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Titre : | Investigation of interspecies oligomerisation of thiol peroxidases using fluorescence-based probes |
Auteurs : | Marie Diedhiou, Auteur ; Bruce Morgan, Promoteur |
Type de document : | Travail de fin d'études |
Editeur : | Woluwe-Saint-Lambert : Haute École Léonard de Vinci, 2022 |
Langues: | Anglais |
Index. décimale : | TFE - Biologie médicale |
Résumé : |
A high concentration of H2O2 can be considered as a threat to the body. The role of thiol peroxidases, like the peroxiredoxin (Prx), is to protect us from this threat among its many roles. Prx carry out a redox reaction which involves the reduction of an H2O2 and the oxidation of its thiol group: the peroxidatic cysteine (CP) and the resolving cysteine (CR). At the end of the cycle, the Prx can be regenerated if the Trx reduces the Prx. These antioxidants are very ubiquitous, and they have the particularity of forming oligomers.
In many reviews Prx have been investigated exclusively as entities capable of self-assembly in homooligomers. Yet there is in most organisms several isoforms of Prx that can form hetero-oligomers. Some research even shows that some Prx have a greater tendency to hetero-oligomerise (1). The question is why does Prx achieve hetero-oligomerisation? To answer this, Bruce Morgan and his team in the Biochemistry Laboratory of the University of Saarland decomposed this question into several axes. During my internship I focused on the following aim: identification of Prx inter-species hetero-oligomers. Which Prx subfamilies and Prx isoforms form which type of hetero-oligomers? In long term, performing heterooligomerisation between different species would enable to find pairs of peroxiredoxins that don't interact with each other. This would allow to learn about the impact of this interaction in the cell if it cant happen. To demonstrate the formation of hetero-oligomers between peroxiredoxin pairs from other organisms, I used yeast deleted for the genes encoding tsa1 and tsa2 as a test cell. If an oligomer is formed, its only because a hetero-oligomerisation happen and not because endogenous cytosolic Prx are present in the cell and react with the probe. To detect the formation of these oligomers, a roGFP2 probe must be used. roGFP2 is excited at 405nm and emits green light when Prx passes on its oxidation. We genetically fused a roGFP2 to a Prx misses its CP and CR. If at least the CP is removed, when added to the cell, H2O2 cannot be reduced. No oxidation of roGFP2 is observed. Oxidation of roGFP2 will only be observed if the roGFP2- Prx forms a hetero-oligomer with the second Prx of the pair which is co-expressed with these two cysteines. These cells are exposed to different concentrations of H2O2 ranging from 10 μM to 500 μM. Although these concentrations do not occur under physiological conditions, it will provide an observable response. The CLARIOstar is used to measure the oxidation of roGFP2 and it is adjuste to 405 nm and 490 nm excitation wavelength. Finally, these results provided insight into the oligomerisation of Prx from different species. Heterooligomerisation is possible for some species for which I did not expect this result such as AtPrx. Both isoforms, AtPrxA and AtPrxB can form hetero-oligomers with ScTsa1 and ScTsa2. Surprisingly we have a higher response when ScTsa is linked to roGFP2 than when AtPrx is linked to roGFP2. Several hypotheses could explain this phenomenon. The first hypothesis is that AtPrx is simply more sensitive to oxidation by minimal concentrations of H2O2 than ScTsa. The second hypothesis is that competition between Trx and roGFP2 prevents ScTsa1 from binding to roGFP2 and transferring its oxidation. It doesn't work for some other species like the two isoforms of AtPrx with HsPrx1/HsPrx2. But this fact could be an advantage if AtPrx is used as a H2O2 sensor in human cell. roGFP2-AtPrx wont interact with the endogenous HsPrx contained in the cell. Studying the amino acid sequence of the Prx binding site and their differences would give an idea of which protein areas are important to allow hetero-oligomerisation. In the short term, this would make it possible to intelligently target the non-formation of inter-species hetero-oligomers. There will be less blindness in choosing crosses between Prx. In the long term, this pair would allow to learn more about the impact of oligomerisation. In reality perhaps, there is no inter-species hetero-oligomerisation at all. Maybe, dimers are made between each species as usual. But then these dimers assemble to form decamers. These dimers may be passing on their oxidation to a nearby roGFP2 dimer. roGFP2 is oxidised and emits light. |
Accès : | Identifiez-vous avant d'accéder au document électronique |
Disponible en ligne : | Oui |
Lieu du stage : | Universität des Saarlandes à Saarbrücken |
Département : | Biologie médicale |
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