Structures quaternaires et fonctions de la Hsp90, protéine de choc thermique de 90 kDa

La Hsp90 est une protéine chaperon qui intervient en conditions physiologiques à des étapes tardives du repliement protéique, selon un cycle de chaperonnage du dimère régulé par l'ATP et des protéines co-chaperons. La Hsp90, qui possède de nombreuses protéines clientes impliquées dans les processus d'oncogenèse, est devenue une cible pour le développement de traitement anti-cancéreux. La Hsp90 s'oligomérise in vitro sous l'influence de la température (oligomères stables) ou des cations divalents (oligomères en équilibre dynamique). Ces oligomères semblent être actifs en conditions de stress. Nous avons démontré que l'activité protectrice de la Hsp90 vis-à-vis d'une protéine cible labile, la tubuline, est ATP-indépendante. Ces travaux ont permis la mise au point d'un test d'activité chaperon permettant le criblage de nouveaux inhibiteurs. Nous avons ensuite caractérisé biochimiquement les stoechiométries d'association des oligomères et avons résolu la structure de l'hexamère de Hsp90 par cryo-microscopie électronique. Cette structure quaternaire en forme de nid pourrait accueillir des protéines cibles. p23, un co-chaperon inhibiteur de l'activité ATPase de la Hsp90, se lie principalement au niveau du dimère. Aha1, un activateur, possède une plus grande affinité pour les oligomères de Hsp90. Ceci suggère un rôle de ces structures quaternaires. Enfin, nous avons démontré qu'en conditions de stress thermique modéré, l'oligomérisation stable est prédominante, et que la présence de nucléotides permet le maintien des oligomères en équilibre dynamique. Cette régulation renforce le caractère fonctionnel des oligomères, qui seraient les espèces actives en tant que chaperon.Hsp90 is a chaperone protein involved in late-stage protein folding under physiological conditions, according to a chaperone cycle of the dimer regulated by ATP and co-chaperone proteins. Hsp90 has many client proteins that are mainly involved in oncogenesis, thus making Hsp90 a target for the development of new anti-cancer drugs. Hsp90 self-oligomerizes in vitro under the influence of temperature (irreversible oligomers) or divalent cations (oligomers in a dynamic equilibrium). These oligomers are supposed to be active as chaperones under stress conditions. We demonstrated that the protective effect of Hsp90 towards a labile protein, tubulin, is ATP-independent. This finding allowed us to design a chaperone activity test for new Hsp90 inhibitors screening. We then characterized biochemically the stoichiometries of the oligomers association, and resolved the Hsp90 hexamer's structure by cryo-electron microscopy. This quaternary structure exhibits a nest-like shape that could bind a substrate protein. p23, a co-chaperone known as an Hsp90 ATPase inhibitor, binds mainly the Hsp90 dimer, whereas Aha1 an activator, has a greater affinity for the oligomeric species. This suggests a function of these quaternary structures. Finally, we demonstrated that under mild heat shock conditions, irreversible oligomers are predominating, and that nucleotide binding reverses the process, maintaining the oligomers in a dynamic equilibrium. This regulation strengthens the functionality of the Hsp90 oligomers, which would be the quaternary structures endowed with chaperone activity.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

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Hsp90 Oligomerization Process: How Can p23 Drive the Chaperone Machineries?

International audienceThe 90-kDa heat shock protein (Hsp90) is a highly flexible dimer that is able to self-associate in the presence of divalent cations or under heat shock. In a previous work, we focused on the Mg2+-induced oligomerization process of Hsp90, and characterized the oligomers. Combining analytical ultracentrifugation, size-exclusion chromatography coupled to multi-angle laser light scattering and high-mass matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we studied the interaction of p23 with both Hsp90 dimer and oligomers. Even if p23 predominantly binds the Hsp90 dimer, we demonstrated, for the first time, that p23 is also able to interact with Hsp90 oligomers, shifting the Hsp90 dimer-oligomers equilibrium toward dimer. Our results showed that the Hsp90:p23 binding stoichiometry decreases with the Hsp90 oligomerization degree. Therefore, we propose a model in which p23 would act as a "protein wedge" regarding the Hsp90 dimer closure and the Hsp90 oligomerization process

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Hsp90 Oligomers Interacting with the Aha1 Cochaperone: An Outlook for the Hsp90 Chaperone Machineries

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