8 research outputs found
The Hsp70-Hsp90 co-chaperone Hop/Stip1 shifts the proteostatic balance from folding towards degradation.
Hop/Stip1/Sti1 is thought to be essential as a co-chaperone to facilitate substrate transfer between the Hsp70 and Hsp90 molecular chaperones. Despite this proposed key function for protein folding and maturation, it is not essential in a number of eukaryotes and bacteria lack an ortholog. We set out to identify and to characterize its eukaryote-specific function. Human cell lines and the budding yeast with deletions of the Hop/Sti1 gene display reduced proteasome activity due to inefficient capping of the core particle with regulatory particles. Unexpectedly, knock-out cells are more proficient at preventing protein aggregation and at promoting protein refolding. Without the restraint by Hop, a more efficient folding activity of the prokaryote-like Hsp70-Hsp90 complex, which can also be demonstrated in vitro, compensates for the proteasomal defect and ensures the proteostatic equilibrium. Thus, cells may act on the level and/or activity of Hop to shift the proteostatic balance between folding and degradation
Heat Shock Protein 9O Interaction Networks
Hsp90 est une chaperone majeure dans la cellule humaine où elle maintient l'homéostasie protéique, notamment en cas de stress cellulaire tel que l'hyperthermie. Même en conditions normales, des centaines de protéines, comprenant entre autres des kinases et des facteurs de transcription, dépendent d'Hsp90 pour être fonctionnelles. L'extraordinaire diversité de cette clientèle relie Hsp90 à la quasi-totalité des processus cellulaires ainsi qu'à des maladies. L'activité d'Hsp90 et ses multiples tâches sont régulées par une batterie de co-chaperones et par des modifications post-traductionnelles qui agissent sur sa dynamique structurelle. Le présent travail regroupe plusieurs projets qui ont pour but d'avancer les connaissances sur le mode de fonctionnement d'Hsp90, avec un accent mis sur les réseaux d'interaction d'Hsp90.
Tout d'abord, nous avons publié une revue des travaux sur les réseaux d'interaction d'Hsp90 dans le contexte de maladies en collaboration avec le groupe de Gabriela Chiosis (MSKCC, New York).
Nous avons ensuite mené un projet dont l'objectif était de décrire ces réseaux dans des cellules cancéreuses. L'existence d'un réseau spécifique dans les cancers, distinct d'un réseau normal dédié aux tâches de ménages, a en effet été postulée pour expliquer la plus grande sensibilité des cellules cancéreuses aux inhibiteurs d'Hsp90. Malheureusement, nous avons découvert certains défauts techniques de l'outil expérimental principal utilisé dans ce projet qui jettent de sérieuses résèrves sur les résultats obtenus avec cet outil, et nous avons été contraint d'abandonner le projet.
Le projet principal de ce travail était de comprendre la fonction de deux sites de phosphorylation d'Hsp90. Après avoir confirmé les études précédentes rapportant que ces sites sont abondamment phosphorylés dans une variété de cellules cancéreuses et normales et de conditions, nous avons découvert que la phosphorylation de ces sites modifie le réseau d'interaction d'Hsp90 et sa sécrétion. Une analyse approfondie nous a permis de démontrer que la structure globale d'Hsp90 est modifiée par ces phosphorylations, et que ce changement structurel pourrait être à l'origine des autres effets observés. Nous proposons ainsi une nouvelle hypothèse pour la fonction de ces sites, pouvant expliquer des effets apparemment sans liens à l'aide d'un seul mécanisme.
Les deux derniers projets ont été conduits en collaboration avec le groupe de Didier Picard de l'Université de Genève. L'un d'eux visait à élucider le rôle de STIPl, une co-chaperone centrale d'Hsp90. Notre contribution a été d'analyser les changements protéiques survenant quand STIPl est éliminé de cellules humaines, et son impact sur le réseau d'interaction d'Hsp90. Nous démontrons une élévation du niveau de plusieurs chaperones, et que l'interaction d'Hsp90 avec ces chaperones est réduite mais pas totalement abrogée. En particulier, le complexe binaire de type procaryotique Hsp70:Hsp90 est moins abondant mais toujours associé en l'absence de STIPl. La présence de STIPl dans le complexe ternaire de type eucaryotique Hsp70:STIP1:Hsp90 en revanche redirige le complexe vers une plus grande utilisation du système de dégradation des protéines. L'activité de STIPl pourrait ainsi moduler l'usage du système de dégradation des protéines par Hsp70 et Hsp90. La deuxième collaboration avec le groupe Picard a été de caractériser des cellules où soit Hsp90a soit Hsp90 ont été éliminés. A priori considéré létal, c'est la première lignée cellulaire dont Hsp90 est absente. Nous montrons que l'élimination de n'importe quel Hsp90 conduit à une augmentation du niveau de plusieurs chaperones, ainsi qu'à une réduction du niveau d'enzymes impliquées dans le métabolisme du cholestérol.
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Hsp90 is a major chaperone in the human cell where it plays a key role in controlling protein homeostasis, notably in response to stresses such as heat shock. Even under normal conditions, hundreds of proteins depend on Hsp90 for proper folding and/or activity, including protein kinases, transcription factors and metabolic enzymes. The incredible diversity and vastness of Hsp90's clientele implicates Hsp90 in virtually all cellular processes as well as in some types of diseases.
The activity of Hsp90 in its thousands of tasks is regulated by an array of auxiliary co-chaperones and by post-translational modifications acting on Hsp90's structure and conformational dynamics. The present work gathers several projects aimed at understanding how Hsp90 fonctions in different contexts, with an emphasis on Hsp90's protein interaction networks.
We started by writing a review on the specific tapie of Hsp90 protein interaction networks in the context of diseases, in collaboration with the group of Gabriela Chiosis (MSKCC, New York). After this, our first research project aimed at characterizing Hsp90's interactions in the context of cancer. Indeed, the existence of a cancer specific Hsp90 "interactome", distinct from the normal Hsp90 network dedicated to housekeeping functions, was proposed to explain the observation that cancer cells are more sensitive to Hsp90 inhibition than normal cells. Unfortunately, we discovered technical flaws in the main affinity reagent that was to be used and we were not able to draw any solid conclusion from our experiments, forcing us to abandon the project.
The next main research project aimed at describing two phosphorylation sites in Hsp90, which are very abundant in vivo, but with no confirmed function. After corroborating early findings that phosphorylation at these sites is invariably high in cancer and normal cell lines under bath normal and stress conditions, we found out that phosphorylation modulates Hsp90's interactions and secretion. Further investigation revealed that these phospho-sites impact the global Hsp90 structural dynamics, which may be the cause for the aforementioned effects. We then developed a new hypothesis on the structural and functional effects of these phospho-sites in light of the existing knowledge. This hypothesis could help reconcile some apparently unrelated phenomena we observed and explain divergent results on this topic in the literature.
The third and fourth research projects were conducted in collaboration with the group of Didier Picard (University of Geneva). The first collaboration aimed at understanding the fundamental role of STIPl, a central Hsp90 co-chaperone. Our contribution was to analyze the proteomic changes occurring after STIPl knockout (KO) in human cell lines, as well as the Hsp90 network in absence of STIPl. The results show that STIPl KO upregulates proteins involved in the protein folding process as well as in the unfolded protein response, providing increased resistance to proteotoxic stresses. Accordingly, the prokaryotic-like Hsp70:Hsp90 complex remains associated and functional in the absence of STIPl. The results of this collaboration show that STIPl is able to drive the Hsp70:Hsp90 complex, directed at protein folding, towards the ternary Hsp70:STIP1:Hsp90 complex which promotes usage of the proteasome and protein degradation to maintain proteostasis.
The second collaboration aimed to characterize HEK293T cell lines where either Hsp90a or Hsp90 are knocked out. Initially thought to be essential, this is the first human cell line missing the cytosolic Hsp90 isoform. Proteomic analysis shows that Hsp90 KO of either isoform results in the upregulation of chaperones and the downregulation of the metabolism of steroids
Phosphorylation in the Charged Linker Modulates Interactions and Secretion of Hsp90β
Hsp90 beta is a major chaperone involved in numerous cellular processes. Hundreds of client proteins depend on Hsp90 beta for proper folding and/or activity. Regulation of Hsp90 beta is critical to coordinate its tasks and is mediated by several post-translational modifications. Here, we focus on two phosphorylation sites located in the charged linker region of human Hsp90 beta, Ser226 and Ser255, which have been frequently reported but whose function remains unclear. Targeted measurements by mass spectrometry indicated that intracellular Hsp90 beta is highly phosphorylated on both sites (>90%). The level of phosphorylation was unaffected by various stresses (e.g., heat shock, inhibition with drugs) that impact Hsp90 beta activity. Mutating the two serines to alanines increased the amount of proteins interacting with Hsp90 beta globally and increased the sensitivity to tryptic cleavage in the C-terminal domain. Further investigation revealed that phosphorylation on Ser255 and to a lesser extent on Ser226 is decreased in the conditioned medium of cultured K562 cells, and that a non-phosphorylatable double alanine mutant was secreted more efficiently than the wild type. Overall, our results show that phosphorylation events in the charged linker regulate both the interactions of Hsp90 beta and its secretion, through changes in the conformation of the chaperone
Phosphorylation in the Charged Linker Modulates Interactions and Secretion of Hsp90β
Hsp90β is a major chaperone involved in numerous cellular processes. Hundreds of client proteins depend on Hsp90β for proper folding and/or activity. Regulation of Hsp90β is critical to coordinate its tasks and is mediated by several post-translational modifications. Here, we focus on two phosphorylation sites located in the charged linker region of human Hsp90β, Ser226 and Ser255, which have been frequently reported but whose function remains unclear. Targeted measurements by mass spectrometry indicated that intracellular Hsp90β is highly phosphorylated on both sites (>90%). The level of phosphorylation was unaffected by various stresses (e.g., heat shock, inhibition with drugs) that impact Hsp90β activity. Mutating the two serines to alanines increased the amount of proteins interacting with Hsp90β globally and increased the sensitivity to tryptic cleavage in the C-terminal domain. Further investigation revealed that phosphorylation on Ser255 and to a lesser extent on Ser226 is decreased in the conditioned medium of cultured K562 cells, and that a non-phosphorylatable double alanine mutant was secreted more efficiently than the wild type. Overall, our results show that phosphorylation events in the charged linker regulate both the interactions of Hsp90β and its secretion, through changes in the conformation of the chaperone
Molecular screening of cancer-derived exosomes by surface plasmon resonance spectroscopy
We report on a generic method to detect and iden- tify the molecular profile of exosomes either derived from cultured cell lines or isolated from biofluids. Exosomes are nanovesicles shed by cells into their microenvironment and carry the molecular identity of their mother cells. These vesi- cles are actively involved in intercellular communication un- der physiological conditions and ultimately in the spread of various diseases such as cancer. As they are accessible in most biofluids (e.g., blood, urine, or saliva), these biological entities are promising tools for cancer diagnostics, offering a non- invasive and remote access to the molecular state of the dis- ease. The composition of exosomes derived from cancer cells depends on the sort and state of the tumor, requiring a screen- ing of multiple antigens to fully characterize the disease. Here, we exploited the capacity of surface plasmon resonance bio- sensing to detect simultaneously multiple exosomal and can- cer biomarkers on exosomes derived from breast cancer cells. We developed an immunosensor surface which provides efficient and specific capture of exosomes, together with their identification through their distinct molecular profiles. The successful analysis of blood samples demonstrated the suit- ability of our bioanalytical procedure for clinical us
Molecular screening of cancer-derived exosomes by surface plasmon resonance spectroscopy
We reporton a generic method to detect and identify the molecular profile of exosomes either derived from cultured cell lines or isolated from biofluids. Exosomes are nanovesicles shed by cells into their microenvironment and carry the molecular identity of their mother cells. These vesicles are actively involved in intercellular communication under physiological conditions and ultimately in the spread of various diseases such as cancer. As they are accessible in most biofluids (e.g., blood, urine, or saliva), these biological entities are promising tools for cancer diagnostics, offering a non-invasive and remote access to the molecular state of the disease. The composition of exosomes derived from cancer cells depends on the sort and state of the tumor, requiring a screening of multiple antigens to fully characterize the disease. Here, we exploited the capacity of surface plasmon resonance biosensing to detect simultaneously multiple exosomal and cancer biomarkers on exosomes derived from breast cancer cells. We developed an immunosensor surface which provides efficient and specific capture of exosomes, together with their identification through their distinct molecular profiles. The successful analysis of blood samples demonstrated the suitability of our bioanalytical procedure for clinical use
Translational reprogramming in response to accumulating stressors ensures critical threshold levels of Hsp90 for mammalian life.
The cytosolic molecular chaperone Hsp90 is essential for eukaryotic life. Although reduced Hsp90 levels correlate with aging, it was unknown whether eukaryotic cells and organisms can tune the basal Hsp90 levels to alleviate physiologically accumulated stress. We have investigated whether and how mice adapt to the deletion of three out of four alleles of the two genes encoding cytosolic Hsp90, with one Hsp90β allele being the only remaining one. While the vast majority of such mouse embryos die during gestation, survivors apparently manage to increase their Hsp90β protein to at least wild-type levels. Our studies reveal an internal ribosome entry site in the 5' untranslated region of the Hsp90β mRNA allowing translational reprogramming to compensate for the genetic loss of Hsp90 alleles and in response to stress. We find that the minimum amount of total Hsp90 required to support viability of mammalian cells and organisms is 50-70% of what is normally there. Those that fail to maintain a threshold level are subject to accelerated senescence, proteostatic collapse, and ultimately death. Therefore, considering that Hsp90 levels can be reduced ≥100-fold in the unicellular budding yeast, critical threshold levels of Hsp90 have markedly increased during eukaryotic evolution
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The Hsp70-Hsp90 co-chaperone Hop/Stip1 shifts the proteostatic balance from folding towards degradation.
Hop/Stip1/Sti1 is thought to be essential as a co-chaperone to facilitate substrate transfer between the Hsp70 and Hsp90 molecular chaperones. Despite this proposed key function for protein folding and maturation, it is not essential in a number of eukaryotes and bacteria lack an ortholog. We set out to identify and to characterize its eukaryote-specific function. Human cell lines and the budding yeast with deletions of the Hop/Sti1 gene display reduced proteasome activity due to inefficient capping of the core particle with regulatory particles. Unexpectedly, knock-out cells are more proficient at preventing protein aggregation and at promoting protein refolding. Without the restraint by Hop, a more efficient folding activity of the prokaryote-like Hsp70-Hsp90 complex, which can also be demonstrated in vitro, compensates for the proteasomal defect and ensures the proteostatic equilibrium. Thus, cells may act on the level and/or activity of Hop to shift the proteostatic balance between folding and degradation