by the UGA stop codon. Thus, Sec decoding requires a specialized translation machinery and is driven by transient interactions between RNA and proteins. Although the main factors of the machinery are now identified, many molecular details remain to be clarified. In order to characterize the interactions between all the known factors, we took advantage of the BRET (Bioluminescence Resonance Energy Transfer) assay. Our in vivo interaction studies allowed us to define the oligomeric state of several factors of the machinery and to identify previously unknown interactions. Sites of interaction between several factors of the machinery were also determined using in vitro approaches. Thus, we propose here a more detailed model of the steps leading to the Sec decoding. In addition, we propose the first structural model of a key protein of the machinery (SECp43), that could be later exploited to elucidate the function of the protein in the translation machinery. Then we focused on the type 4 secretion system of Helicobacter pylori. The H. pylori bacterium is a human pathogen whose presence increases the risk of gastric carcinogenesis. The type 4 secretion system encoded by some strains constitute an aggravating factor of pathogenicity. Due to the emergence of multidrug-resistant H. pylori strains, current treatments to eradicate bacteria become unsuitable. Thus, we propose to adopt an alternative strategy to antibiotics and undertook to develop and characterize molecules capable of inhibiting the virulence of the bacterium. The 1G2 molecule, that targets the Cagα ATPase protein, was identified. The 1G2 molecule inhibits the ATPase activity of Cagα and decreases by almost 50% the proinflammatory response of eukaryotic cells induced by the H. pylori infection. The effectiveness of the molecule was subsequently evaluated on drug-resistant clinical strains of H. pylori, genetically and biochemically characterized. We have demonstrated that the 1G2 molecule was also effective on highly virulent clinical strains. As chronicity of H. pylori infection may lead to the development of gastric cancers, our results suggest the high potential of the 1G2 molecule in the treatment of H. pylori infection. This molecule could serve as a basis for the design of other molecules having a more noticeable effect on the virulence of H. pylori.

Keyphrases: BRET, SEPHS1, SEPHS2, SEPSECS, SECp43, SST4, CagA, CagL, H. pylori, Cagα, Macromolecular complexes, Selenocysteine, Selenoprotein, Protein interaction, T4SS, Virulence inhibitors