Essential Molecular Chaperone Research Articles

Quantitative proteomic analysis reveals unique Hsp90 cycle-dependent client interactions.

Hsp90 is an abundant and essential molecular chaperone that mediates the folding and activation of client proteins in a nucleotide-dependent cycle. Hsp90 inhibition directly or indirectly impacts the function of 10-15% of all proteins due to degradation of client proteins or indirect downstream effects. Due to its role in chaperoning oncogenic proteins, Hsp90 is an important drug target. However, compounds that occupy the ATP-binding pocket and broadly inhibit function have not achieved widespread use due to negative effects. More selective inhibitors are needed; however, it is unclear how to achieve selective inhibition. We conducted a quantitative proteomic analysis of soluble proteins in yeast strains expressing wild-type Hsp90 or mutants that disrupt different steps in the client folding pathway. Out of 2,482 proteins in our sample set (approximately 38% of yeast proteins), we observed statistically significant changes in abundance of 350 (14%) of those proteins (log2 fold change ≥1.5). Of these, 257/350 (∼73%) with the strongest differences in abundance were previously connected to Hsp90 function. Principal component analysis of the entire dataset revealed that the effects of the mutants could be separated into three primary clusters. As evidence that Hsp90 mutants affect different pools of clients, simultaneous co-expression of two mutants in different clusters restored wild-type growth. Our data suggests that the ability of Hsp90 to sample a wide range of conformations allows the chaperone to mediate folding of a broad array of clients and that disruption of conformational flexibility results in client defects dependent on those states.

Iso-mukaadial acetate and ursolic acid acetate bind to Plasmodium Falciparum heat shock protein 70: towards targeting parasite protein folding pathway.

Plasmodium falciparum is the most lethal malaria parasite. P. falciparum Hsp70 (PfHsp70) is an essential molecular chaperone (facilitates protein folding) and is deemed a prospective antimalarial drug target. The present study investigates the binding capabilities of select plant derivatives, iso-mukaadial acetate (IMA) and ursolic acid acetate (UAA), against P. falciparum using an in silico docking approach. The interaction between the ligands and PfHsp70 was evaluated using plasmon resonance (SPR) analysis. Molecular docking, binding free energy analysis and molecular dynamics simulations were conducted towards understanding the mechanisms by which the compounds bind to PfHsp70. The molecular docking results revealed ligand flexibilities, conformations and positions of key amino acid residues and protein-ligand interactions as crucial factors accounting for selective inhibition of Hsp70. The simulation results also suggest protein-ligand van der Waals forces as the driving force guiding the interaction of these compounds with PfHsp70. Of the two compounds, UAA and IMA bound to PfHsp70 within the micromolar range based on surface plasmon resonance (SPR) based binding assay. Our findings pave way for future rational design of new selective compounds targeting PfHsp70.

Cryo-EM reveals how Hsp90 and FKBP immunophilins co-regulate the glucocorticoid receptor

Hsp90 is an essential molecular chaperone responsible for the folding and activation of hundreds of ‘client’ proteins, including the glucocorticoid receptor (GR). Previously, we revealed that Hsp70 and Hsp90 remodel the conformation of GR to regulate ligand binding, aided by co-chaperones. In vivo, the co-chaperones FKBP51 and FKBP52 antagonistically regulate GR activity, but a molecular understanding is lacking. Here we present a 3.01 Å cryogenic electron microscopy structure of the human GR:Hsp90:FKBP52 complex, revealing how FKBP52 integrates into the GR chaperone cycle and directly binds to the active client, potentiating GR activity in vitro and in vivo. We also present a 3.23 Å cryogenic electron microscopy structure of the human GR:Hsp90:FKBP51 complex, revealing how FKBP51 competes with FKBP52 for GR:Hsp90 binding and demonstrating how FKBP51 can act as a potent antagonist to FKBP52. Altogether, we demonstrate how FKBP51 and FKBP52 integrate into the GR chaperone cycle to advance GR to the next stage of maturation.

20-kDa accessory protein (P20) from Bacillus thuringiensis subsp. israelensis ISPC-12: Purification, characterization, solution scattering and structural analysis

The 20-kDa accessory protein (P20) from Bacillus thuringiensis subsp. israelensis (Bti) has been identified as an essential molecular chaperone in the enhancement of Cry11Aa and Cyt1Aa toxins production and their bio-crystallization. Additionally, P20 plays a vital role in suppressing the toxic effect of Cyt toxin on the host bacterium and also enhances insecticidal activity of Cry1Ac protein. Thus, the function of P20 is more specific than that of the chaperones. However, P20 is poorly investigated and insufficiently characterized. In the present study, we recombinantly expressed p20 from local isolate Bti ISPC-12 in heterologous bacterium E. coli and P20 protein was purified to homogeneity. Detailed biochemical and biophysical characterization provides crucial insights about in-vitro behavior as well as spatial conformations of P20 protein. Further, structural modelling and analysis provides insights into three-dimensional organization of the protein and shows that P20 is a non-toxic member of cytolytic (Cyt) toxin family similar to Cyt1Ca, with presence of conserved cytolysin fold. Additionally, solution scattering reveals that P20 is present as a dimer in the solution and probable dimeric assembly of P20 is presented. The findings reported here reveal engaging facts about P20 thereby advancing our understanding about this protein, which will expedite future studies.

Differential Hsp90-dependent gene expression is strain-specific and common among yeast strains

Enhanced phenotypic diversity increases a population's likelihood of surviving catastrophic conditions. Hsp90, an essential molecular chaperone and a central network hub in eukaryotes, has been observed to suppress or enhance the effects of genetic variation on phenotypic diversity in response to environmental cues. Because many Hsp90-interacting genes are involved in signaling transduction pathways and transcriptional regulation, we tested how common Hsp90-dependent differential gene expression is in natural populations. Many genes exhibited Hsp90-dependent strain-specific differential expression in five diverse yeast strains. We further identified transcription factors (TFs) potentially contributing to variable expression. We found that on Hsp90 inhibition or environmental stress, activities or abundances of Hsp90-dependent TFs varied among strains, resulting in differential strain-specific expression of their target genes, which consequently led to phenotypic diversity. We provide evidence that individual strains can readily display specific Hsp90-dependent gene expression, suggesting that the evolutionary impacts of Hsp90 are widespread in nature.

The malaria parasite chaperonin containing TCP-1 (CCT) complex: Data integration with other CCT proteomes.

The multi-subunit chaperonin containing TCP-1 (CCT) is an essential molecular chaperone that functions in the folding of key cellular proteins. This paper reviews the interactome of the eukaryotic chaperonin CCT and its primary clients, the ubiquitous cytoskeletal proteins, actin and tubulin. CCT interacts with other nascent proteins, especially the WD40 propeller proteins, and also assists in the assembly of several protein complexes. A new proteomic dataset is presented for CCT purified from the human malarial parasite, P. falciparum (PfCCT). The CCT8 subunit gene was C-terminally FLAG-tagged using Selection Linked Integration (SLI) and CCT complexes were extracted from infected human erythrocyte cultures synchronized for maximum expression levels of CCT at the trophozoite stage of the parasite's asexual life cycle. We analyze the new PfCCT proteome and incorporate it into our existing model of the CCT system, supported by accumulated data from biochemical and cell biological experiments in many eukaryotic species. Together with measurements of CCT mRNA, CCT protein subunit copy number and the post-translational and chemical modifications of the CCT subunits themselves, a cumulative picture is emerging of an essential molecular chaperone system sitting at the heart of eukaryotic cell growth control and cell cycle regulation.

Investigation of the Expression of Serine Protease in Vibrio vulnificus.

Vibrio vulnificus is a Gram-negative estuarine bacterium that causes infection in immuno-compromised patients, eels, and shrimp. V. vulnificus NCIMB2137, a metalloprotease-negative strain isolated from a diseased eel, produces a 45-kDa chymotrypsin-like alkaline serine protease known as VvsA. The gene encoding vvsA also includes another gene, vvsB with an unknown function; however, it is assumed to be an essential molecular chaperone for the maturation of VvsA. In the present study, we used an in vitro cell-free translation system to examine the maturation pathway of VvsA. We individually expressed the vvsA and vvsB genes and detected their mRNAs. However, the sample produced from vvsA did not exhibit protease activity. A sodium dodecyl sulfate (SDS) analysis detected the VvsB protein, but not the VvsA protein. A Western blotting analysis using a histidine (His)-tag at the amino terminus of proteins also showed no protein production by vvsA. These results suggested the translation, but not the transcription of vvsA. Factors derived from Escherichia coli were used in the in vitro cell-free translation system employed in the present study. The operon of the serine protease gene containing vvsA and vvsB was expressed in E. coli. Although serine proteases were produced, they were cleaved at different sites and no active mature forms were detected. These results indicate that the operon encoding vvsA and vvsB is a gene constructed to be specifically expressed in V. vulnificus.

Structural resemblance of the DNAJA-family protein, Tid1, to the DNAJB-family Hsp40

The specific pair of heat shock protein 70 (Hsp70) and Hsp40 constitutes an essential molecular chaperone system involved in numerous cellular processes, including the proper folding/refolding and transport of proteins. Hsp40 family members are characterized by the presence of a conserved J-domain (JD) that functions as a co-chaperone of Hsp70. Tumorous imaginal disc 1 (Tid1) is a tumor suppressor protein belonging to the DNAJA3 subfamily of Hsp40 and functions as a co-chaperone of the mitochondrial Hsp70, mortalin. In this work, we performed nuclear magnetic resonance spectroscopy to determine the solution structure of JD and its interaction with the glycine/phenylalaninerich region (GF-motif) of human Tid1. Notably, Tid1-JD, whose conformation was consistent with that of the DNAJB1 JD, appeared to stably interact with its subsequent GF-motif region. Collectively with our sequence analysis, the present results demonstrate that the functional and regulatory mode of Tid1 resembles that of the DNAJB1 subfamily members rather than DNAJA1 or DNAJA2 subfamily proteins. Therefore, it is suggested that an allosteric interaction between mortalin and Tid1 is involved in the mitochondrial Hsp70/Hsp40 chaperone system. [BMB Reports 2022; 55(10): 488-493].

A non-traditional crystal-based compound screening method targeting the ATP binding site of Plasmodium falciparum GRP78 for identification of novel nucleoside analogues.

Drug resistance to front-line malarial treatments represents an ongoing threat to control malaria, a vector borne infectious disease. The malarial parasite, Plasmodium falciparum has developed genetic variants, conferring resistance to the current standard therapeutic artemisinin and its derivatives commonly referred to as artemisinin-combination therapies (ACTs). Emergence of multi-drug resistance parasite genotypes is a warning of potential treatment failure, reaffirming the urgent and critical need to find and validate alternate drug targets to prevent the spread of disease. An attractive and novel drug target includes glucose-regulated protein 78 kDa (GRP78, or BiP), an essential molecular chaperone protein involved in the unfolded protein response that is upregulated in ACT treated P. falciparum parasites. We have shown that both sequence and structure are closely related to human GRP78 (hGRP78), a chaperone belonging to the HSP70 class of ATPase proteins, which is often upregulated in cellular stress responses and cancer. By screening a library of nucleoside analogues, we identified eight ‘hit’ compounds binding at the active site of the ATP binding domain of P. falciparum GRP78 using a high-throughput ligand soaking screen using x-ray crystallography. These compounds were further evaluated using protein thermal shift assays to assess target binding activity. The nucleoside analogues identified from our screen provide a starting point for the development of more potent and selective antimalarial inhibitors. In addition, we have established a well-defined, high-throughput crystal-based screening approach that can be applied to many crystallizable P. falciparum proteins for generating anti-Plasmodium specific compounds.

Dominant Negative Mutants of Human Immunodeficiency Virus Type 1 Viral Infectivity Factor (Vif) Disrupt Core-Binding Factor Beta-Vif Interaction.

Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 family members (APOBEC3s) are host restriction factors that inhibit viral replication. Viral infectivity factor (Vif), a human immunodeficiency virus type 1 (HIV-1) accessory protein, mediates the degradation of APOBEC3s by forming the Vif-E3 complex, in which core-binding factor beta (CBFβ) is an essential molecular chaperone. Here, we screened nonfunctional Vif mutants with high affinity for CBFβ to inhibit HIV-1 in a dominant negative manner. We applied the yeast surface display technology to express Vif random mutant libraries, and mutants showing high CBFβ affinity were screened using flow cytometry. Most of the screened Vif mutants containing random mutations of different frequencies were able to rescue APOBEC3G (A3G). In the subsequent screening, three of the mutants restricted HIV-1, recovered G-to-A hypermutation, and rescued APOBEC3s. Among them, Vif-6M showed a cross-protection effect toward APOBEC3C, APOBEC3F, and African green monkey A3G. Stable expression of Vif-6M in T lymphocytes inhibited the viral replication in newly HIV-1-infected cells and the chronically infected cell line H9/HXB2. Furthermore, the expression of Vif-6M provided a survival advantage to T lymphocytes infected with HIV-1. These results suggest that dominant negative Vif mutants acting on the Vif-CBFβ target potently restrict HIV-1. IMPORTANCE Antiviral therapy cannot eliminate HIV and exhibits disadvantages such as drug resistance and toxicity. Therefore, novel strategies for inhibiting viral replication in patients with HIV are urgently needed. APOBEC3s in host cells are able to inhibit viral replication but are antagonized by HIV-1 Vif-mediated degradation. Therefore, we screened nonfunctional Vif mutants with high affinity for CBFβ to compete with the wild-type Vif (wtVif) as a potential strategy to assist with HIV-1 treatment. Most screened mutants rescued the expression of A3G in the presence of wtVif, especially Vif-6M, which could protect various APOBEC3s and improve the incorporation of A3G into HIV-1 particles. Transduction of Vif-6M into T lymphocytes inhibited the replication of the newly infected virus and the chronically infected virus. These data suggest that Vif mutants targeting the Vif-CBFβ interaction may be promising in the development of a new AIDS therapeutic strategy.

Hsp90: From Cellular to Organismal Proteostasis.

Assuring a healthy proteome is indispensable for survival and organismal health. Proteome disbalance and the loss of the proteostasis buffer are hallmarks of various diseases. The essential molecular chaperone Hsp90 is a regulator of the heat shock response via HSF1 and a stabilizer of a plethora of signaling proteins. In this review, we summarize the role of Hsp90 in the cellular and organismal regulation of proteome maintenance.

Identification of HbHSP90 gene family and characterization HbHSP90.1 as a candidate gene for stress response in rubber tree

Heat shock protein 90 (HSP90), an essential molecular chaperone, is triggered in response to stress situations in plants. However, the roles of HSP90 gene family members in rubber tree have not been totally specified. In this study, 7 HbHSP90 genes were identified from rubber tree genome. Classification of HbHSP90 family genes into three groups, namely A, B, and C was based on phylogenetic analysis. The structural and motif analyses showed similar structural features in the same group of HbHSP90 members, but differences between groups. Analysis of cis-regulatory element sequences of HbHSP90 genes indicates that the HbHSP90 gene promoter is rich in drought, temperature, and hormone elements. qRT-PCR analysis showed that the 7 HbHSP90 genes responded in different degrees to temperature, drought and powdery mildew infection, and in particularly, HbHSP90.1 was differentially expressed under both abiotic and biotic stresses. Meanwhile, HbHSP90.1 gene was significantly expressed under the treatment of different phytohormone and H2O2 (Hydrogen Peroxide) treatments, which means that HbHSP90.1 gene performs an essential part in the growth and development of rubber trees. Furthermore, the protein interaction results showed that HbHSP90.1 interacted with HbSGT1b. Subcellular localization showed that both HbHSP90.1 and HbSGT1b located in the nucleus. Taken together, we speculate that HbHSP90.1 interacts with HbSGT1b in the nucleus to respond to rubber tree stress processes. The results of this study provide a solid foundation for further studies on the mechanism of HbHSP90 family genes in the stress resistance response of rubber tree.

The TRiCky Business of Protein Folding in Health and Disease.

Maintenance of the cellular proteome or proteostasis is an essential process that when deregulated leads to diseases like neurological disorders and cancer. Central to proteostasis are the molecular chaperones that fold proteins into functional 3-dimensional (3D) shapes and prevent protein aggregation. Chaperonins, a family of chaperones found in all lineages of organisms, are efficient machines that fold proteins within central cavities. The eukaryotic Chaperonin Containing TCP1 (CCT), also known as Tailless complex polypeptide 1 (TCP-1) Ring Complex (TRiC), is a multi-subunit molecular complex that folds the obligate substrates, actin, and tubulin. But more than folding cytoskeletal proteins, CCT differs from most chaperones in its ability to fold proteins larger than its central folding chamber and in a sequential manner that enables it to tackle proteins with complex topologies or very large proteins and complexes. Unique features of CCT include an asymmetry of charges and ATP affinities across the eight subunits that form the hetero-oligomeric complex. Variable substrate binding capacities endow CCT with a plasticity that developed as the chaperonin evolved with eukaryotes and acquired functional capacity in the densely packed intracellular environment. Given the decades of discovery on the structure and function of CCT, much remains unknown such as the scope of its interactome. New findings on the role of CCT in disease, and potential for diagnostic and therapeutic uses, heighten the need to better understand the function of this essential molecular chaperone. Clues as to how CCT causes cancer or neurological disorders lie in the early studies of the chaperonin that form a foundational knowledgebase. In this review, we span the decades of CCT discoveries to provide critical context to the continued research on the diverse capacities in health and disease of this essential protein-folding complex.

Molecular chaperone GP96 is a potential target to modulate dendritic cell programming and shape anti-tumor immunity

Abstract Dendritic cells (DCs) are professional antigen-presenting cells providing costimulatory signals to adaptive immune cells. Currently, the lack of strategies to improve antigen presentation is a hindrance in tumor immunology. Despite studies focusing on antigen cross-presenting Type-1 DCs (DC1s), a study published by our lab revealed Type-2 DCs (DC2s) with significant survival across multiple human cancers. Our research group has a long-term interest in studying immune chaperone GP96, which is an essential molecular chaperone for TLRs, GARP, and other vital innate receptors. The immune-related client network of GP96 creates opportunities to unmask the roles of multiple receptors on DCs. We have generated DC-specific GP96 deficient mice and reported increased tumor-infiltrating DC2 and delayed tumor development on the spontaneous breast cancer model. However, the mechanism by which GP96 regulates DCs function is under investigation. Using different tumor models, we found that deletion of GP96 on DCs improved immune response and decreased tumor growth. However, macrophage-GP96KO mice showed a loss of benefits observed on DC-GP96KO mice. Also, differential stimulatory/inhibitory molecules on DC1 vs. DC2 were tumor-dependent. To determine the role of DC-intrinsic GP96 in T cell activation, we used in vitro antigen-cross presentation assay. We found that OT-I CD8+ T cell activation was delayed at the initial days of the co-culture with GP96KO DCs but restored in the late phase compared with WT DCs. Collectively, the results show beneficial inflammatory DC2 molecular activation, with DC1 able to cross-present antigens to CD8+ T cells. Our study indicated that targeting GP96 on DCs may contribute to shaping T cell anti-tumor immunity. Supported by grants from NIH NCI (R01:CA193939) and NIH NIAID (U01:AI125859)

Nitrated Hsp90 Supports Glioblastoma Multiforme Cell Survival and Migration

Glioblastoma multiforme (GBM) is the most aggressive malignancy affecting the central nervous system. With no effective pharmacological options, patients undergo repeated surgeries to remove or debulk tumors. New targets for the development of safe and effective therapies are desperately needed. We and others have shown that solid tumors present high levels of protein tyrosine nitration, an oxidative post‐translational modification that occurs in pathological conditions involving oxidative stress but is absent in normal tissues. In many solid tumors tyrosine nitration correlates with poor prognosis, suggesting nitrated proteins may benefit tumor growth. We have previously shown that tyrosine nitration supports survival and migration of GBM cells. Here, we identified the first nitrated protein with pro‐tumorigenic activity, nitrated Heat shock protein 90 (Hsp90). Hsp90 is an essential molecular chaperone present in all cells. We showed that nitration of Hsp90 induces a gain‐of‐function that decreases mitochondrial metabolism in tumor cells. We detected endogenously nitrated Hsp90 (NO2Hsp90) in GBM‐patient derived cell culture models, and in 2D‐ and 3D‐ U87 cell culture models of GBM. To study the role of NO2Hsp90 in supporting GBM cell survival/proliferation, we prevented tyrosine nitration in U87 cells for 48 h to decrease the endogenous levels of nitrated proteins. In these conditions, cell viability decreases by ~25%, while the levels of nitrated proteins decrease by ~50%. We then intracellularly released NO2Hsp90 at levels comparable to those normally present in U87 cells (~1.5% of total cellular Hsp90 calculated by quantitative slot blot), and sustained prevention of endogenous tyrosine nitration for an additional 48 h. Cell viability was assessed at this final timepoint. If NO2Hsp90 is necessary and sufficient to support U87 cell survival/proliferation, intracellular release of the nitrated chaperone to cells containing decreased levels of nitrated proteins should rescue cell viability. Release of unmodified (UM) Hsp90 was used as control. Excitingly, the intracellular release of NO2Hsp90 but not UM Hsp90 significantly increased U87 cell survival/proliferation by ~50%. Intracellular release of NO2Hsp90 to untreated cells that contain endogenous NO2Hsp90 had no effect on cell proliferation but to our surprise, the extracellular release of NO2Hsp90 significantly increased cell numbers after 48 h incubation. This effect was significantly hindered by preventing endogenous tyrosine nitration, suggesting that intracellular nitrated proteins mediate the effect of extracellular NO2Hsp90. It is well described that extracellular Hsp90 (eHsp90) induces GBM motility and invasion. We detected eHsp90 in culture media conditioned by U87 cells by western blot. Using live transwell migration assays, we found that incubation with either extracellular UM‐ or NO2Hsp90 increased U87 cell migration versus control cells that did not receive protein. These results suggest that NO2Hsp90 plays multiple essential roles in GBM biology, posing NO2Hsp90 as a promising tumor‐directed therapeutic target.

Structure of Hsp90-p23-GR reveals the Hsp90 client-remodelling mechanism.

Hsp90 is a conserved and essential molecular chaperone responsible for the folding and activation of hundreds of 'client' proteins1-3. The glucocorticoid receptor (GR) is a model client that constantly depends on Hsp90 for activity4-9. GR ligand binding was previously shown to nr inhibited by Hsp70 and restored by Hsp90, aided by the co-chaperone p2310. However, a molecular understanding of the chaperone-mediated remodelling that occurs between the inactive Hsp70-Hsp90 'client-loading complex' and an activated Hsp90-p23 'client-maturation complex' is lacking for any client, including GR. Here we present a cryo-electron microscopy (cryo-EM) structure of the human GR-maturation complex (GR-Hsp90-p23), revealing that the GR ligand-binding domain is restored to a folded, ligand-bound conformation, while being simultaneously threaded through the Hsp90 lumen. In addition, p23 directly stabilizes native GR using a C-terminal helix, resulting in enhanced ligand binding. This structure of a client bound to Hsp90 in a native conformation contrasts sharply with the unfolded kinase-Hsp90 structure11. Thus, aided by direct co-chaperone-client interactions, Hsp90 can directly dictate client-specific folding outcomes. Together with the GR-loading complex structure12, we present the molecular mechanism of chaperone-mediated GR remodelling, establishing the first, to our knowledge,complete chaperone cycle for anyHsp90 client.

Genetic analysis of Hsp90 function in Cryptococcus neoformans highlights key roles in stress tolerance and virulence.

The opportunistic human fungal pathogen Cryptococcus neoformans has tremendous impact on global health, causing 181,000 deaths annually. Current treatment options are limited, and the frequent development of drug resistance exacerbates the challenge of managing invasive cryptococcal infections. In diverse fungal pathogens, the essential molecular chaperone Hsp90 governs fungal survival, drug resistance, and virulence. Therefore, targeting this chaperone has emerged as a promising approach to combat fungal infections. However, the role of Hsp90 in supporting C. neoformans pathogenesis remains largely elusive due to a lack of genetic characterization. To help dissect the functions of Hsp90 in C. neoformans, we generated a conditional expression strain in which HSP90 is under control of the copper-repressible promoter CTR4-2. Addition of copper to culture medium depleted Hsp90 transcript and protein levels in this strain, resulting in compromised fungal growth at host temperature; increased sensitivity to stressors, including the azole class of antifungals; altered C. neoformans morphology; and impaired melanin production. Finally, leveraging the fact that copper concentrations vary widely in different mouse tissues, we demonstrated attenuated virulence for the CTR4-2p-HSP90 mutant specifically in an inhalation model of Cryptococcus infection. During invasion and establishment of infection in this mouse model, the pathogen is exposed to the relatively high copper concentrations found in the lung as compared to blood. Overall, this work generates a tractable genetic system to study the role of Hsp90 in supporting the pathogenicity of C. neoformans and provides proof-of-principle that targeting Hsp90 holds great promise as a strategy to control cryptococcal infection.

Hsp90 chaperone code and the tumor suppressor VHL cooperatively regulate the mitotic checkpoint

Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes that plays a vital role in protecting and maintaining the functional integrity of deregulated signaling proteins in tumors. We have previously reported that the stability and activity of the mitotic checkpoint kinase Mps1 depend on Hsp90. In turn, Mps1-mediated phosphorylation Hsp90 regulates its chaperone function and is essential for the mitotic arrest. Cdc14-assisted dephosphorylation of Hsp90 is vital for the mitotic exit. Post-translational regulation of Hsp90 function is also known as the Hsp90 “Chaperone Code.” Here, we demonstrate that only the active Mps1 is ubiquitinated on K86, K827, and K848 by the tumor suppressor von Hippel-Lindau (VHL) containing E3 enzyme, in a prolyl hydroxylation-independent manner and degraded in the proteasome. Furthermore, we show that this process regulates cell exit from the mitotic checkpoint. Collectively, our data demonstrates an interplay between the Hsp90 chaperone and VHL degradation machinery in regulating mitosis.

Purification and biochemical characterization of Msi3, an essential Hsp110 molecular chaperone in Candida albicans.

Hsp110s are unique and essential molecular chaperones in the eukaryotic cytosol. They play important roles in maintaining cellular protein homeostasis. Candida albicans is the most prevalent yeast opportunistic pathogen that causes fungal infections in humans. As the only Hsp110 in Candida albicans, Msi3 is essential for the growth and infection of Candida albicans. In this study, we have expressed and purified Msi3 in nucleotide-free state and carried out biochemical analyses. Sse1 is the major Hsp110 in budding yeast S. cerevisiae and the best characterized Hsp110. Msi3 can substitute Sse1 in complementing the temperature-sensitive phenotype of S. cerevisiae carrying a deletion of SSE1 gene although Msi3 shares only 63.4% sequence identity with Sse1. Consistent with this functional similarity, the purified Msi3 protein shares many similar biochemical activities with Sse1 including binding ATP with high affinity, changing conformation upon ATP binding, stimulating the nucleotide-exchange for Hsp70, preventing protein aggregation, and assisting Hsp70 in refolding denatured luciferase. These biochemical characterizations suggested that Msi3 can be used as a model for studying the molecular mechanisms of Hsp110s.

Sequestration of the Transcription Factor STAT3 by the Molecular Chaperone CCT: A Potential Mechanism for Modulation of STAT3 Phosphorylation

Chaperonin Containing Tailless complex polypeptide 1 (CCT) is an essential molecular chaperone required for the folding of the abundant proteins actin and tubulin. The CCT oligomer also folds a range of other proteins and participates in non-folding activities such as providing assembly support for complexes of the von Hippel Lindau tumor suppressor protein and elongins. Here we show that the oncogenic transcription factor STAT3 binds to the CCT oligomer, but does not display the early binding upon translation in rabbit reticulocyte lysate typical of an obligate CCT folding substrate. Consistent with this, depletion of each of the CCT subunits by siRNA targeting indicates that loss of CCT oligomer does not suppress the activation steps of STAT3 upon stimulation with IL-6: phosphorylation, dimerisation and nuclear translocation. Furthermore, the transcriptional activity of STAT3 is not negatively affected by reduction in CCT levels. Instead, loss of CCT oligomer in MCF7 cells leads to an enhancement of STAT3 phosphorylation at Tyr705, implicating a role for the CCT oligomer in the sequestration of non-phosphorylated STAT3. Thus, as CCT is dynamic oligomer, the assembly state and also abundance of CCT oligomer may provide a means to modulate STAT3 phosphorylation.