PhD Defense Paul MONASSA "Proteome-wide analysis of human protein demyristoylation by IpaJ virulence bacterial effectors" (eq. Giglione - B3S dpt)

Paul MONASSA - eq. Giglione - B3S dpt

Title: Proteome-wide analysis of human protein demyristoylation by IpaJ virulence bacterial effectors

Composition du jury :
Dr Nienke BUDDELMEIJER (Rapporteure & Examinatrice)
Dr Joëlle VINH (Rapporteure & Examinatrice)
Dr Boris VAUZEILLES (Examinateur)
Dr Emmanuelle THINON (Examinatrice)
Pr Dominique GUIANVARC’H (Examinatrice)
Pr Nicolas BAYAN (Examinateur)
Dr Thierry MEINNEL (Directeur de thèse)

Abstract:
The disease caused by bacteria of the genus Shigella, known as shigellosis, is responsible for severe dysentery in humans. The infectious process relies in part on the direct injection of bacterial proteins, known as effectors, into intestinal cells. Many of these effectors modify host proteins involved in essential cellular processes through post-translational modifications.
Among the major Shigella effectors is IpaJ, a highly specific cysteine protease. Its mechanism of action involves cleavage of the myristate group attached to the N-terminus of proteins involved in intracellular trafficking, including the small GTPases of the ARF family. However, N-myristoylation is a much more widespread modification, affecting more than 400 proteins in the human proteome, corresponding to approximately 2% of all proteins.
Preliminary experiments to which I contributed showed that IpaJ could act in vitro on a wide range of myristoylated proteins, suggesting that its substrate spectrum may be much broader than initially described. My PhD project therefore focused on developing global proteomic approaches to specifically identify N-myristoylated proteins and determine which of these proteins are most sensitive to IpaJ activity.
To achieve this, I took advantage of new click-chemistry probes developed as part of a collaboration with the CEA. These probes have the advantage of being cleavable, allowing the selected proteins to be released from the enrichment matrix. I showed that they significantly improved the efficiency of the enrichment, notably by increasing the signal-to-noise ratio.
Using three different cell lines, I then developed and optimized an approach to enrich N-myristoylated proteins in each cell type. After validating this strategy, I applied it to cells expressing IpaJ. I found that, in addition to the four ARF proteins previously described as IpaJ substrates, approximately ten other proteins were affected. I focused in particular on a new target, ARF6, and showed that cleavage of its N-myristoyl modification by IpaJ ultimately led to its intracellular degradation.
The IpaJ-mediated inactivation of this group of proteins may therefore contribute to exacerbating the cellular alterations induced by other Shigella effectors, ultimately contributing to host-cell death through apoptosis.
Finally, I also contributed to characterizing the mechanism of action of the cellular enzyme responsible for N-myristoylation, N-myristoyltransferase (NMT). Our work shows that NMT directly interacts with the ribosome, allowing it to protect the newly synthesized N-terminus of the proteins it modifies. NMT may therefore act as a molecular chaperone, transiently protecting this region before the proteins are targeted to membranes.
In conclusion, I discuss a model that reconciles the action of NMT with the cleavage of myristoylated proteins by IpaJ. This model takes into account the apparent limited accessibility of the protein-associated myristate, owing to the hydrophobicity of the lipid chain, as well as its protection by NMT and the membrane targeting promoted by this modification.