PhD defence of Anouchka Gatin – Tuesday, July 19 at 9:30

10 July 2022 par clavaguera
Anouchka Gatin from the CPSysBio group will defend her thesis on Tuesday, July 19 at 9:30.

Anouchka did her thesis under the supervision of Cécile SICARD-ROSELLI:

"Oxidative dimerization of proteins via aromatic amino acids cross-links"

The defense will take place in salle Magat.

Summary in English:

Covalent protein cross-links involving aromatic amino acids has been associated with a numerous of biological processes, as well physiological as pathological. Bi-tyrosine, used as a biomarker of protein oxidation is a typical example: it has been evidenced in a large number if isolated proteins, tissues and biological fluids.
In the context of this study, covalent dimerization via aromatic amino acids will be studied through oxidation of i) isolated proteins, peptides and free amino acids in solution, ii) a protein complex made of human centrin 2 (CEN2) and Xeroderma Pigmentosum group C protein (XPC), then of iii) breast cancer human cells.
Oxidative radicals’ formation, as hydroxyl (•OH) and azide (N3•) radicals will be mediated by irradiation of protein samples in aqueous solution with gamma rays. Radio-induced covalent cross-links will be mainly examined by ultra performance liquid chromatography coupled to mass spectrometry (UPLC-MS). A new analytic methodology based on isotopic labelling and H/D exchanges has been developed in order to precisely determine the chemical nature of the cross-links.
First, tyrosine dimerization study at the amino acid scale allowed to highlight the formation of at least four different bi-tyrosine isomers. The applied analytical approach allowed to identify a new dimers family. These latter result from intramolecular Michael cycloaddition reactions and involve the para position of the tyrosyl radical from which dimerization originated.
In the second part, dimers structural plurality has been confirmed at the protein scale. Co-existence of plentiful Michael additions’ intermediates and final products has been underlined in particular via ion mobility spectrometry (IMS) experiments. While the considered protein possesses also tryptophan residues, tyrosine-tyrosine, tyrosine-tryptophan and tryptophane-tryptophane cross-links were evidenced.
Third, oxidative dimerization behaviour of protein complexes composed of CEN2 and two XPC fragments of different size has been studied. It allowed to evidence for the first time the formation of covalent interprotein cross-links between both partners. In function of the amino acid sequence of the considered XPC fragment (presence or not of tyrosine), the cross-link nature differs: tyrosine-tyrosine or tyrosine-tryptophan. However, whatever the nature of the covalent bridges, both partners covalently bind throughout an oxidative attack. In the context of plural tyrosine-tryptophan cross-links formation, a detailed analysis of the chemical bonds nature has been completed at the amino acid scale. It brought to propose new types of tyrosine-tryptophan bridges.
Finally, in-cellulo approach of CEN2 covalent cross-links detection when exposed to oxidative radicals has been engaged. The conditions of endogenous CEN2 detection and of its potential dimers have been developed by Western-Blot. Cellular extracts enrichment steps by immunoprecipitation and ammonium sulfate precipitation were realized. These preliminary experiments pave the way of the future investigation of in-cellulo covalents cross-links.

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