Hsp70 Isoforms Research Articles

Effects of Acute Hypoxia and Reoxygenation on Physiological and Immune Responses and Redox Balance of Wuchang Bream (Megalobrama amblycephala Yih, 1955).

To study Megalobrama amblycephala adaption to water hypoxia, the changes in physiological levels, innate immune responses, redox balance of M.amblycephala during hypoxia were investigated in the present study. When M. amblycephala were exposed to different dissolved oxygen (DO) including control (DO: 5.5 mg/L) and acute hypoxia (DO: 3.5 and 1.0 mg/L, respectively), hemoglobin (Hb), methemoglobin (MetHb), glucose, Na+, succinatedehydrogenase (SDH), lactate, interferon alpha (IFNα), and lysozyme (LYZ), except hepatic glycogen and albumin gradually increased with the decrease of DO level. When M. amblycephala were exposed to different hypoxia time including 0.5 and 6 h (DO: 3.5 mg/L), and then reoxygenation for 24 h after 6 h hypoxia, Hb, MetHb, glucose, lactate, and IFNα, except Na+, SDH, hepatic glycogen, albumin, and LYZ increased with the extension of hypoxia time, while the above investigated indexes (except albumin, IFNα, and LYZ) decreased after reoxygenation. On the other hand, the liver SOD, CAT, hydrogen peroxide (H2O2), and total ROS were all remained at lower levels under hypoxia stress. Finally, Hif-1α protein in the liver, spleen, and gill were increased with the decrease of oxygen concentration and prolongation of hypoxia time. Interestingly, one Hsp70 isoforms mediated by internal ribozyme entry site (IRES) named junior Hsp70 was only detected in liver, spleen and gill. Taken together, these results suggest that hypoxia affects M. amblycephala physiology and reduces liver oxidative stress. Hypoxia-reoxygenation stimulates M. amblycephala immune parameter expressions, while Hsp70 response to hypoxia is tissue-specific.

Involvement of cell surface 90 kDa heat shock protein (HSP90) in pattern recognition by human monocyte-derived macrophages.

Heat shock proteins (HSPs) are typical intracellular chaperones which also appear on the cell surface and in extracellular milieu. HSP90, which chaperones many proteins involved in signal transduction, is also a regular component of LPS-signaling complexes on Mϕ. As LPS is a prototypical PAMP, we speculated that HSP90 is engaged in pattern recognition by professional phagocytes. In this report, we provide the first evidence, to our knowledge, of the geldanamycin (Ge)-inhibitable HSP90 on the surface of live monocyte-derived Mϕs (hMDMs). Using cytometry and specific Abs, we showed both HSP90 isoforms (α and β) on the surface of human monocytes and hMDMs. The cell-surface HSP90 pool was also labeled with cell-impermeable Ge derivatives. Confocal analysis of hMDMs revealed that HSP90-inhibitor complexes were rapidly clustered on the cell surface and recycled through the endosomal compartment. This finding suggests that the N-terminal (ATPase) domain of HSP90 is exposed and accessible from the extracellular space. To study the role of cell-surface HSP90 in pattern recognition, we used pathogen (PAMPs)- or apoptotic cell-associated molecular patterns (ACAMPs). We showed that blocking the cell-surface HSP90 pool leads to a dramatic decrease in TNF production by monocytes and hMDMs exposed to soluble (TLRs-specific ligands) and particulate [bacteria Staphylococcus aureus (SA) and Porphyromonas gingivalis (PG)] PAMPs. Surprisingly, in hMDMs the functional cell-surface HSP90 was not necessary for the engulfment of either apoptotic neutrophils or bacteria. The presented data suggest that the cell-surface HSP90 is a "signaling complex chaperone," with activity that is essential for cytokine response but not for target engulfment by Mϕ.

Expression dynamics of HSP90 and nitric oxide synthase (NOS) isoforms during heat stress acclimation in Tharparkar cattle.

Six male Tharparkar cattle of 2-3years old were selected for the study. After 15-day acclimation at thermoneutral zone (TNZ) in psychrometric chamber, animals were exposed at 42°C for 6h up to 23days followed by 12days of recovery period. Blood samples were collected during control period at TNZ (days 1, 5, and 12), after heat stress exposure (day 1, immediate heat stress acclimation (IHSA); days 2 to 10, short-term heat stress acclimation (STHSA); days 15 to 23, long-term heat stress acclimation (LTHSA); days 7 and 12, recovery period), and peripheral blood mononuclear cells (PBMCs) were isolated for RNA and protein extraction. The messenger RNA (mRNA) and protein expression in PBMCs were determined by qPCR and western blot, respectively. Samples at TNZ were taken as control. The mRNA expression of HSP90, iNOS, and eNOS was significantly upregulated (P<0.05) on day 1 (ISHA) as compared to control, remained consistent during STHSA, again increased during LTHSA, and finally reduced to basal level during recovery period. The protein expression of HSP90, iNOS, and eNOS were akin to their transcript pattern. PBMC culture study was conducted to study transcriptional abundance of HSP90, iNOS, and eNOS at different temperature-time combinations. The present findings indicate that HSP90, iNOS, and eNOS could possibly play an important role in mitigating thermal insults and confer thermotolerance during long-term heat stress exposure in Tharparkar cattle.

Ritterostatin GN 1N , a Cephalostatin-Ritterazine Bis-steroidal Pyrazine Hybrid, Selectively Targets GRP78.

Natural products discovered by using agnostic approaches, unlike rationally designed leads or those obtained through high-throughput screening, offer the ability to reveal new biological pathways and, hence, serve as an important vehicle to unveil new avenues in drug discovery. The ritterazine-cephalostatin family of natural products displays robust and potent antitumor activities, with sub-nanomolar growth inhibition against multiple cell lines and potent activity in xenograft models. Herein, we used comparative cellular and molecular biological methods to uncover the ritterazine-cephalostatin cytotoxic mode of action (MOA) in human tumor cells. Our findings indicated that, whereas ritterostatin GN 1N , a cephalostatin-ritterazine hybrid, binds to multiple HSP70s, its cellular trafficking confines activity to the endoplasmic reticulum (ER)-based HSP70 isoform, GRP78. This targeting results in activation of the unfolding protein response (UPR) and subsequent apoptotic cell death.

De novo and rare mutations in the HSPA1L heat shock gene associated with inflammatory bowel disease

BackgroundInflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disease of the gastrointestinal tract which includes ulcerative colitis and Crohn's disease. Genetic risk factors for IBD are not well understood.MethodsWe performed a family-based whole exome sequencing (WES) analysis on a core family (Family A) to identify potential causal mutations and then analyzed exome data from a Caucasian pediatric cohort (136 patients and 106 controls) to validate the presence of mutations in the candidate gene, heat shock 70 kDa protein 1-like (HSPA1L). Biochemical assays of the de novo and rare (minor allele frequency, MAF < 0.01) mutation variant proteins further validated the predicted deleterious effects of the identified alleles.ResultsIn the proband of Family A, we found a heterozygous de novo mutation (c.830C > T; p.Ser277Leu) in HSPA1L. Through analysis of WES data of 136 patients, we identified five additional rare HSPA1L mutations (p.Gly77Ser, p.Leu172del, p.Thr267Ile, p.Ala268Thr, p.Glu558Asp) in six patients. In contrast, rare HSPA1L mutations were not observed in controls, and were significantly enriched in patients (P = 0.02). Interestingly, we did not find non-synonymous rare mutations in the HSP70 isoforms HSPA1A and HSPA1B. Biochemical assays revealed that all six rare HSPA1L variant proteins showed decreased chaperone activity in vitro. Moreover, three variants demonstrated dominant negative effects on HSPA1L and HSPA1A protein activity.ConclusionsOur results indicate that de novo and rare mutations in HSPA1L are associated with IBD and provide insights into the pathogenesis of IBD, and also expand our understanding of the roles of HSP70s in human disease.

Transformation of the Non-Selective Aminocyclohexanol-Based Hsp90 Inhibitor into a Grp94-Seletive Scaffold.

Glucose regulated protein 94 kDa, Grp94, is the endoplasmic reticulum (ER) localized isoform of heat shock protein 90 (Hsp90) that is responsible for the trafficking and maturation of toll-like receptors, immunoglobulins, and integrins. As a result, Grp94 has emerged as a therapeutic target to disrupt cellular communication, adhesion, and tumor proliferation, potentially with fewer side effects compared to pan-inhibitors of all Hsp90 isoforms. Although, the N-terminal ATP binding site is highly conserved among all four Hsp90 isoforms, recent cocrystal structures of Grp94 have revealed subtle differences between Grp94 and other Hsp90 isoforms that has been exploited for the development of Grp94-selective inhibitors. In the current study, a structure-based approach has been applied to a Grp94 nonselective compound, SNX 2112, which led to the development of 8j (ACO1), a Grp94-selective inhibitor that manifests ∼440 nM affinity and >200-fold selectivity against cytosolic Hsp90 isoforms.

Design, Synthesis, and Evaluation of Heat Shock Protein 90 Inhibitors in Human Breast Cancer and Its Metastasis.

Despite development of novel cancer drugs, invasive ductal breast carcinoma and its metastasis are still highly morbid. Therefore, new therapeutic approaches are being developed and Hsp90 is an important target for drug design. For this purpose, a series of benzodiazepine derivatives were designed and synthesized as novel Hsp90 inhibitor. Benzodiazepine derivatives anticancer activities were determined by XTT cell proliferation assay against human breast cancer cell line (MCF-7). Effects of the compounds on endothelial function were monitored on human vascular endothelium (HUVEC) cell line as well. In order to determine the anti-proliferative mechanism of the compounds, in silico molecular docking studies were performed between Hsp90 ATPase domain and the benzodiazepine derivatives. Further, these compounds perturbation on Hsp90 ATPase function were tested. Fluorescence binding experiments showed that the derivatives bind Hsp90 effectively. Expression analysis of known cancer drug target genes by PCR array experiments suggest that the benzodiazepine derivatives have remarkable anticancer activity. A representative Benzodiazepine derivative D5 binds Hsp90 with Kd value of 3,93 μM and with estimated free energy of binding -7.99 (kcal/mol). The compound decreases Hsp90 ATPase function and inhibit Hsp90 client protein folding activity. The compound inhibits expression of both Hsp90 isoforms and key proteins (cell cycle receptors; PLK2 and TERT, kinases; PI3KC3 and PRKCE, and growth factors; IGF1, IGF2, KDR, and PDGFRA) on oncogenic pathways. Benzodiazepine derivatives presented here display anticancer activity. The compounds effect on both breast cancer and endothelial cell lines show their potential as drug templates to inhibit breast cancer and its metastasis.

Structural and functional studies of the Leishmania braziliensis mitochondrial Hsp70: Similarities and dissimilarities to human orthologues

Heat shock protein 70 kDa (Hsp70) is a conserved molecular chaperone family involved in several functions related to protein homeostasis. In eukaryotes, Hsp70 homologues are found in all cell compartments. The mitochondrial Hsp70 isoform (mtHsp70) is involved in import of mitochondrial matrix proteins as well as their folding and maturation. Moreover, mtHsp70 has the propensity to self-aggregate, and it depends on the action of the co-chaperone Hsp70-escort protein 1 (Hep1) to be produced functional. Here, we analyze the solution structure and function of mtHsp70 of Leishmania braziliensis (LbmtHsp70). This recombinant protein was obtained folded, in the monomeric state and it has an elongated shape. We observed that LbmtHsp70 suffers thermal aggregation that depends on the protein concentration and is composed of domains with different thermal stabilities. LbmtHsp70 interacted with adenosine nucleotides with a thermodynamic signature different from those reported for human orthologues and interacted, driven by both enthalpy and entropy, with L. braziliensis Hep1 (LbHep1) with a nanomolar dissociation constant. Moreover, LbHep1 stimulated the LbmtHsp70 ATPase activity. Since little is known about mitochondrial Hsp70, particularly in protozoa, we believe that our data are of interest for understanding protozoan Hsp70 machinery.

Characterization of HSP90 isoforms in transformed bovine leukocytes infected with Theileria annulata.

SummaryHSP90 chaperones are essential regulators of cellular function, as they ensure the appropriate conformation of multiple key client proteins. Four HSP90 isoforms were identified in the protozoan parasite Theileria annulata. Partial characterization was undertaken for three and localization confirmed for cytoplasmic (TA12105), endoplasmic reticulum (TA06470), and apicoplast (TA10720) forms. ATPase activity and binding to the HSP90 inhibitor geldanamycin were demonstrated for recombinant TA12105, and all three native forms could be isolated to varying extents by binding to geldanamycin beads. Because it is essential, HSP90 is considered a potential therapeutic drug target. Resistance to the only specific Theileriacidal drug is increasing, and one challenge for design of drugs that target the parasite is to limit the effect on the host. An in vitro cell culture system that allows comparison between uninfected bovine cells and the T. annulata‐infected counterpart was utilized to test the effects of geldanamycin and the derivative 17‐AAG. T. annulata‐infected cells had greater tolerance to geldanamycin than uninfected cells yet exhibited significantly more sensitivity to 17‐AAG. These findings suggest that parasite HSP90 isoform(s) can alter the drug sensitivity of infected host cells and that members of the Theileria HSP90 family are potential targets worthy of further investigation.

ATM is the primary kinase responsible for phosphorylation of Hsp90α after ionizing radiation.

Heat shock protein 90 is a chaperone that plays an essential role in the stabilization of a large number of signal transduction molecules, many of which are associated with oncogenesis. An Hsp90 isoform (Hsp90α) has been shown to be selectively phosphorylated on two N-terminal threonine residues (threonine 5 and 7) and is involved in the DNA damage response and apoptosis. However, the kinase that phosphorylates Hsp90α after ionizing radiation (IR) and its role in post-radiation DNA repair remains unclear. Inasmuch as several proteins of the DNA damage response machinery are Hsp90 clients, the functional consequences of Hsp90α phosphorylation following IR have implications for the design of novel radiosensitizing agents that specifically target the Hsp90α isoform. Here we show that ATM phosphorylates Hsp90α at the T5/7 residues immediately after IR. The kinetics of Hsp90α T5/7 phosphorylation correlate with the kinetics of H2AX S139 phosphorylation (γH2AX). Although Hsp90α is located in both the cytoplasm and nucleus, only nuclear Hsp90α is phosphorylated by ATM after IR. The siRNA mediated knockdown of Hsp90α sensitizes head and neck squamous cell carcinoma cells, lung cancer cells and lung fibroblasts to IR. Furthermore, MEF cells that are Hsp90α null have reduced levels of γH2AX indicating that Hsp90α is important for the formation of γH2AX. Thus, this study provides evidence that Hsp90α is a component of the signal transduction events mediated by ATM following IR, and that Hsp90α loss decreases γH2AX levels. This work supports additional investigation into Hsp90α T5/7 phosphorylation with the goal of developing targeted radiosensitizing therapies.

Responses of the arcto-boreal krill species Thysanoessa inermis to variations in water temperature: coupling Hsp70 isoform expressions with metabolism.

Recent studies have indicated a metabolic temperature sensitivity in both the arcto-boreal krill species Thysanoessa inermis and Thysanoessa raschii that may determine these species' abundance and population persistence at lower latitudes (up to 40° N). T. inermis currently dominates the krill community in the Barents Sea and in the high Arctic Kongsfjord. We aimed to increase the knowledge on the upper thermal limit found in the latter species by estimating the CT50 value (19.7°C) (critical temperature at which 50% of animals are reactive) and by linking metabolic rate measurements with molecular approaches. Optical oxygen sensors were used to measure respiration rates in steps of 2°C (from 0 to 16°C). To follow the temperature-mediated mechanisms of passive response, i.e., as a proxy for molecular stress, molecular chaperone heat shock protein 70 (Hsp70) sequences were extracted from a transcriptome assembly, and the gene expression kinetics were monitored during an acute temperature exposure to 6 or 10°C with subsequent recovery at 4°C. Our results showed upregulation of hsp70 genes, especially the structurally constitutive and mitochondrial isoforms. These findings confirmed the temperature sensitivity of T. inermis and showed that the thermal stress took place before reaching the upper temperature limit estimated by respirometry at 12°C. This study provides a baseline for further investigations into the thermal tolerances of arcto-boreal Thysanoessa spp. and comparisons with other krill species under different climatic regimes, especially Antarctica.

Exposure to tritiated water at an elevated temperature: Genotoxic and transcriptomic effects in marine mussels (M. galloprovincialis)

Temperature is an abiotic factor of particular concern for assessing the potential impacts of radionuclides on marine species. This is particularly true for tritium, which is discharged as tritiated water (HTO) in the process of cooling nuclear institutions. Additionally, with sea surface temperatures forecast to rise 0.5–3.5 °C in the next 30–100 years, determining the interaction of elevated temperature with radiological exposure has never been more relevant. We assessed the tissue-specific accumulation, transcriptional expression of key genes, and genotoxicity of tritiated water to marine mussels at either 15 or 25 °C, over a 7 day time course with sampling after 1 h, 12 h, 3 d and 7d. The activity concentration used (15 MBq L−1) resulted in tritium accumulation that varied with both time and temperature, but consistently produced dose rates (calculated using the ERICA tool) of <20 Gy h−1, i.e. considerably below the recommended guidelines of the IAEA and EURATOM. Despite this, there was significant induction of DNA strand breaks (as measured by the comet assay), which also showed a temperature-dependent time shift. At 15 °C, DNA damage was only significantly elevated after 7 d, in contrast to 25 °C where a similar response was observed after only 3 d. The transcription profiles of two isoforms of hsp70, hsp90, mt20, p53 and rad51 indicated potential mechanisms behind this temperature-induced acceleration of genotoxicity, which may be the result of compromised defence. Specifically, genes involved in protein folding, DNA double strand break repair and cell cycle checkpoint control were upregulated after 3 d HTO exposure at 15 °C, but significantly downregulated when the same exposure occurred at 25 °C. This study is the first to investigate temperature effects on radiation-induced genotoxicity in an ecologically relevant marine invertebrate, Mytilus galloprovincialis. From an ecological perspective, our study suggests that mussels (or similar marine species) exposed to increased temperature and HTO may have a compromised ability to defend against genotoxic stress.

Acute hyperthermic responses of heat shock protein and estrogen receptor mRNAs in rainbow trout hepatocytes

Heat shock proteins (HSPs) are induced upon elevated temperature in fishes. HSPs also function as molecular chaperones for cellular proteins, including steroid hormone receptors. Estrogen receptors (ERs) are critical for the hormone signaling necessary during the liver production of the yolk precursor protein vitellogenin in oviparous vertebrates. Considering the possible regulatory role of HSPs on the ER signaling pathway, the present study characterized the mRNA expression of all known isoforms of HSP70 (hsp70a, hsp70b), HSP90 (hsp90a1a, hsp90a1b, hsp90a2a, hsp90a2b, hsp90b1, hsp90b2), and ERs (erα1, erα2, erβ1, erβ2) in Rainbow Trout hepatocytes following an acute heat shock (1h at 25°C) compared to a control treatment (12°C). The results showed that the mRNA levels of hsp70a, hsp70b, hsp90a1b, hsp90a2a, and hsp90b2 were significantly increased after heat shock, while erα1 mRNA levels were significantly reduced by this treatment. hsp90a1a, hsp90a2b, hsp90b1, erα2, erβ1 and erβ2 were unaffected by this acute hyperthermic treatment. Comparatively, the responses of the two hsp70 isoforms were much greater than the hsp90 isoforms. Acute heat shock treatment of hepatocytes followed by a 24h exposure to 17β-estradiol (E2) exposure also resulted in decreased expression of erα1 mRNA, but not vitellogenin (vtg) mRNA. This study showed that some hsp70 and hsp90 isoforms display a robust response to an acute hyperthermic treatment in Rainbow Trout hepatocytes. Among the transcripts measured here, the erα1 isoform uniquely showed significantly decreased mRNA levels upon acute heat treatment.

Thermal stress induces a distinct transcriptome profile in the Pacific oyster Crassostrea gigas

Oysters are frequently subjected to heat stress during tidal emersion/immersion cycles in their habitats due to attachment on the rocky shore. To understand the effect of temperature elevation on the whole transcriptome over time, the Pacific oyster Crassostrea gigas was exposed to seawater temperature 32°C for 72h from the control 20°C. RNA-seq identified differentially expressed stress responsive transcripts upon thermal stress in the gill tissues of C. gigas. The primary effect of heat stress appears to be significantly induced transcription of molecular chaperones, including members of the heat shock protein (hsp) families, while genes typically associated with protein metabolism, such as those involved in protein degradation (e.g. ATP-dependent proteolysis pathway) and biosynthesis (e.g. ribosomal protein genes), were repressed. In particular, several hsp70 isoforms and a small hsp20 maintained prolonged mRNA expressions for 72h. This study provides preliminary insights into the molecular response of C. gigas to heat stress and suggests a basis for future studies examining molecular adaptation or thermotolerance metabolism in the Pacific oyster.

Conserved effects of salinity acclimation on thermal tolerance and hsp70 expression in divergent populations of threespine stickleback (Gasterosteus aculeatus)

In natural environments, organisms must cope with complex combinations of abiotic stressors. Here, we use threespine stickleback (Gasterosteus aculeatus) to examine how changes in salinity affect tolerance of high temperatures. Threespine stickleback inhabit a range of environments that vary in both salinity and thermal stability making this species an excellent system for investigating interacting stressors. We examined the effects of environmental salinity on maximum thermal tolerance (CTMax) and 70kDa heat shock protein (hsp70) gene expression using divergent stickleback ecotypes from marine and freshwater habitats. In both ecotypes, the CTMax of fish acclimated to 20 ppt was significantly higher compared to fish acclimated to 2 ppt. The effect of salinity acclimation on the expression of hsp70-1 and hsp70-2 was similar in both the marine and freshwater stickleback ecotype. There were differences in the expression profiles of hsp70-1 and hsp70-2 during heat shock, with hsp70-2 being induced earlier and to a higher level compared to hsp70-1. These data suggest that the two hsp70 isoforms may have functionally different roles in the heat shock response. Lastly, acute salinity challenge coupled with heat shock revealed that the osmoregulatory demands experienced during the heat shock response have a larger effect on the hsp70 expression profile than does the acclimation salinity.

HSP-90 Expression as a Predictor of Response to Radiotherapy in Head and Neck Cancer Patients

Introduction and objectivesHSP-90 is an intracellular protein that protects the cell from environmental stress situations. The overexpression of HSP-90 isoforms could serve as a mechanism of resistance to radiotherapy for tumour cells. We studied this effect in a sample of head and neck tumours. MethodsWe included 87 patients diagnosed with oral cavity, oropharynx, larynx and hypopharynx tumours. We studied the expression of the HSP-90 isoforms by real-time PCR on pre-treatment biopsy samples. We analysed the relationship between HSP-90 expression levels and local relapse of the tumour with CRT decision trees. ResultsThe expression levels of the inducible cytosolic isoform (HSP90AA) allowed the definition of two groups of patients with different rates of local relapse. The group with a low expression level showed a 2.9% local relapse rate, while the group with a high expression level showed a 38.2% rate.Survival curves showed differences in time to local relapse for both groups of patients. These differences did not reach statistical significance. ConclusionsRadiotherapy response was related to expression levels of HSP-90 in a sample of head and neck cancer patients. This result could prove useful in the selection of treatments for this group of patients.

Development of Glucose Regulated Protein 94-Selective Inhibitors Based on the BnIm and Radamide Scaffold.

Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum resident of the heat shock protein 90 kDa (Hsp90) family of molecular chaperones. Grp94 associates with many proteins involved in cell adhesion and signaling, including integrins, Toll-like receptors, immunoglobulins, and mutant myocilin. Grp94 has been implicated as a target for several therapeutic areas including glaucoma, cancer metastasis, and multiple myeloma. While 85% identical to other Hsp90 isoforms, the N-terminal ATP-binding site of Grp94 possesses a unique hydrophobic pocket that was used to design isoform-selective inhibitors. Incorporation of a cis-amide bioisostere into the radamide scaffold led to development of the original Grp94-selective inhibitor, BnIm. Structure-activity relationship studies have now been performed on the aryl side chain of BnIm, which resulted in improved analogues that exhibit better potency and selectivity for Grp94. These analogues also manifest superior antimigratory activity in a metastasis model as well as enhanced mutant myocilin degradation in a glaucoma model compared to BnIm.

A Remodeled Hsp90 Molecular Chaperone Ensemble with the Novel Cochaperone Aarsd1 Is Required for Muscle Differentiation.

Hsp90 is the ATP-consuming core component of a very abundant molecular chaperone machine that handles a substantial portion of the cytosolic proteome. Rather than one machine, it is in fact an ensemble of molecular machines, since most mammalian cells express two cytosolic isoforms of Hsp90 and a subset of up to 40 to 50 cochaperones and regulate their interactions and functions by a variety of posttranslational modifications. We demonstrate that the Hsp90 ensemble is fundamentally remodeled during muscle differentiation and that this remodeling is not just a consequence of muscle differentiation but possibly one of the drivers to accompany and to match the vast proteomic changes associated with this process. As myoblasts differentiate into myotubes, Hsp90α disappears and only Hsp90β remains, which is the only isoform capable of interacting with the novel muscle-specific Hsp90 cochaperone Aarsd1L. Artificially maintaining Hsp90α or knocking down Aarsd1L expression interferes with the differentiation of C2C12 myotubes. During muscle differentiation, Aarsd1L replaces the more ubiquitous cochaperone p23 and in doing so dampens the activity of the glucocorticoid receptor, one of the Hsp90 clients relevant to muscle functions. This cochaperone switch protects muscle cells against the inhibitory effects of glucocorticoids and may contribute to preventing muscle wasting induced by excess glucocorticoids.

Proteomic identification of predictive tissue biomarkers of sensitive to neoadjuvant chemotherapy in squamous cervical cancer

AimsThe regimens of neoadjuvant chemotherapy (NAC) in squamous cervical cancer (SCC) frequently use cisplatin combined with paclitaxel. Unfortunately, some cervical cancers show resistance to the principal chemotherapeutic agents in the treatment, decreasing the effectiveness of this therapy. The objective of this study was to search for predictive markers of response to NAC in patients with SCC. Main methodsTwo-dimensional gel electrophoresis (2-DE) accompanied by matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS) was used to analyze and identify differentially expressed proteins in ten cases of advanced cervical cancer patients receiving cisplatin-based NAC. Each of these patients received more than two cycles of NAC. Cell proliferation rate in cisplatin resistant human cervical cancer cell Hela/DDP and its parent cell Hela after treatment with Hsp70 inhibitor and/or cisplatin were tested by MTT assay. Key findingsTwelve protein spots changed in abundance, quantitative comparison of spot volumes showed that seven protein spots were up-regulated and five spots were down-regulated in the NAC non-responders compared to the NAC responders. These proteins are involved in various cellular processes essential for cell metabolism, migration and apoptotic signal transduction. The high-fold changes proteins of stathmin1, Hsp70 and pyruvate kinase isoform M2 were validated by Western blot analysis. Over-expression of Hsp70 inhibits the efficacy of cisplatin. Hsp70 inhibitor enhanced the sensitivity to cisplatin in both Hela and Hela/DDP cells. SignificanceThe study found many candidate proteins involved in chemotherapy resistance/sensitivity, among them Hsp70 might be potential biomarkers to predict the efficacy of chemotherapy for SCC patients.

The role of membrane-bound heat shock Hsp90 proteins in the migration of tumor cells in vitro and the involvement of cell surface heparan sulfate proteoglycans in protein binding to the plasma membrane

The heat-shock protein Hsp90, which is found in the extracellular space and on the cell membrane, plays an important role in cell motility, migration, invasion, and metastasis of tumor cells. Currently, the functional role and molecular mechanisms of Hsp90 binding to the plasma membrane are not clear. Using isoform-specific antibodies against Hsp90, Hsp90α, and Hsp90β, we showed that the membrane-bound Hsp90α and Hsp90β proteins play a significant role in the migration of human fibrosarcoma (HT1080) and glioblastoma (A-172) cells in vitro. Impairment of sulfonation of cellular heparan sulfates, cleavage of cellular heparan sulfates by heparinase I/III, as well as the treatment of cells with heparin, lead to a dramatic reduction in the expression levels of the Hsp90 isoforms. Furthermore, heparin significantly inhibits tumor cell migration. These results demonstrate that two isoforms of membrane-bound Hsp90 are involved in tumor-cell migration in vitro. As well, the cell surface heparan sulfate proteoglycans are shown to play a pivotal role in the “anchoring” of Hsp90α and Hsp90β to the plasma membrane.