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Tumors, as chronic malignant diseases that account for about 20% of all deaths worldwide, are the number one threat to human health. Until now there is no reliable treatment for most types of tumors. Tumorigenesis and cellular carcinogenesis remain difficult challenges due to their complex etiology and unknown mechanisms. As stress process regulating molecules and protein folding promoters, heat shock proteins (HSPs) play an important role in cancer development. Most studies have shown that HSPs are one of the major anticancer drug targets. HSPs are not only modulators of the cellular stress response, but are also closely associated with tumor initiation, progression, and drug resistance, so understanding the mechanism of the HSP family involved in cellular carcinogenesis is an important part of understanding tumorigenesis and enabling anticancer drug development. In this review, we discuss the functions and mechanisms of key members of the HSP family (HSP70, HSP90, and HSP110) in participating in the process of tumorigenesis and cell carcinogenesis, and look forward to the prospect of key members of the HSP family in targeted cancer therapy. [ABSTRACT FROM AUTHOR]

Fungal infections present a significant global health challenge, prompting ongoing research to discover innovative antifungal agents. The 110 kDa heat shock proteins (Hsp110s) are molecular chaperones essential for maintaining cellular protein homeostasis in eukaryotes. Fungal Hsp110s have emerged as a promising target for innovative antifungal strategies. Notably, 2H stands out as a promising candidate in the endeavor to target Hsp110s and combat fungal infections. Our study reveals that 2H exhibits broad-spectrum antifungal activity, effectively disrupting the in vitro chaperone activity of Hsp110 from Candida auris and inhibiting the growth of Cryptococcus neoformans. Pharmacokinetic analysis indicates that oral administration of 2H may offer enhanced efficacy compared to intravenous delivery, emphasizing the importance of optimizing the AUC/MIC ratio for advancing its clinical therapy. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum heat shock protein 70-1 (PfHsp70-1) and PfHsp70-z are essential cytosol localised chaperones of the malaria parasite. The two chaperones functionally interact to drive folding of several parasite proteins. While PfHsp70-1 is regarded as a canonical Hsp70 chaperone, PfHsp70-z belongs to the Hsp110 subcluster. One of the distinctive features of PfHsp70-z is its unique linker segment which delineates it from canonical Hsp70. In the current study, we elucidated the role of the linker in regulating Hsp70 self-association and client selection. Using recombinant forms of PfHsp70-1, PfHsp70-z and E. coli Hsp70 (DnaK) and their respective linker switch mutants we investigated self-association of the chaperones using surface plasmon resonance (SPR) analysis. The effect of the changes on client selectivity was investigated on DnaK and its mutant through co-affinity chromatography coupled to LC-MS analysis. Our findings demonstrated that the linker is important for both Hsp70 self-association and client binding.

Heat shock protein 70 (Hsp70) is a molecular chaperone and central regulator of protein homeostasis (proteostasis). Paramount to this role is Hsp70's binding to client proteins and co-chaperones to produce distinct complexes, such that understanding the protein-protein interactions (PPIs) of Hsp70 is foundational to describing its function and dysfunction in disease. Mounting evidence suggests that these PPIs include both "canonical" interactions, which are universally conserved, and "non-canonical" (or "secondary") contacts that seem to have emerged in eukaryotes. These two categories of interactions involve discrete binding surfaces, such that some clients and co-chaperones engage Hsp70 with at least two points of contact. While the contributions of canonical interactions to chaperone function are becoming increasingly clear, it can be challenging to deconvolute the roles of secondary interactions. Here, we review what is known about non-canonical contacts and highlight examples where their contributions have been parsed, giving rise to a model in which Hsp70's secondary contacts are not simply sites of additional avidity but are necessary and sufficient to impart unique functions. From this perspective, we propose that further exploration of non-canonical contacts will generate important insights into the evolution of Hsp70 systems and inspire new approaches for developing small molecules that tune Hsp70-mediated proteostasis.

Fungal infections present a significant global health challenge, prompting ongoing research to discover innovative antifungal agents. The 110 kDa heat shock proteins (Hsp110s) are molecular chaperones essential for maintaining cellular protein homeostasis in eukaryotes. Fungal Hsp110s have emerged as a promising target for innovative antifungal strategies. Notably, 2H stands out as a promising candidate in the endeavor to target Hsp110s and combat fungal infections. Our study reveals that 2H exhibits broad-spectrum antifungal activity, effectively disrupting the in vitro chaperone activity of Hsp110 from Candida auris and inhibiting the growth of Cryptococcus neoformans. Pharmacokinetic analysis indicates that oral administration of 2H may offer enhanced efficacy compared to intravenous delivery, emphasizing the importance of optimizing the AUC/MIC ratio for advancing its clinical therapy.

Simple Summary: Exploring the impact of heat shock protein (HSP) inhibition in cancer may give new insights into the cellular processes that these molecules sustain to promote cancer survival and may accelerate the discovery of more HSP inhibitors to be introduced in clinical trials. In this study, we explored the expression and role of high-molecular-weight HSP110 in the survival of Primary Effusion Lymphoma (PEL) cells. We found that the proper expression of this HSP is required to prevent lysosomal permeabilization, DNA damage, c-Myc downregulation and STAT3 de-phosphorylation. Indeed, HSP silencing strongly reduces PEL cell survival through the dysregulation of these processes that have been found to be interconnected. Heat shock proteins (HSPs) are highly expressed in cancer cells and represent a promising target in anti-cancer therapy. In this study, we investigated for the first time the expression of high-molecular-weight HSP110, belonging to the HSP70 family of proteins, in Primary Effusion Lymphoma (PEL) and explored its role in their survival. This is a rare lymphoma associated with KSHV, for which an effective therapy remains to be discovered. The results obtained from this study suggest that targeting HSP110 could be a very promising strategy against PEL, as its silencing induced lysosomal membrane permeabilization, the cleavage of BID, caspase 8 activation, downregulated c-Myc, and strongly impaired the HR and NHEJ DNA repair pathways, leading to apoptotic cell death. Since chemical inhibitors of this HSP are not commercially available yet, this study encourages a more intense search in this direction in order to discover a new potential treatment that is effective against this and likely other B cell lymphomas that are known to overexpress HSP110. [ABSTRACT FROM AUTHOR]

Perturbations in the native structure, often caused by stressing cellular conditions, not only impair protein function but also lead to the formation of aggregates, which can accumulate in the cell leading to harmful effects. Some organisms, such as plants, express the molecular chaperone HSP100 (homologous to HSP104 from yeast), which has the remarkable capacity to disaggregate and reactivate proteins. Recently, studies with animal cells, which lack a canonical HSP100, have identified the involvement of a distinct system composed of HSP70/HSP40 that needs the assistance of HSP110 to efficiently perform protein breakdown. As sessile plants experience stressful conditions more severe than those experienced by animals, we asked whether a plant HSP110 could also play a role in collaborating with HSP70/HSP40 in a system that increases the efficiency of disaggregation. Thus, the gene for a putative HSP110 from the cereal Sorghum bicolor was cloned and the protein, named SbHSP110, purified. For comparison purposes, human HsHSP110 (HSPH1/HSP105) was also purified and investigated in parallel. First, a combination of spectroscopic and hydrodynamic techniques was used for the characterization of the conformation and stability of recombinant SbHSP110, which was produced folded. Second, small‐angle X‐ray scattering and combined predictors of protein structure indicated that SbHSP110 and HsHSP110 have similar conformations. Then, the chaperone activities, which included protection against aggregation, refolding, and reactivation, were investigated, showing that SbHSP110 and HsHSP110 have similar functional activities. Altogether, the results add to the structure/function relationship study of HSP110s and support the hypothesis that plants have multiple strategies to act upon the reactivation of protein aggregates. [ABSTRACT FROM AUTHOR]

Heat shock protein 110 (HSP110) is an emerging biological target for development of drug to treat colorectal cancer. In the present study, we designed and synthesized a series of novel small-molecule inhibitors of HSP110 based on previously reported HSP110 modulator (hit 1). Among the novel compounds, molecule 7 exhibited the most improved biological activity in vitro and anticancer efficacy in vivo. It exerted greater anti-proliferative potency against several colorectal cancer cells including HCT116 and SW480 with IC50 values of 11.59 ​μM and 6.05 ​μM, respectively. Molecular docking experiment of 7 with HSP110 protein revealed it bound well with the target in the similar way as compound 1. Mechanistic studies revealed that 7 effectively inhibited STAT3 activity and the expression of STAT3 downstream genes, VEGF, MMP7, MMP9. Moreover, 7 significantly abolished IL-6-induced epithelial-mesenchymal transition (EMT) in HCT116 and SW480 ​cells. Finally, 7 exhibited stronger tumor growth inhibition than lead compound 1 ​at the dose of 5 ​mg/kg (i.p.) in an in vivo HCT116 ​cells xenografted nude mice model. Taken together, we identified 7 as 2nd generation of HSP110 inhibitor which exerts remarkable anti-colorectal cancer activities. This compound might serve as a new lead for developing anti-cancer drugs to treat colorectal cancer.

Whereas assembly of the 20S proteasome core particle (CP) in prokaryotes apparently occurs spontaneously, the efficiency of this process in eukaryotes relies on the dedicated assembly chaperones Ump1, Pba1-Pba2, and Pba3-Pba4. For mammals, it was reported that CP assembly initiates with formation of a complete α-ring that functions as a template for β subunit incorporation. By contrast, we were not able to detect a ring composed only of a complete set of α subunits in S. cerevisiae. Instead, we found that the CP subunits α1, α2, and α4 each form independent small complexes. Purification of such complexes containing α4 revealed the presence of chaperones of the Hsp70/Ssa and Hsp110/Sse families. Consistently, certain small complexes containing α1, α2, and α4 were not formed in strains lacking these chaperones. Deletion of the SSE1 gene in combination with deletions of PRE9 (α3), PBA3, or UMP1 genes resulted in severe synthetic growth defects, high levels of ubiquitin-conjugates, and an accumulation of distinct small complexes with α subunits. Our study shows that Hsp70 and Hsp110 chaperones cooperate to promote the folding of individual α subunits and/or their assembly with other CP subunits, Ump1, and Pba1-Pba4 in subsequent steps. [ABSTRACT FROM AUTHOR]

The mammalian prion protein can form infectious, non-native, and protease resistant aggregates (PrP D ), which cause lethal prion diseases like human Creutzfeldt-Jakob disease. PrP D seeds the formation of new infectious prions by interacting with and triggering the refolding of the normally soluble mammalian prion protein, PrP C , into more PrP D . Refolding of misfolded proteins in the cell is carried out by molecular chaperones such as Grp78. We have recently shown that Grp78 sensitizes PrP D to proteases, indicating structural alterations and leading to its degradation. However, the process of chaperone mediated PrP D disaggregation, the chaperones involved, and the effect of disaggregation on PrP D seeding activity are unclear. We have now monitored the structural modification, disaggregation, and seeding activity of PrP D from two mouse adapted prion strains, 22L and 87V, in the presence of Grp78 and two forms of the Hsp110 disaggregase chaperone family, Hsp105 and Apg-2. We found that both forms of Hsp110 induced similar amounts of disaggregation and structural change in the protease resistant cores of PrP D from both strains. However, 22L PrP D was more susceptible to destabilization and disaggregation by the chaperones than 87V. Surprisingly, despite disaggregation of both strains, only the 22L PrP D aggregates released by the chaperones had seeding activity, with both forms of Hsp110 enhancing the Grp78 mediated release of these aggregates. Our data show that disassembly of PrP D by Grp78 and Hsp110 chaperones can release seeding particles of PrP D in a strain specific manner, potentially facilitating prion replication and spread., Competing Interests: CONFLICT OF INTERST The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Our previous research verified that HSP (heat shock protein) 110 could enhance the anti-tumor effect of HPV16 E749-57 epitope. In this study, to optimize the immunotherapy of this vaccine type, we developed and evaluated the anti-tumor immunity of a nanoparticle vaccine format assembling with E749-57-HSP110 fusion expression plasmid and RGD-GGG-K18 polypeptide. The nanoparticle vaccine was self-assembled from positively charged RGD-GGG-K18 polypeptide and negatively charged fusion expression plasmid pIRES2-3× E7-HSP110-EGFP. The particle size, stability, expression of E749-57-HSP110 fusion protein and the target ability of nanoparticle were determined, respectively. Specific CTL responses were determined by E7 tetramer staining and cytotoxicity assay in TC-1 tumor-bearing mice (CD4/CD8 knockout). The preventive and therapeutic experiments of nanoparticle vaccine were investigated in TC-1 tumor-bearing mice. Results showed that the RGD-GGG-K18 polypeptide and pIRES2-3× E7-HSP110-EGFP plasmid self-assembled nanoparticles about 100 nanometers in diameter when the charge ratios of peptide/plasmid were 2. The nanoparticles effectively entered TC-1 cells directed by RGD target-peptide, and correctly expressed the E7-HSP110 fusion protein. The HSP110 effectively facilitated nanoparticles activating CD8+T cells than nanoparticles without HSP110, including the CD8+ T cell number and the IFN-γ level; in contrast, the CD4+T cells immune response remained indiscriminate among the mice groups. This nanoparticle formulation inhibited tumor growth and prolonged the survival duration in the prophylactic and therapeutic mouse models. Therefore, the RGD-based tumor-targeting nanoparticle expressing E749-57-HSP110 fusion protein can efficiently evoke anti-tumor activity and thus suggests it might be a favorable candidate for cervical cancer immunotherapy.

A study was carried out to evaluate the effect of heat stress on carcass traits and expression of Myostatin,Heat Shock Protein (HSP) 70and HSP110expression in skeletal muscle of Kanni Aadu goat. Twelve female (one year old) Kanni Aadu goats were randomly allocated into two groups of six animals each, control (KC) goats were placed in the thermo neutral zone (TNZ), i.e., control chamber, while the heat stress (KHS) goats were exposed to heat stress in hot chamber (40°C) for 45 days. Major carcass traits remained intact between the groups. Physico-chemical characteristics revealed a significantly (p

Heat shock proteins (HSPs) play oncogenic roles in human tumours. We reported a somatic inactivating mutation of HSP110 (HSP110DE9) in mismatch repair-deficient (dMMR) cancers displaying microsatellite instability (MSI) but did not assess its impact. We evaluated the impact of the Hsp110DE9 mutation on tumour development and the chemotherapy response in a dMMR knock-in mouse model (Hsp110DE9KIMsh2KO mice). The effect of the Hsp110DE9 mutation on tumorigenesis and survival was evaluated in Msh2KO mice that were null (Hsp110wt), heterozygous (Hsp110DE9KI/+), or homozygous (Hsp110DE9KI/KI) for the Hsp110DE9 mutation by assessing tumoral syndrome (organomegaly index, tumour staging) and survival (Kaplan–Meier curves). 5-Fluorouracil (5-FU), which is the backbone of chemotherapy regimens in gastrointestinal cancers and is commonly used in other tumour types but is not effective against dMMR cells in vivo, was administered to Hsp110DE9KI/KI, Hsp110DE9KI/+, and Hsp110wtMsh2KO mice. Hsp110, Ki67 (proliferation marker) and activated caspase-3 (apoptosis marker) expression were assessed in normal and tumour tissue samples by western blotting, immunophenotyping and cell sorting. Hsp110wt expression was drastically reduced or totally lost in tumours from Msh2KOHsp110DE9KI/+ and Msh2KOHsp110DE9KI/KI mice. The Hsp110DE9 mutation did not affect overall survival or tumoral syndrome in Msh2KOHsp110DE9KI/+ and Msh2KOHsp110DE9KI/KI mice but drastically improved the 5-FU response in all cohorts (Msh2KOHsp110DE9KI/KI: P5fu = 0.001; Msh2KOHsp110DE9KI/+: P5fu = 0.005; Msh2KOHsp110wt: P5fu = 0.335). Histopathological examination and cell sorting analyses confirmed major hypersensitization to 5-FU-induced death of both Hsp110DE9KI/KI and Hsp110DE9KI/+ dMMR cancer cells. This study highlights how dMMR tumour cells adapt to HSP110 inactivation but become hypersensitive to 5-FU, suggesting Hsp110DE9 as a predictive factor of 5-FU efficacy. [ABSTRACT FROM AUTHOR]

Osp94 (also known as HSPA4L or HSPH3), a member of the Hsp110/Sse1 family of heat-shock proteins, has a longer C-terminus than found in Hsc70/Hsp70 family proteins, composed of the loop region with a partial substrate-binding domain (SBD) β (L), and the SBDα and the C-terminal extension (H), but the functions of these domains are poorly understood. Here, we found that Osp94 suppressed heat-induced aggregation of luciferase (Luc). Osp94-bound heat-inactivated Luc was reactivated in the presence of rabbit reticulocyte lysate (RRL) and/or a combination of Hsc70 and Hsp40 (also known as HSPA8 and DNAJB1, respectively). Targeted deletion mutagenesis revealed that the SBDβ and H domains of Osp94 are critical for protein disaggregation and RRL-mediated refolding. Reactivation of Hsp90-bound heat-inactivated Luc was abolished in the absence of RRL but compensated for by PA28α (also known as PSME1), a proteasome activator. Interestingly, the LH domain also reactivated heat-inactivated Luc, independently of PA28α. Biotin-tag cross-linking experiments indicated that the LH domain and PA28α interact with Luc bound by Hsp90 during refolding. A chimeric protein in which the H domain was exchanged for PA28α also mediated disaggregation and reactivation of heat-inactivated Luc. These results indicate that Osp94 acts as a holdase, and that the C-terminal region plays a PA28α-like role in the refolding of unfolded proteins. [ABSTRACT FROM AUTHOR]

Background: Ischemia-reperfusion injury (IRI) poses a significant challenge to liver transplantation (LT). The underlying mechanism primarily involves overactivation of the immune system. Heat shock protein 110 (HSP110) functions as a molecular chaperone that helps stabilize protein structures., Methods: An IRI model was established by performing LT on Sprague-Dawley rats, and HSP110 was silenced using siRNA. Hematoxylin-eosin staining, TUNEL, immunohistochemistry, ELISA and liver enzyme analysis were performed to assess IRI following LT. Western blotting and quantitative reverse transcription-polymerase chain reaction were conducted to investigate the pertinent molecular changes., Results: Our findings revealed a significant increase in the expression of HSP110 at both the mRNA and protein levels in the rat liver following LT (P < 0.05). However, when rats were injected with siRNA-HSP110, IRI subsequent to LT was notably reduced (P < 0.05). Additionally, the levels of liver enzymes and inflammatory chemokines in rat serum were significantly reduced (P < 0.05). Silencing HSP110 with siRNA resulted in a marked decrease in M1-type polarization of Kupffer cells in the liver and downregulated the NF-κB pathway in the liver (P < 0.05)., Conclusions: HSP110 in the liver promotes IRI after LT in rats by activating the NF-κB pathway and inducing M1-type polarization of Kupffer cells. Targeting HSP110 to prevent IRI after LT may represent a promising new approach for the treatment of LT-associated IRI., Competing Interests: Competing interest No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article., (Copyright © 2023 First Affiliated Hospital, Zhejiang University School of Medicine in China. Published by Elsevier B.V. All rights reserved.)

Determination of microsatellite instability (MSI) using molecular test and deficient mismatch repair (dMMR) using immunohistochemistry (IHC) has major implications on colorectal cancer (CRC) management. The HSP110 T17 microsatellite has been reported to be more monomorphic than the common markers used for MSI determination. Large deletion of HSP110 T17 has been associated with efficacy of adjuvant chemotherapy in dMMR/MSI CRCs. The aim of this study was to evaluate the interest of HSP110 deletion/expression as a diagnostic tool of dMMR/MSI CRCs and a predictive tool of adjuvant chemotherapy efficacy. All patients with MSI CRC classified by molecular testing were included in this multicenter prospective cohort (n = 381). IHC of the 4 MMR proteins was carried out. HSP110 expression was carried out by IHC (n = 343), and the size of HSP110 T17 deletion was determined by PCR (n = 327). In the 293 MSI CRCs with both tests, a strong correlation was found between the expression of HSP110 protein and the size of HSP110 T17 deletion. Only 5.8% of MSI CRCs had no HSP110 T17 deletion (n = 19/327). HSP110 T17 deletion helped to re-classify 4 of the 9 pMMR/MSI discordance cases as pMMR/MSS cases. We did not observe any correlation between HSP110 expression or HSP110 T17 deletion size with time to recurrence in patients with stage II and III CRC, treated with or without adjuvant chemotherapy. HSP110 is neither a robust prognosis marker nor a predictor tool of adjuvant chemotherapy efficacy in dMMR/MSI CRC. However, HSP110 T17 is an interesting marker, which may be combined with the other pentaplex markers to identify discordant cases between MMR IHC and MSI.

Determination of microsatellite instability (MSI) using molecular test and deficient mismatch repair (dMMR) using immunohistochemistry (IHC) has major implications on colorectal cancer (CRC) management. The HSP110 T 17 microsatellite has been reported to be more monomorphic than the common markers used for MSI determination. Large deletion of HSP110 T 17 has been associated with efficacy of adjuvant chemotherapy in dMMR/MSI CRCs. The aim of this study was to evaluate the interest of HSP110 deletion/expression as a diagnostic tool of dMMR/MSI CRCs and a predictive tool of adjuvant chemotherapy efficacy. All patients with MSI CRC classified by molecular testing were included in this multicenter prospective cohort (n = 381). IHC of the 4 MMR proteins was carried out. HSP110 expression was carried out by IHC (n = 343), and the size of HSP110 T 17 deletion was determined by PCR (n = 327). In the 293 MSI CRCs with both tests, a strong correlation was found between the expression of HSP110 protein and the size of HSP110 T 17 deletion. Only 5.8% of MSI CRCs had no HSP110 T 17 deletion (n = 19/327). HSP110 T 17 deletion helped to re-classify 4 of the 9 pMMR/MSI discordance cases as pMMR/MSS cases. We did not observe any correlation between HSP110 expression or HSP110 T 17 deletion size with time to recurrence in patients with stage II and III CRC, treated with or without adjuvant chemotherapy. HSP110 is neither a robust prognosis marker nor a predictor tool of adjuvant chemotherapy efficacy in dMMR/MSI CRC. However, HSP110 T17 is an interesting marker, which may be combined with the other pentaplex markers to identify discordant cases between MMR IHC and MSI. [ABSTRACT FROM AUTHOR]

Whereas assembly of the 20S proteasome core particle (CP) in prokaryotes apparently occurs spontaneously, the efficiency of this process in eukaryotes relies on the dedicated assembly chaperones Ump1, Pba1-Pba2, and Pba3-Pba4. For mammals, it was reported that CP assembly initiates with formation of a complete α-ring that functions as a template for β subunit incorporation. By contrast, we were not able to detect a ring composed only of a complete set of α subunits in S. cerevisiae. Instead, we found that the CP subunits α1, α2, and α4 each form independent small complexes. Purification of such complexes containing α4 revealed the presence of chaperones of the Hsp70/Ssa and Hsp110/Sse families. Consistently, certain small complexes containing α1, α2, and α4 were not formed in strains lacking these chaperones. Deletion of the SSE1 gene in combination with deletions of PRE9 (α3), PBA3, or UMP1 genes resulted in severe synthetic growth defects, high levels of ubiquitin-conjugates, and an accumulation of distinct small complexes with α subunits. Our study shows that Hsp70 and Hsp110 chaperones cooperate to promote the folding of individual α subunits and/or their assembly with other CP subunits, Ump1, and Pba1-Pba4 in subsequent steps.

Here, we present data on characterisation of the linker of Plasmodium falciparum Hsp110 (PfHsp70-z) relative to the linker of canonical Hsp70s in support of a co-published article [1]. The linker of PfHsp70-z was switched with that of canonical Hsp70s, represented by PfHsp70–1 (cytosolic counterpart of PfHsp70-z) and E. coli Hsp70/DnaK. The datasets represent comparative analyses of PfHsp70-z, PfHsp70–1, and E. coli DnaK, relative to their linker switch mutants; PfHsp70-zLS, PfHsp70–1LS, DnaKLS, respectively. Intrinsic and extrinsic fluorescence spectroscopic analyses were employed to elucidate effects of the mutations on the structural features of the proteins. The structural conformations of the proteins were analysed in the absence as well as presence of nucleotides. In addition, stability of the proteins to stress (pH changes and urea) was also determined. Surface plasmon resonance (SPR) was employed to determine affinity of the proteins for ATP. The relative affinities of PfHsp70-z and PfHsp70–1 for the parasite cytosol localised, J domain co-chaperone, PfHsp40, was determined by SPR analysis. The effect of the linker of PfHsp70-z on the interaction of DnaKLS with DnaJ (a co-chaperone of DnaK), was similarly determined. These data could be used for future investigations involving protein-protein/ligand interactions as described in [1]. The raw data obtained using the various techniques here described are hosted in the Mendeley Data repository at [2].

The presence of an inactivating heat shock protein 110 (HSP110) mutation in colorectal cancers has been correlated with an excellent prognosis and with the ability of HSP110 to favor the formation of tolerogenic (M2‐like) macrophages. These clinical and experimental results suggest a potentially powerful new strategy against colorectal cancer: the inhibition of HSP110. In this work, as an alternative to neutralizing antibodies, Nanofitins (scaffold ~7 kDa proteins) targeting HSP110 were isolated from the screening of a synthetic Nanofitin library, and their capacity to bind (immunoprecipitation, biolayer interferometry) and to inhibit HSP110 was analyzed in vitro and in vivo. Three Nanofitins were found to inhibit HSP110 chaperone activity. Interestingly, they share a high degree of homology in their variable domain and target the peptide‐binding domain of HSP110. In vitro, they inhibited the ability of HSP110 to favor M2‐like macrophages. The Nanofitin with the highest affinity, A‐C2, was studied in the CT26 colorectal cancer mice model. Our PET/scan experiments demonstrate that A‐C2 may be localized within the tumor area, in accordance with the reported HSP110 abundance in the tumor microenvironment. A‐C2 treatment reduced tumor growth and was associated with an increase in immune cells infiltrating the tumor and particularly cytotoxic macrophages. These results were confirmed in a chicken chorioallantoic membrane tumor model. Finally, we showed the complementarity between A‐C2 and an anti‐PD‐L1 strategy in the in vivo and in ovo tumor models. Overall, Nanofitins appear to be promising new immunotherapeutic lead compounds. What's new? Inactivating HSP110 mutations in colorectal cancers have been correlated with excellent prognosis and revealed the ability of HSP110 to favor the formation of tolerogenic macrophages. As an alternative to neutralizing antibodies, here the authors selected Nanofitins (scaffold ~7 kDa‐proteins) able to target HSP110 with subnanomolar affinity. The highest‐affinity Nanofitin, A‐C2, was shown to accumulate within the tumor and reduce tumor growth in vivo through a mechanism involving macrophages. The results also demonstrated A‐C2 complementarity with PD‐L1 checkpoint inhibitors, an immunotherapeutic approach currently in use. Altogether, this work shows the potential of Nanofitins as therapeutic lead compounds. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum expresses two essential cytosol localised chaperones; PfHsp70 -1 and PfHsp70 -z. PfHsp70 -z (Hsp110 homologue) is thought to facilitate nucleotide exchange function of PfHsp70 -1. PfHsp70 -1 is a refoldase, while PfHsp70 -z is restricted to holdase chaperone function. The structural features delineating functional specialisation of these chaperones remain unknown. Notably, PfHsp70 -z possesses a unique linker segment which could account for its distinct functions. Using recombinant forms of PfHsp70 -1, PfHsp70 -z and E. coli Hsp70 (DnaK) as well as their linker switch mutant forms, we explored the effects of the linker mutations by conducting several assays such as circular dichroism, intrinsic and extrinsic fluorescence coupled to biochemical and in cellular analyses. Our findings demonstrate that the linker of PfHsp70 -z modulates global conformation of the chaperone, regulating several functions such as client protein binding, chaperone- and ATPase activities. In addition, as opposed to the flexible linker of PfHsp70 -1, the PfHsp70 -z linker is rigid, thus regulating its notable thermal stability, making it an effective stress buffer. Our findings suggest a crucial role for the linker in streamlining the functions of these two chaperones. The findings further explain how these distinct chaperones cooperate to ensure survival of P. falciparum particularly under the stressful human host environment. [ABSTRACT FROM AUTHOR]

In eukaryotes, Hsp110s are unambiguous cognates of the Hsp70 chaperones, in primary sequence, domain organization, and structure. Hsp110s function as nucleotide exchange factors (NEFs) for the Hsp70s although their apparent loss of Hsp70‐like chaperone activity, nature of interdomain communication, and breadth of domain functions are still puzzling. Here, by combining single‐molecule FRET, small angle X‐ray scattering measurements (SAXS), and MD simulation, we show that yeast Hsp110, Sse1 lacks canonical Hsp70‐like interdomain allostery. However, the protein exhibits unique noncanonical conformational changes within its domains. Sse1 maintains an open‐lid substrate‐binding domain (SBD) in close contact with its nucleotide‐binding domain (NBD), irrespective of its ATP hydrolysis status. To further appreciate such ATP‐hydrolysis‐independent exhaustive interaction between two domains of Hsp110s, NBD‐SBD chimera was constructed between Hsp110 (Sse1) and Hsp70 (Ssa1). In Sse1/Ssa1 chimera, we observed undocking of two domains leading to complete loss of NEF activity of Sse1. Interestingly, chimeric proteins exhibited significantly enhanced ATPase rate of Sse1‐NBD compared to wild‐type protein, implying that intrinsic ATPase activity of the protein remains mostly repressed. Apart from repressing the high ATPase activity of its NBD, interactions between two domains confer thermal stability to Sse1 and play critical role in the (co)chaperoning function of Sse1 in Ssa1‐mediated disaggregation activity. Altogether, Sse1 exhibits a unique interdomain interaction, which is essential for its NEF activity, suppression of high intrinsic ATPase activity, co‐chaperoning activity in disaggregase machinery, and stability of the protein. [ABSTRACT FROM AUTHOR]

Proteins carry out the vast majority of functions in cells, but can only do so when properly folded. Following stress or mutation, proteins can lose their proper fold, resulting in misfolding, inactivity, and aggregation–posing a threat to cellular health. In order to counteract protein aggregation, cells have evolved a remarkable subset of molecular chaperones, called protein disaggregases, which collaboratively possess the ability to forcibly untangle protein aggregates. Here, we review the different chaperone disaggregation machineries present in the human cytosol and their mechanisms of action. Understanding, how these disaggregases function, is both universally and clinically important, as protein aggregation has been linked to multiple, debilitating neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

HSP70 and its evolutionarily diverged co-chaperone HSP110, forms an important node in protein folding cascade. How these proteins maintain the aggregation-prone proteome of malaria parasite in functional state remains underexplored, in contrast to its human orthologs. In this study, we have probed into conformational dynamics of plasmodial HSP70 and HSP110 through multiple μ s MD-simulations (ATP-state) and compared with their respective human counterparts. Simulations covered sampling of 3.4 and 2.8 μs for HSP70 and HSP110, respectively, for parasite and human orthologs. We provide a comprehensive description of the dynamic behaviors that characterize the systems and also introduce a parameter for quantifying protein rigidity. For HSP70, the interspecies comparison reveals enhanced flexibility in IA and IB subdomain within the conserved NBD, lesser solvent accessibility of the interdomain linker and distinct dynamics of the SBDβ of Pf HSP70 in comparison to Hs HSP70. In the case of HSP110, notable contrast in the dynamics of NBD, SBDβ and SBDα was observed between parasite and human ortholog. Although HSP70 and HSP110 are members of the same superfamily, we identified specific differences in the subdomain contacts in NBD, linker properties and interdomain movements in their human and parasite orthologs. Our study suggests that differences in conformational dynamics may translate into species-specific differences in the chaperoning activities of HSP70-HSP110 in the parasite and human, respectively. Dynamical features of Pf HSP70-HSP110 may contribute to the maintenance of proteostasis in the parasite during its intracellular survival in the host. [Display omitted] • Parasite and human HSP70-HSP110 exhibit distinct differences in their conformational dynamics. • The conserved NBD and SBDβ show significant differences in flexibility and motion in both HSP70 and HSP110 orthologs. • Interdomain linker shows differences in length and solvent accessibility across species. • Structural-functional diversity of HSP70-HSP110 may facilitate intracellular survival of the malaria parasite. [ABSTRACT FROM AUTHOR]

Although cancers account for over 16% of all global deaths annually, at present, no reliable therapies exist for most types of the disease. As protein folding facilitators, heat shock proteins (Hsps) play an important role in cancer development. Not surprisingly, Hsps are among leading anticancer drug targets. Generally, Hsp70s are divided into two main subtypes: canonical Hsp70 (Escherichia coli Hsp70/DnaK homologues) and the non-canonical (Hsp110 and Grp170) members. These two main Hsp70 groups are delineated from each other by distinct structural and functional specifications. Non-canonical Hsp70s are considered as holdase chaperones, while canonical Hsp70s are refoldases. This unique characteristic feature is mirrored by the distinct structural features of these two groups of chaperones. Hsp110/Grp170 members are larger as they possess an extended acidic insertion in their substrate binding domains. While the role of canonical Hsp70s in cancer has received a fair share of attention, the roles of non-canonical Hsp70s in cancer development has received less attention in comparison. In the current review, we discuss the structure-function features of non-canonical Hsp70s members and how these features impact their role in cancer development. We further mapped out their interactome and discussed the prospects of targeting these proteins in cancer therapy.

Abstract Protein aggregate reactivation in metazoans is accomplished by the combined activity of Hsp70, Hsp40 and Hsp110 chaperones. Hsp110s support the refolding of aggregated polypeptides acting as specialized nucleotide exchange factors of Hsp70. We have studied how Apg2, one of the three human Hsp110s, regulates the activity of Hsc70 (HspA8), the constitutive Hsp70 in our cells. Apg2 shows a biphasic behavior: at low concentration, it stimulates the ATPase cycle of Hsc70, binding of the chaperone to protein aggregates and the refolding activity of the system, while it inhibits these three processes at high concentration. When the acidic subdomain of Apg2, a characteristic sequence present in the substrate binding domain of all Hsp110s, is deleted, the detrimental effects occur at lower concentration and are more pronounced, which concurs with an increase in the affinity of the Apg2 mutant for Hsc70. Our data support a mechanism in which Apg2 arrests the chaperone cycle through an interaction with Hsc70(ATP) that might lead to premature ATP dissociation before hydrolysis. In this line, the acidic subdomain might serve as a conformational switch to support dissociation of the Hsc70:Apg2 complex. Graphical abstract Unlabelled Image Highlights • Human Apg2 supports protein aggregate reactivation acting as nucleotide exchange factor of Hsc70. • Regulation of Hsc70 binding to aggregates by Apg2 correlates with substrate reactivation. • Apg2 stimulates/inhibits the activity of the Hsc70 system in a concentration-dependent manner. • An Hsc70:Apg2 complex arrests the ATPase cycle in the ADP/ATP exchange process. • The acidic subdomain supports dissociation of the Hsc70:Apg2 complex. [ABSTRACT FROM AUTHOR]

Cellular protein homeostasis (proteostasis) is maintained by a broad network of proteins involved in synthesis, folding, triage, repair and degradation. Chief among these are molecular chaperones and their cofactors that act as powerful protein remodelers. The growing realization that many human pathologies are fundamentally diseases of protein misfolding (proteopathies) has generated interest in understanding how the proteostasis network impacts onset and progression of these diseases. In this minireview, we highlight recent progress in understanding the enigmatic Hsp110 class of heat shock protein that acts as both a potent nucleotide exchange factor to regulate activity of the foldase Hsp70, and as a passive chaperone capable of recognizing and binding cellular substrates on its own, and its integration into the proteostasis network. [ABSTRACT FROM AUTHOR]

Protein aggregates are formed in cells with profoundly perturbed proteostasis, where the generation of misfolded proteins exceeds the cellular refolding and degradative capacity. They are a hallmark of protein conformational disorders and aged and/or environmentally stressed cells. Protein aggregation is a reversible process in vivo , which counteracts proteotoxicities derived from aggregate persistence, but the chaperone machineries involved in protein disaggregation in Metazoa were uncovered only recently. Here we highlight recent advances in the mechanistic understanding of the major protein disaggregation machinery mediated by the Hsp70 chaperone system and discuss emerging alternative disaggregation activities in multicellular organisms. [ABSTRACT FROM AUTHOR]

Abstract: Every colorectal cancer (CRC) patient should be tested for microsatellite instability (MSI) to screen for Lynch syndrome. Evaluation of MSI status involves screening tumor DNA for the presence of somatic deletions in DNA repeats using PCR followed by fragment analysis. While this method may lack sensitivity due to the presence of a high level of germline DNA, which frequently contaminates the core of primary colon tumors, no other method developed to date is capable of modifying the standard PCR protocol to achieve improvement of MSI detection. Here, we describe a new approach developed for the ultra‐sensitive detection of MSI in CRC based on E‐ice‐COLD‐PCR, using HSP110 T17, a mononucleotide DNA repeat previously proposed as an optimal marker to detect MSI in tumor DNA, and an oligo(dT)16 LNA blocker probe complementary to wild‐type genotypes. The HT17 E‐ice‐COLD‐PCR assay improved MSI detection by 20–200‐fold compared with standard PCR using HT17 alone. It presents an analytical sensitivity of 0.1%–0.05% of mutant alleles in wild‐type background, thus greatly improving MSI detection in CRC samples highly contaminated with normal DNA. HT17 E‐ice‐COLD‐PCR is a rapid, cost‐effective, easy‐to‐implement, and highly sensitive method, which could significantly improve the detection of MSI in routine clinical testing. [ABSTRACT FROM AUTHOR]

Synapses must be preserved throughout an organism's lifespan to allow for normal brain function and behavior. Synapse maintenance is challenging given the long distances between the termini and the cell body, reliance on axonal transport for delivery of newly synthesized presynaptic proteins, and high rates of synaptic vesicle exo- and endocytosis. Hence, synapses rely on efficient proteostasis mechanisms to preserve their structure and function. To this end, the synaptic compartment has specific chaperones to support its functions. Without proper synaptic chaperone activity, local proteostasis imbalances lead to neurotransmission deficits, dismantling of synapses, and neurodegeneration. In this review, we address the roles of four synaptic chaperones in the maintenance of the nerve terminal, as well as their genetic links to neurodegenerative disease. Three of these are Hsp40 co-chaperones (DNAJs): Cysteine String Protein alpha (CSPα; DNAJC5), auxilin (DNAJC6), and Receptor-Mediated Endocytosis 8 (RME-8; DNAJC13). These co-chaperones contain a conserved J domain through which they form a complex with heat shock cognate 70 (Hsc70), enhancing the chaperone's ATPase activity. CSPα is a synaptic vesicle protein known to chaperone the t-SNARE SNAP-25 and the endocytic GTPase dynamin-1, thereby regulating synaptic vesicle exocytosis and endocytosis. Auxilin binds assembled clathrin cages, and through its interactions with Hsc70 leads to the uncoating of clathrin-coated vesicles, a process necessary for the regeneration of synaptic vesicles. RME-8 is a co-chaperone on endosomes and may have a role in clathrin-coated vesicle endocytosis on this organelle. These three co-chaperones maintain client function by preserving folding and assembly to prevent client aggregation, but they do not break down aggregates that have already formed. The fourth synaptic chaperone we will discuss is Heat shock protein 110 (Hsp110), which interacts with Hsc70, DNAJAs, and DNAJBs to constitute a disaggregase. Hsp110-related disaggregase activity is present at the synapse and is known to protect against aggregation of proteins such as α-synuclein. Congruent with their importance in the nervous system, mutations of these co-chaperones lead to familial neurodegenerative disease. CSPα mutations cause adult neuronal ceroid lipofuscinosis, while auxilin mutations result in early-onset Parkinson's disease, demonstrating their significance in preservation of the nervous system.

Protein aggregation is intimately associated with cellular stress and is accelerated during aging, disease, and cellular dysfunction. Yeast cells rely on the ATP-consuming chaperone Hsp104 to disaggregate proteins together with Hsp70. Hsp110s are ancient and abundant chaperones that form complexes with Hsp70. Here we provide in vivo data showing that the Saccharomyces cerevisiae Hsp110s Sse1 and Sse2 are essential for Hsp104-dependent protein disaggregation. Following heat shock, complexes of Hsp110 and Hsp70 are recruited to protein aggregates and function together with Hsp104 in the disaggregation process. In the absence of Hsp110, targeting of Hsp70 and Hsp104 to the aggregates is impaired, and the residual Hsp104 that still reaches the aggregates fails to disaggregate. Thus, coordinated activities of both Hsp104 and Hsp110 are required to reactivate aggregated proteins. These findings have important implications for the understanding of how eukaryotic cells manage misfolded and amyloid proteins. [ABSTRACT FROM AUTHOR]

Synapses must be preserved throughout an organism's lifespan to allow for normal brain function and behavior. Synapse maintenance is challenging given the long distances between the termini and the cell body, reliance on axonal transport for delivery of newly synthesized presynaptic proteins, and high rates of synaptic vesicle exo- and endocytosis. Hence, synapses rely on efficient proteostasis mechanisms to preserve their structure and function. To this end, the synaptic compartment has specific chaperones to support its functions. Without proper synaptic chaperone activity, local proteostasis imbalances lead to neurotransmission deficits, dismantling of synapses, and neurodegeneration. In this review, we address the roles of four synaptic chaperones in the maintenance of the nerve terminal, as well as their genetic links to neurodegenerative disease. Three of these are Hsp40 co-chaperones (DNAJs): Cysteine String Protein alpha (CSPα; DNAJC5), auxilin (DNAJC6), and Receptor-Mediated Endocytosis 8 (RME-8; DNAJC13). These co-chaperones contain a conserved J domain through which they form a complex with heat shock cognate 70 (Hsc70), enhancing the chaperone's ATPase activity. CSPα is a synaptic vesicle protein known to chaperone the t-SNARE SNAP-25 and the endocytic GTPase dynamin-1, thereby regulating synaptic vesicle exocytosis and endocytosis. Auxilin binds assembled clathrin cages, and through its interactions with Hsc70 leads to the uncoating of clathrin-coated vesicles, a process necessary for the regeneration of synaptic vesicles. RME-8 is a co-chaperone on endosomes and may have a role in clathrin-coated vesicle endocytosis on this organelle. These three co-chaperones maintain client function by preserving folding and assembly to prevent client aggregation, but they do not break down aggregates that have already formed. The fourth synaptic chaperone we will discuss is Heat shock protein 110 (Hsp110), which interacts with Hsc70, DNAJAs, and DNAJBs to constitute a disaggregase. Hsp110-related disaggregase activity is present at the synapse and is known to protect against aggregation of proteins such as α-synuclein. Congruent with their importance in the nervous system, mutations of these co-chaperones lead to familial neurodegenerative disease. CSPα mutations cause adult neuronal ceroid lipofuscinosis, while auxilin mutations result in early-onset Parkinson's disease, demonstrating their significance in preservation of the nervous system. [ABSTRACT FROM AUTHOR]

Protein misfolding is implicated in numerous neurodegenerative disorders including amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease. A unifying feature of patients with these disorders is the accumulation of deposits comprised of misfolded protein. Aberrant protein folding can cause toxicity through a loss or gain of protein function, or both. An intriguing therapeutic approach to counter these disorders is the application of protein-remodeling factors to resolve these misfolded conformers and return the proteins to their native fold and function. Here, we describe the application of protein-remodeling factors to alleviate protein misfolding in neurodegenerative disease. We focus on Hsp104, Hsp110/Hsp70/Hsp40, NMNAT, and HtrA1, which can prevent and reverse protein aggregation. While many of these protein-remodeling systems are highly promising, their activity can be limited. Thus, engineering protein-remodeling factors to enhance their activity could be therapeutically valuable. Indeed, engineered Hsp104 variants suppress neurodegeneration in animal models, which opens the way to novel therapeutics and mechanistic probes to help understand neurodegenerative disease. [ABSTRACT FROM AUTHOR]

Protein misfolding under stressful environmental conditions cause several cellular problems owing to the disturbed cellular protein homeostasis, which may further lead to neurological disorders like Parkinson's disease (PD), Alzheimer's disease (AD), Amyloid lateral sclerosis and Huntington disease (HD). The presence of cellular defense mechanisms like molecular chaperones and proteasomal degradation systems prevent protein misfolding and aggregation. Molecular chaperones plays primary role in preventing protein misfolding by mediating proper native folding, unfolding and refolding of the polypeptides along with vast number of cellular functions. In past few years, the understanding of molecular chaperone mechanisms has been expanded enormously although implementation to prevent protein aggregation diseases is still deficient. We in this review evaluated major classes of molecular chaperones and their mechanisms relevant for preventing protein aggregation, specific case of α-synuclein aggregation. We also evaluate the molecular chaperone function as a novel therapeutic approach and the chaperone inhibitors or activators as small molecular drug targets. [ABSTRACT FROM AUTHOR]

Proteotoxic stresses and ageing cause breakdown of cellular protein homeostasis, allowing misfolded proteins to form aggregates, which dedicated molecular machines have evolved to solubilize. In bacteria, fungi, protozoa and plants protein disaggregation involves an Hsp70-J-protein chaperone system, which loads and activates a powerful AAA+ ATPase (Hsp100) disaggregase onto protein aggregate substrates. Metazoans lack cytosolic and nuclear Hsp100 disaggregases but still eliminate protein aggregates. This longstanding puzzle of protein quality control is now resolved. Robust protein disaggregation activity recently shown for the metazoan Hsp70-based disaggregases relies instead on a crucial cooperation between two J-protein classes and interaction with the Hsp110 co-chaperone. An expanding multiplicity of Hsp70 and J-protein family members in metazoan cells facilitates different configurations of this Hsp70-based disaggregase allowing unprecedented versatility and specificity in protein disaggregation. Here we review the architecture, operation and adaptability of the emerging metazoan disaggregation system and discuss how this evolved.

Recent studies have indicated that mammalian cells contain a cytosolic protein disaggregation machinery comprised of Hsc70, DnaJ homologs, and Hsp110 proteins, the last of which acts to accelerate a rate-limiting step of nucleotide exchange of Hsc70. We tested the ability of transgenic overexpression of a Thy1 promoterdriven human Hsp110 protein, HspA4L (Apg1), in neuronal cells of a transgenic G85R SOD1YFP ALS mouse strain to improve survival. Notably, G85R is a mutant version of Cu/Zn superoxide dismutase 1 (SOD1) that is unable to reach native form and that is prone to aggregation, with prominent YFP-fluorescent aggregates observed in the motor neurons of the transgenic mice as early as 1 mo of age. The several-fold overexpression of Hsp110 in motor neurons of these mice was associated with an increased median survival from ~5.5 to 7.5 mo and increased maximum survival from 6.5 to 12 mo. Improvement of survival was also observed for a G93A mutant SOD1 ALS strain. We conclude that neurodegeneration associated with cytosolic misfolding and aggregation can be ameliorated by overexpression of Hsp110, likely enhancing the function of a cytosolic disaggregation machinery. [ABSTRACT FROM AUTHOR]

Perturbations in the native structure, often caused by stressing cellular conditions, not only impair protein function but also lead to the formation of aggregates, which can accumulate in the cell leading to harmful effects. Some organisms, such as plants, express the molecular chaperone HSP100 (homologous to HSP104 from yeast), which has the remarkable capacity to disaggregate and reactivate proteins. Recently, studies with animal cells, which lack a canonical HSP100, have identified the involvement of a distinct system composed of HSP70/HSP40 that needs the assistance of HSP110 to efficiently perform protein breakdown. As sessile plants experience stressful conditions more severe than those experienced by animals, we asked whether a plant HSP110 could also play a role in collaborating with HSP70/HSP40 in a system that increases the efficiency of disaggregation. Thus, the gene for a putative HSP110 from the cereal Sorghum bicolor was cloned and the protein, named SbHSP110, purified. For comparison purposes, human HsHSP110 (HSPH1/HSP105) was also purified and investigated in parallel. First, a combination of spectroscopic and hydrodynamic techniques was used for the characterization of the conformation and stability of recombinant SbHSP110, which was produced folded. Second, small-angle X-ray scattering and combined predictors of protein structure indicated that SbHSP110 and HsHSP110 have similar conformations. Then, the chaperone activities, which included protection against aggregation, refolding, and reactivation, were investigated, showing that SbHSP110 and HsHSP110 have similar functional activities. Altogether, the results add to the structure/function relationship study of HSP110s and support the hypothesis that plants have multiple strategies to act upon the reactivation of protein aggregates., (© 2023 Wiley Periodicals LLC.)

Molecular chaperones control the cellular folding, assembly, unfolding, disassembly, translocation, activation, inactivation, disaggregation, and degradation of proteins. In 1989, groundbreaking experiments demonstrated that a purified chaperone can bind and prevent the aggregation of artificially unfolded polypeptides and use ATP to dissociate and convert them into native proteins. A decade later, other chaperones were shown to use ATP hydrolysis to unfold and solubilize stable protein aggregates, leading to their native refolding. Presently, the main conserved chaperone families Hsp70, Hsp104, Hsp90, Hsp60, and small heat-shock proteins (sHsps) apparently act as unfolding nanomachines capable of converting functional alternatively folded or toxic misfolded polypeptides into harmless protease-degradable or biologically active native proteins. Being unfoldases, the chaperones can proofread three-dimensional protein structures and thus control protein quality in the cell. Understanding the mechanisms of the cellular unfoldases is central to the design of new therapies against aging, degenerative protein conformational diseases, and specific cancers. [ABSTRACT FROM AUTHOR]