Heat Shock Protein B8 Research Articles

Small heat shock protein B8: from cell functions to its involvement in diseases and potential therapeutic applications.

Heat shock protein family B (small) member 8 (HSPB8) is a 22 kDa ubiquitously expressed protein belonging to the family of small heat shock proteins. HSPB8 is involved in various cellular mechanisms mainly related to proteotoxic stress response and in other processes such as inflammation, cell division, and migration. HSPB8 binds misfolded clients to prevent their aggregation by assisting protein refolding or degradation through chaperone-assisted selective autophagy. In line with this function, the pro-degradative activity of HSPB8 has been found protective in several neurodegenerative and neuromuscular diseases characterized by protein misfolding and aggregation. In cancer, HSPB8 has a dual role being capable of exerting either a pro-or an anti-tumoral activity depending on the pathways and factors expressed by the model of cancer under investigation. Moreover, HSPB8 exerts a protective function in different diseases by modulating the inflammatory response, which characterizes not only neurodegenerative diseases, but also other chronic or acute conditions affecting the nervous system, such as multiple sclerosis and intracerebellar hemorrhage. Of note, HSPB8 modulation may represent a therapeutic approach in other neurological conditions that develop as a secondary consequence of other diseases. This is the case of cognitive impairment related to diabetes mellitus, in which HSPB8 exerts a protective activity by assuring mitochondrial homeostasis.This review aims to summarize the diverse and multiple functions of HSPB8 in different pathological conditions, focusing on the beneficial effects of its modulation. Drug-based and alternative therapeutic approaches targeting HSPB8 and its regulated pathways will be discussed, emphasizing how new strategies for cell and tissue-specific delivery represent an avenue to advance in disease treatments.

M6A-activated BACH1 exacerbates ferroptosis by epigenetic suppression HSPB1 in severe acute pancreatitis.

Severe acute pancreatitis (SAP) is characterized by acute inflammation of the pancreas. The transcription factor BTB and CNC homology 1 (BACH1) has been implicated in various biological processes, including oxidative stress, apoptosis, and cell cycle regulation. However, its involvement in the pathogenesis of SAP remains relatively understudied. In the present work, our data demonstrated that BACH1 level was significantly increased in SAP patients, cellular, and animal models, while heat shock protein B1 (HSPB1) expression was weakened. Mechanistic assays validated that BACH1 acted as a transcriptional inhibitor of HSPB1. Moreover, HPDE6-C7 cells were stimulated with cerulein (Cer) and LPS to mimic the pathological stages of SAP in vitro. Depletion of BACH1 remarkably improved cell survival and alleviated the oxidative stress, ferroptosis, and inflammatory responses in SAP cell models. However, these changes were dramatically reversed upon co-inhibition of HSPB1. Animal findings confirmed that loss of BACH1 decreased pancreatic injury, inflammatory responses, and ferroptosis, but these effects were weakened by HSPB1 silence. Overall, these findings elucidate that the overexpression of BACH1 favors the ferroptosis and inflammation by transcriptionally inhibiting HSBP1, thereby exacerbating SAP progression.

Mutations in the N-domain of aryl hydrocarbon receptor interacting protein affect interactions with heat shock protein 90β and phosphodiesterase 4A5

The aryl hydrocarbon receptor interacting protein (AIP) is a cytoplasmic molecular co-chaperone and tumour suppressor that assists in protein stability and complex formation involving the aryl hydrocarbon receptor. Germline mutations in the AIP gene predispose to pituitary tumourigenesis with patients exhibiting an aggressive clinical phenotype. Full length AIP proteins harbouring N-domain mutations (R9Q, R16H, V49 M and K103R) were purified from E.coli utilizing a methodology that maintained structural integrity and monomeric stability. Mutations did not significantly affect the thermal stability of the protein and caused no overall disruptive effect in the protein structure. The mutations studied lowered the binding affinity of AIP towards two of its binding partners; heat shock protein 90β and phosphodiesterase 4A5 (PDE4A5). The inhibition of phosphodiesterase activity by AIP was also greatly reduced by all mutants. While previously published data has mainly concentrated on the tetratricopeptide repeats of the C-domain of AIP, we present clear evidence that AIP N-domain mutations play a significant role in two protein:protein interactions with partner proteins. The complex interactome of AIP suggests that any observable change in one or more of its binding partners cannot be disregarded as it may have repercussions on other biochemical pathways.

Structural Characterization of Heat Shock Protein 90β and Molecular Interactions with Geldanamycin and Ritonavir: A Computational Study.

Drug repositioning is an important therapeutic strategy for treating breast cancer. Hsp90β chaperone is an attractive target for inhibiting cell progression. Its structure has a disordered and flexible linker region between the N-terminal and central domains. Geldanamycin was the first Hsp90β inhibitor to interact specifically at the N-terminal site. Owing to the toxicity of geldanamycin, we investigated the repositioning of ritonavir as an Hsp90β inhibitor, taking advantage of its proven efficacy against cancer. In this study, we used molecular modeling techniques to analyze the contribution of the Hsp90β linker region to the flexibility and interaction between the ligands geldanamycin, ritonavir, and Hsp90β. Our findings indicate that the linker region is responsible for the fluctuation and overall protein motion without disturbing the interaction between the inhibitors and the N-terminus. We also found that ritonavir established similar interactions with the substrate ATP triphosphate, filling the same pharmacophore zone.

HSPB8 attenuates lipopolysaccharide‑mediated acute lung injury in A549 cells by activating mitophagy.

Sepsis is a life‑threatening multiple organ failure disease caused by an uncontrolled inflammatory response and can progress to acute lung injury (ALI). Heat‑shock protein B8 (HSPB8) serves a cytoprotective role in multiple types of diseases; however, to the best of our knowledge, the regulatory role of HSPB8 in sepsis‑induced ALI remains unclear. A549 human alveolar type II epithelial cells were treated with lipopolysaccharide (LPS) for 24 h to simulate a sepsis‑induced ALI model. Cell transfection was performed to overexpress HSPB8, and cells were treated with mitochondrial division inhibitor‑1 (Mdivi‑1) for 2 h before LPS induction to assess the underlying mechanism. Protein expression was evaluated using western blotting and an immunofluorescence assay. Cytokines were examined using ELISA assay kits and antioxidant enzymes were examined using their detection kits. Cell apoptosis was detected using flow cytometry. The mitochondrial membrane potential was detected by JC‑1 staining. HSPB8 was upregulated in A549 cells treated with LPS and HSPB8 overexpression attenuated LPS‑induced inflammatory cytokine levels, oxidative stress and apoptosis in A549 cells. LPS inhibited mitophagy and reduced the mitochondrial membrane potential in A549 cells, which was partly inhibited by HSPB8 overexpression. Furthermore, Mdivi‑1 decreased the inhibitory effect of HSPB8 on the inflammatory response, oxidative stress and apoptosis in LPS‑treated A549 cells. In conclusion, HSPB8 overexpression attenuated the LPS‑mediated inflammatory response, oxidative stress and apoptosis in A549 cells by promoting mitophagy, indicating HSPB8 as a potential therapeutic target in sepsis‑induced ALI.

Reduction of Filamin C Results in Altered Proteostasis, Cardiomyopathy, and Arrhythmias.

Many cardiomyopathy-associated FLNC pathogenic variants are heterozygous truncations, and FLNC pathogenic variants are associated with arrhythmias. Arrhythmia triggers in filaminopathy are incompletely understood. We describe an individual with biallelic FLNC pathogenic variants, p.Arg650X and c.970-4A>G, with peripartum cardiomyopathy and ventricular arrhythmias. We also describe clinical findings in probands with FLNC variants including Val2715fs87X, Glu2458Serfs71X, Phe106Leu, and c.970-4A>G with hypertrophic and dilated cardiomyopathy, atrial fibrillation, and ventricular tachycardia. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated. The FLNC truncation, Arg650X/c.970-4A>G, showed a marked reduction in filamin C protein consistent with biallelic loss of function mutations. To assess loss of filamin C, gene editing of a healthy control iPSC line was used to generate a homozygous FLNC disruption in the actin binding domain. Because filamin C has been linked to protein quality control, we assessed the necessity of filamin C in iPSC-CMs for response to the proteasome inhibitor bortezomib. After exposure to low-dose bortezomib, FLNC-null iPSC-CMs showed an increase in the chaperone proteins BAG3, HSP70 (heat shock protein 70), and HSPB8 (small heat shock protein B8) and in the autophagy marker LC3I/II. FLNC null iPSC-CMs had prolonged electric field potential, which was further prolonged in the presence of low-dose bortezomib. FLNC null engineered heart tissues had impaired function after low-dose bortezomib. FLNC pathogenic variants associate with a predisposition to arrhythmias, which can be modeled in iPSC-CMs. Reduction of filamin C prolonged field potential, a surrogate for action potential, and with bortezomib-induced proteasome inhibition, reduced filamin C led to greater arrhythmia potential and impaired function.

Arenobufagin enhances T-cell anti-tumor immunity in colorectal cancer by modulating HSP90β accessibility

BackgroundColorectal cancer (CRC) is a significant public health issue, ranking as one of the predominant cancer types globally in terms of incidence. Intriguingly, Arenobufagin (Are), a compound extracted from toad venom, has demonstrated the potential to inhibit tumor growth effectively. PurposeThis study aimed to explore Are's molecular targets and unravel its antitumor mechanism in CRC. Specifically, we were interested in its impact on immune checkpoint modulation and correlations with HSP90β-STAT3-PD-L1 axis activity. MethodsWe investigated the in vivo antitumor effects of Are by constructing a colorectalcancer subcutaneous xenograft mouse model. Subsequently, we employed single-cell multi-omics technology to study the potential mechanism by which Are inhibits CRC. Utilizing target-responsive accessibility profiling (TRAP) technology, we identified heatshock protein 90β (HSP90β) as the direct target of Are, and confirmed this through a microscale thermophoresis experiment (MST). Further downstream mechanisms were explored through techniques such as co-immunoprecipitation, Western blotting, qPCR, and immunofluorescence. Concurrently, we arrived at the same research conclusion at the organoid level by co-cultivating with immune cells. ResultsWe observed that Are inhibits PD-Ll expression in CRC tumor xenografts at low concentrations. Moreover, TRAP revealed that HSP90β's accessibility significantly decreased upon Are binding. We demonstrated a decrease in the activity of the HSP90β-STAT3-PD-Ll axis following low-concentration Are treatment in vivo. The PDO analysis showed improved enrichment of lymphocytes, particularly T cells, on the PDOs following Are treatment. ConclusionContrary to previous research focusing on the direct cytotoxicity of Are towards tumor cells, our findings indicate that it can also inhibit tumor growth at lower concentrations through the modulation of immune checkpoints. This study unveils a novel anti-tumor mechanism of Are and stimulates contemplation on the dose-response relationship of natural products, which is beneficial for the clinical translational application of Are.

Sonic hedgehog-heat shock protein 90β axis promotes the development of nonalcoholic steatohepatitis in mice

Sonic hedgehog (SHH) and heat shock protein 90β (HSP90β) have been implicated in nonalcoholic steatohepatitis (NASH) but their molecular mechanisms of action remain elusive. We find that HSP90β is a key SHH downstream molecule for promoting NASH process. In hepatocytes, SHH reduces HSP90β ubiquitylation through deubiquitylase USP31, thus preventing HSP90β degradation and promoting hepatic lipid synthesis. HSP90β significantly increases in NASH mouse model, leading to secretion of exosomes enriched with miR-28-5p. miR-28-5p directly targetes and decreases Rap1b levels, which in turn promotes NF-κB transcriptional activity in macrophages and stimulates the expression of inflammatory factors. Genetic deletion, pharmacological inhibition of the SHH-HSP90β axis, or delivery of miR-28-5p to macrophages in the male mice liver, impairs NASH symptomatic development. Importantly, there is a markedly higher abundance of miR-28-5p in NASH patient sera. Taken together, the SHH-HSP90β-miR-28-5p axis offers promising therapeutic targets against NASH, and serum miR-28-5p may serve as a NASH diagnostic biomarker.

The deubiquitinase USP40 preserves endothelial integrity by targeting the heat shock protein HSP90β

Endothelial cell (EC) barrier disruption and inflammation are the pathological hallmarks of vascular disorders and acute infectious diseases and related conditions, including the coronavirus disease 2019 (COVID-19) and sepsis. Ubiquitination plays a critical role in regulating the stability, intracellular trafficking, and enzymatic activity of proteins and is reversed by deubiquitinating enzymes (DUBs). The role of DUBs in endothelial biology is largely unknown. In this study, we report that USP40, a poorly characterized DUB, prevents EC barrier disruption through reductions in the activation of RhoA and phosphorylation of myosin light chain (MLC) and cofilin. Furthermore, USP40 reduces EC inflammation through the attenuation of NF-ĸB activation, ICAM1 expression, and leukocyte-EC adhesion. We further show that USP40 activity and expression are reduced in response to endotoxin challenge. Global depletion of USP40 and EC-targeted USP40 depletion in mice exacerbated experimental lung injury, whereas lentiviral gene transfer of USP40 protected against endotoxin-induced lung injury. Using an unbiased approach, we discovered that the protective effect of USP40 occurs through the targeting of heat shock protein 90β (HSP90β) for its deubiquitination and inactivation. Together, these data reveal a critical protective role of USP40 in vascular injury, identifying a unique mechanistic pathway that profoundly impacts endothelial function via DUBs.

HSP90β regulates EAAT2 expression and participates in ischemia‑reperfusion injury in rats.

Cerebrovascular diseases (CVDs) have become a global public health problem and ischemia‑reperfusion injury, the major cause of neurological impairment exacerbation, is closely related to excitotoxicity. The present study aimed to investigate the effects of changes in heat shock protein (HSP)90β expression and verify whether HSP90β regulates EAAT2 expression in a cerebral ischemia‑reperfusion injury model. Healthy adult Sprague‑Dawley (SD) male rats were used to establish a control group, sham‑operated group, middle cerebral artery occlusion (MCAO) group, empty virus group and lentivirus group. A model of cerebral ischemia‑reperfusion was established using the MCAO method. Lentivirus construction and injection were used to interfere with the expression of HSP90β. The modified neurological severity score was used to assess neurological deficits. Triphenyltetrazolium chloride staining was used to detect infarct areas. Immunofluorescence was used to detect HSP90β expression localization and the expression levels of HSP90β and EAAT2 were determined using western blotting and reverse transcription‑quantitative PCR. An MCAO model was successfully established and it was found that HSP90β, but not HSP90α, was upregulated after MCAO. HSP90β expression coincided with astrocyte markers in the ischemic penumbra area, while no expression was observed in microglia. Inhibition of HSP90β expression improved neurological deficits and alleviated brain injury by increasing EAAT2 expression. These results suggested that HSP90β is involved in the process of cerebral ischemia‑reperfusion injury in rats and that inhibition of HSP90β expression increases EAAT2 levels, conferring a neuroprotective effect in MCAO model rats.

Successful Expression of DNA-Based Vaccine Construct Encoding Human Papillomavirus Type 16 E7 Fused to Heat Shock Protein B1 in Eukaryotic Cells

Successful Expression of DNA-Based Vaccine Construct Encoding Human Papillomavirus Type 16 E7 Fused to Heat Shock Protein B1 in Eukaryotic Cells

A novel HSPB1S139F mouse model of Charcot-Marie-Tooth Disease

Charcot-Marie-Tooth Disease (CMT) is a commonly inherited peripheral polyneuropathy. Clinical manifestations for this disease include symmetrical distal polyneuropathy, altered deep tendon reflexes, distal sensory loss, foot deformities, and gait abnormalities. Genetic mutations in heat shock proteins have been linked to CMT2. Specifically, mutations in the heat shock protein B1 (HSPB1) gene encoding for heat shock protein 27 (Hsp27) have been linked to CMT2F and distal hereditary motor and sensory neuropathy type 2B (dHMSN2B) subtype. The goal of the study was to examine the role of an endogenous mutation in HSPB1 in vivo and to define the effects of this mutation on motor function and pathology in a novel animal model. As sphingolipids have been implicated in hereditary and sensory neuropathies, we examined sphingolipid metabolism in central and peripheral nervous tissues in 3-month-old HspS139F mice. Though sphingolipid levels were not altered in sciatic nerves from HspS139F mice, ceramides and deoxyceramides, as well as sphingomyelins (SMs) were elevated in brain tissues from HspS139F mice. Histology was utilized to further characterize HspS139F mice. HspS139F mice exhibited no alterations to the expression and phosphorylation of neurofilaments, or in the expression of acetylated α-tubulin in the brain or sciatic nerve. Interestingly, HspS139F mice demonstrated cerebellar demyelination. Locomotor function, grip strength and gait were examined to define the role of HspS139F in the clinical phenotypes associated with CMT2F. Gait analysis revealed no differences between HspWT and HspS139F mice. However, both coordination and grip strength were decreased in 3-month-old HspS139F mice. Together these data suggest that the endogenous S139F mutation in HSPB1 may serve as a mouse model for hereditary and sensory neuropathies such as CMT2F.

HSP90β prevents aging-related cataract formation through regulation of the charged multivesicular body protein (CHMP4B) and p53

Cataract is a leading ocular disease causing global blindness. The mechanism of cataractogenesis has not been well defined. Here, we demonstrate that the heat shock protein 90β (HSP90β) plays a fundamental role in suppressing cataractogenesis. HSP90β is the most dominant HSP in normal lens, and its constitutive high level of expression is largely derived from regulation by Sp1 family transcription factors. More importantly, HSP90β is significantly down-regulated in human cataract patients and in aging mouse lenses, whereas HSP90β silencing in zebrafish causes cataractogenesis, which can only be rescued by itself but not other HSP90 genes. Mechanistically, HSP90β can directly interact with CHMP4B, a newly-found client protein involved in control of cytokinesis. HSP90β silencing causes upregulation of CHMP4B and another client protein, the tumor suppressor p53. CHMP4B upregulation or overexpression induces excessive division of lens epithelial cells without proper differentiation. As a result, these cells were triggered to undergo apoptosis due to activation of the p53/Bak-Bim pathway, leading to cataractogenesis and microphthalmia. Silence of both HSP90β and CHMP4B restored normal phenotype of zebrafish eye. Together, our results reveal that HSP90β is a critical inhibitor of cataractogenesis through negative regulation of CHMP4B and the p53-Bak/Bim pathway.

Human Small Heat Shock Protein B8 Inhibits Protein Aggregation without Affecting the Native Folding Process.

Small Heat Shock Proteins (sHSPs) are key components of our Protein Quality Control system and are thought to act as reservoirs that neutralize irreversible protein aggregation. Yet, sHSPs can also act as sequestrases, promoting protein sequestration into aggregates, thus challenging our understanding of their exact mechanisms of action. Here, we employ optical tweezers to explore the mechanisms of action of the human small heat shock protein HSPB8 and its pathogenic mutant K141E, which is associated with neuromuscular disease. Through single-molecule manipulation experiments, we studied how HSPB8 and its K141E mutant affect the refolding and aggregation processes of the maltose binding protein. Our data show that HSPB8 selectively suppresses protein aggregation without affecting the native folding process. This anti-aggregation mechanism is distinct from previous models that rely on the stabilization of unfolded polypeptide chains or partially folded structures, as has been reported for other chaperones. Rather, it appears that HSPB8 selectively recognizes and binds to aggregated species formed at the early stages of aggregation, preventing them from growing into larger aggregated structures. Consistently, the K141E mutation specifically targets the affinity for aggregated structures without impacting native folding, and hence impairs its anti-aggregation activity.

HSP27 modulates tumoural immune evasion by enhancing STAT3-mediated upregulation of PD-L1 and NLRC5 in ovarian cancer.

Multiple preclinical studies have demonstrated that the addition of hyperthermia (HT) to immunotherapy could enhance tumour immunogenicity and stimulate an antitumour immune response, primarily via heat shock proteins (HSPs). However, antitumour immune responses are often impeded by immune evasion mechanisms, such as the overexpression of programmed death-ligand1 (PD-L1) and the loss of major histocompatibility complex class 1 (MHC-1) expression. In this context, we sought to investigate the effect of HT on PD-L1 and NOD-like receptor family CARD domain containing 5 (NLRC5) identified as the key transcriptional activator of MHC-1 genes, and their interaction in ovarian cancer. A coculture of ovarian cancer cell lines (IGROV1 and SKOV3) with peripheral blood mononuclear cells was set up. Then, culture media conditioned with IGROV1 or SKOV3 subjected to HT was tested on untreated cell cultures. Knocking down heat shock protein B1 (HSPB1 or HSP27), heat shock protein A1 (HSPA1 or HSP70), and pharmacological inhibition of STAT3 phosphorylation were performed. Subsequently, we measured expression levels of PD-L1, NLRC5, and proinflammatory cytokines. The correlation between PD-L1 and NLRC5 expression in ovarian cancer was evaluated using the Cancer Genome Atlas database. We found that HT produces a concomitant decrease in PD-L1 and NLRC5 expression in coculture. Notably, however, the conditioned media by heat-shocked cells increases their expression. HSP27 knockdown can reverse this increase. Adding STAT3 phosphorylation inhibitor significantly enhanced the expression inhibition of PD-L1 and NLRC5 induced by HSP27 silencing. Correlation analysis showed a positive correlation in ovarian cancer between NLRC5 and PD-L1. These findings demonstrate that HSP27 modulates PD-L1 and NLRC5 expression through the activation of a common regulator 'STAT3'. Moreover, the positive correlation between PD-L1 and NLRC5 led us to conclude that the upregulation of PD-L1 and the downregulation of MHC class I are two mutually exclusive mechanisms of immune evasion in ovarian cancer.

Celastrol attenuates hepatitis C virus translation and inflammatory response in mice by suppressing heat shock protein 90β.

Hepatitis C virus (HCV) infection is one of the major factors to trigger a sustained hepatic inflammatory response and hence hepatocellular carcinoma (HCC), but direct-acting-antiviral (DAAs) was not efficient to suppress HCC development. Heat shock protein 90 kDa (HSP90) is highly abundant in different types of cancers, and especially controls protein translation, endoplasmic reticulum stress, and viral replication. In this study we investigated the correlation between the expression levels of HSP90 isoforms and inflammatory response marker NLRP3 in different types of HCC patients as well as the effect of a natural product celastrol in suppression of HCV translation and associated inflammatory response in vivo. We identified that the expression level of HSP90β isoform was correlated with that of NLRP3 in the liver tissues of HCV positive HCC patients (R2 = 0.3867, P < 0.0101), but not in hepatitis B virus-associated HCC or cirrhosis patients. We demonstrated that celastrol (3, 10, 30 μM) dose-dependently suppressed the ATPase activity of both HSP90α and HSP90β, while its anti-HCV activity was dependent on the Ala47 residue in the ATPase pocket of HSP90β. Celastrol (200 nM) halted HCV internal ribosomal entry site (IRES)-mediated translation at the initial step by disrupting the association between HSP90β and 4EBP1. The inhibitory activity of celastrol on HCV RNA-dependent RNA polymerase (RdRp)-triggered inflammatory response also depended on the Ala47 residue of HSP90β. Intravenous injection of adenovirus expressing HCV NS5B (pAde-NS5B) in mice induced severe hepatic inflammatory response characterized by significantly increased infiltration of immune cells and hepatic expression level of Nlrp3, which was dose-dependently ameliorated by pretreatment with celastrol (0.2, 0.5 mg/kg, i.p.). This study reveals a fundamental role of HSP90β in governing HCV IRES-mediated translation as well as hepatic inflammation, and celastrol as a novel inhibitor of HCV translation and associated inflammation by specifically targeting HSP90β, which could be developed as a lead for the treatment of HSP90β positive HCV-associated HCC.

Identification of HSPB8 modulators counteracting misfolded protein accumulation in neurodegenerative diseases

AimsThe small Heat Shock Protein B8 (HSPB8) is the core component of the Chaperone-Assisted Selective Autophagy (CASA) complex. This complex selectively targets, transports, and tags misfolded proteins for their recognition by autophagy receptors and insertion into the autophagosome for clearance. CASA is essential to maintain intracellular proteostasis, especially in heart, muscle, and brain often exposed to various types of cell stresses. In neurons, HSPB8 protects against neurotoxicity caused by misfolded proteins in several models of neurodegenerative diseases; by facilitating autophagy, HSPB8 assists misfolded proteins degradation also counteracting proteasome overwhelming and inhibition. Materials and methodsTo enhance HSPB8 protective activity, we screened a library of approximately 120,000 small molecules to identify compounds capable of increasing HSPB8 gene transcription, translation, or protein stability. Key findingsWe found 83 active compounds active in preliminary dose-response assays and further classified them in 19 chemical classes by medicinal chemists' visual inspection. Of these 19 prototypes, 14 induced HSPB8 mRNA and protein levels in SH-SY5Y cells. Out of these 14 compounds, 3 successfully reduced the aggregation propensity of a disease-associated mutant misfolded superoxide dismutase 1 (SOD1) protein in a flow cytometry-based aggregation assay (Flow cytometric analysis of Inclusions and Trafficking (FloIT)) and induced the expression (mRNA and protein) of some autophagy receptors. Notably, the 3 hits were inactive in HSPB8-depleted cells, confirming that their protective activity is mediated by and requires HSPB8. SignificanceThese compounds may be highly relevant for a therapeutic approach in several human disorders, including neurodegenerative diseases, in which enhancement of CASA exerts beneficial activities.

HSPB8 counteracts tumor activity of BRAF- and NRAS-mutant melanoma cells by modulation of RAS-prenylation and autophagy

Cutaneous melanoma is one of the most aggressive and lethal forms of skin cancer. Some specific driver mutations have been described in multiple oncogenes including BRAF and NRAS that are mutated in 60–70% and 15–20% of melanoma, respectively. The aim of this study was to evaluate the role of Small Heat Shock Protein B8 (HSPB8) on cell growth and migration of both BLM (BRAFwt/NRASQ61R) and A375 (BRAFV600E/NRASwt) human melanoma cell lines. HSPB8 is a member of the HSPB family of chaperones involved in protein quality control (PQC) system and contributes to chaperone assisted selective autophagy (CASA) as well as in the regulation of mitotic spindle. In cancer, HSPB8 has anti- or pro-tumoral action depending on tumor type. In melanoma cell lines characterized by low HSPB8 levels, we demonstrated that the restoration of HSPB8 expression causes cell growth arrest, reversion of EMT (Epithelial-Mesenchymal Transition)-like phenotype switching and antimigratory effect, independently from the cell mutational status. We demonstrated that HSPB8 regulates the levels of the active prenylated form of NRAS in NRAS-mutant and NRAS-wild-type melanoma cell lines. Consequently, the inhibition of NRAS impairs the activation of Akt/mTOR pathway inducing autophagy activation. Autophagy can play a dual role in regulating cell death and survival. We have therefore demonstrated that HSPB8-induced autophagy is a crucial event that counteracts cell growth in melanoma. Collectively, our results suggest that HSPB8 has an antitumoral action in melanoma cells characterized by BRAF and NRAS mutations.

HSPB8 facilitates prostate cancer progression via activating the JAK/STAT3 signaling pathway.

Prostate cancer (PC) is a clinically and biologically heterogeneous disease that lacks effective treatment. Heat shock protein B8 (HSPB8) is an important factor in the progression of various types of cancer. However, the clinical significance and biological role of HSPB8 in PC are still unclear. In this study, we determined HSPB8 expression in PC tissues by immunohistochemical staining and explored the in vitro functions of HSPB8 using HSPB8 knockdown DU145 and LNcap PC cell lines. The in vivo effect of HSPB8 was explored by a subcutaneous xenograft mice model. The human phospho-kinase array and signal transducer and activator of transcription (STAT) 3 activator were utilized to explore the potential mechanism of HSPB8-induced PC progression. As a result, we found that HSPB8 was abundantly expressed in PC tissues and cell lines. HSPB8 knockdown inhibited cell proliferation and migration, promoted apoptosis and cycle repression, as well as weakened tumorigenesis ability. Mechanistically, we demonstrated that HSPB8 facilitates the malignant phenotypes of PC by activating the Janus kinase/STAT3 signaling pathway. These results proposed that HSPB8 seems to be an attractive therapeutic target for PC patients.

HSP90β promotes osteoclastogenesis by dual-activation of cholesterol synthesis and NF-κB signaling.

Heat shock protein 90β (Hsp90β, encoded by Hsp90ab1 gene) is the most abundant proteins in the cells and contributes to variety of biological processes including metabolism, cell growth and neural functions. However, genetic evidences showing Hsp90β in vivo functions using tissue specific knockout mice are still lacking. Here, we showed that Hsp90β exerted paralogue-specific role in osteoclastogenesis. Using myeloid-specific Hsp90ab1 knockout mice, we provided the first genetic evidence showing the in vivo function of Hsp90β. Hsp90β binds to Ikkβ and reduces its ubiquitylation and proteasomal degradation, thus leading to activated NF-κB signaling. Meanwhile, Hsp90β increases cholesterol biosynthesis by activating Srebp2. Both pathways promote osteoclastogenic genes expression. Genetic deletion of Hsp90ab1 in osteoclast or pharmacological inhibition of Hsp90β alleviates bone loss in ovariectomy-induced mice. Therefore, Hsp90β is a promising druggable target for the treatment of osteoporosis.