Hsp90 Activity Research Articles

Aha-type co-chaperones: the alpha or the omega of the Hsp90 ATPase cycle?

Heat shock protein 90 (Hsp90) is a dimeric molecular chaperone that plays an essential role in cellular homeostasis. It functions in the context of a structurally dynamic ATP-dependent cycle to promote conformational changes in its clientele to aid stability, maturation, and activation. The client activation cycle is tightly regulated by a cohort of co-chaperone proteins that display specific binding preferences for certain conformations of Hsp90, guiding Hsp90 through its functional ATPase cycle. Aha-type co-chaperones are well-known to robustly stimulate the ATPase activity of Hsp90 but other roles in regulating the functional cycle are being revealed. In this review, we summarize the work done on the Aha-type co-chaperones since the 1990s and highlight recent discoveries with respect to the complexity of Hsp90 cycle regulation.

Нові мішені фармакологічної корекції когнітивних розладів за умов пренатальної дії гіпоксії

В огляді наведено сучасні концепції механізмів пошкоджень клітин головного мозку за умов пренатального впливу хронічної гіпоксії. На основі сучасних даних про механізми адаптації організму до стану гіпоксії пропонуються нові перспективні напрями пошуку ефективних лікарських засобів для фармакологічної корекції наслідків пренатальної гіпоксії. Пренатальна гіпоксія є найпоширенішою причиною смертності та інвалідності новонароджених дітей. Хронічна пренатальна гіпоксія призводить до значних змін у розвитку когнітивних функцій у постнатальному періоді життя, які базуються на морфологічних змінах у структурах мозку, що беруть участь у навчанні та пам'яті. Результати досліджень останніх років внесли великий вклад у розкриття механізмів формування стану гіпоксії різного генезу та індукованих нею порушень метаболічних і функціональних процесів на рівні клітини й субклітинних структур. Визначено ряд морфофункціональних детермінант (специфічні рецептори, регуляторні білки, внутрішньоклітинні ферменти, мітохондріальні іонні канали і т. д.), які беруть безпосередню участь у розвитку термінової та довготривалої адаптації клітини й усього організму до гіпоксії. Аналіз літератури з досліджень молекулярних механізмів дії пренатальної гіпоксії показав, що основою гіпоксичних уражень головного мозку новонароджених є каскад біохімічних і молекулярних процесів, що призводять до ексайтотоксичності, окиснювального та нітрозуючого стресів і запалення. Одночасно з цим за гіпоксії починають функціонувати й компенсаторно­пристосувальні механізми, що підвищують стійкість організму до дефіциту кисню. В огляді особливу увагу приділено обговоренню даних щодо ролі молекулярних детермінант ендогенної нейропротекції, таких як HSP70 і HIF­1a. Транскрипційна система HIF­1 є ключовим регулятором адаптивних механізмів відповідей на гіпоксію на локальному та системному рівнях. Зміни в експресії генів HIF­1 у пренатальному розвитку запускають епігенетичні механізми, що призводять до негативних змін у розвитку нервової системи. Важливим компонентом системи ендогенної нейропротекції є сімейство білків теплового шоку HSP70, які сприяють широкому спектру процесів фолдингу, рефолдингу білків, мембранній транслокації органелярних і секреторних білків, а також здійснюють контроль за активністю регуляторних білків. Вплив HSP на стабілізацію HIF­1 забезпечує активацію процесів проліферації, апоптозу, ангіогенезу за умов гіпоксії. Білки HSP70 / HIF­1a­ системи можуть бути специфічними мішенями для впливу фармакологічних агентів з метою регуляції процесів адаптації організму до гіпоксії. Тому пошук нових ефективних нейропротекторних засобів повинен бути спрямований на активацію HSP70/HIF­1a­системи та використання агентів, здатних забезпечувати модуляцію генів, що кодують синтез білків HSP70 і HIF­1a.

Concentration Heat Shock Protein and Dairy Performances in Heat-Stressed Goats

Thermal shock is expressed through the response of high levels of a thermal shock protein (tolerant gene expression) in this way, the cell gets rid of heat. The current study aims to assess the relation between the concentration of heat shock protein and dairy performances in goats under hot environment. the study performed at dairy goat research farm of Cukurova University. Located in the province of Adana. Milk yields were weekly recorded. The average ambient temperature between 28.7 and 34.9C, THI were higher than 78. HSP60 and HSP70 were 4.4 and 8.7ng/ml, and 13.9 and 18.34ng/ml for Alpine and Saanen goats respectively. The cellular thermoregulatory response directly expressed by an increasing activation and format of HSP60 and HSP70 was demonstrated to be negatively correlated to lactation performances in the thermal stressed goats.

Infusion of Mesenchymal Stem Cells Combined with Epithelial Progenitor Cells Promotes Injured Intestinal Repairing after Hematopoietic Cell Transplantation

Pre-transplant chemoradiotherapy can impair intestinal barrier function and disrupt immune homeostasis, and thus increase the incidence of infection and other related complications. Mesenchymal stem cells (MSCs) have the potential to rescue Inflammatory Bowel Disease and intestinal graft versus host disease owing to their immunosuppressive capabilities. However, limited studies regarding MSC administration has been reported to aggravate T cell-mediated tissue injury. Plus, endothelial progenitor cells (EPCs) could improve endothelium repair, facilitate hematopoietic reconstitution and alleviate complications associated with hematopoietic cell transplantation (HCT). The key goals of this study were to i) identify the role of MSC derived soluble and contact dependent factors and how these affect intestinal injury; ii) investigate the effect of MSC combined EPC therapy for repairing the injured intestine. In this study, BALB/c mice were randomly divided into five groups, namely, total body irradiation only, bone marrow transplantation (BMT), MSC(BMT with 1 × 106MSC infusion), EPC(BMT with 5 × 105 EPCs infusion), and MSC+EPC (BMT with 1 × 106 MSC and 5 × 105 EPCs infusion). Results showed that the best performance of intestine was found in the MSC+EPC treated group, which showed more epithelial and goblet cells, and less apoptosis cells and adjacent crypts fusion in intestine morphology. EPC or MSC only group improved the intestinal injuries slightly compared with BMT group. The higher MECA-32 expression level was found in the intestinal tissue of MSC+EPC and MSC groups compared with other treated groups. The tight junction molecules occludin had highest expression level in the intestinal epithelial cells of MSC+EPC group, followed by MSC group and then EPC groups, but the expression level in BMT group was extremely low. Further study demonstrated that in MSC+EPC group the phosphorylated P38 enhanced heat shock protein HSP27 activation, which promoted cytoskeleton reconstruction and intestinal epithelial cells proliferation; and phosphorylated P38 also down-regulated the expression of apoptosis-related molecule caspase3. Moreover, the multiple effects of MSCs on cellular immunity may reflect their diverse influences on the different T-cell subpopulations. MSC+EPC infusion increased the number of IL-17A secreting cells (Th17 and Tc17) in mesenteric lymph nodes early after HCT, and decreased Th1 cells at day 10 and delayed expanding of Tc1 from day 10 to day 15. The soluble IL-17A in intestinal tissue was significantly increased after MSC+EPC infusion in according with IL-17A secreting cells in mesenteric lymph nodes. Furthermore, the gut bacterial information analyzed by high throughput sequencing uncovered that MSC and MSC+EPC groups had higher bacterial diversity and richness compared with other groups, and the bacterial community structures exhibited big changes regarding different treatment. Co-occurrence analysis demonstrated that the genus Akkermansia inducing PD-1 expression of T cells had significant correlation with phosphorylation of P38 and HSP27 in MSC+EPC and EPC treated groups, which indicated Akkermansia may be a key bacteria participanting the intestinal repairing in MSC and MSC+EPC groups. In conclusion, this study confirmed that the MSC+EPC infusion can obviously repair injured intestine, and gut bacteria Akkermansia correlated with phosphorylation of P38 and HSP27 expression and participated in intestinal repairing. Disclosures No relevant conflicts of interest to declare.

Hsp70 plays a role in programmed cell death during the remodelling of leaves of the lace plant (Aponogeton madagascariensis).

Lace plant leaves utilize programmed cell death (PCD) to form perforations during development. The role of heat shock proteins (Hsps) in PCD during lace plant leaf development is currently unknown. Hsp70 amounts were measured throughout lace plant leaf development, and the results indicate that it is highest before and during PCD. Increased Hsp70 amounts correlate with raised anthocyanin content and caspase-like protease (CLP) activity. To investigate the effects of Hsp70 on leaf development, whole plants were treated with either of the known regulators of PCD [reactive oxygen species (ROS) or antioxidants] or an Hsp70 inhibitor, chlorophenylethynylsulfonamide (PES-Cl). ROS treatment significantly increased Hsp70 2-fold and CLP activity in early developing leaves, but no change in anthocyanin and the number of perforations formed was observed. Antioxidant treatment significantly decreased Hsp70, anthocyanin, and CLP activity in early leaves, resulting in the fewest perforations. PES-Cl (25 μM) treatment significantly increased Hsp70 4-fold in early leaves, while anthocyanin, superoxide, and CLP activity significantly declined, leading to fewer perforations. Results show that significantly increased (4-fold) or decreased Hsp70 amounts lead to lower anthocyanin and CLP activity, inhibiting PCD induction. Our data support the hypothesis that Hsp70 plays a role in regulating PCD at a threshold in lace plant leaf development. Hsp70 affects anthocyanin content and caspase-like protease activity, and helps regulate PCD during the remodelling of leaves of lace plant, Aponogeton madagascariensis.

NECA derivatives exploit the paralog-specific properties of the site 3 side pocket of Grp94, the endoplasmic reticulum Hsp90

The hsp90 chaperones govern the function of essential client proteins critical for normal cell function as well as cancer initiation and progression. Hsp90 activity is driven by ATP, which binds to the N-terminal domain and induces large conformational changes that are required for client maturation. Inhibitors targeting the ATP-binding pocket of the N-terminal domain have anticancer effects, but most bind with similar affinity to cytosolic Hsp90α and Hsp90β, endoplasmic reticulum Grp94, and mitochondrial Trap1, the four cellular hsp90 paralogs. Paralog-specific inhibitors may lead to drugs with fewer side effects. The ATP-binding pockets of the four paralogs are flanked by three side pockets, termed sites 1, 2, and 3, which differ between the paralogs in their accessibility to inhibitors. Previous insights into the principles governing access to sites 1 and 2 have resulted in development of paralog-selective inhibitors targeting these sites, but the rules for selective targeting of site 3 are less clear. Earlier studies identified 5'N-ethylcarboxamido adenosine (NECA) as a Grp94-selective ligand. Here we use NECA and its derivatives to probe the properties of site 3. We found that derivatives that lengthen the 5' moiety of NECA improve selectivity for Grp94 over Hsp90α. Crystal structures reveal that the derivatives extend further into site 3 of Grp94 compared with their parent compound and that selectivity is due to paralog-specific differences in ligand pose and ligand-induced conformational strain in the protein. These studies provide a structural basis for Grp94-selective inhibition using site 3.

Heat Shock Protein 90 Inhibitors: An Update on Achievements, Challenges, and Future Directions.

Hsp90 is one of the most important chaperones involved in regulating the maturation of more than 300 client proteins, many of which are closely associated with refractory diseases, including cancer, neurodegenerative diseases, and viral infections. Clinical Hsp90 inhibitors bind to the ATP pocket in the N-terminal domain of Hsp90 and subsequently suppress the ATPase activity of Hsp90. Recently, with the increased understanding of the discrepancies in the isoforms of Hsp90 and the modes of Hsp90-co-chaperone-client complex interactions, some new strategies for Hsp90 inhibition have emerged. Novel Hsp90 inhibitors that offer selective suppression of Hsp90 isoforms or specific disruption of Hsp90-co-chaperone protein-protein interactions are expected to show with satisfactory efficacy and safety profiles. This review summarizes the recent progress in Hsp90 inhibitors. Additionally, Hsp90 inhibitory strategies are emphasized in this review.

Targeting Extracellular Heat Shock Protein 70 Ameliorates Doxorubicin-Induced Heart Failure Through Resolution of Toll-Like Receptor 2-Mediated Myocardial Inflammation.

BackgroundHeart failure (HF) is one of the most significant causes of morbidity and mortality for the cardiovascular risk population. We found previously that extracellular HSP70 (heat shock protein) is an important trigger in cardiac hypertrophy and fibrosis, which are associated with the development of heart dysfunction. However, the potential role of HSP70 in response to HF and whether it could be a target for the therapy of HF remain unknown.Methods and ResultsAn HF mouse model was generated by a single IP injection of doxorubicin at a dose of 15 mg/kg. Ten days later, these mice were treated with an HSP70 neutralizing antibody for 5 times. We observed that doxorubicin treatment increased circulating HSP70 and expression of HSP70 in myocardium and promoted its extracellular release in the heart. Blocking extracellular HSP70 activity by its antibody significantly ameliorated doxorubicin‐induced left ventricular dilation and dysfunction, which was accompanied by a significant inhibition of cardiac fibrosis. The cardioprotective effect of the anti‐HSP70 antibody was largely attributed to its ability to promote the resolution of myocardial inflammation, as evidenced by its suppression of the toll‐like receptor 2–associated signaling cascade and modulation of the intracellular distribution of the p50 and p65 subunits of nuclear factor‐κB.ConclusionsExtracellular HSP70 serves as a noninfectious inflammatory factor in the development of HF, and blocking extracellular HSP70 activity may provide potential therapeutic benefits for the treatment of HF.

Use of Acid Ceramidase and Sphingosine Kinase Inhibitors as Antiviral Compounds Against Measles Virus Infection of Lymphocytes in vitro.

As structural membrane components and signaling effector molecules sphingolipids influence a plethora of host cell functions, and by doing so also the replication of viruses. Investigating the effects of various inhibitors of sphingolipid metabolism in primary human peripheral blood lymphocytes (PBL) and the human B cell line BJAB we found that not only the sphingosine kinase (SphK) inhibitor SKI-II, but also the acid ceramidase inhibitor ceranib-2 efficiently inhibited measles virus (MV) replication. Virus uptake into the target cells was not grossly altered by the two inhibitors, while titers of newly synthesized MV were reduced by approximately 1 log (90%) in PBL and 70–80% in BJAB cells. Lipidomic analyses revealed that in PBL SKI-II led to increased ceramide levels, whereas in BJAB cells ceranib-2 increased ceramides. SKI-II treatment decreased sphingosine-1-phosphate (S1P) levels in PBL and BJAB cells. Furthermore, we found that MV infection of lymphocytes induced a transient (0.5–6 h) increase in S1P, which was prevented by SKI-II. Investigating the effect of the inhibitors on the metabolic (mTORC1) activity we found that ceranib-2 reduced the phosphorylation of p70 S6K in PBL, and that both inhibitors, ceranib-2 and SKI-II, reduced the phosphorylation of p70 S6K in BJAB cells. As mTORC1 activity is required for efficient MV replication, this effect of the inhibitors is one possible antiviral mechanism. In addition, reduced intracellular S1P levels affect a number of signaling pathways and functions including Hsp90 activity, which was reported to be required for MV replication. Accordingly, we found that pharmacological inhibition of Hsp90 with the inhibitor 17-AAG strongly impaired MV replication in primary PBL. Thus, our data suggest that treatment of lymphocytes with both, acid ceramidase and SphK inhibitors, impair MV replication by affecting a number of cellular activities including mTORC1 and Hsp90, which alter the metabolic state of the cells causing a hostile environment for the virus.

Hsp90 Chaperones Bluetongue Virus Proteins and Prevents Proteasomal Degradation.

The molecular chaperone machinery is important for the maintenance of protein homeostasis within the cells. The principle activities of the chaperone machinery are to facilitate protein folding and organize conformationally dynamic client proteins. Prominent among the members of the chaperone family are heat shock protein 70 (Hsp70) and 90 (Hsp90). Like cellular proteins, viral proteins depend upon molecular chaperones to mediate their stabilization and folding. Bluetongue virus (BTV), which is a model system for the Reoviridae family, is a nonenveloped arbovirus that causes hemorrhagic disease in ruminants. This constitutes a significant burden upon animals of commercial significance, such as sheep and cattle. Here, for the first time, we examined the role of chaperone proteins in the viral lifecycle of BTV. Using a combination of molecular, biochemical, and microscopic techniques, we examined the function of Hsp90 and its relevance to BTV replication. We demonstrate that Hsp70, the chaperone that is commonly usurped by viral proteins, does not influence virus replication, while Hsp90 activity is important for virus replication by stabilizing BTV proteins and preventing their degradation via the ubiquitin-proteasome pathway. To our knowledge this is the first report showing the involvement of Hsp90 as a modulator of BTV infection.IMPORTANCE Protein chaperones are instrumental for maintaining protein homeostasis, enabling correct protein folding and organization; prominent members include heat shock proteins 70 and 90. Virus infections place a large burden on this homeostasis. Identifying and understanding the underlying mechanisms that facilitate Bluetongue virus replication and spread through the usurpation of host factors is of primary importance for the development of intervention strategies. Our data identify and show that heat shock protein 90, but not heat shock protein 70, stabilizes bluetongue virus proteins, safeguarding them from proteasomal degradation.

Heat shock-induced chaperoning by Hsp70 is enabled in-cell.

Recent work has shown that weak protein-protein interactions are susceptible to the cellular milieu. One case in point is the binding of heat shock proteins (Hsps) to substrate proteins in cells under stress. Upregulation of the Hsp70 chaperone machinery at elevated temperature was discovered in the 1960s, and more recent studies have shown that ATPase activity in one Hsp70 domain is essential for control of substrate binding by the other Hsp70 domain. Although there are several denaturant-based assays of Hsp70 activity, reports of ATP-dependent binding of Hsp70 to a globular protein substrate under heat shock are scarce. Here we show that binding of heat-inducible Hsp70 to phosphoglycerate kinase (PGK) is remarkably different in vitro compared to in-cell. We use fluorescent-labeled mHsp70 and ePGK, and begin by showing that mHsp70 passes the standard β-galactosidase assay, and that it does not self-aggregate until 50°C in presence of ATP. Yet during denaturant refolding or during in vitro heat shock, mHsp70 shows only ATP-independent non-specific sticking to ePGK, as evidenced by nearly identical results with an ATPase activity-deficient K71M mutant of Hsp70 as a control. Addition of Hsp40 (co-factor) or Ficoll (crowder) does not reduce non-specific sticking, but cell lysate does. Therefore, Hsp70 does not act as an ATP-dependent chaperone on its substrate PGK in vitro. In contrast, we observe only specific ATP-dependent binding of mHsp70 to ePGK in mammalian cells, when compared to the inactive Hsp70 K71M mutant. We hypothesize that enhanced in-cell activity is not due to an unknown co-factor, but simply to a favorable shift in binding equilibrium caused by the combination of crowding and osmolyte/macromolecular interactions present in the cell. One candidate mechanism for such a favorable shift in binding equilibrium is the proven ability of Hsp70 to bind near-native states of substrate proteins in vitro. We show evidence for early onset of binding in-cell. Our results suggest that Hsp70 binds PGK preemptively, prior to its full unfolding transition, thus stabilizing it against further unfolding. We propose a "preemptive holdase" mechanism for Hsp70-substrate binding. Given our result for PGK, more proteins than one might think based on in vitro assays may be chaperoned by Hsp70 in vivo. The cellular environment thus plays an important role in maintaining proper Hsp70 function.

Targeting Hsp70 facilitated protein quality control for treatment of polyglutamine diseases.

The polyglutamine (polyQ) diseases are a group of nine fatal, adult-onset neurodegenerative disorders characterized by the misfolding and aggregation of mutant proteins containing toxic expansions of CAG/polyQ tracts. The heat shock protein 90 and 70 (Hsp90/Hsp70) chaperone machinery is a key component of cellular protein quality control, playing a role in the regulation of folding, aggregation, and degradation of polyQ proteins. The ability of Hsp70 to facilitate disaggregation and degradation of misfolded proteins makes it an attractive therapeutic target in polyQ diseases. Genetic studies have demonstrated that manipulation of Hsp70 and related co-chaperones can enhance the disaggregation and/or degradation of misfolded proteins in models of polyQ disease. Therefore, the development of small molecules that enhance Hsp70 activity is of great interest. However, it is still unclear if currently available Hsp70 modulators can selectively enhance disaggregation or degradation of misfolded proteins without perturbing other Hsp70 functions essential for cellular homeostasis. This review discusses the multifaceted role of Hsp70 in protein quality control and the opportunities and challenges Hsp70 poses as a potential therapeutic target in polyQ disease.

Diagnostic value of chaperone activity of heat shock proteins 70 in coronary atherosclerosis

Aim. The aim of the research was to study the role of chaperone activity heat shock proteins (HSP) 70 in pathogenesis and diagnostic in patients with coronary atherosclerosis. Materials and methods. We examined 354 patients with coronary heart disease, who had coronary atherosclerosis of varying degrees, according to coronary angiography (was performed by the Judkins technique). The severity of coronary atherosclerosis was determined on the basis of the Gensini index. According to the Gensini index, patients were divided into 2 groups: GS0 - 152 patients without signs of coronary atherosclerosis, GS1 - 202 patients with coronary lesions. Chaperone activity was determined by thermodynamic method. Results. The study showed significant differences in the level of chaperone activity HSP70 in patients with different severity of coronary atherosclerosis. The correlation analysis revealed a significant negative relationship between chaperone activity HSP70 and the Gensini index. The cut-off value of chaperone activity of HSP70, by which can be judged on the presence or absence of coronary atherosclerosis, is establish. Conclusion. The revealed threshold of chaperone activity can be considered as a possible marker of the severity of coronary atherosclerosis.

Structural studies of the Hsp70/Hsp90 organizing protein of Plasmodium falciparum and its modulation of Hsp70 and Hsp90 ATPase activities

HOP is a cochaperone belonging to the foldosome, a system formed by the cytoplasmic Hsp70 and Hsp90 chaperones. HOP acts as an adapter protein capable of transferring client proteins from the first to the second molecular chaperone. HOP is a modular protein that regulates the ATPase activity of Hsp70 and Hsp90 to perform its function. To obtain more detailed information on the structure and function of this protein, we produced the recombinant HOP of Plasmodium falciparum (PfHOP). The protein was obtained in a folded form, with a high content of α-helix secondary structure. Unfolding experiments showed that PfHOP unfolds through two transitions, suggesting the presence of at least two domains with different stabilities. In addition, PfHOP primarily behaved as an elongated dimer in equilibrium with the monomer. Small-angle X-ray scattering data corroborated this interpretation and led to the reconstruction of a PfHOP ab initio model as a dimer. Finally, the PfHOP protein was able to inhibit and to stimulate the ATPase activity of the recombinant Hsp90 and Hsp70–1, respectively, of P. falciparum. Our results deepened the knowledge of the structure and function of PfHOP and further clarified its participation in the P. falciparum foldosome.

Natural compounds as potential Hsp90 inhibitors for breast cancer-Pharmacophore guided molecular modelling studies

Breast cancer is one of the major impediments affecting women globally. The ATP-dependant heat shock protein 90 (Hsp90) forms the central component of molecular chaperone machinery that predominantly governs the folding of newly synthesized peptides and their conformational maturation. It regulates the stability and function of numerous client proteins that are frequently upregulated and/or mutated in cancer cells, therefore, making Hsp90 inhibition a promising therapeutic strategy for the development of new efficacious drugs to treat breast cancer. In the present in silico investigation, a structure-based pharmacophore model was generated with hydrogen bond donor, hydrogen bond acceptor and hydrophobic features complementary to crucial residues Ala55, Lys58, Asp93, Ile96, Met98 and Thr184 directed at inhibiting the ATP-binding activity of Hsp90. Subsequently, the phytochemical dataset of 3210 natural compounds was screened to retrieve the prospective inhibitors after rigorous validation of the model pharmacophore. The retrieved 135 phytocompounds were further filtered by drug-likeness parameters including Lipinski’s rule of five and ADMET properties, then investigated via molecular docking-based scoring. Molecular interactions were assessed using Genetic Optimisation for Ligand Docking program for 95 drug-like natural compounds against Hsp90 along with two clinical drugs as reference compounds – Geldanamycin and Radicicol. Docking studies revealed three phytochemicals are better than the investigated clinical drugs. The reference and hit compounds with dock scores of 48.27 (Geldanamycin), 40.90 (Radicicol), 73.04 (Hit1), 72.92 (Hit2) and 68.12 (Hit3) were further validated for their binding stability through molecular dynamics simulations. We propose that the non-macrocyclic scaffolds of three identified phytochemicals might aid in the development of novel therapeutic candidates against Hsp90-driven cancers.

The Complex Phosphorylation Patterns that Regulate the Activity of Hsp70 and Its Cochaperones.

Proteins must fold into their native structure and maintain it during their lifespan to display the desired activity. To ensure proper folding and stability, and avoid generation of misfolded conformations that can be potentially cytotoxic, cells synthesize a wide variety of molecular chaperones that assist folding of other proteins and avoid their aggregation, which unfortunately is unavoidable under acute stress conditions. A protein machinery in metazoa, composed of representatives of the Hsp70, Hsp40, and Hsp110 chaperone families, can reactivate protein aggregates. We revised herein the phosphorylation sites found so far in members of these chaperone families and the functional consequences associated with some of them. We also discuss how phosphorylation might regulate the chaperone activity and the interaction of human Hsp70 with its accessory and client proteins. Finally, we present the information that would be necessary to decrypt the effect that post-translational modifications, and especially phosphorylation, could have on the biological activity of the Hsp70 system, known as the “chaperone code”.

HSP70 improves the viability of cryopreserved Paeonia lactiflora pollen by regulating oxidative stress and apoptosis-like programmed cell death events

Heat shock proteins (HSPs) have many positive stabilizing effects on the proteins of stressed cells. This study investigated the effect of HSP70 on cryopreserved pollen. HSP70 was differentially expressed in Paeonia lactiflora and Magnolia denudate pollen before and after exposure to liquid nitrogen (LN). In this study, cryopreserved pollen samples of P. lactiflora ‘Fen Yu Nu’, ‘Zi Feng Chao Yang’ and ‘Hong Pan Tuo Jing’ were incubated with HSP70 at six concentrations, and the pollen germination, oxidative stress and apoptosis-like programmed cell death indicators were measured after LN storage. HSP70 significantly improved pollen viability after LN storage, and the concentration of HSP70 that was required varied from cultivar to cultivar, ranging from 0.5 to 10 μg/mL. Compared to the activity of the control treatment without HSP70, the activity of superoxide dismutase (SOD) increased, the contents of reactive oxygen species (ROS) and malondialdehyde (MDA) decreased, and the Ca2+ level, apoptosis rate and caspase-3-like activity decreased depending on the appropriate concentration of HSP70 added. Pollen germination was negatively correlated with MDA content, Ca2+ level, and caspase-3-like activity and positively correlated with SOD activity. In conclusion, HSP70 improved the viability of pollen after cryopreservation by improving the activity of antioxidant enzymes, reducing the contents of ROS and MDA, and affecting the Ca2+ signal to inhibit apoptosis-like programmed cell death events. This report is the first time to describe the application of HSP70 for improving cryopreserved pollen viability by regulating oxidative and apoptosis-like programmed cell death events, and provide a novel insight into the mechanisms of HSP70 action in cryopreservation. After the pollen was rewarmed and then incubated with appropriate concentrations of exogenous HSP70, it significantly increasing the post-freezing germination of the pollen. Which by affecting oxidative stress and apoptosis-like events, the ROS level, MDA content, intracellular Ca2+ concentration, apoptosis rate and caspase-3-like enzyme activity were reduced, and SOD activity was increased, thereby significantly increasing the post-freezing germination of the pollen.

Nascent Polypeptide Domain Topology and Elongation Rate Direct the Cotranslational Hierarchy of Hsp70 and TRiC/CCT

Cotranslational protein folding requires assistance from elaborate ribosome-associated chaperone networks. It remains unclear how the changing information in a growing nascent polypeptide dictates the recruitment of functionally distinct chaperones. Here, we used ribosome profiling to define the principles governing the cotranslational action of the chaperones TRiC/CCT and Hsp70/Ssb. We show that these chaperones are sequentially recruited to specific sites within domain-encoding regions of select nascent polypeptides. Hsp70 associates first, binding select sites throughout domains, whereas TRiC associates later, upon the emergence of nearly complete domains that expose an unprotected hydrophobic surface. This suggests that transient topological properties of nascent folding intermediates drive sequential chaperone association. Moreover, cotranslational recruitment of both TRiC and Hsp70 correlated with translation elongation slowdowns. We propose that the temporal modulation of the nascent chain structural landscape is coordinated with local elongation rates to regulate the hierarchical action of Hsp70 and TRiC for cotranslational folding.

Enhancement of the Anti-Aggregation Activity of a Molecular Chaperone Using a Rationally Designed Post-Translational Modification.

Protein behavior is closely regulated by a plethora of post-translational modifications (PTMs). It is therefore desirable to develop approaches to design rational PTMs to modulate specific protein functions. Here, we report one such method, and we illustrate its successful implementation by potentiating the anti-aggregation activity of a molecular chaperone. Molecular chaperones are a multifaceted class of proteins essential to protein homeostasis, and one of their major functions is to combat protein misfolding and aggregation, a phenomenon linked to a number of human disorders. In this work, we conjugated a small-molecule inhibitor of the aggregation of α-synuclein, a process associated with Parkinson’s disease (PD), to a specific cysteine residue on human Hsp70, a molecular chaperone with five free cysteines. We show that this regioselective conjugation augments in vitro the anti-aggregation activity of Hsp70 in a synergistic manner. This Hsp70 variant also displays in vivo an enhanced suppression of α-synuclein aggregation and its associated toxicity in a Caenorhabditis elegans model of PD.

The Lethality of [Pazopanib + HDAC Inhibitors] Is Enhanced by Neratinib.

Sarcomas are a diverse set of malignancies. For soft tissue sarcomas, the kinase and chaperone inhibitor pazopanib is a standard of care therapeutic. Previously, we demonstrated that HDAC inhibitors enhanced pazopanib lethality against sarcoma and other tumor cell types in vitro and in vivo. The present studies defined mechanisms of drug-combination resistance. Exposure of sarcoma and PDX ovarian carcinoma cells to [pazopanib + entinostat] caused a prolonged activation of ERBB1 and transient/prolonged activations of ERBB2, c-KIT, and c-MET, in a cell-specific fashion. The activities of mTORC1, mTORC2, GRP78, HSP90, and HSP70 were reduced, expression of Beclin1 and ATG5 enhanced, and the ATM-AMPK-ULK1-ATG13-Beclin1/ATG5 pathway activated. Inhibition of ERBB1/2/4 using neratinib or of c-MET using crizotinib significantly enhanced [pazopanib + entinostat] lethality. For neratinib with [pazopanib + entinostat], this effect correlated with reduced phosphorylation and expression of ERBB1, ERBB2, c-KIT, and c-MET and reduced expression, regardless of mutational status, of N-RAS and K-RAS. [Pazopanib + entinostat + neratinib] reduced the phosphorylation of the Hippo pathway proteins MST1/3/4 and MOB1 whereas this treatment increased the phosphorylation of LATS1, YAP, and TAZ. The activation of ATM, ULK-1, and eIF2α was further enhanced by [pazopanib + entinostat + neratinib] as was the expression of ATG5 and Beclin1. Compared to other manipulations, knock down of eIF2α or over-expression of BCL-XL significantly reduced killing by the three-drug interaction. In vivo, pazopanib and entinostat, and also neratinib and entinostat, both combined to significantly suppress the growth of sarcoma tumors.