Hsp90 Function Research Articles

Design, synthesis and bioevaluation of inhibitors targeting HSP90-CDC37 protein-protein interaction based on a hydrophobic core

HSP90-CDC37 protein-protein interaction (PPI) works as a kinase specific-molecular chaperone system to regulate the maturation of kinases. Currently, selectively disrupting HSP90-CDC37 PPI, rather than the direct inhibition of the ATPase function of HSP90, is emerging as a promising strategy for cancer therapy by specifically blocking the maturation of kinases. However, due to the limited understanding of HSP90-CDC37 binding interface, design of small molecule inhibitors targeting HSP90-CDC37 PPI is challenging. In this work, based on the binding mode of compound 11 (previously reported by our group), we discovered a hydrophobic pocket centered on Phe213, which was previously unknown, contributing to the binding affinity of HSP90-CDC37 PPI inhibitors. A series of hydrophobic substituted inhibitors were utilized to confirm the importance of Phe213 hydrophobic core. Finally, we obtained an optimum compound DDO-5994 (exhibited an ideal binding pattern on hydrophobic core) with improved binding affinity (KD=5.52μM) and antiproliferative activity (IC50=6.34μM). Both invitro and invivo assays confirmed DDO-5994 as a promising inhibitor to exhibit ideal antitumor efficacy through blocking HSP90-CDC37 PPI.

Hsp90 function is required for stable transcription of the baculovirus transactivator ie-1 gene

A molecular chaperone heat shock protein 90 (Hsp90) is required for efficient infection by several viruses. Hsp90 has been recently implicated in baculovirus infection, but its exact role remains obscure. This study investigated the effect of 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), an Hsp90-specific inhibitor, on Bombyx mori nucleopolyhedrovirus (BmNPV) infection. The 17-AAG treatment significantly decreased the production of budded viruses and occlusion bodies in BmNPV-infected Bombyx mori cultured cells. Immunoblot and SDS-PAGE analyses showed that the expression of early and delayed early gene products, DBP and BRO, was delayed and dysregulated, and the very late gene product POLH was almost completely diminished. RT-qPCR experiments revealed that 17-AAG treatment did not affect initiation of the immediate early gene ie-1 expression, but the expression decreased by ∼50 % during the late stage of infection. 17-AAG treatment also decreased ie-1 promoter activity by ∼50 %. In addition, the expression of delayed early and late genes was dysregulated and inhibited, respectively. These results indicated that Hsp90 function is required for stable ie-1 transcription. Inhibiting Hsp90 function negatively affects ie-1 expression, resulting in dysregulation of delayed early genes and a severe decrease in late and very late gene expression.

Methylation of HSP70 Orchestrates Its Binding to and Stabilization of BCL2 mRNA and Renders Pancreatic Cancer Cells Resistant to Therapeutics.

Pancreatic cancer is a lethal disease owing to its intrinsic and acquired resistance to therapeutic modalities. The altered balance between pro- and antiapoptosis signals within cancer cells is critical to therapeutic resistance. However, the molecular mechanisms underlying increased antiapoptosis signals remain poorly understood. In this study, we report that PRMT1 expression is increased in pancreatic cancer tissues and is associated with higher tumor grade, increased aggressiveness, and worse prognosis. PRMT1 overexpression increased arginine methylation of HSPs of 70 kDa (HSP70); this methylation enhanced HSP70 binding and stabilization of BCL2 mRNA through AU-rich elements in 3'-untranslated region and consequentially increased BCL2 protein expression and protected cancer cells from apoptosis induced by cellular stresses and therapeutics. RNA binding and regulation function of HSP70 was involved in pancreatic cancer drug resistance and was dependent on protein arginine methylation. These findings not only reveal a novel PRMT1-HSP70-BCL2 signaling axis that is crucial to pancreatic cancer cell survival and therapeutic resistance, but they also provide a proof of concept that targeted inhibition of this axis may represent a new therapeutic strategy. SIGNIFICANCE: This study demonstrates that a PRMT1-mediated stabilization of BCL2 mRNA contributes to therapeutic resistance in pancreatic cancer and that targeting this pathway could overcome said resistance.

Genome-wide identification and structural analysis of heat shock protein gene families in the marine rotifer Brachionus spp.: Potential application in molecular ecotoxicology

Heat shock proteins (Hsp) are class of conserved and ubiquitous stress proteins present in all living organisms from primitive to higher level. Various studies have demonstrated multiple cellular functions of Hsp in living organisms as an important biomarker in response to abiotic and biotic stressors including temperature, salinity, pH, hypoxia, environmental pollutants, and pathogens. However, full understanding on the mechanism and pathway involved in the induction of Hsp still remains challenging, especially in aquatic invertebrates. In this study, the entire Hsp family and subfamily members in the marine rotifers Brachionus spp., one of the cosmopolitan ecotoxicological model organisms, have been genome-widely identified. In Brachionus spp. Hsp family was comprised of Hsp10, small hsp (sHsp), Hsp40, Hsp60, Hsp70/105, and Hsp90, with highest number of genes found within Hsp40 DnaJ homolog subfamily C members. Also, the differences in the orientation of the conserved motifs within Hsp family may have induced differences in transcriptional gene modulation in response to thermal stress in Brachionus koreanus. Overall, Hsp family-specific domains were highly conserved in all three Brachionus spp., relative to Homo sapiens and across other animal taxa and these findings will be helpful for future ecotoxicological studies focusing on Hsps.

Hsp90 regulates HDAC3-dependent gene transcription while HDAC3 regulates the functions of Hsp90

Deregulated DNA methylation and post-translational histone modifications are majorly associated with cancer progression. Histone modification regulates the gene expression patterns that contribute to the emergence of sporadic cancers. Histone deacetylases (HDACs) act as erasers of acetylation marks, and their functions are often deregulated in cancer. Since non-histone proteins can also act as substrates for HDACs, identifying their involvement in vital regulatory molecules contributing to cancer progression is essential. Hsp90 is a cancer chaperone that contributes to kinase evolution and, thus, cellular adaptations. Acetylated Hsp90 loses its chaperoning functions and client protein interactions. Robust cell proliferation is one of the hallmarks of cancer. However, Hsp90 involvement in cancer promoting gene transcription is less understood. Using human breast cancer cells, we demonstrate that nuclear Hsp90 functions are regulated by HDAC3, while Hsp90 regulates HDAC3 nuclear translocation. Pharmacological inhibition of Hsp90 decreased the HDAC3 nuclear translocation and increased the gene expression relevant to epithelial to mesenchymal transition. Further, inhibition of HDAC3 resulted in the nuclear accumulation of acetylated Hsp90. Additionally, Hsp90 inhibition affected the global histone acetylation and methylation patterns, whereas HDAC3 inhibition exhibited less impact. Our results display a novel regulatory mechanism mediated by Hsp90 and HDAC3 in tumor cells. Considering that Hsp90 and histone deacetylase inhibitors are emerging as novel anticancer agents, our findings may have clinical relevance.

Fungal mycotoxin penisuloxazin A, a novel C-terminal Hsp90 inhibitor and characteristics of its analogues on Hsp90 function related to binding sites

Hsp90 is a promising drug target for cancer therapy. However, toxicity and moderate effect are limitations of current inhibitors owing to broad protein degradation. The fungal mycotoxin penisuloxazin A (PNSA) belongs to a new epipolythiodiketopiperazines (ETPs) possessing a rare 3H-spiro[benzofuran-2,2′-piperazine] ring system. PNSA bound to cysteine residues C572/C598 of CT-Hsp90 with disulfide bonds and inhibits Hsp90 activity, resulting in apoptosis and growth inhibition of HCT116 cells in vitro and in vivo. We identified that analogues PEN-A and HDN-1 bound to C572/C597 and C572 of CT-Hsp90α respectively, with binding pattern very similar to PNSA. These ETPs exhibited different effects on ATPase activity, dimerization formation and selectivity on client protein of Hsp90, indicating client recognition of Hsp90 can be exactly regulated by different sites of Hsp90. Our findings not only offer new chemotypes for anticancer drug development, but also help to better understand biological function of Hsp90 for exploring inhibitor with some client protein bias.

Identification and Characterization of Three Heat Shock Protein 90 (Hsp90) Homologs in the Brown Planthopper.

Hsp90 (heat shock protein 90) chaperone machinery is considered to be a key regulator of proteostasis under both physiological and stress growth conditions in eukaryotic cells. The high conservation of both the sequence and function of Hsp90 allows for the utilization of various species to explore new phenotypes and mechanisms. In this study, three Hsp90 homologs were identified in the brown planthopper (BPH), Nilaparvata lugens: cytosolic NlHsp90, endoplasmic reticulum (ER) NlGRP94 and mitochondrial NlTRAP1. Sequence analysis and phylogenetic construction showed that these proteins belonged to distinct classes consistent with the predicted localization and suggested an evolutionary relationship between NlTRAP1 and bacterial HtpG (high-temperature protein G). Temporospatial expression analyses showed that NlHsp90 was inducible under heat stress throughout the developmental stage, while NlGRP94 was only induced at the egg stage. All three genes had a significantly high transcript level in the ovary. The RNA interference-mediated knockdown of NlHsp90 its essential role in nymph development and oogenesis under physiological conditions. NlGRP94 was also required during the early developmental stage and played a crucial role in oogenesis, fecundity and late embryogenesis. Notably, we first found that NlHsp90 and NlGRP94 were likely involved in the cuticle structure of female BPH. Together, our research revealed multifunctional roles of Hsp90s in the BPH.

Persistent Activation of mRNA Translation by Transient Hsp90 Inhibition

The heat shock protein 90 (Hsp90) chaperone functions as a protein-folding buffer and plays a role promoting the evolution of new heritable traits. To better understand how Hsp90 can affect mRNA translation, we screen more than 1,600 factors involved in mRNA regulation for physical interactions with Hsp90 in human cells. The mRNA binding protein CPEB2 strongly binds Hsp90 via its prion domain. In a yeast model, transient inhibition of Hsp90 results in persistent activation of a CPEB translation reporter even in the absence of exogenous CPEB that persists for 30 generations after the inhibitor is removed. Ribosomal profiling reveals that some endogenous yeast mRNAs, including HAC1, show a persistent change in translation efficiency following transient Hsp90 inhibition. Thus, transient loss of Hsp90 function can promote a nongenetic inheritance of a translational state affecting specific mRNAs, introducing a mechanism by which Hsp90 can promote phenotypic variation.

Hsp70 Is a Potential Therapeutic Target for Echovirus 9 Infection.

Echovirus is an important cause of viral pneumonia and encephalitis in infants, neonates, and young children worldwide. However, the exact mechanism of its pathogenesis is still not well understood. Here, we established an echovirus type 9 infection mice model, and performed two-dimensional gel electrophoresis (2DE) and tandem mass spectrometry (MS/MS)-based comparative proteomics analysis to investigate the differentially expressed host proteins in mice brain. A total of 21 differentially expressed proteins were identified by MS/MS. The annotation of the differentially expressed proteins by function using the UniProt and GO databases identified one viral protein (5%), seven cytoskeletal proteins (33%), six macromolecular biosynthesis and metabolism proteins (28%), two stress response and chaperone binding proteins (9%), and five other cellular proteins (25%). The subcellular locations of these proteins were mainly found in the cytoskeleton, cytoplasm, nucleus, mitochondria, and Golgi apparatus. The protein expression profiles and the results of quantitative RT-PCR in the detection of gene transcripts were found to complement each other. The differential protein interaction network was predicted using the STRING database. Of the identified proteins, heat shock protein 70 (Hsp70), showing consistent results in the proteomics and transcriptomic analyses, was analyzed through Western blotting to verify the reliability of differential protein expression data in this study. Further, evaluation of the function of Hsp70 using siRNA and quercetin, an inhibitor of Hsp70, showed that Hsp70 was necessary for the infection of echovirus type 9. This study revealed that echovirus infection could cause the differential expression of a series of host proteins, which is helpful to reveal the pathogenesis of viral infection and identify therapeutic drug targets. Additionally, our results suggest that Hsp70 could be a useful therapeutic host protein target for echovirus infection.

Chemical Perturbation of Oncogenic Protein Folding: from the Prediction of Locally Unstable Structures to the Design of Disruptors of Hsp90-Client Interactions.

Protein folding quality control in cells requires the activity of a class of proteins known as molecular chaperones. Heat shock protein-90 (Hsp90), a multidomain ATP driven molecular machine, is a prime representative of this family of proteins. Interactions between Hsp90, its co-chaperones, and client proteins have been shown to be important in facilitating the correct folding and activation of clients. Hsp90 levels and functions are elevated in tumor cells. Here, we computationally predict the regions on the native structures of clients c-Abl, c-Src, Cdk4, B-Raf and Glucocorticoid Receptor, that have the highest probability of undergoing local unfolding, despite being ordered in their native structures. Such regions represent potential ideal interaction points with the Hsp90-system. We synthesize mimics spanning these regions and confirm their interaction with partners of the Hsp90 complex (Hsp90, Cdc37 and Aha1) by Nuclear Magnetic Resonance (NMR). Designed mimics selectively disrupt the association of their respective clients with the Hsp90 machinery, leaving unrelated clients unperturbed and causing apoptosis in cancer cells. Overall, selective targeting of Hsp90 protein-protein interactions is achieved without causing indiscriminate degradation of all clients, setting the stage for the development of therapeutics based on specific chaperone:client perturbation.

The Yeast Hsp70 Cochaperone Ydj1 Regulates Functional Distinction of Ssa Hsp70s in the Hsp90 Chaperoning Pathway.

Heat-shock protein (Hsp) 90 assists in the folding of diverse sets of client proteins including kinases and growth hormone receptors. Hsp70 plays a major role in many Hsp90 functions by interacting and modulating conformation of its substrates before being transferred to Hsp90s for final maturation. Each eukaryote contains multiple members of the Hsp70 family. However, the role of different Hsp70 isoforms in Hsp90 chaperoning actions remains unknown. Using v-Src as an Hsp90 substrate, we examined the role of each of the four yeast cytosolic Ssa Hsp70s in regulating Hsp90 functions. We show that the strain expressing stress-inducible Ssa3 or Ssa4, and the not constitutively expressed Ssa1 or Ssa2, as the sole Ssa Hsp70 isoform reduces v-Src-mediated growth defects. The study shows that although different Hsp70 isoforms interact similarly with Hsp90s, v-Src maturation is less efficient in strains expressing Ssa4 as the sole Hsp70. We further show that the functional distinction between Ssa2 and Ssa4 is regulated by its C-terminal domain. Further studies reveal that Ydj1, which is known to assist substrate transfer to Hsp70s, interacts relatively weakly with Ssa4 compared with Ssa2, which could be the basis for poor maturation of the Hsp90 client in cells expressing stress-inducible Ssa4 as the sole Ssa Hsp70. The study thus reveals a novel role of Ydj1 in determining the functional distinction among Hsp70 isoforms with respect to the Hsp90 chaperoning action.

Mutations in the Hsp90 N Domain Identify a Site that Controls Dimer Opening and Expand Human Hsp90α Function in Yeast

Hsp90 is a highly conserved molecular chaperone important for the activity of many client proteins. Hsp90 has an N-terminal ATPase domain (N), a middle domain (M) that interacts with clients and a C-terminal dimerization domain (C). “Closing” of dimers around clients is regulated by ATP binding, co-chaperones, and post-translational modifications. ATP hydrolysis coincides with release of mature client and resetting the reaction cycle. Humans have two Hsp90s: hHsp90α and hHsp90β. Although 85% identical, hHsp90β supports Hsp90 function in yeast much better than hHsp90α. Determining the basis of this difference would provide important insight into functional specificity of seemingly redundant Hsp90s, and the evolution of eukaryotic Hsp90 systems and clientele. Here, we found host co-chaperones Sba1, Cpr6 and Cpr7 inhibited hHsp90α function in yeast, and we identified mutations clustering in the N domain that considerably improved hHsp90α function in yeast. The strongest of these rescuer mutations accelerated nucleotide-dependent lid closing, N–M domain docking, and ATPase. It also disrupted binding to Sba1, which prolongs the closed state, and promoted N–M undocking and lid opening. Our data suggest the rescuer mutations improve function of hHsp90α in yeast by accelerating return to the open state. Our findings imply hHsp90α occupies the closed state too long to function effectively in yeast, and define an evolutionarily conserved region of the N domain involved in resetting the Hsp90 reaction cycle.

Molecular characterization of GRP94 and HSP90α from Trachinotus ovatus, Linnaeus 1758 and their expression responses to various levels of stocking density stress and Cryptocaryon irritans infection

Heat shock proteins (HSPs) are a class of molecular chaperones with a wide range of biological activities. To understand the stress responses of 94-kDa glucose-regulated protein (GRP94) and heat shock protein 90 alpha (HSP90α) under stocking density stress and C. irritans infection, we obtained their cDNA sequences from T. ovatus and performed bioinformatic analysis on their structures. Meanwhile, the tissue-specific expression patterns of these genes were also detected by qPCR. The ToGRP94 cDNA contains an open reading frame of 2394 bp, which encodes a protein of 797 amino acids, while ToHSP90α contains an open reading frame of 2190 bp, which encodes a protein of 730 amino acids. Amino acid sequence analysis showed that these proteins have highly conserved characteristic family domains or motifs, such as the heat shock hsp90 protein family signature (Y [k/s] NKEIFLRE). ToGRP94 was recently related to S. dumerili GRP94 with amino acid sequence homology up to 96.86%, and ToHSP90α had a higher evolutionary correlation with M. armatus and A. testudineus HSP90α with sequence homology of 98.49% and 97.67%, respectively. ToGRP94 and ToHSP90α mRNAs were widely expressed in all tissues tested under normal conditions. ToGRP94 mRNA was most strongly expressed in gill tissue followed by fin and kidney tissues, while ToHSP90α was most strongly expressed in blood tissues followed by brain, kidney and gill tissues. Under stocking density stress, ToGRP94 mRNA in the liver was significantly higher in the LD group, but it did not change in the kidney and spleen tissues with increasing stocking density; however, the expression level of ToHSP90α gene increased significantly in liver, spleen, and kidney tissues under high stocking densities, which indicates that ToHSP90α was closely related to density chronic stress. After C. irritans infection, the expression of ToGRP94 was significantly increased in liver, spleen, and kidney tissues. Similarly, ToHSP90α gene expression was significantly increased in liver and kidney tissues, but there was no difference in spleen tissue. The results indicate that these two genes may be involved in the immune response of T. ovatus. In summary, ToHSP90α would be involved in chronic stress and the immune response, while ToGRP94 would be mainly involved in the immune regulatory response. These results may provide a reference for further research studying the function of fish HSP90 and its mechanism under environmental stress.

Development of a novel truncated deguelin derivative possessing nitric oxide donor as a potential anti-lung cancer agent

Lung cancer is the leading cause of cancer death in the world. Natural product deguelin and its truncated analogs have been reported to be potential therapeutic agents for lung cancer. In order to improve the potency, a novel truncated deguelin derivative (4) possessing nitric oxide (NO) donor was designed and synthesized. The biological evaluation showed that hybrid 4 exerted potent activity with an IC50 value of 0.41 μM in H1299 cells. Mechanism studies showed that it arrested the cell cycle at G2/M phase and suppressed Hsp90 function. In addition, hybrid 4 demonstrated potent inhibitory activity on the migration and invasion of lung cancer cells. Together, the promising results warrant further development of hybrid 4 as a potential anticancer agent for the treatment of lung cancer.

Hsp60 Post-translational Modifications: Functional and Pathological Consequences.

Hsp60 is a chaperone belonging to the Chaperonins of Group I and typically functions inside mitochondria in which, together with the co-chaperonin Hsp10, maintains protein homeostasis. In addition to this canonical role, Hsp60 plays many others beyond the mitochondria, for instance in the cytosol, plasma-cell membrane, extracellular space, and body fluids. These non-canonical functions include participation in inflammation, autoimmunity, carcinogenesis, cell replication, and other cellular events in health and disease. Thus, Hsp60 is a multifaceted molecule with a wide range of cellular and tissue locations and functions, which is noteworthy because there is only one hsp60 gene. The question is by what mechanism this protein can become multifaceted. Likely, one factor contributing to this diversity is post-translational modification (PTM). The amino acid sequence of Hsp60 contains many potential phosphorylation sites, and other PTMs are possible such as O-GlcNAcylation, nitration, acetylation, S-nitrosylation, citrullination, oxidation, and ubiquitination. The effect of some of these PTMs on Hsp60 functions have been examined, for instance phosphorylation has been implicated in sperm capacitation, docking of H2B and microtubule-associated proteins, mitochondrial dysfunction, tumor invasiveness, and delay or facilitation of apoptosis. Nitration was found to affect the stability of the mitochondrial permeability transition pore, to inhibit folding ability, and to perturb insulin secretion. Hyperacetylation was associated with mitochondrial failure; S-nitrosylation has an impact on mitochondrial stability and endothelial integrity; citrullination can be pro-apoptotic; oxidation has a role in the response to cellular injury and in cell migration; and ubiquitination regulates interaction with the ubiquitin-proteasome system. Future research ought to determine which PTM causes which variations in the Hsp60 molecular properties and functions, and which of them are pathogenic, causing chaperonopathies. This is an important topic considering the number of acquired Hsp60 chaperonopathies already cataloged, many of which are serious diseases without efficacious treatment.

S-Glutathionylation of human inducible Hsp70 reveals a regulatory mechanism involving the C-terminal α-helical lid

Heat shock protein 70 (Hsp70) proteins are a family of ancient and conserved chaperones. Cysteine modifications have been widely detected among different Hsp70 family members in vivo, but their effects on Hsp70 structure and function are unclear. Here, we treated HeLa cells with diamide, which typically induces disulfide bond formation except in the presence of excess GSH, when glutathionylated cysteines predominate. We show that in these cells, HspA1A (hHsp70) undergoes reversible cysteine modifications, including glutathionylation, potentially at all five cysteine residues. In vitro experiments revealed that modification of cysteines in the nucleotide-binding domain of hHsp70 is prevented by nucleotide binding but that Cys-574 and Cys-603, located in the C-terminal α-helical lid of the substrate-binding domain, can undergo glutathionylation in both the presence and absence of nucleotide. We found that glutathionylation of these cysteine residues results in unfolding of the α-helical lid structure. The unfolded region mimics substrate by binding to and blocking the substrate-binding site, thereby promoting intrinsic ATPase activity and competing with binding of external substrates, including heat shock transcription factor 1 (Hsf1). Thus, post-translational modification can alter the structure and regulate the function of hHsp70.

Two-step mechanism of J-domain action in driving Hsp70 function.

J-domain proteins (JDPs), obligatory Hsp70 cochaperones, play critical roles in protein homeostasis. They promote key allosteric transitions that stabilize Hsp70 interaction with substrate polypeptides upon hydrolysis of its bound ATP. Although a recent crystal structure revealed the physical mode of interaction between a J-domain and an Hsp70, the structural and dynamic consequences of J-domain action once bound and how Hsp70s discriminate among its multiple JDP partners remain enigmatic. We combined free energy simulations, biochemical assays and evolutionary analyses to address these issues. Our results indicate that the invariant aspartate of the J-domain perturbs a conserved intramolecular Hsp70 network of contacts that crosses domains. This perturbation leads to destabilization of the domain-domain interface—thereby promoting the allosteric transition that triggers ATP hydrolysis. While this mechanistic step is driven by conserved residues, evolutionarily variable residues are key to initial JDP/Hsp70 recognition—via electrostatic interactions between oppositely charged surfaces. We speculate that these variable residues allow an Hsp70 to discriminate amongst JDP partners, as many of them have coevolved. Together, our data points to a two-step mode of J-domain action, a recognition stage followed by a mechanistic stage.

DCZ5248, a novel dual inhibitor of Hsp90 and autophagy, exerts antitumor activity against colon cancer.

Hsp90 is a potential therapeutic target for tumor, as it maintains the stability of a variety of proteins related to tumor development and progression. Autophagy is a self-degradation process to maintain cellular homeostasis and autophagy inhibitors can suppress tumor growth. In this study, we identified DCZ5248, a triazine derivative, was a dual inhibitor of both Hsp90 and late-autophagy with potent antitumor activity against colon cancer cells in vitro and in vivo. We showed that DCZ5248 (0.1-10 μM) induced dose-dependent degradation of Hsp90 client proteins (AKT, CDK4, CDK6 and RAF-1) in HCT 116 colon cancer cells through a proteasome-dependent pathway. Meanwhile, DCZ5248 (0.3 μM) induced cytoplasmic vacuole formation, LC3 II conversion, p62 protein upregulation, and inhibited autophagy at the late stage in the colon cancer cell lines tested. We further revealed that the inhibition of autophagy was achieved by impairing lysosomal functions through induction of lysosomal acidification and attenuation of lysosomal cathepsin activity. The modulation of autophagy by DCZ5248 was independent of Hsp90 inhibition as the autophagy inhibition was not blocked by Hsp90 knockdown. Importantly, inhibition of both Hsp90 function and autophagy by DCZ5248 induced G1-phase cell cycle arrest, apoptosis, and exerted potent antitumor activity against colon cancer cells both in vitro and in vivo. These findings demonstrate that DCZ5248 is a novel dual inhibitor of Hsp90 and autophagy with potential for colon cancer therapy.

The Proteasome Governs Fungal Morphogenesis via Functional Connections with Hsp90 and cAMP-Protein Kinase A Signaling.

Protein homeostasis is critical for proliferation and viability of all organisms. For Candida albicans, protein homeostasis also modulates the transition between yeast and filamentous forms, which is critical for virulence. A key regulator of morphogenesis is the molecular chaperone Hsp90, which mediates proteostasis under physiological and stress conditions. Hsp90 regulates morphogenesis by repressing cyclic AMP-protein kinase A (cAMP-PKA) signaling, such that inhibition of Hsp90 causes filamentation in the absence of an inducing cue. We explored the effect of perturbation of another facet of protein homeostasis and discovered that morphogenesis is also regulated by the proteasome, a large 33-subunit protein complex consisting of a 20S catalytic core and two 19S regulatory particles, which controls degradation of intracellular proteins. We identified a conserved role of the proteasome in morphogenesis as pharmacological inhibition of the proteasome induced filamentation of C. albicans and the related species Candida dubliniensis, Candida tropicalis, Candida krusei, and Candida parapsilosis For C. albicans, genetic depletion of any of 29 subunits of the 19S or 20S particle induced filamentation. Filaments induced by inhibition of either the proteasome or Hsp90 have shared structural characteristics, such as aberrant nuclear content, and shared genetic dependencies, such as intact cAMP-PKA signaling. Consistent with a functional connection between these facets of protein homeostasis that modulate morphogenesis, we observed that proteasome inhibition results in an accumulation of ubiquitinated proteins that overwhelm Hsp90 function, relieving Hsp90-mediated repression of morphogenesis. Together, our findings provide a mechanism whereby interconnected facets of proteostasis regulate C. albicans morphogenesis.IMPORTANCE Fungi cause life-threatening infections and pose a serious threat to human health as there are very few effective antifungal drugs. Candida albicans is a major human fungal pathogen and cause of morbidity and mortality in immunocompromised individuals. A key trait that enables C. albicans virulence is its ability to transition between yeast and filamentous forms. Understanding the mechanisms regulating this virulence trait can facilitate the development of much-needed, novel therapeutic strategies. A key regulator of morphogenesis is the molecular chaperone Hsp90, which is crucial for proteostasis. Here, we expanded our understanding of how proteostasis regulates fungal morphogenesis and identified the proteasome as a repressor of filamentation in C. albicans and related species. Our work suggests that proteasome inhibition overwhelms Hsp90 function, thereby inducing morphogenesis. This work provides a foundation for understanding the role of the proteasome in fungal virulence and offers potential for targeting the proteasome to disarm fungal pathogens.

HSP90 inhibitor 17AAG attenuates sevoflurane-induced neurotoxicity in rats and human neuroglioma cells via induction of HSP70

Background17AAG has been extensively studied for its antitumor effects that protect cells from lethal stress by maintaining protein stability. The role of 17AAG in sevoflurane-induced neuronal injury has never been studied. We aim to investigate the effect of 17AAG on sevoflurane-induced neurotoxicity in vivo and in vitro.MethodsSevoflurane-induced hippocampal neuron injury model was established in aged Sprague–Dawley rats. Pretreatment of vehicle or 17AAG was administered prior to sevoflurane inhalation. H4 neuroglioma cells were pretreated with vehicle or 17AAG and exposed to sevoflurane. Apoptosis, oxidative stress, expression of interleukin-6 (IL-6), and activation of the nuclear factor-κB (NF-κB) signaling pathway in H4 cells were examined by Hoechst assay, flow cytometry, Western blot, and immunofluorescent staining. RNA interference against HSPA1A was performed to test the function of HSP70 in neuroprotection.ResultsExogenous 17AAG reduced sevoflurane-induced apoptosis and oxidative stress in rat hippocampal neurons and in H4 cells. In H4 cells, 17AAG suppressed sevoflurane-induced upregulation of IL-6 and activation of NF-κB signaling. 17AAG enhanced sevoflurane-induced upregulation of HSP70 in rat hippocampal neurons and in H4 cells. Conversely, silencing of HSPA1A in H4 cells blocked the cytoprotective effect of 17AAG against sevoflurane-induced apoptosis and oxidative stress, and prevented upregulation of IL-6 and activation of NF-κB signaling.Conclusions17AAG protects against sevoflurane-induced neurotoxicity in vivo and in vitro via HSP70-dependent inhibition of apoptosis, oxidative stress, and pro-inflammatory signaling pathway.