Hsc70 promotes anti-tumor immunity by targeting PD-L1 for lysosomal degradation.

Immune checkpoint inhibition targeting the PD-1/PD-L1 pathway has become a powerful clinical strategy for treating cancer, but its efficacy is complicated by various resistance mechanisms. One of the reasons for the resistance is the internalization and recycling of PD-L1 itself upon antibody binding. The inhibition of lysosome-mediated degradation of PD-L1 is critical for preserving the amount of PD-L1 recycling back to the cell membrane. In this study, we find that Hsc70 promotes PD-L1 degradation through the endosome-lysosome pathway and reduces PD-L1 recycling to the cell membrane. This effect is dependent on Hsc70-PD-L1 binding which inhibits the CMTM6-PD-L1 interaction. We further identify an Hsp90α/ß inhibitor, AUY-922, which induces Hsc70 expression and PD-L1 lysosomal degradation. Either Hsc70 overexpression or AUY-922 treatment can reduce PD-L1 expression, inhibit tumor growth and promote anti-tumor immunity in female mice; AUY-922 can further enhance the anti-tumor efficacy of anti-PD-L1 and anti-CTLA4 treatment. Our study elucidates a molecular mechanism of Hsc70-mediated PD-L1 lysosomal degradation and provides a target and therapeutic strategies for tumor immunotherapy.

PI3K/HSCB axis facilitates FOG1 nuclear translocation to promote erythropoiesis and megakaryopoiesis.

Erythropoiesis and megakaryopoiesis are stringently regulated by signaling pathways. However, the precise molecular mechanisms through which signaling pathways regulate key transcription factors controlling erythropoiesis and megakaryopoiesis remain partially understood. Herein, we identified heat shock cognate B (HSCB), which is well known for its iron-sulfur cluster delivery function, as an indispensable protein for friend of GATA 1 (FOG1) nuclear translocation during erythropoiesis of K562 human erythroleukemia cells and cord-blood-derived human CD34+CD90+hematopoietic stem cells (HSCs), as well as during megakaryopoiesis of the CD34+CD90+HSCs. Mechanistically, HSCB could be phosphorylated by phosphoinositol-3-kinase (PI3K) to bind with and mediate the proteasomal degradation of transforming acidic coiled-coil containing protein 3 (TACC3), which otherwise detained FOG1 in the cytoplasm, thereby facilitating FOG1 nuclear translocation. Given that PI3K is activated during both erythropoiesis and megakaryopoiesis, and that FOG1 is a key transcription factor for these processes, our findings elucidate an important, previously unrecognized iron-sulfur cluster delivery independent function of HSCB in erythropoiesis and megakaryopoiesis.

Impact of histone deacetylase inhibition and arimoclomol on heat shock protein expression and disease biomarkers in primary culture models of familial ALS.

Protein misfolding and mislocalization are common themes in neurodegenerative disorders, including motor neuron disease, and amyotrophic lateral sclerosis (ALS). Maintaining proteostasis is a crosscutting therapeutic target, including the upregulation of heat shock proteins (HSP) to increase chaperoning capacity. Motor neurons have a high threshold for upregulating stress-inducible HSPA1A, but constitutively express high levels of HSPA8. This study compared the expression of these HSPs in cultured motor neurons expressing three variants linked to familial ALS: TAR DNA binding protein 43 kDa (TDP-43)G348C, fused in sarcoma (FUS)R521G, or superoxide dismutase I (SOD1)G93A. All variants were poor inducers of Hspa1a, and reduced levels of Hspa8 mRNA and protein, indicating multiple compromises in chaperoning capacity. To promote HSP expression, cultures were treated with the putative HSP coinducer, arimoclomol, and class I histone deacetylase inhibitors, to promote active chromatin for transcription, and with the combination. Treatments had variable, often different effects on the expression of Hspa1a and Hspa8, depending on the ALS variant expressed, mRNA distribution (somata and dendrites), and biomarker of toxicity measured (histone acetylation, maintaining nuclear TDP-43 and the neuronal Brm/Brg-associated factor chromatin remodeling complex component Brg1, mitochondrial transport, FUS aggregation). Overall, histone deacetylase inhibition alone was effective on more measures than arimoclomol. As in the FUS model, arimoclomol failed to induce HSPA1A or preserve Hspa8 mRNA in the TDP-43 model, despite preserving nuclear TDP-43 and Brg1, indicating neuroprotective properties other than HSP induction. The data speak to the complexity of drug mechanisms against multiple biomarkers of ALS pathogenesis, as well as to the importance of HSPA8 for neuronal proteostasis in both somata and dendrites.

Chaperones Hsc70 and Hsp70 play distinct roles in the replication of bocaparvovirus minute virus of canines.

Minute virus of canines (MVC) belongs to the genus Bocaparvovirus (formerly Bocavirus) within the Parvoviridae family and causes serious respiratory and gastrointestinal symptoms in neonatal canines worldwide. A productive viral infection relies on the successful recruitment of host factors for various stages of the viral life cycle. However, little is known about the MVC-host cell interactions. In this study, we identified that two cellular proteins (Hsc70 and Hsp70) interacted with NS1 and VP2 proteins of MVC, and both two domains of Hsc70/Hsp70 were mediated for their interactions. Functional studies revealed that Hsp70 was induced by MVC infection, knockdown of Hsc70 considerably suppressed MVC replication, whereas the replication was dramatically promoted by Hsp70 knockdown. It is interesting that low amounts of overexpressed Hsp70 enhanced viral protein expression and virus production, but high amounts of Hsp70 overexpression weakened them. Upon Hsp70 overexpressing, we observed that the ubiquitination of viral proteins changed with Hsp70 overexpression, and proteasome inhibitor (MG132) restored an accumulation of viral proteins. In addition, we verified that Hsp70 family inhibitors remarkably decreased MVC replication. Overall, we identified Hsc70 and Hsp70 as interactors of MVC NS1 and VP2 proteins and were involved in MVC replication, which may provide novel targets for anti-MVC approach.

Identification and validation of cuproptosis and disulfidptosis related genes in colorectal cancer.

Colorectal cancer, the third most prevalent malignant cancer, is associated with poor prognosis. Recent studies have investigated the mechanisms underlying cuproptosis and disulfidptosis in colorectal cancer. However, whether genes linked to these processes impact the prognosis of colorectal cancer patients through analogous mechanisms remains unclear. In this study, we developed a model of cuproptosis and disulfidptosis in colorectal cancer and concurrently explored the role of the pivotal model gene HSPA8 in colorectal cancer cell lines. Our results revealed a positive correlation between cuproptosis and disulfidptosis, both of which are emerging as protective factors for the prognosis of CRC patients. Consequently, a prognostic model encompassing HSPA8, PDCL3, CBX3, ATP6V1G1, TAF1D, RPL4, and RPL14 was constructed. Notably, the key gene in our model, HSPA8, exhibited heightened expression and was validated as a protective prognostic factor in colorectal cancer, exerting inhibitory effects on colorectal cancer cell proliferation. This study offers novel insights into the interplay between cuproptosis and disulfidptosis. The application of the prognostic model holds promise for more effectively predicting the overall survival of colorectal cancer patients.

Experimental and computational investigation of the effect of Hsc70 structural variants on inhibiting amylin aggregation.

The misfolding and aggregation of human islet amyloid polypeptide (hIAPP), also known as amylin, have been implicated in the pathogenesis of type 2 diabetes (T2D). Heat shock proteins, specifically, heat shock cognate 70 (Hsc70), are molecular chaperones that protect against hIAPP misfolding and inhibits its aggregation. Nevertheless, there is an incomplete understanding of the mechanistic interactions between Hsc70 domains and hIAPP, thus limiting their potential therapeutic role in diabetes. This study investigates the inhibitory capacities of different Hsc70 variants, aiming to identify the structural determinants that strike a balance between efficacy and cytotoxicity. Our experimental findings demonstrate that the ATPase activity of Hsc70 is not a pivotal factor for inhibiting hIAPP misfolding. We underscore the significance of the C-terminal substrate-binding domain of Hsc70 in inhibiting hIAPP aggregation, emphasizing that the removal of the lid subdomain diminishes the inhibitory effect of Hsc70. Additionally, we employed atomistic discrete molecular dynamics simulations to gain deeper insights into the interaction between Hsc70 variants and hIAPP. Integrating both experimental and computational findings, we propose a mechanism by which Hsc70's interaction with hIAPP monomers disrupts protein-protein connections, primarily by shielding the ß-sheet edges of the Hsc70-ß-sandwich. The distinctive conformational dynamics of the alpha helices of Hsc70 potentially enhance hIAPP binding by obstructing the exposed edges of the ß-sandwich, particularly at the ß5-ß8 region along the alpha helix interface. This, in turn, inhibits fibril growth, and similar results were observed following hIAPP dimerization. Overall, this study elucidates the structural intricacies of Hsc70 crucial for impeding hIAPP aggregation, improving our understanding of the potential anti-aggregative properties of molecular chaperones in diabetes treatment.

Salidroside directly activates HSC70, revealing a new role for HSC70 in BDNF signalling and neurogenesis after cerebral ischemia.

Salidroside, a principal bioactive component of Rhodiola crenulata, is neuroprotective across a wide time window in stroke models. We investigated whether salidroside induced neurogenesis after cerebral ischemia and aimed to identify its primary molecular targets. Rats, subjected to transient 2 h of middle cerebral artery occlusion (MCAO), received intraperitoneal vehicle or salidroside ± intracerebroventricular HSC70 inhibitor VER155008 or TrkB inhibitor ANA-12 for up to 7 days. MRI, behavioural tests, immunofluorescent staining and western blotting measured effects of salidroside. Reverse virtual docking and enzymatic assays assessed interaction of salidroside with purified recombinant HSC70. Salidroside dose-dependently decreased cerebral infarct volumes and neurological deficits, with maximal effects by 50 mg/kg/day. This dose also improved performance in beam balance and Morris water maze tests. Salidroside significantly increased BrdU+/nestin+, BrdU+/DCX+, BrdU+/NeuN+, BrdU-/NeuN+ and BDNF+ cells in the peri-infarct cortex, with less effect in striatum and no significant effect in the subventricular zone. Salidroside was predicted to bind with HSC70. Salidroside dose-dependently increased HSC70 ATPase and HSC70-dependent luciferase activities, but it did not activate HSP70. HSC70 immunoreactivity concentrated in the peri-infarct cortex and was unchanged by salidroside. However, VER155008 prevented salidroside-dependent increases of neurogenesis, BrdU-/NeuN+ cells and BDNF+ cells in peri-infarct cortex. Salidroside also increased BDNF protein and p-TrkB/TrkB ratio in ischemic brain, changes prevented by VER155008 and ANA-12, respectively. Additionally, ANA-12 blocked salidroside-dependent neurogenesis and increased BrdU-/NeuN+ cells in the peri-infarct cortex. Salidroside directly activates HSC70, thereby stimulating neurogenesis and neuroprotection via BDNF/TrkB signalling after MCAO. Salidroside and similar activators of HSC70 might provide clinical therapies for ischemic stroke.

BAG5 regulates HSPA8-mediated protein folding required for sperm head-tail coupling apparatus assembly.

Teratozoospermia is a significant cause of male infertility, but the pathogenic mechanism of acephalic spermatozoa syndrome (ASS), one of the most severe teratozoospermia, remains elusive. We previously reported Spermatogenesis Associated 6 (SPATA6) as the component of the sperm head-tail coupling apparatus (HTCA) required for normal assembly of the sperm head-tail conjunction, but the underlying molecular mechanism has not been explored. Here, we find that the co-chaperone protein BAG5, expressed in step 9-16 spermatids, is essential for sperm HTCA assembly. BAG5-deficient male mice show abnormal assembly of HTCA, leading to ASS and male infertility, phenocopying SPATA6-deficient mice. In vivo and in vitro experiments demonstrate that SPATA6, cargo transport-related myosin proteins (MYO5A and MYL6) and dynein proteins (DYNLT1, DCTN1, and DNAL1) are misfolded upon BAG5 depletion. Mechanistically, we find that BAG5 forms a complex with HSPA8 and promotes the folding of SPATA6 by enhancing HSPA8's affinity for substrate proteins. Collectively, our findings reveal a novel protein-regulated network in sperm formation in which BAG5 governs the assembly of the HTCA by activating the protein-folding function of HSPA8.

Plasmodium falciparum J-dot localized J domain protein A8iJp modulates the chaperone activity of human HSPA8.

Plasmodium falciparum renovates the host erythrocyte to survive during intraerythrocytic development. This renovation requires many parasite proteins to unfold and move outside the parasitophorous vacuolar membrane, and chaperone-regulated protein folding becomes essential for the exported proteins to function. We report on a type-IV J domain protein (JDP), PF3D7_1401100, which we found to be processed before export and trafficked inside the lumen of parasite-derived structures known as J-dots. We found this protein to have holdase activity, as well as stimulate the ATPase and aggregation suppression activity of the human HSP70 chaperone HsHSPA8; thus, we named it "HSPA8-interacting J protein" (A8iJp). Moreover, we found a subset of HsHSPA8 to co-localize with A8iJp inside the infected human erythrocyte. Our results suggest that A8iJp modulates HsHSPA8 chaperone activity and may play an important role in host erythrocyte renovation.

Mfn2/Hsc70 Complex Mediates the Formation of Mitochondria-Lipid Droplets Membrane Contact and Regulates Myocardial Lipid Metabolism.

The heart primarily derives its energy through lipid oxidation. In cardiomyocytes, lipids are stored in lipid droplets (LDs) and are utilized in mitochondria, although the structural and functional connections between these two organelles remain largely unknown. In this study, visible evidence have presented indicating that a complex is formed at the mitochondria-LD membrane contact (MLC) site, involving mitochondrion-localized Mfn2 and LD-localized Hsc70. This complex serves to tether mitochondria to LDs, facilitating the transfer of fatty acids (FAs) from LDs to mitochondria for ß-oxidation. Reduction of Mfn2 induced by lipid overload inhibits MLC, hinders FA transfer, and results in lipid accumulation. Restoring Mfn2 reinstates MLC, alleviating myocardial lipotoxicity under lipid overload conditions both in-vivo and in-vitro. Additionally, prolonged lipid overload induces Mfn2 degradation through the ubiquitin-proteasome pathway, following Mfn2 acetylation at the K243 site. This leads to the transition from adaptive lipid utilization to maladaptive lipotoxicity. The experimental findings are supported by clinical data from patients with obesity and age-matched non-obese individuals. These translational results make a significant contribution to the molecular understanding of MLC in the heart, and offer new insights into its role in myocardial lipotoxicity.

RPL35A promotes the progression of cholangiocarcinoma by mediating HSPA8 ubiquitination.

BACKGROUND: Cholangiocarcinoma (CCA) is a biliary epithelial malignant tumor with an increasing incidence worldwide. Therefore, further understanding of the molecular mechanisms of CCA progression is required to identify new therapeutic targets. METHODS: The expression of RPL35A in CCA and para-carcinoma tissues was detected by immunohistochemical staining. IP-MS combined with Co-IP identified downstream proteins regulated by RPL35A. Western blot and Co-IP of CHX or MG-132 treated CCA cells were used to verify the regulation of HSPA8 protein by RPL35A. Cell experiments and subcutaneous tumorigenesis experiments in nude mice were performed to evaluate the effects of RPL35A and HSPA8 on the proliferation, apoptosis, cell cycle, migration of CCA cells and tumor growth in vivo. RESULTS: RPL35A was significantly upregulated in CCA tissues and cells. RPL35A knockdown inhibited the proliferation and migration of HCCC-9810 and HUCCT1 cells, induced apoptosis, and arrested the cell cycle in G1 phase. HSPA8 was a downstream protein of RPL35A and overexpressed in CCA. RPL35A knockdown impaired HSPA8 protein stability and increased HSPA8 protein ubiquitination levels. RPL35A overexpression promoted CCA cell proliferation and migration. HSPA8 knockdown inhibited CCA cell proliferation and migration, and reversed the promoting effect of RPL35A. Furthermore, RPL35A promoted tumor growth in vivo. In contrast, HSPA8 knockdown suppressed tumor growth, while was able to restore the effects of RPL35A overexpression. CONCLUSION: RPL35A was upregulated in CCA tissues and promoted the progression of CCA by mediating HSPA8 ubiquitination.

Raltitrexed induces apoptosis through activating ROS-mediated ER stress by impeding HSPA8 expression in prostate cancer cells.

Prostate cancer is the most common malignant tumor in males, which frequently develops into castration-resistant prostate cancer (CRPC). CRPC metastasis is the main reason for its high mortality rate. At present, it lacks effective treatment for patients with CRPC. Raltitrexed (RTX) has been shown to be effective in the treatment of colorectal cancer. However, the effect of RTX on prostate cancer and the underlying mechanism remain unknown. In the current study, we found that RTX could dose-dependently inhibit proliferation, migration, colony formation and induce apoptosis in DU145 and PC-3 cells. RTX also increased ROS generation in prostate cancer cells. Pretreatment with N-acetyl-L-cysteine (NAC) significantly prevented RTX-induced cell apoptosis and endoplasmic reticulum (ER) stress signaling activation in prostate cancer cells. Additionally, we found RTX-induced ROS generation and ER stress activation depended on the expression of heat shock protein family A member 8 (HSPA8). Over-expression of HSPA8 could alleviate RTX-induced cell apoptosis, ROS generation and ER stress signaling activation. Finally, our study also showed that RTX attenuated the tumor growth of prostate cancer in the DU145 xenograft model and significantly downregulated HSPA8 expression and activated ER stress signaling pathway in tumor tissues. Our study is the first to reveal that RTX induces prostate cancer cells apoptosis through inhibiting the expression of HSPA8 and further inducing ROS-mediated ER stress pathway action. This study suggests that RTX may be a novel promising candidate drug for prostate cancer therapy.

HSPA8-mediated stability of the CLPP protein regulates mitochondrial autophagy in cisplatin-resistant ovarian cancer cells.

Currently, platinum agents remain the mainstay of chemotherapy for ovarian cancer (OC). However, cisplatin (DDP) resistance is a major reason for chemotherapy failure. Thus, it is extremely important to elucidate the mechanism of resistance to DDP. Here, we establish two DDP-resistant ovarian cancer cell lines and find that caseinolytic protease P (CLPP) level is significantly downregulated in DDP-resistant cell lines compared to wild-type ovarian cancer cell lines (SK-OV-3 and OVcar3). Next, we investigate the functions of CLPP in DDP-resistant and wild-type ovarian cancer cells using various assays, including cell counting kit-8 assay, western blot analysis, immunofluorescence staining, and detection of reactive oxygen species (ROS) and apoptosis. Our results show that CLPP knockdown significantly increases the half maximal inhibitory concentration (IC 50) and mitophagy of wild-type SK-OV-3 and OVcar3 cells, while CLPP overexpression reduces the IC 50 values and mitophagy of DDP-resistant SK-OV-3 and OVcar3 cells. Next, we perform database predictions and confirmation experiments, which show that heat shock protein family A member 8 (HSPA8) regulates CLPP protein stability. The dynamic effects of the HSPA8/CLPP axis in ovarian cancer cells are also examined. HSPA8 increases mitophagy and the IC 50 values of SK-OV-3 and OVcar3 cells but inhibits their ROS production and apoptosis. In addition, CLPP partly reverses the effects induced by HSPA8 in SK-OV-3 and OVcar3 cells. In conclusion, CLPP increases DDP resistance in ovarian cancer by inhibiting mitophagy and promoting cellular stress. Meanwhile, HSPA8 promotes the degradation of CLPP protein by regulating its stability.

Hemin competitively inhibits HSPA8 ATPase activity mitigating its foldase function.

Hemolysis in red blood cells followed by hemoglobin degradation results in high hemin levels in the systemic circulation. Such a level of hemin is disastrous for cells and tissues and is considerably responsible for the pathologies of diseases like severe malaria. Hemin's hydrophobic chemical nature and structure allow it to bind several proteins leading to their functional modification. Such modifications in physiologically relevant proteins can have a high impact on various cellular processes. HSPA8 is a chaperone that has a protective role in oxidative stress by aiding protein refolding. Through ATPase activity assays we found that hemin can competitively inhibit ATP hydrolysis by the chaperone HSPA8. Hemin as such does not affect the structural integrity of the protein which is inferred from CD spectroscopy and Gel filtration but it hinders the ATP-dependent foldase function of the chaperone. HSPA8 was not able to cause the refolding of the model protein lysozyme in the presence of hemin. The loss in HSPA8 function was due to competition between hemin and ATP as the chaperone was able to regain the foldase function when the concentration of ATP was gradually increased with hemin present at the inhibitory concentration. In-silico studies to establish the competition for the specific binding site revealed that ATP was unable to replace hemin from the ATP binding pocket of HSPA8 and was forced to form a non-specific and unstable complex. In-vitro isothermal calorimetry revealed that the affinity of ATP for binding to HSPA8 was reduced 22 folds in the presence of hemin. The prevention of HSPA8's cytoprotective function by hemin can be a major factor contributing to the overall cellular damage during hemin accumulation in the case of severe malaria and other hemolytic diseases.

Targeted Degradation of Signal Transduction and Activator of Transcription 3 by Chaperone-Mediated Autophagy Targeting Chimeric Nanoplatform.

Chaperone-mediated autophagy (CMA) is a lysosomal-dependent proteolysis pathway for the degradation of cytosolic proteins. However, exploiting CMA-mediated proteolysis to degrade proteins of interest in cancer therapy has not been widely applied. In this study, we develop a CMA-targeting chimera (CMATAC) to efficiently and specifically degrade signal transduction and activator of transcription 3 (STAT3) in tumor cells. CMATAC consists of STAT3 and heat shock cognate 70 kDa protein (HSC70) targeting peptides connected by a linker. To efficiently deliver CMATACs into tumor cells, lipid nanoparticles (LNPs) are used to encapsulate CMATACs (nCMATACs) and decorated with an insulin-like growth factor 2 receptor (IGF2R) targeting peptide (InCMATACs) to achieve tumor targeting and precise delivery. The CMA pathway is activated in tumor cells by a fasting-mimicking diet (FMD). Furthermore, FMD treatment strongly enhances the cellular uptake and tumor accumulation of InCMATACs by upregulating the IGF2R expression. As a result, InCMATACs efficiently degrade STAT3 protein in both A549 and HCC827 tumor cells and inhibit tumor growths in vivo. This study demonstrates that InCMATACs can be used for selective proteolysis in cancer therapy.

Arginine methylation of HSPA8 by PRMT9 inhibits ferroptosis to accelerate hepatitis B virus-associated hepatocellular carcinoma progression.

BACKGROUND: The hepatitis B virus X (HBx) protein is an established cause of hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC). Whether arginine methylation regulates ferroptosis involved in HBx-induced HCC progression has not been reported. This study aimed to explore whether HBx-regulated protein arginine methyltransferase 9 (PRMT9) mediates the involvement of ferroptosis in the development of HCC. METHODS AND RESULTS: HBx inhibited ferroptosis through promoting PRMT9 expression in HCC cells. PRMT9 suppressed ferroptosis to accelerate HCC progression in vivo. PRMT9 targeted HSPA8 and enhanced arginine methylation of HSPA8 at R76 and R100 to regulate ferroptosis in HCC. HSPA8 overexpression altered the transcriptome profile of HepG2 cells, in particular, ferroptosis and immune-related pathways were significantly enriched by differentially expressed genes, including CD44. HSPA8 overexpression up-regulated CD44 expression and knockdown of CD44 significantly reversed the inhibition of ferroptosis caused by PRMT9 overexpression. CONCLUSIONS: In conclusion, HBx/PRMT9/HSPA8/CD44 axis is a vital signal pathway regulating ferroptosis in HCC cells. This study provides new opportunities and targets for the treatment of HBV-induced HCC.

Heat shock protein family A member 8 is a prognostic marker for bladder cancer: Evidences based on experiments and machine learning.

Heat shock protein member 8 (HSPA8) is one of the most abundant chaperones in eukaryotic cells, but its biological roles in bladder cancer (BC) are largely unclear. First, we observed that HSPA8 was abundant in both cell lines and tissues of BC, and the HSPA8-high group had poorer T stages and overall survival (OS) than the HSPA8-low group in the TCGA patients. Next, when we knocked down HSPA8 in BC cells, the growth and migration abilities were significantly decreased, the apoptosis rates were significantly increased, and the Ki67 fluorescence intensity was decreased in BC cells. Moreover, caspase 3 was significantly decreased with overexpression of HSPA8 in BC cells. After that, a machine learning prognostic model was created based on the expression of HSPA8 by applying LASSO Cox regression in TCGA and GEO patients. The model indicated that the low-risk (LR) group with BC had better tumour stages, lymphovascular invasion, and OS than the high-risk (HR) group. Additionally, the risk score was demonstrated to be an independent risk factor for the prognosis of BC by univariate and multivariate Cox analyses. Moreover, the HR group showed a greater rate of TP53 mutations and was mostly enriched in the ECM-receptor interaction pathway than the LR group. Importantly, lower CD8+ T-cell and NK cell infiltration, higher immune exclusion scores, higher expression of PD-L1 and CTLA4 and poorer immune checkpoint therapy effects were found in the HR group. These findings demonstrated how crucial HSPA8 plays a role in determining the prognosis of bladder cancer.

HSPA8 acts as an amyloidase to suppress necroptosis by inhibiting and reversing functional amyloid formation.

Ultra-stable fibrous structure is a hallmark of amyloids. In contrast to canonical disease-related amyloids, emerging research indicates that a significant number of cellular amyloids, termed 'functional amyloids', contribute to signal transduction as temporal signaling hubs in humans. However, it is unclear how these functional amyloids are effectively disassembled to terminate signal transduction. RHIM motif-containing amyloids, the largest functional amyloid family discovered thus far, play an important role in mediating necroptosis signal transduction in mammalian cells. Here, we identify heat shock protein family A member 8 (HSPA8) as a new type of enzyme - which we name as 'amyloidase' - that directly disassembles RHIM-amyloids to inhibit necroptosis signaling in cells and mice. Different from its role in chaperone-mediated autophagy where it selects substrates containing a KFERQ-like motif, HSPA8 specifically recognizes RHIM-containing proteins through a hydrophobic hexapeptide motif N(X1)φ(X3). The SBD domain of HSPA8 interacts with RHIM-containing proteins, preventing proximate RHIM monomers from stacking into functional fibrils; furthermore, with the NBD domain supplying energy via ATP hydrolysis, HSPA8 breaks down pre-formed RHIM-amyloids into non-functional monomers. Notably, HSPA8's amyloidase activity in disassembling functional RHIM-amyloids does not require its co-chaperone system. Using this amyloidase activity, HSPA8 reverses the initiator RHIM-amyloids (formed by RIP1, ZBP1, and TRIF) to prevent necroptosis initiation, and reverses RIP3-amyloid to prevent necroptosis execution, thus eliminating multi-level RHIM-amyloids to effectively prevent spontaneous necroptosis activation. The discovery that HSPA8 acts as an amyloidase dismantling functional amyloids provides a fundamental understanding of the reversibility nature of functional amyloids, a property distinguishing them from disease-related amyloids that are unbreakable in vivo.

Molecular mechanism of the carboxyl terminus of Hsc70-interacting protein in TAU hyperphosphorylation induced by AlCl3 in N2a cells.

Aluminum (Al) is recognized as a neurotoxin. Studies have confirmed that the neurotoxicity induced by Al may be related to tau hyperphosphorylation. Phosphorylated tau is degraded through the ubiquitin-proteasome pathway (UPP), in which the carboxyl terminus of Hsc70-interacting protein (CHIP) plays an important role. However, whether the CHIP plays a role in regulating tau hyperphosphorylation induced by Al is yet to be determined. The purpose of this study was to explore the molecular mechanism of the CHIP in tau hyperphosphorylation induced by AlCl3 in N2a cells. Mouse neuroblastoma cells (N2a) were exposed to different concentrations of AlCl3 (0, 0.5, 1, and 2 mM) and treated with CHIP/CHIP shRNA/CHIP (ΔU-box)/CHIP (ΔTPR) plasmid transfection. The cell viability was determined by the CCK-8 kit. Protein expression was detected by Western blot. The interaction between CHIP and AlCl3 exposure on the proteins was analyzed by factorial design ANOVA. The results showed that Al can cause tau hyperphosphorylation, mainly affecting the pThr231, pSer262, and pSer396 sites of tau in N2a cells. UPP is involved in the degradation of tau hyperphosphorylation induced by Al in N2a cells, of which CHIP may be the main regulatory target. Both the U-box and TPR domains of CHIP are indispensable and play an important role in the regulation of tau hyperphosphorylation induced by AlCl3 in N2a cells.

Association between HSPA8 Gene Variants and Ischemic Stroke: A Pilot Study Providing Additional Evidence for the Role of Heat Shock Proteins in Disease Pathogenesis.

HSPA8 is involved in many stroke-associated cellular processes, playing a pivotal role in the protein quality control system. Here we report the results of the pilot study aimed at determining whether HSPA8 SNPs are linked to the risk of ischemic stroke (IS). DNA samples from 2139 Russians (888 IS patients and 1251 healthy controls) were genotyped for tagSNPs (rs1461496, rs10892958, and rs1136141) in the HSPA8 gene using probe-based PCR. SNP rs10892958 of HSPA8 was associated with an increased risk (risk allele G) of IS in smokers (OR = 1.37; 95% CI = 1.07-1.77; p = 0.01) and patients with low fruit and vegetable consumption (OR = 1.36; 95% CI = 1.14-1.63; p = 0.002). SNP rs1136141 of HSPA8 was also associated with an increased risk of IS (risk allele A) exclusively in smokers (OR = 1.68; 95% CI = 1.23-2.28; p = 0.0007) and in patients with a low fruit and vegetable intake (OR = 1.29; 95% CI = 1.05-1.60; p = 0.04). Sex-stratified analysis revealed an association of rs10892958 HSPA8 with an increased risk of IS in males (risk allele G; OR = 1.30; 95% CI = 1.05-1.61; p = 0.01). Thus, SNPs rs10892958 and rs1136141 in the HSPA8 gene represent novel genetic markers of IS.