Hsp90 Co-chaperone Research Articles

Unveiling the Role of Sorghum RPAP3 in the Function of R2TP Complex: Insights into Protein Assembly in Plants.

The chaperone R2TP has multiple subunits that assist in the proper folding, assembly, and stabilization of various protein complexes in cells and its study can offer valuable insights into the regulation and maintenance of protein assemblies in plant systems. The 'T' component of R2TP is Tah1 in yeast, consisting of 111 residues, while its counterpart in humans is RPAP3, with 665 residues. RPAP3 acts as a co-chaperone of Hsp90 and facilitates interactions between RUVBL proteins and other complex components, enhancing the recruitment of client proteins by the R2TP complex. These facts further underscore the relevance of studying this complex in different organisms. The putative gene corresponding to the RPAP3 in Sorghum bicolor, a monocotyledon plant, was cloned, and the protein (396 residues) purified for biochemical characterization. SbRPAP3 exists as a folded monomer and has a RPAP3 domain, which is present in human RPAP3 but absent in yeast Tah1. SbRPAP3 retains its functional capabilities, including binding with RUVBLs, Hsp90, and Hsp70. By elucidating the role of RPAP3 in plant R2TP complex, we can further comprehend the molecular mechanisms underlying plant-specific protein assembly and contribute to advancements in plant biology and biotechnological applications.

Abstract P1025: Hsp70 Co-chaperones Regulate Sarcomeric Proteostasis In Hypertrophic Cardiomyopathy

Significance: Recent Genome Wide Association Studies (GWAS) identified non-sarcomeric variants in hypertrophic cardiomyopathy (HCM) with three of the top 10 variant loci in or near genes that encode for co-chaperones of HSP70: BAG3, DNAJC18 and HSPB7. These variants are also present in dilated cardiomyopathy (DCM), but have an opposing effect on cardiac function, leading us to hypothesize that GWAS variants in HSP70 co-chaperones can affect the proteostasis of contractile proteins in the HCM heart. As a first step, we determined the effects each HSP70 co-chaperone knockdown (KD) had on sarcomeric and z-disc protein expression and the poly-ubiquitinated state of cardiomyocyte-specific proteins. Methods and Results: Transduction of human iPSC-CMs (iPSC-CMs) with adenoviral vectors driving shRNA expression against BAG3, DNAJC18, HSPB7, or scramble (control) yielded transduction efficiencies > 75% and >80% reduction in target transcript 4 days post-transduction. BAG3 KD increased poly-ubiquitinated proteins by 38+/-9% (p<0.001) and reduced steady state expression of sarcomeric and z-disc proteins, including: MyBP-C (p<0.0001), actin (p<0.0001), cardiac troponin I (p<0.05), tropomyosin (TPM1) (p<0.01), myosin light chain 3 (MYL3) (p<0.0001), filamin C (p<0.05), and alpha-actinin-2 (p<0.0001). DNAJC18 KD increased poly-ubiquitinated protein by 66+/-17% (p<0.01) but did not impact sarcomere protein expression. Conversely, HSPB7 KD reduced poly-ubiquitinated proteins by 21+/-7% (p<0.01) and increased protein expression of MyBP-C (p<0.01), TPM1 (p<0.05), and myosin light chain 2 (p<0.05). DNAJC18 KD increased BAG3 and HSPB7 protein expression levels by ~2-fold (p<0.001) without increasing transcript levels, suggesting BAG3 and HSPB7 may be protein clients of DNAJC18. Conclusions: HSP70 co-chaperones that have an established genetic association with human HCM play an important role in cardiomyocyte proteostasis. Multiple sarcomeric and z-disc proteins appear to be clients of BAG3, while BAG3 and HSPB7 themselves may be clients of DNAJC18. Targeting this axis could be therapeutic for patients with HCM that possess genetic variants in sarcomeric proteins.

The Crystal Structure of the Hsp90-LA1011 Complex and the Mechanism by Which LA1011 May Improve the Prognosis of Alzheimer's Disease.

Functional changes in chaperone systems play a major role in the decline of cognition and contribute to neurological pathologies, such as Alzheimer's disease (AD). While such a decline may occur naturally with age or with stress or trauma, the mechanisms involved have remained elusive. The current models suggest that amyloid-β (Aβ) plaque formation leads to the hyperphosphorylation of tau by a Hsp90-dependent process that triggers tau neurofibrillary tangle formation and neurotoxicity. Several co-chaperones of Hsp90 can influence the phosphorylation of tau, including FKBP51, FKBP52 and PP5. In particular, elevated levels of FKBP51 occur with age and stress and are further elevated in AD. Recently, the dihydropyridine LA1011 was shown to reduce tau pathology and amyloid plaque formation in transgenic AD mice, probably through its interaction with Hsp90, although the precise mode of action is currently unknown. Here, we present a co-crystal structure of LA1011 in complex with a fragment of Hsp90. We show that LA1011 can disrupt the binding of FKBP51, which might help to rebalance the Hsp90-FKBP51 chaperone machinery and provide a favourable prognosis towards AD. However, without direct evidence, we cannot completely rule out effects on other Hsp90-co-chaprone complexes and the mechanisms they are involved in, including effects on Hsp90 client proteins. Nonetheless, it is highly significant that LA1011 showed promise in our previous AD mouse models, as AD is generally a disease affecting older patients, where slowing of disease progression could result in AD no longer being life limiting. The clinical value of LA1011 and its possible derivatives thereof remains to be seen.

Dnajb11-Kidney Disease Develops from Reduced Polycystin-1 Dosage but not Unfolded Protein Response in Mice.

Heterozygous DNAJB11 mutation carriers manifest with small cystic kidneys and renal failure in adulthood. Recessive cases with prenatal cystic kidney dysplasia were recently described. Our in vitro and mouse model studies investigate the proposed disease mechanism as an overlap of autosomal-dominant polycystic kidney disease and autosomal-dominant tubulointerstitial kidney disease pathogenesis. We find that DNAJB11 loss impairs cleavage and maturation of the autosomal-dominant polycystic kidney disease protein polycystin-1 (PC1) and results in dosage-dependent cyst formation in mice. We find that Dnajb11 loss does not activate the unfolded protein response, drawing a fundamental contrast with the pathogenesis of autosomal-dominant tubulointerstitial kidney disease. We instead propose that fibrosis in DNAJB11 -kidney disease may represent an exaggerated response to polycystin-dependent cysts. Patients with heterozygous inactivating mutations in DNAJB11 manifest with cystic but not enlarged kidneys and renal failure in adulthood. Pathogenesis is proposed to resemble an overlap of autosomal-dominant polycystic kidney disease (ADPKD) and autosomal-dominant tubulointerstitial kidney disease (ADTKD), but this phenotype has never been modeled in vivo . DNAJB11 encodes an Hsp40 cochaperone in the endoplasmic reticulum: the site of maturation of the ADPKD polycystin-1 (PC1) protein and of unfolded protein response (UPR) activation in ADTKD. We hypothesized that investigation of DNAJB11 would shed light on mechanisms for both diseases. We used germline and conditional alleles to model Dnajb11 -kidney disease in mice. In complementary experiments, we generated two novel Dnajb11-/- cell lines that allow assessment of PC1 C-terminal fragment and its ratio to the immature full-length protein. Dnajb11 loss results in a profound defect in PC1 cleavage but with no effect on other cystoproteins assayed. Dnajb11-/- mice are live-born at below the expected Mendelian ratio and die at a weaning age with cystic kidneys. Conditional loss of Dnajb11 in renal tubular epithelium results in PC1 dosage-dependent kidney cysts, thus defining a shared mechanism with ADPKD. Dnajb11 mouse models show no evidence of UPR activation or cyst-independent fibrosis, which is a fundamental distinction from typical ADTKD pathogenesis. DNAJB11 -kidney disease is on the spectrum of ADPKD phenotypes with a PC1-dependent pathomechanism. The absence of UPR across multiple models suggests that alternative mechanisms, which may be cyst-dependent, explain the renal failure in the absence of kidney enlargement.

Differential localization of dengue virus protease affects cell homeostasis and triggers to thrombocytopenia

Thrombocytopenia is one of the symptoms of many virus infections which is the "hallmark" in the case of dengue virus. In this study, we show the differential localization of existing two forms of dengue virus protease, i.e., NS2BNS3 to the nucleus and NS3 to the nucleus and mitochondria. We also report a nuclear transcription factor, erythroid differentiation regulatory factor 1 (EDRF1), as the substrate for this protease. EDRF1 regulates the expression and activity of GATA1, which in turn controls spectrin synthesis. Both GATA1 and spectrins are required for platelet formation. On the other hand, we found that the mitochondrial activities will be damaged by NS3 localization which cleaves GrpEL1, a co-chaperone of mitochondrial Hsp70. Levels of both EDRF1 and GrpEL1 were found to deteriorate in dengue virus-infected clinical samples. Hence, we conclude that NS2BNS3-mediated EDRF1 cleavage and the NS3-led mitochondrial dysfunction account for thrombocytopenia.

The stability and function of human cochaperone Hsp40/DNAJA1 are affected by zinc removal and partially restored by copper

The imbalance in metal homeostasis can be associated with several human diseases, and exposure to increasing concentrations of metals promotes cell stress and toxicity. Therefore, understanding the cytotoxic effect of metal imbalance is important to unravel the biochemical mechanism of homeostasis and the action of potential protective proteins against metal toxicity. Several studies, including gene deletion in yeast, provide evidence indicating the possible indirect involvement of cochaperones from the Hsp40/DNAJA family in metal homeostasis, possibly through modulating the activity of Hsp 70.This work first investigated the effect of zinc and copper on the conformation and function of the human Hsp40 cochaperone DNAJA1, a zinc-binding protein. DNAJA1 was capable to complement the phenotype of a yeast strain deleted of the ydj1 gene, which was more sensitive to the presence of zinc and copper than the wild-type strain. To gain further insight about the role of the DNAJA family in metal binding, the recombinant human DNAJA1 protein was studied. Zinc removal from DNAJA1 affected both its stability and ability to act as a chaperone, i.e., to protect other proteins from aggregation. The reintroduction of zinc restored the native properties of DNAJA1 and, surprisingly, the addition of copper partially restored the native properties.

DNAJB6 isoform specific knockdown: Therapeutic potential for limb girdle muscular dystrophy D1

Dominant missense mutations in DNAJB6, a co-chaperone of HSP70, cause limb girdle muscular dystrophy (LGMD) D1. No treatments are currently available. Two isoforms exist, DNAJB6a and DNAJB6b, each with distinct localizations in muscle. Mutations reside in both isoforms, yet evidence suggests that DNAJB6b is primarily responsible for disease pathogenesis. Knockdown treatment strategies involving both isoforms carry risk, as DNAJB6 knockout is embryonic lethal. We therefore developed an isoform-specific knockdown approach using morpholinos. Selective reduction of each isoform was achieved invitro in primary mouse myotubes and human LGMDD1 myoblasts, as well as invivo in mouse skeletal muscle. To assess isoform specific knockdown in LGMDD1, we created primary myotube cultures from a knockin LGMDD1 mouse model. Using mass spectrometry, we identified an LGMDD1 protein signature related to protein homeostasis and myofibril structure. Selective reduction of DNAJB6b levels in LGMDD1 myotubes corrected much of the proteomic disease signature toward wild type levels. Additional invivo functional data is required to determine if selective reduction of DNAJB6b is a viable therapeutic target for LGMDD1.

Transcriptional regulation of Sis1 promotes fitness but not feedback in the heat shock response.

The heat shock response (HSR) controls expression of molecular chaperones to maintain protein homeostasis. Previously, we proposed a feedback loop model of the HSR in which heat-denatured proteins sequester the chaperone Hsp70 to activate the HSR, and subsequent induction of Hsp70 deactivates the HSR (Krakowiak et al., 2018; Zheng et al., 2016). However, recent work has implicated newly synthesized proteins (NSPs) - rather than unfolded mature proteins - and the Hsp70 co-chaperone Sis1 in HSR regulation, yet their contributions to HSR dynamics have not been determined. Here, we generate a new mathematical model that incorporates NSPs and Sis1 into the HSR activation mechanism, and we perform genetic decoupling and pulse-labeling experiments to demonstrate that Sis1 induction is dispensable for HSR deactivation. Rather than providing negative feedback to the HSR, transcriptional regulation of Sis1 by Hsf1 promotes fitness by coordinating stress granules and carbon metabolism. These results support an overall model in which NSPs signal the HSR by sequestering Sis1 and Hsp70, while induction of Hsp70 - but not Sis1 - attenuates the response.

Molecular Chaperones' Potential against Defective Proteostasis of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neuronal degenerative condition identified via a build-up of mutant aberrantly folded proteins. The native folding of polypeptides is mediated by molecular chaperones, preventing their pathogenic aggregation. The mutant protein expression in ALS is linked with the entrapment and depletion of chaperone capacity. The lack of a thorough understanding of chaperones' involvement in ALS pathogenesis presents a significant challenge in its treatment. Here, we review how the accumulation of the ALS-linked mutant FUS, TDP-43, SOD1, and C9orf72 proteins damage cellular homeostasis mechanisms leading to neuronal loss. Further, we discuss how the HSP70 and DNAJ family co-chaperones can act as potential targets for reducing misfolded protein accumulation in ALS. Moreover, small HSPB1 and HSPB8 chaperones can facilitate neuroprotection and prevent stress-associated misfolded protein apoptosis. Designing therapeutic strategies by pharmacologically enhancing cellular chaperone capacity to reduce mutant protein proteotoxic effects on ALS pathomechanisms can be a considerable advancement. Chaperones, apart from directly interacting with misfolded proteins for protein quality control, can also filter their toxicity by initiating strong stress-response pathways, modulating transcriptional expression profiles, and promoting anti-apoptotic functions. Overall, these properties of chaperones make them an attractive target for gaining fundamental insights into misfolded protein disorders and designing more effective therapies against ALS.

Targeting co-chaperones of HSP70 to modulate Myosin Binding Protein C turnover in hypertrophic cardiomyopathy.

Rationale: Hypertrophic Cardiomyopathy (HCM) is an inherited cardiovascular disease that affects 1 in 500 people. Over half of familial HCM cases arise from MyBP-C haploinsufficiency, thus stabilizing MyBP-C protein expression is an appealing therapeutic approach. Recently, two GWAS studies identified multiple HCM risk alleles. Three of the top 10 are localized near or in gene loci that code for HSP70 co-chaperones, highlighting the important role of the HSP70 system in HCM. Hypothesis: Co-chaperones of HSP70, (BAG3, DNAJC18, and HSPB7) regulate the expression and stability of sarcomeric and z-disc proteins, specifically MyBP-C. Methods/Results: Proximity labeling was performed to identify which components of the HSP70 co-chaperone network interact with or lie in close proximity to MyBP-C. Human iPSC-CMs (hiPSC-CMs) were transduced with a Turbo-ID-MYBPC3 adenoviral (AdV) construct, treated with biotin, and biotinylated proteins were captured by streptavidin beads. HSPA1A (i.e. HSP70), BAG3, DNAJC13, HSPB7, HSPB1, and CRYAB were identified by mass spectrometry as proximity proteins to wild-type MyBP-C but were absent in controls (AdV-MyBP-C without Turbo). hiPSC-CMs were transfected with AdV-shRNA to knock down HSP70 co-chaperone expression (BAG3, DNAJC18, or HSPB7). DNAJC18 was included because of the risk allele identified in GWAS, even though DNAJC18 was not identified as a direct interactor with MyBP-C during proximity labeling. Adv transfection efficiency was > 70% (p<0.0001) and resulted in a >75% decrease (p<0.01) in co-chaperone transcript expression and a >50% decrease (p<0.001) in protein. BAG3 knockdown decreased the steady state protein expression of MyBP-C (p<0.0001) and a majority of other sarcomeric proteins. Utilizing a cycloheximide (CHX) chase assay to measure protein ½ life, MyBP-C protein degradation was greatly accelerated by BAG3 knockdown compared to scrambled shRNA 24 hours post-CHX (p<0.0001). DNAJC18 knockdown had no significant effects on steady state protein expression of MyBP-C or other sarcomeric proteins, while HSPB7 knockdown increased MyBP-C (p<0.01), myosin (p<0.01), tropomyosin (p<0.05), and myosin light chain 2 (p<0.05) protein compared to scrambled shRNA. DNAJC18 and HSPB7 knockdown each increased BAG3 protein expression (p<0.01), while BAG3 knockdown reduced DNAJC18 protein (p<0.0001) but had no effect on HSPB7. Conclusion: MyBP-C interacts with and is stabilized by BAG3. MyBP-C may also be a direct client for HSPB7, but further studies are needed. Knockdown experiments of DNAJC13, HSBP1 and CRYAB, identified as proximity proteins of MyBP-C by Turbo-ID, are also underway. The HSP70 co-chaperone network may serve a therapeutic target to normalize MyBP-C levels in the context of MyBP-C haploinsufficiency. Institutional Start Up Funds This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identifying the role of HSP70 co-chaperones in modulating sarcomeric proteostasis

Background: Hypertrophic cardiomyopathy (HCM) is the most common genetically inherited cardiovascular disease that affects one in 500 people. Variants in MYBPC3 result in premature termination codons, ultimately causing haploinsufficiency in MyBP-C protein–a hallmark of familial HCM pathophysiology. Recent GWAS reports identify risk alleles for HCM that are concordant with left ventricular (LV) functional traits of decreased left ventricular chamber volumes and increased ejection fraction, and in contrast are protective alleles for dilated cardiomyopathy. Several of the top HCM risk alleles encode for co-chaperones of HSP70. However, how these co-chaperones directly modulate sarcomeric protein turnover and HCM pathophysiology is unknown. Hypothesis: We hypothesize that HSP70 co-chaperones identified by recent GWAS regulate contractility by affecting sarcomeric and Z-disc steady state expression. Methods and Results: Human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) were transduced with GFP-tagged adenovirus (AdV) expressing shRNA targeted against BAG3, DNAJC18, HSPB7, or scrambled shRNA (control) and transduction efficiency was assessed by flow cytometry for GFP expression. A >70% transduction efficiency was achieved with each viral construct (p<0.0001). Viral transduction and knockdown of HSP70 co-chaperone did not induce cellular toxicity. BAG3, DNAJC18, and HSPB7 were reduced by ≥50% at both the transcript and protein levels as measured via RT-qPCR and western blot. BAG3 KD reduced MyBP-C protein levels (p<0.0001) by 53%, along with most other profiled sarcomeric and Z-disk proteins (p<0.05). DNAJC18 had no significant effect on MyBP-C protein levels, but caused a decrease in tropomyosin (p<0.0001) and myosin light chain 3 (p<0.0001). HSPB7 KD increased MyBP-C protein levels (p<0.01) by 46%. Myosin (p<0.01), tropomyosin (p<0.05), and myosin light chain 2 (p<0.05) also showed increased expression upon HSPB7 knockdown. Conclusion: GWAS-identified HSP70 co-chaperones BAG3, DNAJC18, and HSPB7 regulate the steady state expression of sarcomeric and Z-disc proteins. Further studies will be needed to elucidate whether regulation is occurring at the transcription, translational, or posttranslational level, and whether therapeutic modulation of these co-chaperones can be leveraged to stabilize MyBP-C and other sarcomeric and Z-disc proteins in patients with familial HCM. Institutional/internal grants This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Role of heat shock proteins in oncogenesis and strategy for treating cancers using Drosophila model

A cluster of closely stored molecules that are engaged in different cellular processes is the mammalian HSP90 protein family. HSP90 works with HSP70 but is not dependent on the cochaperone of HSP90. HSPs are molecular chaperones required to fold, stabilize, and degrade proteins that are responsible for multiple tumor types. Hence targeting HSP90 and HSP70 presents a striking strategy for treating cancers. The present review article provides an overview of the structure, mode of action as well as role of HSP90 and HSP70. They are part of key regulatory processes that directly influence oncogenic pathways. As Drosophila melanogaster acts as an appropriate model system for research studies involving the above-stated disorders, emphasis has been put on summarizing some of the research studies in oncogenesis.

Abstract 4555: Aryl-hydrocarbon receptor inhibitors in combination with anticancer agents, especially proteasome pathway inhibitors, in a complex spheroid screen using patient-derived cell lines can result in greater-than additive cytotoxicity

Abstract Three aryl-hydrocarbon receptor (AhR) selective inhibitors, BAY-2416964, GNF351 and CH-223191, were assayed alone and in combination with 25 approved or investigational anticancer agents in complex spheroids including tumor cells, endothelial cells, and mesenchymal stem cells. The tumor cell lines were from the National Cancer Institute’s Patient-Derived Models Repository (PDMR) collection (https://pdmr.cancer.gov/models/database.htm). The AhR is a ligand-activated helix-loop-helix transcription factor of the bHLH-PAS family. It is involved in the regulation of responses to planar aromatic hydrocarbons by activating the transcription of xenobiotic-metabolizing enzymes such as cytochrome P450 or by acting as an E3 ligase. Thus, the AhR activates detoxification pathways that dispose of small molecule toxins and damaged proteins. In the absence of xenobiotics, the AhR is located within the cytoplasm in complex with two Hsp90 molecules and co-chaperones. Upon xenobiotic binding, the AhR translocates to the nucleus and binds to the AhR nuclear translocator protein to form an active transcription factor complex and promote gene transcription. As single agents, the three AhR inhibitors showed little cytotoxicity at concentrations up to 10 µM. Combinations of AhR inhibitors with anticancer agents including doxorubicin, cisplatin, SN38, venetoclax, selinexor, and etoposide (https://dtp.cancer.gov/organization/dscb/obtaining/default.htm) produced primarily additive cytotoxicity in complex spheroids after a 7-day exposure. However, unexpected greater-than-additive effects were observed with AhR inhibitors in combination with proteasome pathway inhibitors. Bortezomib, a direct inhibitor of the chymotrypsin-like protease of the 26S proteasome, and pevonedistat, a NEDD8-activating enzyme inhibitor, produced additive or greater-than-additive cytotoxicity in some complex spheroids. However, the combination of BAY-2416964 with TAK-243, a ubiquitin activating enzyme inhibitor, produced profound greater-than-additive cytotoxicity in more than half of the 28 PDMR cell lines tested as complex spheroids. The combination resulted in an increase of 1- to 3-logs of excess cytotoxicity in malignancies including bladder cancer, pancreatic cancer, colon cancer, NSCLC, SCLC and glioblastoma. In vivo studies are under discussion. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: BEVERLY A. TEICHER, Jeffrey A. Moscow, Joel Morris, James H. Doroshow, Thomas S. Dexheimer, Nathan P. Coussens, Thomas Silvers, Rene Delosh, Zahra Davoudi, Russel Reinhart, Chad Ogle, Eric Jones. Aryl-hydrocarbon receptor inhibitors in combination with anticancer agents, especially proteasome pathway inhibitors, in a complex spheroid screen using patient-derived cell lines can result in greater-than additive cytotoxicity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4555.

Structural and biochemical insights into FKBP51 as a Hsp90 co-chaperone.

The FK506-binding protein 51 (FKBP51) is a high-molecular-weight immunophilin that emerged as an important drug target for stress-related disorders, chronic pain, and obesity. It has been implicated in a plethora of molecular pathways but remains best characterized as a co-chaperone of Hsp90 in the steroid hormone receptor (SHR) maturation cycle. However, the mechanistic and structural basis for the regulation of SHRs by FKBP51 and the usually antagonistic function compared withits closest homolog FKBP52 remains enigmatic. Herewe review recent structural and biochemical studies of FKBPs as regulators in the Hsp90 machinery. These advances provide important insights into the roles of FKBP51 and FKBP52 in SHR regulation.

Hypoviral-regulated HSP90 co-chaperone p23 (CpCop23) determines the colony morphology, virulence, and viral response of chestnut blight fungus Cryphonectria parasitica.

We previously identified a protein spot that showed down-regulation in the presence of Cryphonectria hypovirus 1 (CHV1) and tannic acid supplementation as a Hsp90 co-chaperone p23 gene (CpCop23). The CpCop23-null mutant strain showed retarded growth with less aerial mycelia and intense pigmentation. Conidia of the CpCop23-null mutant were significantly decreased and their viability was dramatically diminished. The CpCop23-null mutant showed hypersensitivity to Hsp90 inhibitors. However, no differences in responsiveness were observed after exposure to other stressors such as temperature, reactive oxygen species, and high osmosis, the exception being cell wall-disturbing agents. A severe reduction in virulence was observed in the CpCop23-null mutant. Interestingly, viral transfer to the CpCop23-null mutant from CHV1-infected strain via anastomosis was more inefficient than a comparable transfer with the wild type as a result of decreased hyphal branching of the CpCop23-null mutant around the peripheral region, which resulted in less fusion of the hyphae. The CHV1-infected CpCop23-null mutant exhibited recovered mycelial growth with less pigmentation and sporulation. The CHV1-transfected CpCop23-null mutant demonstrated almost no virulence, that is, even less than that of the CHV1-infected wild type (UEP1), a further indication that reduced virulence of the mutant is not attributable exclusively to the retarded growth but rather is a function of the CpCop23 gene. Thus, this study indicates that CpCop23 plays a role in ensuring appropriate mycelial growth and development, spore viability, responses to antifungal drugs, and fungal virulence. Moreover, the CpCop23 gene acts as a host factor that affects CHV1-infected fungal growth and maintains viral symptom development.

Molecular dynamics study of the mechanism of the client unfolded protein delivery from HSP40 to HSP70.

Heat Shock Protein (HSP) 70 is one of the molecular chaperones that supports protein folding. HSP70 is attracting attention as a target for drug discovery because of its involvement in cancer, neurodegenerative diseases, and so on. HSP70 consists of the nucleotide-binding domain (NBD) and the substrate-binding domain (SBD), with a flexible-linker region between the NBD and SBD. The SBD has two regions, SBD-α, which contains alpha-helices, and SBD-β, which contains beta-sheets. In the HSP70 chaperone cycle, unfolded client proteins are delivered by a J-domain protein (JDP), such as a co-chaperone HSP40. The J-domain in the JDP binds to the ATP-bound HSP70 state when the substrate is delivered to HSP70. After that, the JDP stimulates the ATP hydrolysis of HSP70, and the conformational change to the ADP-bound HSP70 form. Then the HSP70 helps the client protein folding with the nucleotide exchange factor molecular cooperation. However, the complex structure of JDP and HSP70 has not been solved, and the mechanism of substrate transfer has not been in detail yet. In this research, to clarify the mechanism of the substrate transfer process from HSP40 to HSP70, we performed molecular dynamics simulations of HSP40, HSP70, and HSP70 with the J-domain. J-domain in the HSP40 has two forms, open and closed. We modeled the complex structure of JDP and HSP70 using the open form of the HSP40. We found that SBD-β of HSP70 locates between two J-domains of HSP40, and HSP40 fluctuates. Therefore, the SBD-β would scoop the substrate that binds to the HSP40. Also, we found that the J-domain binding induced the fluctuations around the ATP binding site and the substrate-binding site from the simulation of HSP70 and the J-domain complex structure.

Plk4 Is a Novel Substrate of Protein Phosphatase 5.

The conserved Ser/Thr protein phosphatase 5 (PP5) is involved in the regulation of key cellular processes, including DNA damage repair and cell division in eukaryotes. As a co-chaperone of Hsp90, PP5 has been shown to modulate the maturation and activity of numerous oncogenic kinases. Here, we identify a novel substrate of PP5, the Polo-like kinase 4 (Plk4), which is the master regulator of centriole duplication in animal cells. We show that PP5 specifically interacts with Plk4, and is able to dephosphorylate the kinase in vitro and in vivo, which affects the interaction of Plk4 with its partner proteins. In addition, we provide evidence that PP5 and Plk4 co-localize to the centrosomes in Drosophila embryos and cultured cells. We demonstrate that PP5 is not essential; the null mutant flies are viable without a severe mitotic phenotype; however, its loss significantly reduces the fertility of the animals. Our results suggest that PP5 is a novel regulator of the Plk4 kinase in Drosophila.

Balanced activities of Hsp70 and the ubiquitin proteasome system underlie cellular protein homeostasis.

To counteract proteotoxic stress and cellular aging, protein quality control (PQC) systems rely on the refolding, degradation and sequestration of misfolded proteins. In Saccharomyces cerevisiae the Hsp70 chaperone system plays a central role in protein refolding, while degradation is predominantly executed by the ubiquitin proteasome system (UPS). The sequestrases Hsp42 and Btn2 deposit misfolded proteins in cytosolic and nuclear inclusions, thereby restricting the accessibility of misfolded proteins to Hsp70 and preventing the exhaustion of limited Hsp70 resources. Therefore, in yeast, sequestrase mutants show negative genetic interactions with double mutants lacking the Hsp70 co-chaperone Fes1 and the Hsp104 disaggregase (fes1Δ hsp104Δ, ΔΔ) and suffering from low Hsp70 capacity. Growth of ΔΔbtn2Δ mutants is highly temperature-sensitive and results in proteostasis breakdown at non-permissive temperatures. Here, we probed for the role of the ubiquitin proteasome system in maintaining protein homeostasis in ΔΔbtn2Δ cells, which are affected in two major protein quality control branches. We show that ΔΔbtn2Δ cells induce expression of diverse stress-related pathways including the ubiquitin proteasome system to counteract the proteostasis defects. Ubiquitin proteasome system dependent degradation of the stringent Hsp70 substrate firefly Luciferase in the mutant cells mirrors such compensatory activities of the protein quality control system. Surprisingly however, the enhanced ubiquitin proteasome system activity does not improve but aggravates the growth defects of ΔΔbtn2Δ cells. Reducing ubiquitin proteasome system activity in the mutant by lowering the levels of functional 26S proteasomes improved growth, increased refolding yield of the Luciferase reporter and attenuated global stress responses. Our findings indicate that an imbalance between Hsp70-dependent refolding, sequestration and ubiquitin proteasome system-mediated degradation activities strongly affects protein homeostasis of Hsp70 capacity mutants and contributes to their severe growth phenotypes.

Multiple Targeting of HSP Isoforms to Challenge Isoform Specificity and Compensatory Expression.

Heat shock proteins (HSPs) are molecular chaperones that assist in protein folding, trafficking, and metabolism. Intracellular chaperone functions of HSPs had been well-investigated, but extracellular and exosomal HSPs have been recently found. Exosomal HSPs are intercellularly transferred, while extracellular HSPs play cytokine-like roles called chaperokines. We have shown that exosomal HSPs play key roles in intercellular communication between tongue carcinoma and tumor-associated macrophages in the tumor microenvironment. Notably, HSP90 isoforms consist of HSP90alpha, HSP90beta, mitochondrial TRAP1, and GRP94 in the endoplasmic reticulum. Moreover, many pseudogenes of HSP90 can be transcribed into RNA. Besides, the function of HSP90 is defined by their cochaperones, such as CDC37 or AHA1. Therefore, isoform-specific small interfering RNA (siRNA) is necessary for precisely targeting each HSP90 isoform and cochaperone. Nevertheless, we often encountered compensatory expression of HSP90 isoforms in the knockdown studies. Here, we provide dual and triple knockdown methods to target multiple RNA for challenging isoform-specific roles and compensatory expression of intracellular, extracellular, and exosomal HSPs.

Phosphorylation of the Hsp90 Co-Chaperone Hop Changes its Conformational Dynamics and Biological Function

The molecular chaperones Hsp90 and Hsp70 and their regulatory co-chaperone Hop play a key role at the crossroads of the folding pathways of numerous client proteins by forming fine-tuned multiprotein complexes. Alterations of the biomolecules involved may functionally impact the chaperone machinery: here, we integrate simulations and experiments to unveil how Hop conformational fitness and interactions can be controlled by the perturbation of just one residue. Specifically, we unveil how mechanisms mediated by Hop residue Y354 control Hop open and closed states, which affect binding of Hsp70/Hsp90. Phosphorylation or mutation of Hop-Y354 are shown to favor structural ensembles that are indeed not optimal for stable interactions with Hsp90 and Hsp70. This disfavors cellular accumulation of the stringent Hsp90 clients glucocorticoid receptor and the viral tyrosine kinase v-Src, with detrimental effects on v-Src activity. Our results show how the post-translational modification of a specific residue in Hop provides a regulation mechanism for the larger chaperone complex of which it is part. In this framework, the effects of one single alteration are amplified at the cellular level through the perturbation of protein-interaction networks.