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Pro-survival stress-inducible chaperone HSP110 is the only HSP for which a mutation has been found in a cancer. Multicenter clinical studies demonstrated a direct association between HSP110 inactivating mutation presence and excellent prognosis in colorectal cancer patients. Here, we have combined crystallographic studies on human HSP110 and in silico modeling to identify HSP110 inhibitors that could be used in colorectal cancer therapy. Two molecules (foldamers 33 and 52), binding to the same cleft of HSP110 nucleotide-binding domain, were selected from a chemical library (by co-immunoprecipitation, AlphaScreening, Interference-Biolayer, Duo-link). These molecules block HSP110 chaperone anti-aggregation activity and HSP110 association to its client protein STAT3, thereby inhibiting STAT3 phosphorylation and colorectal cancer cell growth. These effects were strongly decreased in HSP110 knockdown cells. Foldamer’s 33 ability to inhibit tumor growth was confirmed in two colorectal cancer animal models. Although tumor cell death (apoptosis) was noted after treatment of the animals with foldamer 33, no apparent toxicity was observed, notably in epithelial cells from intestinal crypts. Taken together, we identified the first HSP110 inhibitor, a possible drug-candidate for colorectal cancer patients whose unfavorable outcome is associated to HSP110.

The deposition of highly ordered fibrillar-type aggregates into inclusion bodies is a hallmark of neurodegenerative diseases such as Parkinson’s disease. The high stability of such amyloid fibril aggregates makes them challenging substrates for the cellular protein quality-control machinery 1 , 2 . However, the human HSP70 chaperone and its co-chaperones DNAJB1 and HSP110 can dissolve preformed fibrils of the Parkinson’s disease-linked presynaptic protein α-synuclein in vitro 3 , 4 . The underlying mechanisms of this unique activity remain poorly understood. Here we use biochemical tools and nuclear magnetic resonance spectroscopy to determine the crucial steps of the disaggregation process of amyloid fibrils. We find that DNAJB1 specifically recognizes the oligomeric form of α-synuclein via multivalent interactions, and selectively targets HSP70 to fibrils. HSP70 and DNAJB1 interact with the fibril through exposed, flexible amino and carboxy termini of α-synuclein rather than the amyloid core itself. The synergistic action of DNAJB1 and HSP110 strongly accelerates disaggregation by facilitating the loading of several HSP70 molecules in a densely packed arrangement at the fibril surface, which is ideal for the generation of ‘entropic pulling’ forces. The cooperation of DNAJB1 and HSP110 in amyloid disaggregation goes beyond the classical substrate targeting and recycling functions that are attributed to these HSP70 co-chaperones and constitutes an active and essential contribution to the remodelling of the amyloid substrate. These mechanistic insights into the essential prerequisites for amyloid disaggregation may provide a basis for new therapeutic interventions in neurodegeneration. The molecular steps that lead to the disaggregation of amyloid fibrils are shown to involve the synergistic action of HSP70 and its co-chaperones DNAJB1 and HSP110.

Accumulation of aggregation‐prone misfolded proteins disrupts normal cellular function and promotes ageing and disease. Bacteria, fungi and plants counteract this by solubilizing and refolding aggregated proteins via a powerful cytosolic ATP‐dependent bichaperone system, comprising the AAA+ disaggregase Hsp100 and the Hsp70‐Hsp40 system. Metazoa, however, lack Hsp100 disaggregases. We show that instead the Hsp110 member of the Hsp70 superfamily remodels the human Hsp70‐Hsp40 system to efficiently disaggregate and refold aggregates of heat and chemically denatured proteins in vitro and in cell extracts. This Hsp110 effect relies on nucleotide exchange, not on ATPase activity, implying ATP‐driven chaperoning is not required. Knock‐down of nematode Caenorhabditis elegans Hsp110, but not an unrelated nucleotide exchange factor, compromises dissolution of heat‐induced protein aggregates and severely shortens lifespan after heat shock. We conclude that in metazoa, Hsp70‐Hsp40 powered by Hsp110 nucleotide exchange represents the crucial disaggregation machinery that reestablishes protein homeostasis to counteract protein unfolding stress. Aggregation of misfolded proteins disrupts cellular homeostasis leading to ageing and disease. In metazoa, the nucleotide exchange factor Hsp110 acts together with the human Hsp70‐Hsp40 chaperone system to promote protein disaggregation and longevity.

An efficient protein disaggregation system uncovered in metazoan cells requires transient interactions between J-protein co-chaperones of classes A and B, which synergistically boost HSP70-dependent disaggregation activity, providing a flexible further level of regulation for metazoan protein quality control, with direct relevance to human diseases such as age-related neurodegeneration. A novel protein quality control mechanism Microbes eliminate toxic intracellular protein aggregates thanks to HSP100 disaggregases, which are missing from animal cells. Now Bernd Bukau and colleagues have discovered an efficient protein disaggregation system in metazoan cells, which requires transient interactions between J-protein co-chaperones of classes A and B, which synergistically boost HSP70-dependent disaggregation activity. This system provides a flexible further level of regulation for metazoan protein quality control, with direct relevance to human diseases such as age-related neurodegeneration. Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states 1 , 2 . Healthy metazoan cells effectively eliminate intracellular protein aggregates 3 , 4 , indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems 5 , 6 , and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro 4 , 7 . This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.

Bacteria, fungi, protozoa, chromista and plants all harbor homologues of Hsp104, a AAA+ ATPase that collaborates with Hsp70 and Hsp40 to promote protein disaggregation and reactivation. Curiously, however, metazoa do not possess an Hsp104 homologue. Thus, whether animal cells renature large protein aggregates has long remained unclear. Here, it is established that mammalian cytosol prepared from different sources possesses a potent, ATP-dependent protein disaggregase and reactivation activity, which can be accelerated and stimulated by Hsp104. This activity did not require the AAA+ ATPase, p97. Rather, mammalian Hsp110 (Apg-2), Hsp70 (Hsc70 or Hsp70) and Hsp40 (Hdj1) were necessary and sufficient to slowly dissolve large disordered aggregates and recover natively folded protein. This slow disaggregase activity was conserved to yeast Hsp110 (Sse1), Hsp70 (Ssa1) and Hsp40 (Sis1 or Ydj1). Hsp110 must engage substrate, engage Hsp70, promote nucleotide exchange on Hsp70, and hydrolyze ATP to promote disaggregation of disordered aggregates. Similarly, Hsp70 must engage substrate and Hsp110, and hydrolyze ATP for protein disaggregation. Hsp40 must harbor a functional J domain to promote protein disaggregation, but the J domain alone is insufficient. Optimal disaggregase activity is achieved when the Hsp40 can stimulate the ATPase activity of Hsp110 and Hsp70. Finally, Hsp110, Hsp70 and Hsp40 fail to rapidly remodel amyloid forms of the yeast prion protein, Sup35, or the Parkinson's disease protein, alpha-synuclein. However, Hsp110, Hsp70 and Hsp40 enhanced the activity of Hsp104 against these amyloid substrates. Taken together, these findings suggest that Hsp110 fulfils a subset of Hsp104 activities in mammals. Moreover, they suggest that Hsp104 can collaborate with the mammalian disaggregase machinery to rapidly remodel amyloid conformers.

Here, the presentation of lipid antigens by CD1d is shown to induce retrograde anti-inflammatory signalling in intestinal epithelial cells, resulting in the production of IL-10. Anti-inflammatory IL-10 in the intestinal mucosa Intestinal epithelial cells (IECs) are crucial to mucosal homeostasis, serving as a physical barrier and regulating the responses of mucosal immune cells to environmental factors. This paper shows that CD1d, a glycoprotein involved in the presentation of lipid antigens, induces a self-reinforcing pathway of protective mucosal immunity within the intestinal epithelium. The pathway is mediated by regulatory cytokines and heat shock proteins, and interference with any molecules involved (including CD1d, IL-10 and HSP110) is associated with uncontrolled intestinal inflammation. These findings could have relevance for inflammatory bowel disease and similar conditions. The mechanisms by which mucosal homeostasis is maintained are of central importance to inflammatory bowel disease. Critical to these processes is the intestinal epithelial cell (IEC), which regulates immune responses at the interface between the commensal microbiota and the host 1 , 2 . CD1d presents self and microbial lipid antigens to natural killer T (NKT) cells, which are involved in the pathogenesis of colitis in animal models and human inflammatory bowel disease 3 , 4 , 5 , 6 , 7 , 8 . As CD1d crosslinking on model IECs results in the production of the important regulatory cytokine interleukin (IL)-10 (ref. 9 ), decreased epithelial CD1d expression—as observed in inflammatory bowel disease 10 , 11 —may contribute substantially to intestinal inflammation. Here we show in mice that whereas bone-marrow-derived CD1d signals contribute to NKT-cell-mediated intestinal inflammation, engagement of epithelial CD1d elicits protective effects through the activation of STAT3 and STAT3-dependent transcription of IL-10, heat shock protein 110 (HSP110; also known as HSP105), and CD1d itself. All of these epithelial elements are critically involved in controlling CD1d-mediated intestinal inflammation. This is demonstrated by severe NKT-cell-mediated colitis upon IEC-specific deletion of IL-10, CD1d, and its critical regulator microsomal triglyceride transfer protein (MTP) 12 , 13 , as well as deletion of HSP110 in the radioresistant compartment. Our studies thus uncover a novel pathway of IEC-dependent regulation of mucosal homeostasis and highlight a critical role of IL-10 in the intestinal epithelium, with broad implications for diseases such as inflammatory bowel disease.

Hsp70 molecular chaperones function in protein folding in a manner dependent on regulation by co‐chaperones. Hsp40s increase the low intrinsic ATPase activity of Hsp70, and nucleotide exchange factors (NEFs) remove ADP after ATP hydrolysis, enabling a new Hsp70 interaction cycle with non‐native protein substrate. Here, we show that members of the Hsp70‐related Hsp110 family cooperate with Hsp70 in protein folding in the eukaryotic cytosol. Mammalian Hsp110 and the yeast homologues Sse1p/2p catalyze efficient nucleotide exchange on Hsp70 and its orthologue in Saccharomyces cerevisiae , Ssa1p, respectively. Moreover, Sse1p has the same effect on Ssb1p, a ribosome‐associated isoform of Hsp70 in yeast. Mutational analysis revealed that the N‐terminal ATPase domain and the ultimate C‐terminus of Sse1p are required for nucleotide exchange activity. The Hsp110 homologues significantly increase the rate and yield of Hsp70‐mediated re‐folding of thermally denatured firefly luciferase in vitro . Similarly, deletion of SSE1 causes a firefly luciferase folding defect in yeast cells under heat stress in vivo . Our data indicate that Hsp110 proteins are important components of the eukaryotic Hsp70 machinery of protein folding.

Heat shock proteins (HSPs) are a kind of molecular chaperone that helps protein folding, which is closely related to cancer. However, the association between HSPs and clear cell renal clear cell carcinoma (ccRCC) is uncertain. We explored the prognostic value of HSP110, HSP90, HSP70 and HSP60 families in ccRCC and their role in tumor immune microenvironment. The data obtained from the Cancer Genome Atlas (TCGA) were applied to determine the differential expression of HSPs in normal tissues and ccRCC. We comprehensively analyzed the prognostic value of HSPs in ccRCC and constructed a prognostic signature. We further explored the differences of tumor immune microenvironment and targeted therapy based on the signature. Cell proliferation, invasion and metastasis were detected by CCK8 assay, wound healing and transwell. Three clusters were identified with differences in overall survival and tumor stage. 6-gene signature (HSPA8, HSP90B1, HSPA7, HSPA12B, HSPA4L, HSPA1L) was identified to predict ccRCC patients’ prognosis. The signature was confirmed in the internal cohort. Survival analysis, receiver operating characteristic (ROC) curve, univariate and multivariate COX regression analysis demonstrated the accuracy and independence of signature. The expression of HSPA7, HSPA8 and HSP90B1 were validated with quantitative real-time PCR. Our signature played a pivotal role in predicting tumor immune microenvironment, immune checkpoint gene expression, drug sensitivity, and tumor mutational burden (TMB) in patients with ccRCC. Our cellular experiments confirmed HSPA7 promotes the proliferation, invasion and metastasis of ccCRC cells. The HSPs signature identified in this study could serve as potential biomarkers for predicting prognosis and treatment response in ccRCC patients. It may provide new ideas for the current research on targeted therapy and immunotherapy strategies for ccRCC patients.

Primary tumors may create the premetastatic niche in secondary organs for subsequent metastasis. Humoral immunity contributes to the progression of certain cancers, but the roles of B cells and their derived antibodies in premetastatic niche formation are poorly defined. Using a mouse model of spontaneous lymph node metastasis of breast cancer, we show that primary tumors induced B cell accumulation in draining lymph nodes. These B cells selectively promoted lymph node metastasis by producing pathogenic IgG that targeted glycosylated membrane protein HSPA4, and activated the HSPA4-binding protein ITGB5 and the downstream Src/NF-κB pathway in tumor cells for CXCR4/SDF1α-axis-mediated metastasis. High serum anti-HSPA4 IgG was correlated with high tumor HSPA4 expression and poor prognosis of breast cancer subjects. Our findings identify a key role for tumor-educated B cells and their derived antibodies in lymph node premetastatic niche formation, providing potential targets for cancer intervention.B cells facilitate breast cancer metastasis to lymph nodes through production of antibodies targeting a protein on the surface of cancer cells that stimulates tumor dissemination.

Hsp70 is a key cellular system counteracting protein misfolding and aggregation, associated with stress, ageing, and disease. Hsp70 solubilises aggregates and aids protein refolding through substrate binding and release cycles regulated by co-chaperones: J-domain proteins (JDPs) and nucleotide exchange factors (NEFs). Here, we elucidate the collaborative impact of Hsp110 NEFs and different JDP classes throughout Hsp70-dependent aggregate processing. We show that Hsp110 plays a major role at initial stages of disaggregation, determining its final efficacy. The NEF catalyses the recruitment of thick Hsp70 assemblies onto aggregate surface, which modifies aggregates into smaller species more readily processed by chaperones. Hsp70 stimulation by Hsp110 is much stronger with class B than class A JDPs and requires the auxiliary interaction between class B JDP and the Hsp70 EEVD motif. Furthermore, we demonstrate for the first time that Hsp110 disrupts the JDP-Hsp70 interaction. Such destabilisation of chaperone complexes at the aggregate surface might improve disaggregation, but also lead to the inhibition above the sub-stoichiometric Hsp110 optimum. Thus, balanced interplay between the co-chaperones and Hsp70 is critical to unlock its disaggregating potential. For proteins to accurately carry out their role in the cell, they must first be precisely folded into specific 3D shapes. Stress, aging or disease can interfere with this delicate process, leading to misfolded proteins clumping together and causing damage. In response, the cell can deploy ‘chaperones’ which disentangle these aggregates and ensure that proteins recover their proper structure. Chaperones from the Hsp70 protein family, for example, are crucial for cell survival, especially under biologically stressful conditions. Yet Hsp70 proteins cannot perform their role without the assistance of co-chaperones such as Hsp110; why this is the case, however, has remained unclear. To investigate this question, Sztangierska et al. used a variety of biochemical assays to test how purified human and yeast Hsp70, Hsp110 and other co-chaperones could bind aggregates and recover misfolded proteins. The role of each protein was examined at every stage of the disaggregation process – from the initial aggregate binding, through chaperone-driven changes in aggregate structure to the final protein folding. The experiments revealed that Hsp110 helps draw Hsp70 to the aggregate surface, breaking down the protein ‘clump’ into smaller pieces which are more easily processed by other chaperones. The results also showed that the various co-chaperones compete for Hsp70 binding; too much of one might interfere with another, emphasizing the need for balance between chaperones for optimal disaggregation. Overall, these results clarify the role of Hsp110 in the Hsp70 system and reveal several mechanistic details of the protein rescue process. Further experiments will be needed to fully understand these dynamics and identify how they may be relevant to conditions in which harmful protein aggregates are observed, such as Parkinson’s or Alzheimer’s disease.