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Master thesis

Caractérisation biochimique et fonctionnelle des glutarédoxines de classe I chez Populus trichocarpa

Abstract : Glutaredoxins (Grxs) are small ubiquitous proteins implicated in numerous biological processes through their disulfide bonds reduction capacity, especially glutathione adducts, and to bind ironsulfur clusters. In higher plants, this multigenic family's members (30 to 40 genes) are distributed through 4 classes. The work done during this internship aimed at the biochemical characterization of GrxC1, C2, C3 and C4 (class I) from Populus trichocarpa to complete the data already obtained on the poplar GrxS12 and the GrxC5 of Arabidopsis thaliana, to attempt to elucidate the catalytic mechanism at stake as well as to understand the role of the 3rd cysteine present in the C-terminal region in PtGrxC1 and PtGrxC2. These proteins and their respective cysteinic mutants have been produced in recombinant form in the heterolog system Escherichia coli and then purified. The measurement of the free thiol groups and the mass spectrometry experiences habe revealed that these 4 proteins are produced oxydized and that, as for PtGrxS12 and AtGrxC5, the catalytic cysteine of PtGrxC3 and C4 is stably glutathionylated. The redox state analysis of the wild type isoforms suggest the implication of the C-ter cysteine of PtGrxC1 and C2 in a covalent dimer's formation, of which the properties should be explored further. The activities of the wild type isoforms have been assessed in vitro for 2 classical substrates for the Grxs, dehydroascorbate and hydroxyéthyldisulfide. The reduction capacity of insulin disulfide bonds has also been assessed. The results suggest that these 4 Grxs use a mechanism previously described as monothiol, as GrxS12 and GrxC5, which implicate only one cysteine over the 2 or 3 present. Finaly, the Western blot analysis of the Grxs abundance in several organs shows that PtGrxC4 is expressed in most of the analysed tissues, particularly in stems and fruits.
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Master thesis
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Submitted on : Tuesday, May 22, 2018 - 9:09:58 AM
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Bruno Dastillung. Caractérisation biochimique et fonctionnelle des glutarédoxines de classe I chez Populus trichocarpa. Sciences de l'environnement. 2011. ⟨hal-01796799⟩



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