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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.

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. \\[V{\\text{O}}_{2\\text{max} }\\] 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 \\[V{\\text{O}}_{2\\text{max} }\\] 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.

In human β-thalassaemiaerythroblasts, HSP70 is sequestered in the cytoplasm by the excess of free α-globin chains and can no longer protect the master transcriptional factor of erythropoiesis GATA-1 from caspase-3 cleavage; transduction of a nuclear-targeted HSP70 or a caspase-3 uncleavable GATA-1 mutant restored maturation of erythropoiesis. HSP70 a target in β-thalassemia During normal human erythroid cell maturation, the chaperone protein HSP70 translocates to the nucleus where it protects the master transcriptional factor of erythropoiesis, GATA1, from caspase-3 cleavage. Here Jean-Benoît Arlet et al . show that in erythroblasts from patients with the inherited haemoglobinopathy β-thalassemia major (β-TM), HSP70 is sequestered in the cytoplasm by the excess of free α-globin chains that accumulate in these cells. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA1 mutant restores maturation of β-TM erythroblasts. The discovery of a mechanism contributing to the ineffective erythropoiesis seen in β-TM suggests a rationale for possible targeted therapies for β-TM β-Thalassaemia major (β-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of β-globin chains of haemoglobin, leading to the accumulation of free α-globin chains that form toxic aggregates 1 , 2 . Despite extensive knowledge of the molecular defects causing β-TM, little is known of the mechanisms responsible for the ineffective erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage 3 , 4 , 5 , 6 . We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation 7 . Although erythroid transcription factor GATA-1, the master transcriptional factor of erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase-3 cleavage 8 . The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation 9 . Here we show in vitro that during the maturation of human β-TM erythroblasts, HSP70 interacts directly with free α-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA-1 mutant restores terminal maturation of β-TM erythroblasts, which may provide a rationale for new targeted therapies of β-TM.

PurposeAutophagy and heat shock protein (HSP) response are proteostatic systems involved in the acute and adaptive responses to exercise. These systems may upregulate sequentially following cellular stress including acute exercise, however, currently few data exist in humans. This study investigated the autophagic and HSP responses to acute intense lower body resistance exercise in peripheral blood mononuclear cells (PBMCs) with and without branched-chain amino acids (BCAA) supplementation.MethodsTwenty resistance-trained males (22.3 ± 1.5 yr; 175.4 ± .7 cm; 86.4 ± 15.6 kg) performed a bout of intense lower body resistance exercise and markers of autophagy and HSP70 were measured immediately post- (IPE) and 2, 4, 24, 48, and 72 h post-exercise. Prior to resistance exercise, 10 subjects were randomly assigned to BCAA supplementation of 0.22 g/kg/d for 5 days pre-exercise and up to 72 h following exercise while the other 10 subjects consumed a placebo (PLCB).ResultsThere were no difference in autophagy markers or HSP70 expression between BCAA and PLCB groups. LC3II protein expression was significantly lower 2 and 4 h post-exercise compared to pre-exercise. LC3II: I ratio was not different at any time point compared to pre-exercise. Protein expression of p62 was lower IPE, 2, and 4 h post-exercise and elevated 24 h post-exercise. HSP70 expression was elevated 48 and 72 h post-exercise.ConclusionsAutophagy and HSP70 are upregulated in PBMCs following intense resistance exercise with autophagy increasing initially post-exercise and HSP response in the latter period. Moreover, BCAA supplementation did not affect this response.

The ubiquitin–proteasome system (UPS) is the main cellular proteolytic system responsible for the degradation of normal and abnormal (e.g. oxidised) proteins. Under catabolic conditions characterised by chronic inflammation, the UPS is activated resulting in proteolysis, muscle wasting and impaired muscle function. Milk proteins provide sulphur-containing amino acid and have been proposed to affect muscle inflammation. However, the response of the UPS to aseptic inflammation and protein supplementation is largely unknown. The aim of this study was to investigate how milk protein supplementation affects UPS activity and skeletal muscle function under conditions of aseptic injury induced by intense, eccentric exercise. In a double-blind, cross-over, repeated measures design, eleven men received either placebo (PLA) or milk protein concentrate (PRO, 4×20 g on exercise day and 20 g/d for the following 8 days), following an acute bout of eccentric exercise (twenty sets of fifteen eccentric contractions at 30°/s) on an isokinetic dynamometer. In each trial, muscle biopsies were obtained from the vastus lateralis muscle at baseline, as well as at 2 and 8 d post exercise, whereas blood samples were collected before exercise and at 6 h, 1 d, 2 d and 8 d post exercise. Muscle strength and soreness were assessed before exercise, 6 h post exercise and then daily for 8 consecutive days. PRO preserved chymotrypsin-like activity and attenuated the decrease of strength, facilitating its recovery. PRO also prevented the increase of NF-κB phosphorylation and HSP70 expression throughout recovery. We conclude that milk PRO supplementation following exercise-induced muscle trauma preserves proteasome activity and attenuates strength decline during the pro-inflammatory phase.

Chronic glutamine supplementation reduces exercise-induced intestinal permeability and inhibits the NF-κB pro-inflammatory pathway in human peripheral blood mononuclear cells. These effects were correlated with activation of HSP70. The purpose of this paper is to test if an acute dose of oral glutamine prior to exercise reduces intestinal permeability along with activation of the heat shock response leading to inhibition of pro-inflammatory markers. Physically active subjects (N=7) completed baseline and exercise intestinal permeability tests, determined by the percent ratio of urinary lactulose (5 g) to rhamnose (2 g). Exercise included two 60-min treadmill runs at 70 % of VO2max at 30 °C after ingestion of glutamine (Gln) or placebo (Pla). Plasma levels of endotoxin and TNF-α, along with peripheral blood mononuclear cell (PBMC) protein expression of HSP70 and IκBα, were measured pre- and post-exercise and 2 and 4 h post-exercise. Permeability increased in the Pla trial compared to that at rest (0.06±0.01 vs. 0.02±0.018) and did not increase in the Gln trial. Plasma endotoxin was lower at the 4-h time point in the Gln vs. 4 h in the Pla (6.715±0.046 pg/ml vs. 7.952± 1.11 pg/ml). TNF-α was lower 4 h post-exercise in the Gln vs. Pla (1.64±0.09 pg/ml vs. 1.87±0.12 pg/ml). PBMC expression of IκBα was higher 4 h post-exercise in the Gln vs. 4 h in the Pla (1.29±0.43 vs. 0.8892±0.040). HSP70 was higher pre-exercise and 2 h post-exercise in the Gln vs. Pla (1.35 ± 0.21 vs. 1.000±0.000 and 1.65±0.21 vs. 1.27±0.40). Acute oral glutamine supplementation prevents an exercise-induced rise in intestinal permeability and suppresses NF-κB activation in peripheral blood mononuclear cells.

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.

Among the heat shock proteins (HSP), HSP27, HSP70 and HSP90 are the most studied stress-inducible HSPs, and are induced in response to a wide variety of physiological and environmental insults, thus allowing cells to survive to lethal conditions based on their powerful cytoprotective functions. Different functions of HSPs have been described to explain their cytoprotective functions, including their most basic role as molecular chaperones, that is to regulate protein folding, transport, translocation and assembly, especially helping in the refolding of misfolded proteins, as well as their anti-apoptotic properties. In cancer cells, the expression and/or activity of the three HSPs is abnormally high, and is associated with increased tumorigenicity, metastatic potential of cancer cells and resistance to chemotherapy. Associating with key apoptotic factors, they are powerful anti-apoptotic proteins, having the capacity to block the cell death process at different levels. Altogether, the properties suggest that HSP27, HSP70 and HSP90 are appropriate targets for modulating cell death pathways. In this review, we summarize the role of HSP90, HSP70 and HSP27 in apoptosis and the emerging strategies that have been developed for cancer therapy based on the inhibition of the three HSPs.

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.

Tendinopathies are diseases that often entail long-term treatment consisting of analgesics, physiotherapy, orthotics, and sparing. The aim of this study was to investigate the effect of a single application of a high-energy PEMF (pulsed electromagnetic field) on pain perception and blood born inflammation parameters. 34 patients were randomly assigned to a verum group (10 min PEMF, 0,78 T) or a placebo group (10 min sham condition). Prior to and up to one week after the patient blinded treatment (t1-t5), local pain state was assessed by means of algometry as pain pressure threshold (PPT). Accordingly, heat-shock protein 70 (HSP70) levels were analysed. Statistical analyses included 2‑way ANOVA (2 × 5). The clinical trial was registered (DRKS00031321). After randomization and drop-out (verum n = 17, placebo n = 13) baseline-analyses did not reveal significant between-group differences for PPT (p = 0,096), for HSP70 (p = 0,524), or any other sample characteristics (p > 0,05). Pain reduction during one week of observation showed to be significantly higher (p = 0,045, η  = 0,013) for the PEMF group (PPT: +83 bis +139%) compared to the placebo group (PPT: +10 bis +36%). There were no HSP70 associated effects. A single bout of high energy PEMF led to an immediate pain relief in tendinopathy patients lasting at least for one week, but the hypothesized underlying HSP70 associated inflammatory pathway could not be confirmed.