12 research outputs found
Structures quaternaires et fonctions de la Hsp90, protéine de choc thermique de 90 kDa
La Hsp90 est une protĂ©ine chaperon qui intervient en conditions physiologiques Ă des Ă©tapes tardives du repliement protĂ©ique, selon un cycle de chaperonnage du dimĂšre rĂ©gulĂ© par l'ATP et des protĂ©ines co-chaperons. La Hsp90, qui possĂšde de nombreuses protĂ©ines clientes impliquĂ©es dans les processus d'oncogenĂšse, est devenue une cible pour le dĂ©veloppement de traitement anti-cancĂ©reux. La Hsp90 s'oligomĂ©rise in vitro sous l'influence de la tempĂ©rature (oligomĂšres stables) ou des cations divalents (oligomĂšres en Ă©quilibre dynamique). Ces oligomĂšres semblent ĂȘtre actifs en conditions de stress. Nous avons dĂ©montrĂ© que l'activitĂ© protectrice de la Hsp90 vis-Ă -vis d'une protĂ©ine cible labile, la tubuline, est ATP-indĂ©pendante. Ces travaux ont permis la mise au point d'un test d'activitĂ© chaperon permettant le criblage de nouveaux inhibiteurs. Nous avons ensuite caractĂ©risĂ© biochimiquement les stoechiomĂ©tries d'association des oligomĂšres et avons rĂ©solu la structure de l'hexamĂšre de Hsp90 par cryo-microscopie Ă©lectronique. Cette structure quaternaire en forme de nid pourrait accueillir des protĂ©ines cibles. p23, un co-chaperon inhibiteur de l'activitĂ© ATPase de la Hsp90, se lie principalement au niveau du dimĂšre. Aha1, un activateur, possĂšde une plus grande affinitĂ© pour les oligomĂšres de Hsp90. Ceci suggĂšre un rĂŽle de ces structures quaternaires. Enfin, nous avons dĂ©montrĂ© qu'en conditions de stress thermique modĂ©rĂ©, l'oligomĂ©risation stable est prĂ©dominante, et que la prĂ©sence de nuclĂ©otides permet le maintien des oligomĂšres en Ă©quilibre dynamique. Cette rĂ©gulation renforce le caractĂšre fonctionnel des oligomĂšres, qui seraient les espĂšces 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
Optimized Protocol for Protein Macrocomplexes Stabilization Using the EDC, 1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide, Zero-Length Cross-Linker
International audienc
Investigation of CLPH oligomerization properties by exclusion chromatography, electron microscopy and High-Mass MALDI mass spectrometry
Poste
Analysis of molecular dynamics by 3D cryo-EM: the examples of HSP90 and Ribosomal complexes
Conférence présentée par D. ThomasInternational audienc
The Hsp90 Oligomers : âFrom Biophysical and Biochemical Characterization to the structure resolution, a multiple approch study
Poste
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
Integrative study of the aggregation of amyloid peptide, PHF6 a test case.
Poster présenté par C. Garnie
Hsp90 Oligomers Interacting with the Aha1 Cochaperone: An Outlook for the Hsp90 Chaperone Machineries
The 90-kDa heat shock protein (Hsp90)
is a highly flexible dimer
able to self-associate in the presence of divalent cations or under
heat shock. This study investigated the relationship between Hsp90
oligomers and the Hsp90 cochaperone Aha1 (activator of Hsp90 ATPase).
The interactions of Aha1 with Hsp90 dimers and oligomers were evaluated
by ultracentrifugation, size-exclusion chromatography coupled to multiangle
laser light scattering and high-mass matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry. Hsp90 dimer was able to bind up
to four Aha1 molecules, and Hsp90 oligomers are also able to interact
with Aha1. The binding of Aha1 did not interfere with the Hsp90 oligomerization
process. Except for Hsp90 dimer, the stoichiometry of the interaction
remained constant, at 2 Aha1 molecules per Hsp90 dimer, regardless
of the degree of Hsp90 oligomerization. Moreover, Aha1 predominantly
bound to Hsp90 oligomers. Thus, the ability of Hsp90 oligomers to
bind the Aha1 ATPase activator reinforces their role within the Hsp90
chaperone machineries