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Titre : | Characterization of the influence of protein disulfide isomerases on redox sensors roGFP2 in Saccharomyces cerevisiae |
Auteurs : | Romane SCHILS, Auteur ; B. MORGAN, Promoteur |
Type de document : | Travail de fin d'études |
Editeur : | Bruxelles : Institut Paul Lambin, 2020 |
Langues: | Français |
Index. décimale : | TFE - Biologie médicale |
Résumé : |
Traditional sensors used in order to measure the glutathione redox-couple are often composed on roGFP2. roGFP2 form a chromophore when it gets oxidized which will emit fluorescence. With these sensors, the glutathione pool has been studied in different compartment of yeast cell. However, the endoplasmic reticulum has a much more oxidized environment than the cytosol with oxidative protein folding and enzymes contributing to this oxidizing environment. Several oxidizing proteins present in the ER such as protein disulfide isomerase (Pdi) could be able to oxidize roGFP2. Therefore, the aim of this project was to determinate if Pdis are able to oxidize directly roGPF2. This would mean that traditional sensors do not only measure the glutathione pool and that previous data are incorrect.
For this project, different systems have been set up in Saccharomyces cerevisiae cytosol which environment is well-known. With a process of yeast transformation, a plasmid with the gene coding for roGFP2 is inserted in the cytosol. Additional to this, another plasmid either with PDI or with no gene coding for Pdi is targeted to the cytosol. The different measurement has been realised on a 96-wells plate reader. The cells were treated with H2O2 in order to produce GSSG which will supposedly oxidized Pdi. During these experiments, the degree of roGFP2 is measured by fluorescence using CLARIOSTAR. The comparison of the results between yeast strains with Pdi and the strains without it will give the information on the effect of Pdi on roGFP2 oxidation state. Indeed, if the degree of roGFP2 oxidation is higher with the presence of Pdi, it would mean that roGFP2 get oxidized by Pdi. The different results obtained demonstrate no evident difference between the yeast strains with and without Pdi. Three different yeast strains (WT, Δglr1, Δgrx1Δgrx2Δglr1) in association with three different Pdis (Pdi1, Mpd1, Mpd2) have been tested. All of these results are similar to each other: no difference in roGFP2 oxidation state. To summarize, based on all the results obtained, there is no clear evidence of a link between roGFP2 and PDIs. However, there are not enough results to determinate with certitude that Pdi are incapable to oxidize roGFP2. Additional experiments have to be realised to answer this question. |
Accès : | Identifiez-vous avant d'accéder au document électronique |
Disponible en ligne : | Oui |
Lieu du stage : | laboratory of the Biochemistry Department |
Département : | Biologie médicale |
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TFE biologie médicale Adobe Acrobat PDF |