HSF1 deficiency and impaired HSP90-dependent protein folding are hallmarks of aneuploid human cells.
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ABSTRACT:
SUBMITTER: Donnelly N
PROVIDER: S-EPMC4253526 | biostudies-literature | 2014 Oct
REPOSITORIES: biostudies-literature
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Project description:Heat shock factor 1 (HSF1) initiates a broad transcriptional response to proteotoxic stress while also mediating a cancer-specific transcriptional program. HSF1 is thought to be regulated by molecular chaperones, including Heat Shock Protein 90 (HSP90). HSP90 is proposed to sequester HSF1 in unstressed cells, but visualization of this interaction in vivo requires protein crosslinking. In this report, we show that HSP90 binding to HSF1 depends on HSP90 conformation and is only readily visualized for the ATP-dependent, N-domain dimerized chaperone, a conformation only rarely sampled by mammalian HSP90. We have used this mutationally fixed conformation to map HSP90 binding sites on HSF1. Further, we show that ATP-competitive, N-domain targeted HSP90 inhibitors disrupt this interaction, resulting in the increased duration of HSF1 occupancy of the hsp70 promoter and significant prolongation of both the constitutive and heat-induced HSF1 transcriptional activity. While our data do not support a role for HSP90 in sequestering HSF1 monomers to suppress HSF1 transcriptional activity, our findings do identify a noncanonical role for HSP90 in providing dynamic modulation of HSF1 activity by participating in removal of HSF1 trimers from heat shock elements in DNA, thus terminating the heat shock response.
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Project description:Conformational diseases are often caused by mutations, altering protein folding and stability in vivo. We review here our recent work on the effects of mutations on the human phosphoglycerate kinase 1 (hPGK1), with a particular focus on thermodynamics and kinetics of protein folding and misfolding. Expression analyses and in vitro biophysical studies indicate that disease-causing mutations enhance protein aggregation propensity. We found a strong correlation among protein aggregation propensity, thermodynamic stability, cooperativity and dynamics. Comparison of folding and unfolding properties with previous reports in PGKs from other species suggests that hPGK1 is very sensitive to mutations leading to enhance protein aggregation through changes in protein folding cooperativity and the structure of the relevant denaturation transition state for aggregation. Overall, we provide a mechanistic framework for protein misfolding of hPGK1, which is insightful to develop new therapeutic strategies aimed to target native state stability and foldability in hPGK1 deficient patients.
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Project description:Myosin folding and assembly in striated muscle are mediated by the general chaperones Hsc70 and Hsp90 and involve a myosin-specific co-chaperone related to the Caenorhabditis elegans gene unc-45. Two unc-45 genes are found in vertebrates, a general cell isoform, unc45a, and a striated muscle-specific isoform, unc45b. We have investigated the role of both isoforms of mouse Unc45 in myosin folding using an in vitro synthesis and folding assay. A smooth muscle myosin motor domain (MD) fused to green fluorescent protein (GFP) (MD::GFP) was used as substrate, and folding was measured by native gel electrophoresis and functional assays. In the absence of Unc45, the MD::GFP chimera folds poorly. Addition of either Unc45a or Unc45b dramatically enhances the folding in a reaction that is dependent on Hsp90 ATPase activity. Unc45a is more effective than Unc45b with a higher apparent affinity and greater extent of folding. The Unc45-Hsp90 chaperone complex acts late in the folding pathway and promotes motor domain maturation after release from the ribosome. Unc45a behaves kinetically as an activator of the folding reaction by stimulating the rate of the Hsp90-dependent folding by >20-fold with an apparent K(act) of 33 nm. This analysis of vertebrate Unc45 isoforms clearly demonstrates a direct role for Unc45 in Hsp90-mediated myosin motor domain folding and highlights major differences between the isoforms in substrate specificity and mechanism.
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Project description:Fever is a universal response to infection, and opportunistic pathogens such as Candida albicans have evolved complex circuitry to sense and respond to heat. Here we harness RNA-seq and ChIP-seq to discover that the heat shock transcription factor, Hsf1, binds distinct motifs in nucleosome-depleted promoter regions to regulate heat shock genes and genes involved in virulence in C. albicans. Consequently, heat shock increases C. albicans host cell adhesion, damage and virulence. Hsf1 activation depends upon the molecular chaperone Hsp90 under basal and heat shock conditions, but the effects are opposite and in part controlled at the level of Hsf1 expression and DNA binding. Finally, we demonstrate that Hsp90 regulates global transcription programs by modulating nucleosome levels at promoters of stress-responsive genes. Thus, we describe a mechanism by which C. albicans responds to temperature via Hsf1 and Hsp90 to orchestrate gene expression and chromatin architecture, thereby enabling thermal adaptation and virulence.
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Project description:The molecular chaperone heat shock protein 90 (HSP90) facilitates the appropriate folding of various oncogenic proteins and is necessary for the survival of some cancer cells. HSP90 is therefore an attractive drug target, but the efficacy of HSP90 inhibitor may be limited by HSP90 inhibition induced feedback mechanisms. Through pooled RNA interference screens, we identified that heat shock factor 1(HSF1) is a sensitizer of HSP90 inhibitor. A striking combinational effect was observed when HSF1 knockdown plus with HSP90 inhibitors treatment in various cancer cell lines and tumor mouse models. Interestingly, HSF1 is highly expressed in hepatocellular carcinoma (HCC) patient samples and HCC is sensitive to combinational treatment, indicating a potential indication for the combinational treatment. To understand the mechanism of the combinational effect, we identified that a HSF1-target gene DEDD2 is involved in attenuating the effect of HSP90 inhibitors. Thus, the transcriptional activities of HSF1 induced by HSP90 inhibitors provide a feedback mechanism of limiting the HSP90 inhibitor's activity, and targeting HSF1 may provide a new avenue to enhance HSP90 inhibitors activity in human cancers.
| S-EPMC3757240 | biostudies-literature
Project description:Mitogen-activated protein kinases (MAPKs) and heat shock proteins (HSPs) are ubiquitous proteins that are functional mediators in both normal and stressed states of the cell. In this study, we performed heat stress (37 °C) experiments on turbot kidney (TK) cells. Heat stress expression patterns of HSP90, as well as the expression and phosphorylation levels of extracellular-regulated signal kinases (ERKs) and the transcription factor HSF1 and c-Fos, were examined. The results show that heat stress activates ERK1/2 and HSF1, and induces HSP90 gene expression in TK cells. Inhibition of ERK activation attenuates heat stress-induced HSP90 gene expression. The double luciferase reporter gene experiment showed that HSF1 is an important transcription factor for heat-induced HSP90 gene expression. Likewise, c-Fos does not directly regulate the heat-induced expression of HSP90 in turbot kidney cells. To our knowledge, this is the first study to report a signaling pathway that regulates the heat shock response in turbot cells. Our results may facilitate an understanding of the underlying molecular mechanisms of the cellular stress response in marine fish.
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Project description:CLL is a disease characterized by chromosomal deletions, acquired copy number changes and aneuploidy. Recent studies have shown that overexpression of Heat Shock Factor (HSF) 1 in aneuploid tumor cells can overcome deficiencies in heat shock protein (HSP) 90-mediated protein folding and restore protein homeostasis. Interestingly, several independent studies have demonstrated that HSF1 expression and activity also affects the chaperoning of HSP90 kinase clients, although the mechanism underlying this observation is unclear. Here, we determined how HSF1 regulates HSP90 function using CLL as a model system. We report that HSF1 is overexpressed in CLL and treatment with triptolide (a small molecule inhibitor of HSF1) induces apoptosis in cultured and primary CLL B-cells. We demonstrate that knockdown of HSF1 or its inhibition with triptolide results in the reduced association of HSP90 with its kinase co-chaperone cell division cycle 37 (CDC37), leading to the partial depletion of HSP90 client kinases, Bruton's Tyrosine Kinase (BTK), c-RAF and cyclin-dependent kinase 4 (CDK4). Treatment with triptolide or HSF1 knockdown disrupts the cytosolic complex between HSF1, p97, HSP90 and the HSP90 deacetylase- Histone deacetylase 6 (HDAC6). Consequently, HSF1 inhibition results in HSP90 acetylation and abrogation of its chaperone function. Finally, tail vein injection of Mec-1 cells into Rag2-/-IL2R?c-/- mice followed by treatment with minnelide (a pro-drug of triptolide), reduced leukemia, increased survival and attenuated HSP90-dependent survival signaling in vivo. In conclusion, our study provides a strong rationale to target HSF1 and test the activity of minnelide against human CLL.
| S-EPMC4741638 | biostudies-literature
Project description:Background: Proteins fold robustly and reproducibly in vivo, but many cannot fold in vitro in isolation from cellular components. Despite the remarkable progress that has been achieved by the artificial intelligence approaches in predicting the protein native conformations, the pathways that lead to such conformations, either in vitro or in vivo, remain largely unknown. The slow progress in recapitulating protein folding pathways in silico may be an indication of the fundamental deficiencies in our understanding of folding as it occurs in nature. Here we consider the possibility that protein folding in living cells may not be driven solely by the decrease in Gibbs free energy and propose that protein folding in vivo should be modeled as an active energy-dependent process. The mechanism of action of such a protein folding machine might include direct manipulation of the peptide backbone. Methods: To show the feasibility of a protein folding machine, we conducted molecular dynamics simulations that were augmented by the application of mechanical force to rotate the C-terminal amino acid while simultaneously limiting the N-terminal amino acid movements. Results: Remarkably, the addition of this simple manipulation of peptide backbones to the standard molecular dynamics simulation indeed facilitated the formation of native structures in five diverse alpha-helical peptides. Steric clashes that arise in the peptides due to the forced directional rotation resulted in the behavior of the peptide backbone no longer resembling a freely jointed chain. Conclusions: These simulations show the feasibility of a protein folding machine operating under the conditions when the movements of the polypeptide backbone are restricted by applying external forces and constraints. Further investigation is needed to see whether such an effect may play a role during co-translational protein folding in vivo and how it can be utilized to facilitate folding of proteins in artificial environments.
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Project description:Post-heat shock refolding of luciferase requires chaperones. Expression of a dominant negative HSF1 mutant (dnHSF1), which among other effects depletes cells of HSF1-regulated chaperones, blocked post-heat shock refolding of luciferase targeted to the cytoplasm, nucleus, or peroxisomes, while refolding of endoplasmic reticulum (ER)-targeted luciferase was inhibited by about 50 %. Luciferase refolding in the cytoplasm could be partially restored by expression of HSPA1A and fully by both HSPA1A and DNAJB1. For full refolding of ER luciferase, HSPA1A expression sufficed. Neither nuclear nor peroxisomal refolding was rescued by HSPA1A. A stimulatory effect of DNAJB1 on post-heat shock peroxisomal luciferase refolding was seen in control cells, while refolding in the cytoplasm or nucleus in control cells was inhibited by DNAJB1 expression in the absence of added HSPA1A. HSPB1 also improved refolding of peroxisomal luciferase in control cells, but not in dnHSF1 expressing cells. HSP90, HSPA5, HSPA6, and phosphomevalonate kinase (of which the synthesis is also downregulated by dnHSF1) had no effect on peroxisomal refolding in either control or chaperone-depleted cells. The chaperone requirement for post-heat shock refolding of peroxisomal luciferase in control cells is thus unusual in that it can be augmented by DNAJB1 or HSPB1 but not by HSPA1A; in dnHSF1 expressing cells, expression of none of the (co)-chaperones tested was effective, and an as yet to be identified, HSF1-regulated function is required.
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Project description:Diffusion on a low-dimensional free-energy surface is a remarkably successful model for the folding dynamics of small single-domain proteins. Complicating the interpretation of both simulations and experiments is the expectation that the effective diffusion coefficient D will in general depend on the position along the folding coordinate, and this dependence may vary for different coordinates. Here we explore the position dependence of D, its connection to protein internal friction, and the consequences for the interpretation of single-molecule experiments. We find a large decrease in D from unfolded to folded, for reaction coordinates that directly measure fluctuations in Cartesian configuration space, including those probed in single-molecule experiments. In contrast, D is almost independent of Q, the fraction of native amino acid contacts: Near the folded state, small fluctuations in position cause large fluctuations in Q, and vice versa for the unfolded state. In general, position-dependent free energies and diffusion coefficients for any two good reaction coordinates that separate reactant, product, and transition states, are related by a simple transformation, as we demonstrate. With this transformation, we obtain reaction coordinates with position-invariant D. The corresponding free-energy surfaces allow us to justify the assumptions used in estimating the speed limit for protein folding from experimental measurements of the reconfiguration time in the unfolded state, and also reveal intermediates hidden in the original free-energy projection. Lastly, we comment on the design of future single-molecule experiments that probe the position dependence of D directly.
| S-EPMC2824289 | biostudies-literature