Stress-inducible Heat Shock Protein Research Articles

The Stress-Inducible Heat Shock Protein 70 (HSPA1A) as A Novel Biomarker in Lung Tumors: A Promoting Study

Heat shock proteins (HSPs) are a group of intracellular proteins that promote proteins folding and unfolding under normal and/or stress conditions. In addition to their intracellular location, HSPs are found on the plasma membranes of stressed, but not normal, cells and in the extracellular milieu where they can trigger an immune response. For instance, the inducible form of heat shock protein 70 (HSPA1A) was found to be overexpressed, intra or extracellularly, by many types of stressed cells. In our study, we aim to investigate the levels of HSPA1A in the serum of untreated lung tumor patients and its expression in the tissues derived from lung tumors and chronic obstructive pulmonary diseases (COPD) and healthy tissues. Analyses of serum and tissue samples were performed by utilizing ELISA assay and immunohistochemical staining, respectively. The results showed significant differences in the mean levels of serum HSPA1A of the patients (1.66± 0.165) as compared to healthy control (0.938±0.330). Significant differences were also found between these levels in the serum of smoker and nonsmoker patients (2.006±0.342 and 1.353± 0.067, respectively). The protein levels in the patient's serum were also found to be 29.5% correlated to smoking history. No significant differences were found in terms of gender and age within the two groups of patients and healthy volunteers. Our results showed HSPA1A protein levels to be correlated to gender (13.5%) and age in patients and healthy volunteers (14.8% and 0.4%, respectively). In the tissues of the clinicopathological groups, HSPA1A was found to be overexpressed in benign (IRS= 8.5) and malignant tumors (IRS= 8.9) in comparison with both COPD (IRS= 4.4) and healthy tissues (IRS= 0.00). Our pilot results could support the suggestion of HSPA1A as a novel biomarker in lung tumors, as proposed by previous studies.

Transcriptome analysis uncovers the key pathways and candidate genes related to the treatment of Echinococcus granulosus protoscoleces with the repurposed drug pyronaridine

BackgroundCystic echinococcosis (CE) is a life-threatening zoonosis caused by the larval form of Echinococcus granulosus tapeworm. Our previous study showed that an approved drug pyronaridine (PND) is highly effective against CE, both in vitro and in an animal model. To identify possible target genes, transcriptome analysis was performed with E. granulosus sensu stricto protoscoleces treated with PND.ResultsA total of 1,321 genes were differentially expressed in protoscoleces treated with PND, including 541 upregulated and 780 downregulated genes. Gene ontology and KEGG analyses revealed that the spliceosome, mitogen-activated protein kinase (MAPK) pathway and ATP-binding cassette (ABC) transporters were the top three enriched pathways. Western blot analysis showed that PND treatment resulted in a dose-dependent increase in protein expression levels of EgMKK1 (MKK3/6-like) and EgMKK2 (MEK1/2-like), two members of MAPK cascades. Interestingly, several heat shock protein (HSP) genes were greatly downregulated including stress-inducible HSPs and their constitutive cognates, and some of them belong to Echinococcus-specific expansion of HSP70.ConclusionsPND has a great impact on the spliceosome, MAPK pathway and ABC transporters, which may underline the mechanisms by which PND kills E. granulosus protoscoleces. In addition, PND downregulates HSPs expression, suggesting a close relationship between the drug and HSPs.

Potential Role of Hsp70 and Activated NK Cells for Prediction of Prognosis in Glioblastoma Patients.

Despite rapid progress in the treatment of many cancers, glioblastoma remains a devastating disease with dismal prognosis. The aim of this study was to identify chaperone- and immune-related biomarkers to improve prediction of outcome in glioblastoma. Depending on its intra- or extracellular localization the major stress-inducible heat shock protein 70 (Hsp70) fulfills different tasks. In the cytosol Hsp70 interferes with pro-apoptotic signaling pathways and thereby protects tumor cells from programmed cell death. Extracellular Hsp70 together with pro-inflammatory cytokines are reported to stimulate the expression of activatory NK cell receptors, recognizing highly aggressive human tumor cells that present Hsp70 on their cell surface. Therefore, intra-, extracellular and membrane-bound Hsp70 levels were assessed in gliomas together with activatory NK cell receptors. All gliomas were found to be membrane Hsp70-positive and high grade gliomas more frequently show an overexpression of Hsp70 in the nucleus and cytosol. Significantly elevated extracellular Hsp70 levels are detected in glioblastomas with large necrotic areas. Overall survival (OS) is more favorable in patients with low Hsp70 serum levels indicating that a high Hsp70 expression is associated with an unfavorable prognosis. The data provide a first hint that elevated frequencies of activated NK cells at diagnosis might be associated with a better clinical outcome.

Differential Effects of STCH and Stress-Inducible Hsp70 on the Stability and Maturation of NKCC2.

Mutations in the Na-K-2Cl co-transporter NKCC2 lead to type I Bartter syndrome, a life-threatening kidney disease. We previously showed that export from the ER constitutes the limiting step in NKCC2 maturation and cell surface expression. Yet, the molecular mechanisms involved in this process remain obscure. Here, we report the identification of chaperone stress 70 protein (STCH) and the stress-inducible heat shock protein 70 (Hsp70), as two novel binding partners of the ER-resident form of NKCC2. STCH knock-down increased total NKCC2 expression whereas Hsp70 knock-down or its inhibition by YM-01 had the opposite effect. Accordingly, overexpressing of STCH and Hsp70 exerted opposite actions on total protein abundance of NKCC2 and its folding mutants. Cycloheximide chase assay showed that in cells over-expressing STCH, NKCC2 stability and maturation are heavily impaired. In contrast to STCH, Hsp70 co-expression increased NKCC2 maturation. Interestingly, treatment by protein degradation inhibitors revealed that in addition to the proteasome, the ER associated degradation (ERAD) of NKCC2 mediated by STCH, involves also the ER-to-lysosome-associated degradation pathway. In summary, our data are consistent with STCH and Hsp70 having differential and antagonistic effects with regard to NKCC2 biogenesis. These findings may have an impact on our understanding and potential treatment of diseases related to aberrant NKCC2 trafficking and expression.

Improving the Efficacy of Checkpoint Blockade Inhibitors in Breast Cancer by Combining with Radiation Therapy

Large cohorts of recent clinical studies have established that increased levels of tumor-infiltrating lymphocytes (TILs) in patients with the triple-negative breast cancer (TNBC) subtype predicted better clinical outcomes compared to other subtypes. These observations led to the hypothesis that women with TNBC may respond to checkpoint blockade therapy. However, early results from these trials using checkpoint inhibitors alone or with chemotherapy has shown very little promise in breast cancer patients, despite the remarkable long-lasting responses in other hard to treat malignancies such as non-small cell lung and melanoma. Although the outcome falls short of the expectation, we hypothesized that radiation therapy can be utilized to increase the efficacy of check point blockade inhibitors of the stress-induced heat shock protein 70 (HSP70) in order to reverse immunosuppression and induce neo-antigens to improve the outcomes of TNBC. To test the effectiveness of a combination of checkpoint blockade inhibitors and radiation therapy, this study utilized 6-week old female BALB/c 4T1 tumor-bearing mice as a model for TNBC. Combination treatment of HSP70 inhibitors and targeted irradiation was performed prior to tumor resection and data collection. Specifically, Radiation Therapy doses were planned and delivered using a Small Animal Radiation Platform (SARRP) equipped with on-board cone-beam CT. A single fraction of 17 Gy (Two fractions of 11.5 Gy) doses were delivered based on EQD2 calculation (alpha Beta ratio = 4). To achieve highly localized dose delivery, an x-ray collimator was used to focus the broad beam from a 225 kVp x-ray tube. Our preliminary data provide evidence that targeting HSP70 in combination with radiation therapy has dual activity on the tumor and the immunosuppressive microenvironment. Furthermore, reversal of HSP70 blockade significantly reduces the immunosuppressive MDSC infiltration in lungs by more than 50% compared to lungs from control mice. This also led to increased tumor infiltration of effector T cells from 1-2% in controls to 12% in lungs from mice treated with HSP70 inhibitor in combination with radiation. Interestingly, T cell infiltration is further increased to 20% when check point blockade inhibitor is combined with HSP70 inhibitor and radiation. Therefore, we showed that HSP70 regulates three fundamental processes; i) protecting tumor cells from cytotoxic cell death, ii) serving as a powerful tumor antigen attracting TILs, and iii) modulating immunosuppressive MDSCs. In summary, our studies elucidate the molecular mechanism by which HSP70 cytoprotects tumor cells from cytotoxic agents while regulating the immunosuppressive MDSCs which limits the anti-tumor activity of TILs. They also provide evidence that blocking HSP70 potentiates the efficacy of the checkpoint blockade particularly when combined with radiation in syngeneic mouse models representing TNBC subtype.

Abstract 6377: Curcumin analog GO-Y030 diminishes cancer stem cell population by inhibiting interaction of HSP70 with its substrates

Abstract It has been established that a cancer tissue contains, in general, a small subpopulation of cancer stem cells (CSCs) having tumor initiation capacity. Because CSCs are resistant to chemoradiotherapy and play important roles in cancer relapse and metastasis, strategies targeting CSCs are crucial for effective anticancer therapies. Curcumin (diferuloylmethane) isolated from the rhizome of the plant turmeric (Curcuma longa) is one of the most widely characterized phytochemicals and well known to suppress cancer cell proliferation, invasion, angiogenesis, and metastasis by regulating multiple cell signaling pathways. However, its bioavailability is limited due to poor absorption. To improve this disadvantage, Ohori et al. (Mol Cancer Ther 2006;5:2563-71.) synthesized a new curcumin analog GO-Y030 (1,5-bis(3,5-bis(methoxymethoxy)phenyl)-1,4-pentadiene-3-one), which has an about 30-fold greater growth-inhibitory effect than curcumin. In the current study, we verified the potency of GO-Y030 to act against CSC population. We first discovered that GO-Y030 could dose-dependently suppress a sphere-forming ability in human HuH7 hepatocellular carcinoma and human PC3 prostatic carcinoma cell lines. Interestingly, the specific inhibitor of the stress-inducible heat shock protein (HSP) 70 (also called HSP72, HSP70-1, or HSPA1A) exhibited effects similar to GO-Y030, i.e., inhibition of CSC sphere formation and upregulation of HSP70 and HSP40 protein expression. As this result suggested that target molecules of GO-Y030 might be HSP70 and its co-chaperones such as HSP40, we next performed in vitro HSP70/HSP40-mediated refolding activity assay and revealed that GO-Y030 efficiently inhibited HSP70/HSP40 chaperone activity. Finally, performing the substrate-binding assay, we have proven that GO-Y030 reduces the substrate-recognition and binding activity of HSP70. HSPs are molecular chaperones that function to prevent denaturation or unfolding of client proteins when they are threatened by stress or high temperature. Since CSCs by nature adapt to oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum stress by enhancing synthesis of protein-folding chaperones, the function of HSP70/HSP40 is important for the maintenance of CSC population. Our data suggest that GO-Y030 should impair stress tolerance in CSCs by inhibiting the interaction of HSP70 with substrate proteins and disrupting the function of HSP70/HSP40 and thus should finally contribute to reduction of CSC population. Citation Format: Maya Suzuki, Yohei Yamamoto, Aki Nishijima, Hiroyuki Shibata, Yasufumi Omori. Curcumin analog GO-Y030 diminishes cancer stem cell population by inhibiting interaction of HSP70 with its substrates [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6377.

Heat Shock Protein 70 as a Double Agent Acting Inside and Outside the Cell: Insights into Autoimmunity.

Heat shock proteins (Hsp) are a diverse group of constitutive and/or stress-induced molecules that are categorized into several classes on the basis of their molecular weight. Mammalian Hsp have been mostly regarded as intracellular chaperones that mediate a range of essential cellular functions, including proper folding of newly synthesized polypeptides, refolding of denatured proteins, protein transport, and stabilization of native proteins’ structures. The well-characterized and highly evolutionarily conserved, stress-inducible 70-kDa heat shock protein (Hsp70), is a key molecular chaperone that is overexpressed in the cell in response to stress of various origin. Hsp70 exhibits an immunosuppressive activity via, e.g., downregulation of the nuclear factor-kappa B (NF-κB) activation, and pharmacological induction of Hsp70 can ameliorate the autoimmune arthritis development in animal models. Moreover, Hsp70 might be passively or actively released from the necrotic or stressed cells, respectively. Highly immunogenic extracellular Hsp70 has been reported to impact both the innate and adaptive immune responses, and to be implicated in the autoimmune reaction. In addition, preclinical studies revealed that immunization with highly conserved Hsp70 peptides could be regarded as a potential treatment target for autoimmune arthritis, such as the rheumatoid arthritis, via induction of antigen-specific regulatory T helper cells (also called Treg). Here, a dual role of the intra- and extracellular Hsp70 is presented in the context of the autoimmune reaction.

Short exposure to cadmium disrupts the olfactory system of zebrafish (Danio rerio) – Relating altered gene expression in the olfactory organ to behavioral deficits

Fish strongly rely on olfaction as a variety of essential behaviors such as foraging and predator avoidance are mediated by the olfactory system. Cadmium (Cd) is known to impair olfaction and accumulate in the olfactory epithelium (OE) and bulb (OB) of fishes. In the present study, the acute toxicity of Cd on olfaction in zebrafish (Danio rerio) was characterized on the molecular and behavioral level. To this end, quantitative real-time PCR was performed in order to analyze the expression of selected genes in both the OE and OB. Moreover, the response of zebrafish to an alarm cue was investigated. Following 24 h of exposure to Cd, the expression of genes associated with olfactory sensory neurons was reduced in the OE. Furthermore, the antioxidant genes peroxiredoxin 1 (prdx1) and heme oxygenase 1 (hmox1), as well as the metallothionein 2 gene (mt2) were upregulated in the OE, whereas hmox1 and the stress-inducible heat shock protein 70 gene (hsp70) were upregulated in the OB upon exposure to Cd. Following stimulation with a conspecific skin extract, zebrafish displayed a considerable disruption of the antipredator behavior with increasing Cd concentration. Taken together, Cd impaired olfaction in zebrafish, thereby disrupting the antipredator response, which is crucial for the survival of individuals. Cellular stress followed by disruption of olfactory sensory neurons may have contributed to the observed behavioral deficits.

The kinase PERK represses translation of the G-protein–coupled receptor LGR5 and receptor tyrosine kinase ERBB3 during ER stress in cancer cells

As a branch of the unfolded protein response, protein kinase R-like endoplasmic reticulum kinase (PERK) represses global translation in response to endoplasmic reticulum (ER) stress. This pathophysiological condition is associated with the tumor microenvironment in cancer. Previous findings in our lab have suggested that PERK selectively represses translation of some mRNAs, but this possibility awaits additional investigation. In this study, we show that a stem-cell marker protein, leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), is rapidly depleted in colon cancer cells during ER stress, an effect that depended on the PERK-mediated translational repression. Indeed, the PERK inhibition led to the accumulation of premature, underglycosylated forms of LGR5, which were produced only at low levels during proper PERK activation. Unlike the mature LGR5 form, which is constitutively degraded regardless of PERK activation, the underglycosylated LGR5 exhibited a prolonged half-life and accumulated inside the cells without being expressed on the cell surface. We also found that Erb-B2 receptor tyrosine kinase 3 (ERBB3) is subjected to a similarly-regulated depletion by PERK, whereas the epidermal growth factor receptor (EGFR), stress-inducible heat-shock protein family A (Hsp70) member 5 (HSPA5), and anterior gradient 2 protein-disulfide isomerase family member (AGR2) were relatively. insensitive to the PERK-mediated repression of translation. These results indicate that LGR5 and ERBB3 are targets for PERK-mediated translational repression during ER stress.

Prion disease is accelerated in mice lacking stress-induced heat shock protein 70 (HSP70)

Prion diseases are a group of incurable neurodegenerative disorders that affect humans and animals via infection with proteinaceous particles called prions. Prions are composed of PrPSc, a misfolded version of the cellular prion protein (PrPC). During disease progression, PrPSc replicates by interacting with PrPC and inducing its conversion to PrPSc As PrPSc accumulates, cellular stress mechanisms are activated to maintain cellular proteostasis, including increased protein chaperone levels. However, the exact roles of several of these chaperones remain unclear. Here, using various methodologies to monitor prion replication (i.e. protein misfolding cyclic amplification and cellular and animal infectivity bioassays), we studied the potential role of the molecular chaperone heat shock protein 70 (HSP70) in prion replication in vitro and in vivo Our results indicated that pharmacological induction of the heat shock response in cells chronically infected with prions significantly decreased PrPSc accumulation. We also found that HSP70 alters prion replication in vitro More importantly, prion infection of mice lacking the genes encoding stress-induced HSP70 exhibited accelerated prion disease progression compared with WT mice. In parallel with HSP70 being known to respond to endogenous and exogenous stressors such as heat, infection, toxicants, and ischemia, our results indicate that HSP70 may also play an important role in suppressing or delaying prion disease progression, opening opportunities for therapeutic intervention.

Cytosolic Hsp70 as a biomarker to predict clinical outcome in patients with glioblastoma

IntroductionThe major stress-inducible heat shock protein 70 (Hsp70) is induced after different stress stimuli. In tumors, elevated intracellular Hsp70 levels were associated on the one hand with radio- and chemotherapy resistance and on the other hand with a favorable outcome for patients. This study was undertaken to investigate cytosolic Hsp70 (cHsp70) as a potential biomarker for progression free (PFS) and overall survival (OS) in patients with primary glioblastomas (GBM).MethodsThe cHsp70 expression in tumor tissue of 60 patients diagnosed with primary GBM was analyzed by immunohistochemistry. The cHsp70 expression was correlated to the PFS and OS of the patients.ResultsA high cHsp70 expression was associated with a prolonged PFS (hazard ratio = 0.374, p = 0.001) and OS (hazard ratio = 0.416, p = 0.014) in GBM patients treated according to the standard Stupp protocol with surgery, radiotherapy and temozolomide.ConclusionsThese data suggest that the intracellular Hsp70 expression might serve as a prognostic marker in patients with primary GBM.

Профилактическое введение Hsp70 противодействует прогрессированию нейродегенерации в нигростриатной системе в модели болезни Паркинсона у крыс

Parkinson’s disease (PD) is a chronic progressive neurodegenerative disease, which still remains incurable. Hence it is essential to develop new neuroprotective approaches aiming to stop or slow down the neurodegeneration during PD. The literature data evidence that stress-inducible heat shock protein 70 kDa (Hsp70) possesses therapeutic potential in conformational neurodegenerative diseases. The current study evaluated the effect of the prophylactic administration Hsp70 into substantia nigra pars compacta (SNpc) on the progression of neurodegeneration in the rat model of PD, induced by microinjections of the proteasome inhibitor lactaсystin into SNpc. The model reproduced the main characteristics of the clinical stage of PD such as loss of 61% of dopaminergic neurons in the SNpc and 65% of its striatal axons, the decrease in levels of tyrosine hydroxylase, vesicular monoamine transporter 2 and Hsp70, as well as the appearance of motor dysfunction (disturbances in gait and fine movements of the fore-limbs, mouth and tongue, development of sensorimotor deficits of limbs). It was established for the first time that prophylactic administration of Hsp70 into SNpc counteracted the neurodegenerative process and evoked the decrease in the loss of dopaminergic neurons of SNpc (1.8-fold) and its striatal axons (2.5-fold) compared with lactacystin alone, the restoration of tyrosine hydroxylase and vesicular monoamine transporter 2 levels to baseline, the prevention of disturbances of fine motor abilities and sensorimotor deficits of limbs and the attenuation in gait disturbances. Our data may provide the basis for the development of new non-invasive approaches (thermal preconditioning, physical loading, nanomedical technologies, etc) to prophylactic increase in Hsp70 amount in brain aiming to protect from the PD and other neurodegenerative diseases.

Thermal stress induces heat shock protein 70 and apoptosis during embryo development in a Neotropical freshwater fish.

Fish embryos are particularly vulnerable to temperature changes, with the effects varying with developmental stage. The major aim of the present study was to analyse the relationship between apoptosis and heat shock protein (HSP) 70 during embryo development under thermal stress conditions. To this end, Prochilodus lineatus embryos at the blastopore closure stage were subjected to one of three thermal treatments for 1h (Group 1, 25°C (control); Group 2, 20°C; Group 3, 30°C) and then examined at 0, 4 and 8h posttreatment (h.p.t.). The viability of embryos was highest in Group 1 (81.33±16.65%), followed by Group 3 and Group 2 (75.33±12.10% and 68.67±16.86% respectively), with significant difference between Groups 1 and 2 (P<0.05). At 0h.p.t., embryos subjected to thermal stress (Group 3) had a significantly higher number of terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL)- and caspase-3-labelled cells, and a lower number of HSP70-positive cells than those in the control group. At 4h.p.t., there was a decrease in the TUNEL reaction and an increase in HSP70 in embryos in Group 3. At 8h.p.t., the size of Group 3 embryos was significantly smaller than that of Group 1 embryos. The results indicate a cytoprotective role for HSP70, regulating caspase-3-mediated apoptosis during embryo development of P. lineatus; however, this mechanism is not effective in controlling embryo viability and larval malformations.

Study of Age-Related Changes in Compensatory Processes in the Model of Neurodegeneration of the Nigrostriatal System in Rats

It is generally accepted that old age is the main risk factor for the development and progression of Parkinson’s disease (PD). However, there is currently no data available that experimentally confirm the age dependence of the neurodegeneration progression rate and the activity of compensatory processes in the nigrostriatal system in the development of PD. The present study applies a model of nigrostriatal system neurodegeneration in rats of different ages, which was developed using the microinjections with proteasome inhibitor lactacystin (LC) into the substantia nigra pars compacta (SNpc). The model reproduces the main pathomorphological signs of PD with great reliability. It has been demonstrated that the administration of LC to old rats, compared to young and middle-aged ones, causes more pronounced neurodegenerative changes in the nigrostriatal system which are associated with impairments in fine motor function, a decrease in the growth of the stress-inducible heat shock protein Hsp70 in surviving neurons of SNpc, and a decrease in contents of tyrosine hydroxylase and the vesicular monoamine transporter 2. The data obtained suggest that the aging-related decrease in Hsp70 expression together with a decrease in the efficiency of compensatory processes is a significant factor which determines the progression of pathology in the nigrostriatal system in PD model in old rats. The age-related loss of compensatory mechanisms observed may be one of the causes of the rapid PD progression in the elderly.

Pregnancy History Influences the Level of Autophagy in Peripheral Blood Mononuclear Cells From Pregnant Women.

Maternal immune responses are altered during pregnancy and differ between nulliparous and multiparous women. The influence of a prior gestation on autophagy in peripheral blood mononuclear cells (PBMCs) from pregnant women has not been determined and is the subject of this investigation. Peripheral blood mononuclear cells were isolated from 212 pregnant women and immediately lysed in the presence of protease inhibitors, and the extent of autophagy was determined by quantitation of the concentration of p62 (sequestosome-1) in the lysates by enzyme-linked immunosorbent assay (ELISA). In PBMCs, the p62 level is inversely related to the extent of autophagy. The level of the stress-inducible 70-kDa heat shock protein (hsp70), an inhibitor of autophagy, was also measured in the lysates by ELISA. Data were analyzed by the Spearman rank correlation, Mann-Whitney U test, or Kruskal-Wallis test, as appropriate. The p62 concentration in PBMCs increased (autophagy decreased) with the number of previous live ( P = .0322), preterm ( P = .0143), or term ( P = .0418) deliveries. The p62 level was lower (autophagy higher) in women with a prior spontaneous pregnancy loss but no deliveries as compared to women with their first conception ( P = .0087). The intracellular hsp70 concentration correlated with the p62 level ( P < .0001). Multiparity is associated with a reduced level of autophagy in PBMCs. Dysregulated autophagy might be one mechanism leading to spontaneous abortion in nulliparous women.

Inducible HSP70 antagonizes cisplatin‑induced cell apoptosis through inhibition of the MAPK signaling pathway in HGC‑27 cells.

Inducible heat shock protein 70 (HSP70; also known as HSPA1 or HSP72) is implicated in cancer. As a stress-inducible heat shock protein, HSP70 is highly expressed in a variety of cancers and correlates with metastasis, chemotherapy resistance and tumor prognosis. The present study demonstrated that suppression of HSP70 through the specific inhibitor pifithrin-µ or by HSP70 knockdown enhanced cisplatin-induced apoptosis in HGC-27 gastric cancer cells. By contrast, upregulation of HSP70 through transfection of a HSP70 overexpressing plasmid decreased cisplatin-induced HGC-27 cell apoptosis. In exploring the underlying molecular mechanisms, the present results revealed that HSP70 antagonized cisplatin-induced HGC-27 cell apoptosis by regulating the mitogen-activated protein kinase (MAPK) signaling pathway. In addition, suppressing the MAPK pathway enhanced cisplatin-induced HGC-27 cell apoptosis. Collectively, the present findings suggest that inhibition of HSP70 expression enhanced the sensitivity of HGC-27 cells to cisplatin via the MAPK signaling pathway, and that HSP70 may serve as a potential therapeutic target in gastric cancer.

P209L mutation in Bag3 does not cause cardiomyopathy in mice.

Bcl-2-associated athanogene 3 (BAG3) is a cochaperone protein and a central player of the cellular protein quality control system. BAG3 is prominently expressed in the heart and plays an essential role in cardiac protein homeostasis by interacting with chaperone heat shock proteins (HSPs) in large, functionally distinct multichaperone complexes. The BAG3 mutation of proline 209 to leucine (P209L), which resides in a critical region that mediates the direct interaction between BAG3 and small HSPs (sHSPs), is associated with cardiomyopathy in humans. However, the mechanism by which the BAG3 P209L missense mutation leads to cardiomyopathy remains unknown. To determine the molecular basis underlying the cardiomyopathy caused by the BAG3 P209L mutation, we generated a knockin (KI) mouse model in which the endogenous Bag3 gene was replaced with mutant Bag3 containing the P215L mutation, which is equivalent to the human P209L mutation. We performed physiological, histological, and biochemical analyses of Bag3 P209L KI mice to determine the functional, morphological, and molecular consequences of the P209L mutation. We found that Bag3 P209L KI mice exhibited normal cardiac function and morphology up to 16 mo of age. Western blot analysis further revealed that levels of sHSPs, stress-inducible HSPs, ubiquitinated proteins, and autophagy were unaffected in P209L mutant mouse hearts. In conclusion, the P209L mutation in Bag3 does not cause cardiomyopathy in mice up to 16 mo of age under baseline conditions. NEW & NOTEWORTHY Bcl-2-associated athanogene 3 (BAG3) P209L mutation is associated with human cardiomyopathy. A recent study reported that transgenic mice overexpressing human BAG3 P209L in cardiomyocytes have cardiac dysfunction. In contrast, our P209L mice that express mutant BAG3 at the same level as that of wild-type mice displayed no overt phenotype. Our results suggest that human cardiomyopathy may result from species-specific requirements for the conserved motif that is disrupted by P209L mutation or from genetic background-dependent effects.

Imaging of angiogenesis of human umbilical vein endothelial cells by uptake of exosomes secreted from hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a typical hyper-vascular tumor, so the understanding the mechanisms of angiogenesis in HCC is very important for its treatment. However, the influence of the exosomes secreted from HCC cells (HCC-exosomes) on angiogenesis remains poorly understood. We herein examined the effects of the exosomes secreted from HepG2 cells (HepG2-exosomes) on the lumen formation of human umbilical vein endothelial cells (HUVECs) by the imaging of angiogenesis. The degree of lumen formation of HUVECs was dependent on the number of HepG2-exosomes. The HepG2-exosomes expressed NKG2D, an activating receptor for immune cells, and HSP70, a stress-induced heat shock protein associated with angiogenesis through the vascular endothelial growth factor (VEGF) receptor. In addition, the HepG2-exosomes contained several microRNAs (miRNAs) reported to exist in the serum of HCC patients. These results suggest that the HCC-exosomes play an important role in angiogenesis. Further studies on the function of HCC-exosomes may provide a new target for HCC treatment.

HspA1A, a 70‐kDa heat shock protein, contains several distinct lipid‐binding sites

HspA1A is a stress‐inducible seventy‐kilodalton heat shock protein (Hsp70). This molecular chaperone plays critical roles in protein homeostasis and cellular survival. In addition to its role in protein homeostasis, HspA1A also localizes at different cellular membranes in stressed and cancer cells, where it binds to lipids, including phosphatidylserine (PS) and Bis(Monoacylglycero)Phosphate (BMP). Although these interactions have major physiological consequences their mechanistic details remain elusive and unexplored. We have recently hypothesized that the lipid‐binding sites for PS and BMP are distinct and that the lipid‐binding and chaperone functions do not overlap. To test these hypotheses, we used a mutation from Tryptophan to Phenylalanine (W90F), which has been described to affect the binding of HspA1A to BMP, and a Lysine to Alanine (K71A), which is known to abolish the hydrolysis of ATP. We first determined whether these mutations alter HspA1A's stability, intracellular and membrane localization, as well as its ATPase and refolding functions using recombinant proteins and ex vivo experiments. Our results revealed that the W90F mutation does not significantly alter any of the tested functions, but the K71A abolishes both the ATPase and refolding activities, as expected. We then used the lipid vesicle sedimentation (LVS) method and quantified the binding of the wild‐type and mutated HspA1A to both PS and BMP. These experiments revealed that the W90F‐HspA1A binding to BMP was 50% less than the WT, when 0.4 mM of lipids were used, while the loss‐of‐binding was only 10% when 1 and 4 mM of lipids were used. Differently, we found that the W90F mutation does not affect the binding to PS under any of the conditions tested. Furthermore, the K71A mutation did not significantly change the binding of HspA1A to PS or BMP under any of the conditions tested. We verified these results using Surface Plasmon Resonance for both mutations and determined that the W90F‐HspA1A associates with BMP five times faster and dissociates 18 times faster than the WT. These results reveal that the W90F mutation alters the kinetics of the interaction with BMP, but does not alleviate the binding, while the K71A does not affect the binding to any of the lipids tested. Together these results strongly support the prediction that the lipid‐binding sites for PS and BMP are distinct and that the lipid‐binding and chaperone functions do not overlap.Support or Funding InformationNIGMS SC3GM121226This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Serum heat shock protein 70 levels as a biomarker for inflammatory processes in multiple sclerosis.

BackgroundInflammatory and neurodegenerative processes are hallmarks of multiple sclerosis (MS). The synthesis of the major stress-inducible heat shock protein 70 (Hsp70) is induced by inflammation.ObjectiveThe purpose of this study is to determine whether Hsp70 in serum can serve as a potential biomarker to distinguish inflammatory and neurodegenerative processes in MS.MethodsSerum was obtained from 94 patients: 26 clinically isolated syndrome (CIS), 40 relapsing–remitting MS (RRMS), 19 secondary progressive MS (SPMS), and nine primary progressive MS (PPMS). As controls, serum samples were collected from patients with non-inflammatory neurological diseases (NINDs, n = 41), other inflammatory neurological diseases (OINDs, n = 28) and healthy donors (HDs, n = 114). Serum levels of Hsp70 were quantified using the enzyme-linked immunosorbent assay detecting free and liposomal Hsp70 (lipHsp70 ELISA).ResultsPatients with MS displayed significantly higher Hsp70 serum levels than HDs (p < 0.001) and significantly lower levels than OINDs (p = 0.001). A subgroup analysis revealed that Hsp70 serum levels of CIS/RRMS patients are significantly higher than those of patients with progressive MS (SPMS/PPMS) (p < 0.05).ConclusionInflammation causes the release of Hsp70 into the blood. As CIS/RRMS are associated with higher Hsp70 serum levels than progressive MS, serum Hsp70 levels might provide a marker for inflammatory processes.