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Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone which is essential in eukaryotes. It is required for the activation and stabilization of a wide variety of client proteins and many of them are involved in important cellular pathways. Since Hsp90 affects numerous physiological processes such as signal transduction, intracellular transport, and protein degradation, it became an interesting target for cancer therapy. Structurally, Hsp90 is a flexible dimeric protein composed of three different domains which adopt structurally distinct conformations. ATP binding triggers directionality in these conformational changes and leads to a more compact state. To achieve its function, Hsp90 works together with a large group of cofactors, termed co-chaperones. Co-chaperones form defined binary or ternary complexes with Hsp90, which facilitate the maturation of client proteins. In addition, posttranslational modifications of Hsp90, such as phosphorylation and acetylation, provide another level of regulation. They influence the conformational cycle, co-chaperone interaction, and inter-domain communications. In this review, we discuss the recent progress made in understanding the Hsp90 machinery.

Hsp70 chaperone systems are very versatile machines present in nearly all living organisms and in nearly all intracellular compartments. They function in many fundamental processes through their facilitation of protein (re)folding, trafficking, remodeling, disaggregation, and degradation. Hsp70 machines are regulated by co-chaperones. J-domain containing proteins (JDPs) are the largest family of Hsp70 co-chaperones and play a determining role functionally specifying and directing Hsp70 functions. Many features of JDPs are not understood; however, a number of JDP experts gathered at a recent CSSI-sponsored workshop in Gdansk (Poland) to discuss various aspects of J-domain protein function, evolution, and structure. In this report, we present the main findings and the consensus reached to help direct future developments in the field of Hsp70 research.

The ubiquitous heat shock protein 70 (HSP70) family consists of ATP-dependent molecular chaperones, which perform numerous cellular functions that affect almost all aspects of the protein life cycle from synthesis to degradation 1 – 3 . Achieving this broad spectrum of functions requires precise regulation of HSP70 activity. Proteins of the HSP40 family, also known as J-domain proteins (JDPs), have a key role in this process by preselecting substrates for transfer to their HSP70 partners and by stimulating the ATP hydrolysis of HSP70, leading to stable substrate binding 3 , 4 . In humans, JDPs constitute a large and diverse family with more than 40 different members 2 , which vary in their substrate selectivity and in the nature and number of their client-binding domains 5 . Here we show that JDPs can also differ fundamentally in their interactions with HSP70 chaperones. Using nuclear magnetic resonance spectroscopy 6 , 7 we find that the major class B JDPs are regulated by an autoinhibitory mechanism that is not present in other classes. Although in all JDPs the interaction of the characteristic J-domain is responsible for the activation of HSP70, in DNAJB1 the HSP70-binding sites in this domain are intrinsically blocked by an adjacent glycine-phenylalanine rich region—an inhibition that can be released upon the interaction of a second site on DNAJB1 with the HSP70 C-terminal tail. This regulation, which controls substrate targeting to HSP70, is essential for the disaggregation of amyloid fibres by HSP70–DNAJB1, illustrating why no other class of JDPs can substitute for class B in this function. Moreover, this regulatory layer, which governs the functional specificities of JDP co-chaperones and their interactions with HSP70s, could be key to the wide range of cellular functions of HSP70. The binding and activation of HSP70 by class B J-domain proteins is subject to an autoinhibitory regulatory mechanism that controls substrate targeting to HSP70 and is required for the disaggregation of amyloid fibres.

J-domain chaperones are involved in the efficient handover of misfolded/partially folded proteins to Hsp70 but also function independently to protect against cell death. Due to their high flexibility, the mechanism by which they regulate the Hsp70 cycle and how specific substrate recognition is performed remains unknown. Here we focus on DNAJB6b, which has been implicated in various human diseases and represents a key player in protection against neurodegeneration and protein aggregation. Using a variant that exists mainly in a monomeric form, we report the solution structure of an Hsp40 containing not only the J and C-terminal substrate binding (CTD) domains but also the functionally important linkers. The structure reveals a highly dynamic protein in which part of the linker region masks the Hsp70 binding site. Transient interdomain interactions via regions crucial for Hsp70 binding create a closed, autoinhibited state and help retain the monomeric form of the protein. Detailed NMR analysis shows that the CTD (but not the J domain) self-associates to form an oligomer comprising ∼35 monomeric units, revealing an intricate balance between intramolecular and intermolecular interactions. The results shed light on the mechanism of autoregulation of the Hsp70 cycle via conserved parts of the linker region and reveal the mechanism of DNAJB6b oligomerization and potentially antiaggregation.

ObjectiveExercise training is recently considered as a trend in adjuvant therapies for cancer patients, but its mechanisms need to be scrutinized further. This study is aimed to test the hypothesis that the patients who perform the high-intensity interval exercise training (HIIT) during hormone therapy would show improvements in low-grade inflammation and HSP70 compared to the controls receiving standard care.MethodsFifty two non-metastatic and hormone-responsive breast cancer patients were randomly assigned to high-intensity interval exercise (HIIT) (n = 26) and usual care (n = 26) groups. The HIIT groups participated in a high-intensity interval training protocol on a treadmill 3 days/week for 12 weeks. The training intensity was determined according to the predicted maximal heart rate. Demographic characteristics and medical history were collected via an interviewer-administered questionnaire at the baseline visit. Body fat was estimated based on skinfold thickness measured with calipers on the participant’s nonsurgery side at the triceps, suprailiac crest. \\[VO_2max \\] was estimated by 1-Mile Rockport Walk Test. Blood samples were collected 48 h before starting the exercise protocol and 48 h after the last exercise session. TNF-α, IL-6, IL-1β, IL-10, and HSP70 levels in serum were measured using the enzyme-linked immunosorbent assay (ELISA) method according to the manufacture’s instruction. Supernatant cytokine concentrations were determined by ELISA for IL-4 and IFN-γ. The data were analyzed by ANCOVA test that the pretest values were considered as covariate at P ≤ 0.05.ResultsHIIT improved \\[VO_2max \\] in the HIIT group compared to the usual care group (P = 0.002). The serum levels of TNF-α (P = 0.001), IL-6 (P = 0.007), and IL-10 (P = 0.001) were lower in the HIIT group. The level of IL-4 (P = 0.050) in the stimulated peripheral blood mononuclear cells significantly increased in the HIIT group compared to the usual care group. Furthermore, the serum level of the HSP70 was significantly higher in the HIIT group in comparison to the usual care group (P = 0.050). The TNF-α/IL-10 (P = 0.050) and IL-6/IL-10 (P = 0.042) ratios were lower in the HIIT group.ConclusionThe results of this study indicated that HIIT has positive impacts on the cardiorespiratory fitness and inflammatory cytokines in the breast cancer patients undergoing hormone therapy.

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.

Hsp90 is a conserved and essential molecular chaperone responsible for the folding and activation of hundreds of ‘client’ proteins 1 – 3 . The glucocorticoid receptor (GR) is a model client that constantly depends on Hsp90 for activity 4 – 9 . GR ligand binding was previously shown to be inhibited by Hsp70 and restored by Hsp90, aided by the co-chaperone p23 10 . However, a molecular understanding of the chaperone-mediated remodelling that occurs between the inactive Hsp70–Hsp90 ‘client-loading complex’ and an activated Hsp90–p23 ‘client-maturation complex’ is lacking for any client, including GR. Here we present a cryo-electron microscopy (cryo-EM) structure of the human GR-maturation complex (GR–Hsp90–p23), revealing that the GR ligand-binding domain is restored to a folded, ligand-bound conformation, while being simultaneously threaded through the Hsp90 lumen. In addition, p23 directly stabilizes native GR using a C-terminal helix, resulting in enhanced ligand binding. This structure of a client bound to Hsp90 in a native conformation contrasts sharply with the unfolded kinase–Hsp90 structure 11 . Thus, aided by direct co-chaperone–client interactions, Hsp90 can directly dictate client-specific folding outcomes. Together with the GR-loading complex structure 12 , we present the molecular mechanism of chaperone-mediated GR remodelling, establishing the first, to our knowledge, complete chaperone cycle for any Hsp90 client. Studies based on cryo-electron microscopy structures of Hsp90 chaperone complexes reveal the molecular mechanism of the chaperone-mediated maturation of the human glucocorticoid receptor.

J-domain proteins (JDPs) are the largest family of chaperones in most organisms, but much of how they function within the network of other chaperones and protein quality control machineries is still an enigma. Here, we report on the latest findings related to JDP functions presented at a dedicated JDP workshop in Gdansk, Poland. The report does not include all (details) of what was shared and discussed at the meeting, because some of these original data have not yet been accepted for publication elsewhere or represented still preliminary observations at the time.

The present study aims to explore the potential biomarker application of salivary heat shock 70 kDa protein in detecting thermal stress in dairy animals noninvasively. The study spans for 45 days during the mid-summer season (April–May), involving twelve multiparous non-pregnant adult Jersey crossbred cows by randomly allocating them into groups (six animals in each group). The control animals were maintained in the shed, whereas the thermal stress group animals were exposed to environment heat between 10:00 h to 16.00 h and they were feed and watered ad libitum. During the experimental period, the hematobiochemical, physiological, behavioural, nutritional and production responses were recorded and the whole blood and saliva were collected fortnightly. Results revealed significant increase in WBC, AST, ALP, blood urea nitrogen, triglycerides, cholesterol, HDL, blood and salivary cortisol, respiratory rate, rectal temperature, skin temperature of neck, lumbar and forelimb regions, standing time, salivary and blood HSP70 mRNA expression and their protein concentrations in heat stressed animals. In addition, RBC, haemoglobin, MCV, PCV, platelet, platelet-large cell ratio (PLCR), lying time, feed intake, milk yield and rumination time were significantly decreased in thermally stress animals. Furthermore, ROC curve analysis revealed the biomarker potential of these significantly altered parameters with 100% sensitivity and specificity for predicting environmental heat stress in dairy cows with AUC and Youden’s – index of 1.00 except platelet. Moreover, salivary HSP70 demonstrated significant correlation with these biomarkers. Noteworthily, salivary HSP70 also exhibited strong association with blood HSP70 and salivary cortisol. Furthermore, salivary HSP70 revealed 100% sensitivity and specificity in discriminating the dairy cattle succumbed to heat stress from healthy. In conclusion, the present study provides a newer insight into the multifaceted roles of HSP70 and identified salivary heat shock 70 kDa protein as a potential, reliable and more sensitive non-invasive biomarker for identifying environmental heat stress in dairy cattle. Graphical Abstract

Ubiquitin (Ub) and Ub-like proteins (Ubls) such as NEDD8 are best known for their function as covalent modifiers of other proteins but they are also themselves subject to post-translational modifications including phosphorylation. While functions of phosphorylated Ub (pUb) have been characterized, the consequences of Ubl phosphorylation remain unclear. Here we report that NEDD8 can be phosphorylated at S65 - the same site as Ub - and that S65 phosphorylation affects the structural dynamics of NEDD8 and Ub in a similar manner. While both pUb and phosphorylated NEDD8 (pNEDD8) can allosterically activate the Ub ligase Parkin, they have different protein interactomes that in turn are distinct from those of unmodified Ub and NEDD8. Among the preferential pNEDD8 interactors are HSP70 family members and we show that pNEDD8 stimulates HSP70 ATPase activity more pronouncedly than unmodified NEDD8. Our findings highlight the general importance of Ub/NEDD8 phosphorylation and support the notion that the function of pUb/pNEDD8 does not require their covalent attachment to other proteins. Both ubiquitin and NEDD8 can be phosphorylated, but the biological role of NEDD8 phosphorylation remains unclear. Here, the authors identify similarities and differences of ubiquitin and NEDD8 phosphorylation, showing that phosphorylated NEDD8 has a distinct interactome and regulates HSP70 proteins.