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Unveiling Ubiquitination as a New Regulatory Mechanism of the ESCRT-III Protein CHMP1B

Abstract : I did my thesis in the group of Dr. Marie-Odile Fauvarque who implements strategies of molecular genetics on human cell culture models and in the Drosophila fly for the identification and study of the function of proteins in intracellular signaling. In this context, my work aimed to produce fundamental knowledge about the ubiquitin system in the control of the endocytic trafficking, in particular of membrane receptors involved in the inflammatory response (TNFR, ILR) or cell differentiation and growth (EGFR). I was particularly interested in the role of the complex formed by the interaction between an endocytic protein, CHMP1B, and the ubiquitin protease UBPY (synonym USP8). CHMP1B is a member of the ESCRT-III family that controls the biogenesis of intraluminal vesicles (ILVs) at the late endosomes to form multivesicular bodies (MVBs) Conformational change and polymerization at lipidic membrane processes are needed for CHMP1B function. MVBs fuse with the lysosomes, thus ensuring the proteolysis of the internalized receptors and the stoppage of the intracellular signaling. Alternatively, the receptors may be returned to the plasma membrane from early or late endosomes via recycling vesicles. Intracellular trafficking and receptor sorting in these different subcellular compartments play a major role in the activation, duration and termination of intracellular signals. The covalent bond of one or more ubiquitin (a highly conserved polypeptide of 76 amino acids) at the receptors is a major signal triggering their internalization. By hydrolyzing this ubiquitin, UBPY can stop the internalization of receptors at the plasma membrane, or promote their entry into the MVB. UBPY would thus play two opposing roles on the stability of the receptors depending on its level of action in the cell. The interaction between the two proteins CHMP1B and UBPY had been described in the literature in the two-hybrid system in yeast or by co-immunoprecipitation from cell lysates. However, the team's work showed no strong interaction between the domains of interaction of these two proteins in vitro and the function of this interaction in the endocytosis process had only been partially elucidated.During my thesis, I confirmed the existence of the CHMP1B-UBPY in cellulo complex, which is located mainly at the level of late endosomes. I determined the region involved in this interaction and proved that the existence of this complex makes possible the stabilization of both proteins into the cells. I then demonstrated the existence of monomeric and dimeric ubiquitinated forms of CHMP1B in which the binding of a molecule of ubiquitin to one of the two lysines of a flexible loop of the protein likely induces and/or stabilize a conformational conformation. In addition, UBPY hydrolyses this ubiquitin and promotes the accumulation of CHMP1B oligomers which are devoid of ubiquitin. Finally, the treatment of cells by EGF, which binds to EGFR and causes its internalization, induces transient recruitment of ubiquitinated CHMP1B dimers to the membranes. Analysis of the intracellular trafficking of EGFR and the morphogenesis of Drosophila wing in different genetic contexts has also shown that the ubiquitination of CHMP1B is essential to its function. My work has allowed me to formulate a completely new hypothesis in which the ubiquitination of CHMP1B induces an open conformation of the protein incapable of polymerizing in this state which is recruited in the form of dimers to the membrane of the endosomes and there the presence of UBPY induces the deubiquitination and the concomitant polymerization of CHMP1B, most probably in hetero-complexes with other members of the ESCRT-III family acting in concert for deformation and scission of the membranes.
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Submitted on : Monday, January 15, 2018 - 4:59:34 PM
Last modification on : Tuesday, March 29, 2022 - 3:10:29 AM
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  • HAL Id : tel-01684692, version 1



Xènia Crespo-Yañez. Unveiling Ubiquitination as a New Regulatory Mechanism of the ESCRT-III Protein CHMP1B. Cellular Biology. Université Grenoble Alpes, 2017. English. ⟨NNT : 2017GREAV035⟩. ⟨tel-01684692⟩



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