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 Hsp70 system is essential for maintaining protein homeostasis and comprises a central Hsp70 and two accessory proteins that belong to the J-domain protein (JDP) and nucleotide exchange factor families. Posttranslational modifications offer a means to tune the activity of the system. We explore how phosphorylation of specific residues of the J-domain of DNAJA2, a class A JDP, regulates Hsc70 activity using biochemical and structural approaches. Among these residues, we find that pseudophosphorylation of Y10 and S51 enhances the holding/folding balance of the Hsp70 system, reducing cochaperone collaboration with Hsc70 while maintaining the holding capacity. Truly phosphorylated J domains corroborate phosphomimetic variant effects. Notably, distinct mechanisms underlie functional impacts of these DNAJA2 variants. Pseudophosphorylation of Y10 induces partial disordering of the J domain, whereas the S51E substitution weakens essential DNAJA2-Hsc70 interactions without a large structural reorganization of the protein. S51 phosphorylation might be class-specific, as all cytosolic class A human JDPs harbor a phosphorylatable residue at this position.

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.

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.

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.

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.

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.

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.
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.
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.
RPL35A was upregulated in CCA tissues and promoted the progression of CCA by mediating HSPA8 ubiquitination.