Comprehensive characterization of the Hsp70 interactome reveals novel client proteins and interactions mediated by post-translational modifications
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Saccharomyces Cerevisiae (ncbitaxon:4932)
SUBMITTER: 
Andrew W Truman
PROVIDER: MSV000090347 | MassIVE |
SECONDARY ACCESSION(S): PXD036849
REPOSITORIES: MassIVE
Similar Datasets
Project description:Yeast DNA microarray was used to assess and compare the global expression profile of strains harboring different family members of the major cytosolic Hsp70 family. Viability of a yeast strain deleted for all genes encoding members of the Hsp70-Ssa family (Ssa1/2/3/4) was maintained by the presence of a single Ssa family member expressed ectopically from a plasmid vector. The Hsp70-Ssa family constitutes the main source of Hsp70 molecular chaperone activity in the yeast cell. A yeast cell must actively express a member of this family to remain viable. Hsp70-Ssa are highly conserved both within yeast and amongst other species. Ssa1 and 2 are 97% identical at amino acid level and 80% identical to Ssa3 and 4. The aim of this study was to attribute specific functions to single Ssa family members by identifying specific genes or gene families whose expression was altered in the presence (or absence) of Ssa family members.
2012-12-31 | GSE32433 | GEO
Project description:Yeast DNA microarray was used to assess and compare the global expression profile of strains harboring different family members of the major cytosolic Hsp70 family. Viability of a yeast strain deleted for all genes encoding members of the Hsp70-Ssa family (Ssa1/2/3/4) was maintained by the presence of a single Ssa family member expressed ectopically from a plasmid vector. The Hsp70-Ssa family constitutes the main source of Hsp70 molecular chaperone activity in the yeast cell. A yeast cell must actively express a member of this family to remain viable. Hsp70-Ssa are highly conserved both within yeast and amongst other species. Ssa1 and 2 are 97% identical at amino acid level and 80% identical to Ssa3 and 4. The aim of this study was to attribute specific functions to single Ssa family members by identifying specific genes or gene families whose expression was altered in the presence (or absence) of Ssa family members. Eight independent RNA samples were pooled to represent a single biological sample for expression analysis. For example, the single sample analyzed for cells harboring only Ssa1 is a pooled sample of eight independent RNA extractions. Hybridization was performed for cells harboring either Ssa1, Ssa2, Ssa3 or Ssa4 as the sole cytosolic Hsp70-Ssa family member. Gene expression profiles of Ssa2/3/4 were all compared to Ssa1.
2012-12-31 | E-GEOD-32433 | biostudies-arrayexpress
Project description:Hsp70 are ubiquitous, versatile molecular chaperones that cyclically interact with substrate protein(s). The initial step requires synergistic interaction of a substrate and a J-domain protein (JDP) cochaperone, via its J-domain, with Hsp70 to stimulate hydrolysis of its bound ATP. This hydrolysis drives conformational changes in Hsp70 that stabilize substrate binding. However, because of the transient nature of substrate and JDP interactions, this key step is not well understood. Here we leverage a well characterized Hsp70 system specialized for iron-sulfur cluster biogenesis, which like many systems, has a JDP that binds substrate on its own. Utilizing an ATPase-deficient Hsp70 variant, we isolated a Hsp70- JDP-substrate tripartite complex. Complex formation and stability depended on residues previously identified as essential for bipartite interactions: JDP-substrate, Hsp70-substrate and J-domain-Hsp70. Computational docking based on the established J-domain-Hsp70(ATP) interaction placed the substrate close to its predicted position in the peptide-binding cleft, with the JDP having the same architecture as when in a bipartite complex with substrate. Together, our results indicate that the structurally rigid JDP- substrate complex recruits Hsp70(ATP) via precise positioning of J-domain and substrate at their respective interaction sites - resulting in functionally high affinity (i.e., avidity). The exceptionally high avidity observed for this specialized system may be unusual because of the rigid architecture of its JDP and the additional JDP-Hsp70 interaction site uncovered in this study. However, functionally important avidity driven by JDP-substrate interactions is likely sufficient to explain synergistic ATPase stimulation and efficient substrate trapping in many Hsp70 systems.
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Project description:Protein homeostasis and cellular fitness in the presence of proteotoxic stress is promoted by heat shock factor 1 (HSF1), which controls basal and stress-induced expression of molecular chaperones and other targets. The major heat shock proteins Hsp70 and Hsp90 in turn participate in a negative feedback loop that ensures appropriate coordination of the heat shock response with environmental conditions. Features of this regulatory circuit in the budding yeast Saccharomyces cerevisiae have been recently defined, most notably regarding direct interaction between Hsf1 and the constitutively expressed Hsp70 Ssa1. We sought to further explore the complex nature of Ssa1/Hsf1 regulation. Ssa1 is found to interact independently with both the previously defined CE2 site in the Hsf1 carboxyl-terminal transcriptional activation domain as well as a novel site that we identify within the amino-terminal activation domain. Consistent with both sites bearing a recognition signature for Hsp70, we demonstrate that Ssa1 contacts Hsf1 using its substrate binding domain and abolishing either regulatory site results in loss of Ssa1 association. Removing Hsp70 regulation of Hsf1 results in global dysregulation of Hsf1 transcriptional activity, with synergistic effects when both sites are disrupted together on both gene expression and cellular fitness. Finally, we find that Hsp70 interacts with both transcriptional activation domains of Hsf1 in the related yeast Lachancea kluyveri, implying a conserved mechanism of regulation to promote cellular proteostasis.
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Project description:Molecular chaperones including the heat-shock protein 70-kilodalton (HSP70) family and the J-domain containing protein (JDP) co-chaperones maintain homeostatic balance in eukaryotic cells through regulation of the proteome. The expansive JDP family helps direct specific HSP70 functions, and yet loss of single JDP-encoding genes is widely tolerated by mammalian cells, suggesting a high degree of redundancy. Using a genetic screen of DJPs in human cancer cell lines, we found the RNA recognition motif (RRM) containing DNAJC17 to be pan-essential. Knockdown of DNAJC17 leads to relatively few conserved changes in the abundance of individual mRNAs, but instead deranges gene expression through exon skipping, primarily of genes involved in cell cycle progression. Concordant with cell viability experiments, the C-terminal portions of DNAJC17 are dispensable for restoring splicing and G2-M progression. Our findings identify essential cellular JDPs and suggest that diversification in JDP structure extends the HSP70-JDP system to control divergent processes such as RNA splicing.
2023-06-20 | GSE235379 | GEO
Project description:From preliminary experiments, HSP70 deficient MEF cells display moderate thermotolerance to a severe heatshock of 45.5 degrees after a mild preshock at 43 degrees, even in the absence of hsp70 protein. We would like to determine which genes in these cells are being activated to account for this thermotolerance. Keywords: thermal stress, heat shock response, knockout, cell culture, hsp70
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Project description: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. Similarly to budding yeast (ref), human cell lines 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 an alternate proteostatic equilibrium. Thus, Hop may be actionable for cells to shift the proteostatic balance between folding and degradation.
2020-11-09 | PXD012774 | Pride
Project description:The major heat shock protein Hsp70 has been shown to form a complex with a scaffold protein Bag3, linking it to multiple signaling pathways. Via these interactions, the Hsp70-Bag3 module functions as a proteotoxicity sensor that controls cell signaling. Here, as a tool to identify signaling pathways regulated by this complex, we utilized JG-98, an allosteric inhibitor of Hsp70 that blocks its interaction with Bag3. Gene expression profiling followed by the pathway analysis indicated that a set of signaling pathways including the unfolded protein response (UPR) was activated by JG-98. Surprisingly, only the translation initiation factor eIF2a-associated branch of the UPR was activated under these conditions, while other UPR branches mediating induction of ER chaperones were not induced, suggesting that the response was not related to ER proteotoxicity and thus to ER-associated kinase PERK1. Indeed, induction of the UPR genes under these conditions was dependent on activation of a distinct cytoplasmic eIF2a kinase, HRI. We demonstrated that the Hsp70-Bag3 complex directly interacted with HRI and regulated phosphorylation of eIF2a upon induction of cytoplasmic proteotoxicity. Therefore, we uncovered a novel signaling response, which regulates cell death upon the buildup of abnormal protein species in cytoplasm via an Hsp70-Bag3-HRI-eIF2a axis.
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Project description:Proteasome inhibitor (PI) resistance remains a central challenge in multiple myeloma. To identify pathways mediating resistance, we first map proteasome-associated genetic co-dependencies. We identify cytosolic heat shock protein 70 (HSP70) chaperones as a potential target, consistent with proposed mechanisms of myeloma tumor cells overcoming PI-induced stress. These results led us to explore allosteric HSP70 inhibitors as myeloma therapeutics. We show these compounds exhibit increased efficacy against both acquired and intrinsic PI-resistant myeloma models, unlike HSP90 inhibition. Surprisingly, shotgun and pulsed-SILAC proteomics reveal that JG’s overcome PI resistance not via the expected mechanism of inhibiting cytosolic HSP70s, but instead through mitochondrial-localized HSP70, HSPA9, destabilizing the 55S mitoribosome. Analysis of myeloma patient data further supports strong effects of global proteostasis capacity, and particularly HSPA9 expression, on PI response. Our results characterize dynamics of myeloma proteostasis networks under therapeutic pressure while further motivating investigation of HSPA9 as a specific target in PI-resistant disease.
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Project description:Parkinsonâs disease (PD) and Dementia with Lewy bodies (DLB) are the two most common examples of synucleinopathies, i.e. human disorders that display intracellular deposition of the Alpha-synuclein (aSYN) protein. The two forms of aSYN deposits, intracellular Lewy bodies and Lewy neurites, are in the PD brain primarily found in the substantia nigra whereas in DLB brains they are diffusely spread in cortical and limbic areas. We are currently analyzing the protective effects on aSYN pathology by the molecular chaperone hsp70 in mice overexpressing wildtype human aSYN. Moreover, transgenic mice overexpressing hsp70 have been cross-bred with the aSYN mouse line. Intriguingly, the aSYN / hsp70 double transgenics display a pronounced reduction in biochemically assessed aSYN aggregation. Gene expression of these mice will not only provide insight into the disease mechanisms of aSYN pathology but its prevention by the overexpression of hsp70, leading to new potential avenues of treatment. Project 1: To investigate and compare alterations in gene expression profiles of brains from wild-type mice and mice overexpressing wildtype human aSYN, human hsp70 and aSYN / hsp70, respectively. The comparison of the expression profiles of the groups of mice: aSYN, Hsp70, aSYN/ Hsp70 and non-transgenic littermates will help to identify the genes that are involved in the mediation of aggregation toxicity of aSYN expression as well as the genes that may mediate an attenuation of aSYN pathology in hsp70-coexpressors. Project 1: Selection of animals Mice of 4 month old (n=6) of age of each background (aSYN, Hsp70, aSYN/ Hsp70 and non-transgenic littermates) will be used for RNA-extraction for a total of 24 mice. Preparation of samples For the proposed experiments, we will use the recommended procedures, as outlined by the consortium, for extracting and purifying RNA from whole brain homogenate. Briefly, RNA will be extracted by the trizol method and purified by using the RNeasy MinElute Cleanup (QIAGEN Inc., USA). RNA labeling and hybridization procedures will be carried out by the microarray consortium. SPECIAL NOTE: The samples will be sent upon availability. Gene Expression Microarray Analysis Six mice will be included in each group per timepoint to generate statistically testable data and limit the possible confound of individual mouse gene expression variations. The genome expression will be evaluated through the use of the GeneChip® Mouse Genome 430 2.0 Array (Affymetrix) for each individual mouse constituting a total of 24 arrays for the proposed study. Gene expression data interpretation will be assisted by the Microarray Consortium Center bioinformatics core.
2008-06-13 | E-GEOD-4758 | biostudies-arrayexpress