Rationale: Since oncogene expression products often exhibit upregulation or abnormally activated activity, developing a technique to regulate abnormal protein levels represent a viable approach for treating tumors and protein abnormality-related diseases. Methods: We first screened out eMIATAC components with high targeted degradation efficiency and explored the mechanism by which eMIATAC induced target protein degradation, and verified the degradation efficiency of the target protein by protein imprinting and flow cytometry. Next, we recombined eMIATAC with some controllable elements to verify the regulatable degradation performance of the target protein. Subsequently, we constructed eMIATAC that can express targeted degradation of AKT1 and verified its effect on GBM cell development in vitro and in vivo. Finally, we concatenated eMIATAC with CAR sequences to construct CAR-T cells with low BATF protein levels and verified the changes in their anti-tumor efficacy. Results: we developed a system based on the endosome-microautophagy-lysosome pathway for degrading endogenous proteins: endosome-MicroAutophagy TArgeting Chimera (eMIATAC), dependent on Vps4A instead of lysosomal-associated membrane protein 2A (LAMP2A) to bind to the chaperone Hsc70 and the protein of interest (POI). The complex was then transported to the lysosome by late endosomes, where degradation occurred similarly to microautophagy. The eMIATACs demonstrated accuracy, efficiency, reversibility, and controllability in degrading the target protein EGFP. Moreover, eMIATAC exhibited excellent performance in knocking down POI when targeting endogenous proteins in vivo and in vitro. Conclusions: The eMIATACs could not only directly knock down abnormal proteins for glioma treatment but also enhance the therapeutic effect of CAR-T cell therapy for tumors by knocking down T cell exhaustion-related proteins. The newly developed eMIATAC system holds promise as a novel tool for protein knockdown strategies. By enabling direct control over endogenous protein levels, eMIATAC has the potential to revolutionize treatment for cancer and genetic diseases.

The process of viruses entering host cells is complex, involving multiple aspects of the molecular organization of the cell membrane, viral proteins, the interaction of receptor molecules, and cellular signaling. Most viruses depend on endocytosis for uptake, when viruses reach the appropriate location, they are released from the vesicles, undergo uncoating, and release their genomes. Heat shock cognate protein 70(HSC70): also known as HSPA8, a protein involved in mediating clathrin-mediated endocytosis (CME), is involved in various viral entry processes. In this mini-review, our goal is to provide a summary of the function of HSC70 in viral entry. Understanding the interaction networks of HSC70 with viral proteins helps to provide new directions for targeted therapeutic strategies against viral infections.

Our recent work has uncovered a novel function of HSPA8 as an amyloidase, capable of dismantling the RHIM-containing protein fibrils to suppress necroptosis. However, the impact of HSPA8 inhibitors on cancer regression via necroptosis remains unexplored. In this study, we conducted a comprehensive investigation to assess the potential of HSPA8 inhibitors in enhancing necroptosis both in vitro and in vivo. Our findings indicate that pharmacologic inhibition of HSPA8, achieved either through VER (VER-155008) targeting the nucleotide binding domain or pifithrin-μ targeting the substrate binding domain of HSPA8, significantly potentiates necroptosis induced by diverse treatments in cellular assays. These inhibitors effectively disrupt the binding of HSPA8 to the RHIM protein, impeding its regulatory function on RHIM amyloid formation. Importantly, HSPA8 inhibitors significantly enhanced cancer cell sensitivity to microtubule-targeting agents (MTAs) in vitro, while reversing chemoresistance and facilitating tumor regression by augmenting necroptosis in vivo. Our findings suggest a promising therapeutic approach to cancer through necroptosis modulation via HSPA8 targeting, particularly in combination with MTA drugs for enhanced treatment efficacy.

Objective To elucidate the role of chaperone-mediated autophagy (CMA) in alleviating emotional dysfunction in mice with sepsis-associated encephalopathy (SAE). Methods The SAE mouse model was established by cecal ligation and perforation (CLP). The severity of sepsis was assessed using the sepsis severity score (MSS). Emotional function in SAE mice was assessed by the open-field test and elevated plus-maze. The expression levels of cognitive heat shock cognate protein 70 (HSC70), lysosomal-associated membrane protein 2A (LAMP2A) and high mobility group box 1 protein B1 (HMGB1) were detected using Western blotting. Co-localization of LAMP2A in the hippocampal neurons was observed by immunofluorescence. The release of inflammatory factors interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) was measured using ELISA. Following 12 hours post-CLP, mice were orally administered resveratrol at a dose of 30 mg/kg once daily until day 14. Results The mortality rate of CLP mice was 45.83% 24 days post CLP, and all surviving mice exhibited emotional disturbances. 24 hours after CLP, a significant decrease in HSC70 and LAMP2A expression in hippocampal neurons was observed, indicating impaired CMA activity. Meanwhile, HMGB1 and inflammatory cytokines (IL-6 and TNF-α) levels increased. After resveratrol treatment, an increase of HSC70 and LAMP2A expression, and a decrease of HMGB1 expression and inflammatory cytokine release were observed, suggesting enhanced CMA activity and reduced neuroinflammation. Behavioral tests showed that emotional dysfunction was improved in SAE mice after resveratrol treatment. Conclusion CMA activity of hippocampal neurons in SAE mice is significantly reduced, leading to emotional dysfunction. Resveratrol can alleviate neuroinflammation and emotional dysfunction in SAE mice by promoting CMA and inhibiting the expression of HMGB1 and the release of inflammatory factors.

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/β 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.

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.

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.

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.

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.

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.