HSP90 as a regulator of extracellular matrix dynamics.

The extracellular matrix (ECM) is a dynamic and organised extracellular network assembled from proteins and carbohydrates exported from the cell. The ECM is critical for multicellular life, providing spatial and temporal cellular cues to maintain tissue homeostasis. Consequently, ECM production must be carefully balanced with turnover to ensure homeostasis; ECM dysfunction culminates in disease. Hsp90 is a molecular chaperone central to protein homeostasis, including in the ECM. Intracellular and extracellular Hsp90 isoforms collaborate to regulate the levels and status of proteins in the ECM via multiple mechanisms. In so doing, Hsp90 regulates ECM dynamics, and changes in Hsp90 levels or activity support the development of ECM-related diseases, like cancer and fibrosis. Consequently, Hsp90 levels may have prognostic value, while inhibition of Hsp90 may have therapeutic potential in conditions characterised by ECM dysfunction.

Molecular mapping of platelet hyperreactivity in diabetes: the stress proteins complex HSPA8/Hsp90/CSK2α and platelet aggregation in diabetic and normal platelets.

The molecular understanding of the pathophysiological changes elicited by diabetes in platelets may help in further elucidating the involvement of this pseudo-cell in the increased risk of developing cardiovascular disease and thrombosis in diabetic subjects. We aimed to investigate the differential characteristics of platelets from diabetic patients and nondiabetic controls to unveil the molecular mechanisms behind the increased platelet reactivity in diabetes. We compared platelets from diabetic and control subjects by 2 dimensional-electrophoresis followed by mass spectrometry. Changes in selected differential proteins were validated by immunoprecipitation assays and western blot. Platelet aggregation was measured by light transmittance aggregometry induced by collagen and ADP, and dynamic coagulation analysis of whole blood was measured by thromboelastometry. We observed significant differences in proteins related to platelet aggregation, cell migration, and cell homeostasis. Subjects with diabetes showed higher platelet aggregation and thrombogenicity and higher contents of the stress-related protein complex HSPA8/Hsp90/CSK2α than nondiabetic subjects. Changes in the chaperones HSPA8 and Hsp90, and in CSK2α protein contents correlated with changes in platelet aggregation and blood coagulation activity. In conclusion, the complex HSPA8/Hsp90/CSK2α is involved in diabetes-related platelet hyperreactivity. The role of the HSPA8/Hsp90/CSK2α complex may become a molecular target for the development of future preventive and therapeutic strategies for platelet dysfunction associated with diabetes and its complications.

Heat shock induces the nuclear accumulation of YAP1 via SRC.

Yes-associated protein 1 (YAP1), a co-transcription activator, shuttles between the cytoplasm and the nucleus. Phosphorylation by large tumor suppressor kinases (LATS1/2) is the major determinant of YAP1 subcellular localization. Unphosphorylated YAP1 interacts with transcription factors in the nucleus and regulates gene transcription, while phosphorylated YAP1 is trapped in the cytoplasm and is degraded. We found that when U2OS and HeLa cells are exposed to 42 °C, YAP1 enters the nucleus within 30 min and returns to the cytoplasm at 4 h. SRC and HSP90 are involved in nuclear accumulation and return to the cytoplasm, respectively. Upon heat shock, LATS2 forms aggregates including protein phosphatase 1 and is dephosphorylated and inactivated. SRC activation is necessary for the formation of aggregates, while HSP90 is required for their dissociation. YAP1 is involved in heat shock-induced NF-κB signaling. Mechanistically, YAP1 is implicated in strengthening the interaction between RELA and DPF3, a component of SWI/SNF chromatin remodeling complex, in response to heat shock. Thus, YAP1 plays a role as a thermosensor.

The Role of Heat Shock Protein-90 in the Pathogenesis of Birt-Hogg-Dubé and Tuberous Sclerosis Complex Syndromes.

Birt-Hogg-Dubé (BHD) and tuberous sclerosis (TS) syndromes share many clinical features. These two diseases display distinct histologic subtypes of renal tumors: chromophobe renal cell carcinoma and renal angiomyolipoma, respectively. Early work suggested a role for mTOR dysregulation in the pathogenesis of these two diseases, however their detailed molecular link remains elusive. Interestingly, a growing number of case reports describe renal angiomyolipoma in BHD patients, suggesting a common molecular origin. The BHD-associated proteins FNIP1/2 and the TS protein Tsc1 were recently identified as regulators of the molecular chaperone Hsp90. Dysregulation of Hsp90 activity has previously been reported to support tumorigenesis, providing a potential explanation for the overlapping phenotypic manifestations in these two hereditary syndromes.

HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation.

Recently, it has been found that the CacyBP/SIP protein acts as HSP90 co-chaperone and exhibits chaperone properties itself. Namely, CacyBP/SIP has been shown to protect citrate synthase from aggregation and to recover the activity of thermally denatured luciferase in vitro. In the present work, we have analyzed the influence of CacyBP/SIP on aggregation of α-synuclein, a protein present in Lewy bodies of Parkinson's disease brain. By applying a thioflavin T (ThT) fluorescence assay, we have found that CacyBP/SIP protects α-synuclein from aggregation and that the fragment overlapping the N-terminal part and the CS domain of CacyBP/SIP is crucial for this activity. This protective effect of CacyBP/SIP has been confirmed by results obtained using high-speed ultracentrifugation followed by dot-blot and by transmission electron microscopy (TEM). Interestingly, CacyBP/SIP exhibits the protective effect only at the initial phase of α-synuclein aggregation. In addition, we have found that, in HEK293 cells overexpressing CacyBP/SIP, there are less α-synuclein inclusions than in control ones. Moreover, these cells are more viable when treated with rotenone, an agent that mimics PD pathology. By applying proximity ligation assay (PLA) on HEK293 cells and in vitro assays with the use of purified recombinant proteins, we have found that CacyBP/SIP directly interacts with α-synuclein. Altogether, in this work, we show for the first time that CacyBP/SIP is able to protect α-synuclein from aggregation in in vitro assays. Thus, our results point to an important role of CacyBP/SIP in the pathology of Parkinson's disease and other synucleinopathies.

New insights into molecular chaperone TRAP1 as a feasible target for future cancer treatments.

Tumor necrosis factor receptor-associated protein 1 (TRAP1), a molecular chaperone, is a major member of the mitochondrial heat shock protein 90 (Hsp90) family. Studies have shown that TRAP1 can prevent hypoxia-induced damage to cardiomyocytes, maintain cardiomyocytes viability and mitochondrial membrane potential, and protect cardiomyocytes. In addition, it can also protect astrocytes from ischemic damage in vitro. In recent years, there have been many new discoveries in tumors. The abnormal expression of TRAP1 is closely related to the occurrence and development of various tumors. TRAP1 protein seems to be a central regulatory protein, involved in the activation of various oncogenic proteins and signaling pathways, and has a balanced function at tumor transformation and the intersection of different metabolic processes. Targeting its chaperone activity and molecular interactions can destroy the metabolism and survival adaptability of tumor cells, paving the way for the development of highly selective mitochondrial anti-tumor drugs. Moreover, the combination of TRAP1 inhibition and current traditional cancer therapies has shown promising applications. These findings have important implications for the diagnosis and treatment of tumors. Therefore, we reviewed the recently identified functions of the molecular chaperone TRAP1 in cancer development and progression, as well as the discovery and recent advances in selective TRAP1 inhibitors as anticancer drug therapies, opening up new attractive prospects for exploring strategies for targeting TRAP1 as a tumor cell target.

The highly interrelated GHRH, p53, and Hsp90 universe.

p53 universe is composed of a complex regulatory network, destined to counteract multifarious challenges threatening cell survival. Imbalance in those responses may result in human disease associated with inevitable consequences. The present work delivers our view of the corresponding phenomena, by involving the endothelium defender in meticulously orchestrated events against inflammatory stimuli. Immersing into the great depths of p53 cosmos may lead to promising therapies against devastating disorders, including acute respiratory distress syndrome.

Hsp90 and complex birth defects: A plausible mechanism for the interaction of genes and environment.

How genes and environment interact to cause birth defects is not well understood, but key to developing new strategies to modify risk. The threshold model has been proposed to represent this complex interaction. This model stipulates that while environmental exposure or genetic mutation alone may not result in a defect, factors in combination increase phenotypic variability resulting in more individuals crossing the disease threshold where birth defects manifest. Many environmental factors that contribute to birth defects induce widespread cellular stress and misfolding of proteins. Yet, the impact of the stress response on the threshold model is not typically considered in discephering the etiology of birth defects. This mini-review will explore a potential mechanism for gene-environment interactions co-opted from studies of evolution. This model stipulates that heat shock proteins that mediate the stress response induced by environmental factors can influence the number of individuals that cross disease thresholds resulting in increased incidence of birth defects. Studies in the field of evolutionary biology have demonstrated that heat shock proteins and Hsp90 in particular provide a link between environmental stress, genotype and phenotype. Hsp90 is a highly expressed molecular chaperone that assists a wide variety of protein clients with folding and conformational changes needed for proper function. Hsp90 also chaperones client proteins with potentially deleterious amino acid changes to suppress variation caused by genetic mutations. However, upon exposure to stress, Hsp90 abandons its normal physiological clients and is diverted to assist with the misfolded protein response. This can impact the activity of signaling pathways that involve Hsp90 clients as well as unmask suppressed protein variation, essentially creating complex traits in a single step. In this capacity Hsp90 acts as an evolutionary capacitor allowing stored variation to accumulate and then become expressed in times of stress. This mechanism provides a substrate which natural selection can act upon at the population level allowing survival of the species with selective pressure. However, at the level of the individual, this mechanism can result in simultaneous expression of deleterious variants as well as reduced activity of a variety of Hsp90 chaperoned pathways, potentially shifting phenotypic variability over the disease threshold resulting in birth defects.

Heat Shock Protein 90 is Required for cAMP-Induced Differentiation in Rat Primary Schwann Cells.

Schwann cells (SCs) play an important role in producing myelin for rapid neurotransmission in the peripheral nervous system. Activation of the differentiation and myelination processes in SCs requires the expression of a series of transcriptional factors including Sox10, Oct6/Pou3f1, and Egr2/Krox20. However, functional interactions among several transcription factors are poorly defined and the important components of the regulatory network are still unknown. Until now, available evidence suggests that SCs require cAMP signaling to initiate the myelination program. Heat shock protein 90 (Hsp90) is known as a chaperone required to stabilize ErbB2 receptor. In recent years, it was reported that cAMP transactivated the ErbB2/ErbB3 signaling in SCs. However, the relationship between Hsp90 and cAMP-induced differentiation in SCs is undefined. Here we investigated the role of Hsp90 during cAMP-induced differentiation of SCs using Hsp90 inhibitor, geldanamycin and Hsp90 siRNA transfection. Our results showed that dibutyryl-cAMP (db-cAMP) treatment upregulated Hsp90 expression and led to nuclear translocation of Gab1/ERK, the downstream signaling pathway of the ErbB2 signaling mechanism in myelination. The expression of myelin-related genes and nuclear translocation of Gab1/ERK following db-cAMP treatment was inhibited by geldanamycin pretreatment and Hsp90 knockdown. These findings suggest that Hsp90 might play a role in cAMP-induced differentiation via stabilization of ErbB2 and nuclear translocation of Gab1/ERK in SCs.

Heat Shock Protein 90 Ensures the Integrity of Rubella Virus p150 Protein and Supports Viral Replication.

Two viral nonstructural proteins, p150 and p90, are expressed in rubella virus (RUBV)-infected cells and mediate viral genome replication, presumably using various host machineries. Molecular chaperones are critical host factors for the maintenance of cellular proteostasis, and certain viral proteins use this chaperone system. The RUBV p150 and p90 proteins are generated from a precursor polyprotein, p200, via processing by the protease activity of its p150 region. This processing is essential for RUBV genome replication. Here we show that heat shock protein 90 (HSP90), a molecular chaperone, is an important host factor for RUBV genome replication. The treatment of RUBV-infected cells with the HSP90 inhibitors 17-allylamino-17-desmethoxygeldanamycin (17-AAG) and ganetespib suppressed RUBV genome replication. HSP90α physically interacted with p150, but not p90. Further analyses into the mechanism of action of the HSP90 inhibitors revealed that HSP90 activity contributes to p150 functional integrity and promotes p200 processing. Collectively, our data demonstrate that RUBV p150 is a client of the HSP90 molecular chaperone and that HSP90 functions as a key host factor for RUBV replication.IMPORTANCE Accumulating evidence indicates that RNA viruses use numerous host factors during replication of their genomes. However, the host factors involved in rubella virus (RUBV) genome replication are largely unknown. In this study, we demonstrate that the HSP90 molecular chaperone is needed for the efficient replication of the RUBV genome. Further, we reveal that HSP90 interacts with RUBV nonstructural protein p150 and its precursor polyprotein, p200. HSP90 contributes to the stability of p150 and the processing of p200 via its protease domain in the p150 region. We conclude that the cellular molecular chaperone HSP90 is a key host factor for functional maturation of nonstructural proteins for RUBV genome replication. These findings provide novel insight into this host-virus interaction.

The Hsp90 isoforms from S. cerevisiae differ in structure, function and client range.

The molecular chaperone Hsp90 is an important regulator of proteostasis. It has remained unclear why S. cerevisiae possesses two Hsp90 isoforms, the constitutively expressed Hsc82 and the stress-inducible Hsp82. Here, we report distinct differences despite a sequence identity of 97%. Consistent with its function under stress conditions, Hsp82 is more stable and refolds more efficiently than Hsc82. The two isoforms also differ in their ATPases and conformational cycles. Hsc82 is more processive and populates closed states to a greater extent. Variations in the N-terminal ATP-binding domain modulate its dynamics and conformational cycle. Despite these differences, the client interactomes are largely identical, but isoform-specific interactors exist both under physiological and heat shock conditions. Taken together, changes mainly in the N-domain create a stress-specific, more resilient protein with a shifted activity profile. Thus, the precise tuning of the Hsp90 isoforms preserves the basic mechanism but adapts it to specific needs.

Effect of heat stress and Hsp90 inhibition on T-type calcium currents and voltage-dependent potassium currents in leydig cells.

Heat can trigger testicular damage and impair fertility. Leydig cells produce testosterone in response to stimulation by luteinizing hormone (LH), which induces Ca2+ entry and K+ efflux through ion channels in their plasma membrane. Considering that mechanisms coordinating the Leydig cell responses to hyperthermic stress remain unclear; the present study analyzed the effects of heat stress (HS, 43°C, 15 min) and inhibition of Hsp90 on T-type calcium currents and voltage-dependent potassium currents (VKC) in mice Leydig cells. Results show that HS reduced the VKC steady state currents at +80 mV (45.3%) and maximum conductance (71.5%), as well as increased the activation time constant (31.7%) and the voltage for which half the channels are open (30%). Hsp90 inhibition did not change the VKC currents. T-type calcium currents were not affected by HS or Hsp90 inhibition. In conclusion, HS can slow the activation, reduce the currents and voltage dependence of the VKC, suggesting a possible role of these currents in the response to hyperthermic stress in Leydig cells.

The role of heat shock protein 90 in regulating pain, opioid signaling, and opioid antinociception.

Heat shock protein 90 (Hsp90) is one of the central signal transduction regulators of the cell. Via client interactions with hundreds of proteins, including receptors, receptor regulatory kinases, and downstream signaling regulators, Hsp90 has a crucial and wide-ranging impact on signaling in response to numerous drugs with impacts on resultant physiology and behavior. Despite this importance, however, Hsp90 has barely been studied in the context of pain and the opioid receptor system, leaving open the possibility that Hsp90 could be manipulated to improve pain therapeutic outcomes, a current area of massive medical need. In this review, we will highlight the known roles of Hsp90 in directly regulating the initiation and maintenance of the pain state. We will also explore how Hsp90 regulates signaling and antinociceptive responses to opioid analgesic drugs, with a special emphasis on ERK MAPK signaling. Understanding this new and growing area will improve our understanding of how Hsp90 regulates signaling and physiology, and also may provide new ways to treat pain, and perhaps reduce the severe impact of the ongoing opioid addiction and overdose crisis.

Hsp90 of E. coli modulates assembly of FtsZ, the bacterial tubulin homolog.

Heat shock protein 90 (Hsp90) is a highly conserved molecular chaperone involved in ATP-dependent client protein remodeling and activation. It also functions as a protein holdase, binding and stabilizing clients in an ATP-independent process. Hsp90 remodels over 300 client proteins and is essential for cell survival in eukaryotes. In bacteria, Hsp90 is a highly abundant protein, although very few clients have been identified and it is not essential for growth in many bacterial species. We previously demonstrated that in Escherichia coli, Hsp90 causes cell filamentation when expressed at high levels. Here, we have explored the cause of filamentation and identified a potentially important client of E. coli Hsp90 (Hsp90Ec), FtsZ. We observed that FtsZ, a bacterial tubulin homolog essential for cell division, fails to assemble into FtsZ rings (divisomes) in cells overexpressing Hsp90Ec Additionally, Hsp90Ec interacts with FtsZ and inhibits polymerization of FtsZ in vitro, in an ATP-independent holding reaction. The FtsZ-Hsp90Ec interaction involves residues in the client-binding region of Hsp90Ec and in the C-terminal tail of FtsZ, where many cell-division proteins and regulators interact. We observed that E. coli deleted for the Hsp90Ec gene htpG turn over FtsZ more rapidly than wild-type cells. Additionally, the length of ΔhtpG cells is reduced compared to wild-type cells. Altogether, these results suggest that Hsp90Ec is a modulator of cell division, and imply that the polypeptide-holding function of Hsp90 may be a biologically important chaperone activity.

Dual RNA-Seq Unveils Pseudomonas plecoglossicida htpG Gene Functions During Host-Pathogen Interactions With Epinephelus coioides.

Pseudomonas plecoglossicida is a temperature-dependent opportunistic pathogen which is associated with a variety of diseases in fish. During the development of "white nodules" disease, the expression of htpG in P. plecoglossicida was found to be significantly up-regulated at its virulent temperature of 18°C. The infection of htpG-RNAi strain resulted in the onset time delay, reduction in mortality and infection symptoms in spleen of Epinephelus coioides, and affected the bacterial tissue colonization. In order to reveal the effect of htpG silencing of P. plecoglossicida on the virulence regulation in P. plecoglossicida and immune response in E. coioides, dual RNA-seq was performed and a pathogen-host integration network was constructed. Our results showed that infection induced the expression of host genes related to immune response, but attenuated the expression of bacterial virulence genes. Novel integration was found between host immune genes and bacterial virulence genes, while IL6, IL1R2, IL1B, and TLR5 played key roles in the network. Further analysis with GeneMANIA indicated that flgD and rplF might play key roles during the htpG-dependent virulence regulation, which was in accordance with the reduced biofilm production, motility and virulence in htpG-RNAi strain. Meanwhile, IL6 and IL1B were found to play key roles during the defense against P. plecoglossicida, while CELA2, TRY, CPA1, CPA2, and CPB1 were important targets for P. plecoglossicida attacking to the host.

Association of TRAP1 with infliximab-induced mucosal healing in Crohn's disease.

BACKGROUND AND AIM: Anti-tumor necrosis factor (TNF) agents, such as infliximab (IFX), have been increasingly used to induce and maintain disease remission in patients with Crohn's disease (CD). Despite a considerable non-response rate, little is known about the genetic predictors of response to anti-TNF therapy in CD. Our aim in this study was to investigate the genetic factors associated with response to anti-TNF therapy in patients with CD. METHODS: We performed a two-stage genome-wide association study (GWAS) to identify loci influencing the response to IFX among Korean patients with CD, comprising 42 good responders with mucosal healing and 70 non-responders. The achievement of mucosal healing was assessed by endoscopy and imaging. The functional significance of TRAP1 (TNF receptor associated protein 1) was examined using dextran sodium sulfate-induced colitis model in TRAP1 transgenic mice. RESULTS: The GWAS identified rs2158962, an intronic single nucleotide polymorphism (SNP) of TRAP1, significantly associated with mucosal healing (odds ratio = 4.94; Pcombined  = 1.35 × 10-7 ). In the dextran sodium sulfate-induced acute colitis, TRAP1 transgenic mice showed a better response to IFX than the wild-type mice. CONCLUSIONS: The TRAP1 gene is associated with mucosal healing in CD patients following IFX therapy. Identifying the genetic predictors of mucosal healing to anti-TNF therapy can prevent patients from exposure to ineffective therapies.

Heterologous Hsp90 promotes phenotypic diversity through network evolution.

Biological processes in living cells are often carried out by gene networks in which signals and reactions are integrated through network hubs. Despite their functional importance, it remains unclear to what extent network hubs are evolvable and how alterations impact long-term evolution. We investigated these issues using heat shock protein 90 (Hsp90), a central hub of proteostasis networks. When native Hsp90 in Saccharomyces cerevisiae cells was replaced by the ortholog from hypersaline-tolerant Yarrowia lipolytica that diverged from S. cerevisiae about 270 million years ago, the cells exhibited improved growth in hypersaline environments but compromised growth in others, indicating functional divergence in Hsp90 between the two yeasts. Laboratory evolution shows that evolved Y. lipolytica-HSP90-carrying S. cerevisiae cells exhibit a wider range of phenotypic variation than cells carrying native Hsp90. Identified beneficial mutations are involved in multiple pathways and are often pleiotropic. Our results show that cells adapt to a heterologous Hsp90 by modifying different subnetworks, facilitating the evolution of phenotypic diversity inaccessible to wild-type cells.

HSP90-incorporating chaperome networks as biosensor for disease-related pathways in patient-specific midbrain dopamine neurons.

Environmental and genetic risk factors contribute to Parkinson's Disease (PD) pathogenesis and the associated midbrain dopamine (mDA) neuron loss. Here, we identify early PD pathogenic events by developing methodology that utilizes recent innovations in human pluripotent stem cells (hPSC) and chemical sensors of HSP90-incorporating chaperome networks. We show that events triggered by PD-related genetic or toxic stimuli alter the neuronal proteome, thereby altering the stress-specific chaperome networks, which produce changes detected by chemical sensors. Through this method we identify STAT3 and NF-κB signaling activation as examples of genetic stress, and phospho-tyrosine hydroxylase (TH) activation as an example of toxic stress-induced pathways in PD neurons. Importantly, pharmacological inhibition of the stress chaperome network reversed abnormal phospho-STAT3 signaling and phospho-TH-related dopamine levels and rescued PD neuron viability. The use of chemical sensors of chaperome networks on hPSC-derived lineages may present a general strategy to identify molecular events associated with neurodegenerative diseases.

The morphological and functional significance of the NOS/NO system in the respiratory, osmoregulatory, and contractile organs of the African lungfish.

This review aims to summarize the changes of the NOS/NO system which occur in the lungs, gills, kidney, heart, and myotomal muscle of air breathing fish of the genus Protopterus, i.e. P. dolloi and P. annectens, in relation to the switch from freshwater to aestivation, and vice-versa. The modifications of NOS and its partners Akt and Hsp-90, and HIF-1α, detected by immunohistochemical and molecular biology methods, are discussed together with the apoptosis rate, evaluated by TUNEL. We hypothesize that these molecular components are key elements of the stress-induced signal transduction/integration networks which allow the lungfish to overcome the dramatic environmental challenges experienced at the beginning, during, and at the end of the dry season.

Heat shock protein 70 and heat shock protein 90 synergistically increase hepatitis B viral capsid assembly.

Hepatitis B virus (HBV) infection can cause chronic liver diseases, cirrhosis, and hepatocellular carcinoma (HCC). Heat shock proteins (Hsps) are important factors in the formation of the HBV capsid and in genome replication during the viral life cycle. Hsp90 is known to promote capsid assembly. However, the functional roles of Hsp70 in HBV capsid assembly with Hsp90 have not been studied so far. Using microscale thermophoresis analyses and in vitro nucleocapsid formation assays, we found that Hsp70 bound to a HBV core protein dimer and facilitated HBV capsid assembly. Inhibition of Hsp70 by methylene blue (MB) led to a decrease in capsid assembly. Moreover, Hsp70 inhibition reduced intracellular capsid formation and HBV virus particle number in HepG2.2.15 cells. Furthermore, we examined synergism between Hsp70 and Hsp90 on HBV capsid formation in vitro. Our results clarify the role of Hsp70 in HBV capsid formation via an interaction with core dimers and in synergistically promoting capsid assembly with Hsp90.