Cytosolic calcium transients are a determinant of contraction-induced HSP72 transcription in single skeletal muscle fibers.

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Stary, Creed M; Hogan, Michael C

2016-05-15

The intrinsic activating factors that induce transcription of heat shock protein 72 (HSP72) in skeletal muscle following exercise remain unclear. We hypothesized that the cytosolic Ca(2+) transient that occurs with depolarization is a determinant. We utilized intact, single skeletal muscle fibers from Xenopus laevis to test the role of the cytosolic Ca(2+) transient and several other exercise-related factors (fatigue, hypoxia, AMP kinase, and cross-bridge cycling) on the activation of HSP72 transcription. HSP72 and HSP60 mRNA levels were assessed with real-time quantitative PCR; cytosolic Ca(2+) concentration ([Ca(2+)]cyt) was assessed with fura-2. Both fatiguing and nonfatiguing contractions resulted in a significant increase in HSP72 mRNA. As expected, peak [Ca(2+)]cyt remained tightly coupled with peak developed tension in contracting fibers. Pretreatment with N-benzyl-p-toluene sulfonamide (BTS) resulted in depressed peak developed tension with stimulation, while peak [Ca(2+)]cyt remained largely unchanged from control values. Despite excitation-contraction uncoupling, BTS-treated fibers displayed a significant increase in HSP72 mRNA. Treatment of fibers with hypoxia (Po2: skeletal muscle depolarization provides a sufficient activating stimulus for HSP72 transcription. Metabolic or mechanical factors associated with fatigue development and cross-bridge cycling likely play a more limited role. Copyright © 2016 the American Physiological Society.

Combined activity of post-exercise concentrations of NA and eHsp72 on human neutrophil function: role of cAMP.

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Giraldo, Esther; Hinchado, María D; Ortega, Eduardo

2013-09-01

Extracellular heat shock proteins of 72 kDa (eHsp72) and noradrenaline (NA) can act as "danger signals" during exercise-induced stress by activating neutrophil function (chemotaxis, phagocytosis, and fungicidal capacity). In addition, post-exercise concentrations of NA increase the expression and release of Hsp72 by human neutrophils, and adrenoreceptors and cAMP are involved in the stimulation of neutrophils by eHsp72. This suggests an interaction between the two molecules in the modulation of neutrophils during exercise-induced stress. Given this context, the aim of the present investigation was to study the combined activity of post-exercise circulating concentrations of NA and eHsp72 on the neutrophil phagocytic process, and to evaluate the role of cAMP as intracellular signal in these effects. Results showed an accumulative stimulation of chemotaxis induced by NA and eHsp72. However, while NA and eHsp72, separately, stimulate the phagocytosis and fungicidal activity of neutrophils, when they act together they do not modify these capacities of neutrophils. Similarly, post-exercise concentrations of NA and eHsp72 separately increased the intracellular level of cAMP, but NA and eHsp72 acting together did not modify the intracellular concentration of cAMP. These results confirm that cAMP can be involved in the autocrine/paracrine physiological regulation of phagocytosis and fungicidal capacity of human neutrophils mediated by NA and eHsp72 in the context of exercise-induced stress. Copyright © 2013 Wiley Periodicals, Inc.

Influence of exercise on NA- and Hsp72-induced release of IFNγ by the peritoneal suspension of macrophages and lymphocytes from genetically obese Zucker rats.

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Martín-Cordero, L; García, J J; Hinchado, M D; Bote, E; Ortega, E

2013-03-01

Regular physical exercise is recognized as a nonpharmacological therapeutic strategy in the treatment of metabolic syndrome, and has been proposed for improving obesity, diabetic status, insulin resistance, and immune response. The aim of the present study was to evaluate the effect of a regular exercise program (treadmill running, 5 days/week for 14 weeks at 35 cm/s for 35 min in the last month) on the release of the pro-inflammatory cytokine interferon gamma (IFNγ) by peritoneal cells (macrophages and lymphocytes) from obese Zucker rats (fa/fa) in response to noradrenaline (NA) and heat shock proteins of 72 kDa (Hsp72), and the possible adaptation due to training for a bout acute exercise (a single session of 25-35 min at 35 cm/s). In healthy (lean Fa/fa) and obese animals, peritoneal cells released greater concentrations of IFNγ in response to Hsp72 and lower concentrations in response to NA. The regular exercise training protocol, evaluated in the obese animals, produced a clear change in the regulation of the release of IFNγ. Peritoneal immune cells from trained animals released more IFNγ in response to NA, but there was a reduction in the release of IFNγ in response to Hsp72. In the obese animals, regular exercise caused a change in the inhibitory effect of NA (which now becomes stimulatory) and the stimulatory effect of Hsp72e (which now becomes inhibitory) in relation to the release of IFNγ. This reflects that Hsp72, induced by the prior release of NA following exercise-induced stress, plays a role in the homeostatic balance of release of IFNγ by peritoneal immune cells in obese animals during exercise.

Chronic probiotic supplementation with or without glutamine does not influence the eHsp72 response to a multi-day ultra-endurance exercise event.

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Marshall, Hannah; Chrismas, Bryna Catherine Rose; Suckling, Craig Anthony; Roberts, Justin D; Foster, Josh; Taylor, Lee

2017-08-01

Probiotic and glutamine supplementation increases tissue Hsp72, but their influence on extracellular Hsp72 (eHsp72) has not been investigated. The aim of this study was to investigate the effect of chronic probiotic supplementation, with or without glutamine, on eHsp72 concentration before and after an ultramarathon. Thirty-two participants were split into 3 independent groups, where they ingested probiotic capsules (PRO; n = 11), probiotic + glutamine powder (PGLn; n = 10), or no supplementation (CON; n = 11), over a 12-week period prior to commencement of the Marathon des Sables (MDS). eHsp72 concentration in the plasma was measured at baseline, 7 days pre-race, 6-8 h post-race, and 7 days post-race. The MDS increased eHsp72 concentrations by 124% (F [1,3] = 22.716, p 0.05). In conclusion, the MDS caused a substantial increase in eHsp72 concentration, indicating high levels of systemic stress. However, chronic PRO or PGLn supplementation did not affect eHsp72 compared with control pre- or post-MDS. Given the role of eHsp72 in immune activation, the commercially available supplements used in this study are unlikely to influence this cascade.

Up-regulation of Hsp72 and keratin16 mediates wound healing in streptozotocin diabetic rats

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Rasha R. Ahmed

2015-01-01

Full Text Available BACKGROUND: Impaired wound healing is a complication of diabetes and a serious problem in clinical practice. We previously found that whey protein (WP was able to regulate wound healing normally in streptozotocin (STZ-dia-betic models. This subsequent study was designed to assess the effect of WP on heat shock protein-72 (Hsp72 and keratin16 (Krt16 expression during wound healing in diabetic rats. METHODS: WP at a dosage of 100 mg/kg of body weight was orally administered daily to wounded normal and STZ-diabetic rats for 8 days. RESULTS: At day 4, the WP-treated diabetic wound was significantly reduced compared to that in the corresponding control. Diabetic wounded rats developed severe inflammatory infiltration and moderate capillary dilatation and regeneration. Treated rats had mild necrotic formation, moderate infiltration, moderate to severe capillary dilatation and regeneration, in addition to moderate epidermal formation. Hsp72 and Krt16 densities showed low and dense activity in diabetic wounded and diabetic wounded treated groups, respectively. At day 8, WP-treatment of diabetic wounded animals revealed great amelioration with complete recovery and closure of the wound. Reactivity of Hsp72 and Krt16 was reversed, showing dense and low, or medium and low, activity in the diabetic wounded and diabetic wounded treated groups, respectively. Hsp72 expression in the pancreas was found to show dense reactivity with WP-treated diabetic wound rats. CONCLUSION: This data provides evidence for the potential impact of WP in the up-regulation of Hsp72 and Krt16 in T1D, resulting in an improved wound healing process in diabetic models.

Effects of habitual exercise on the eHsp72-induced release of inflammatory cytokines by macrophages from obese Zucker rats.

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Garcia, J J; Martin-Cordero, L; Hinchado, M D; Bote, M E; Ortega, E

2013-06-01

Regular exercise is a good non-pharmacological treatment of metabolic syndrome in that it improves obesity, diabetes, and inflammation. The 72 kDa extracellular heat shock protein (eHsp72) is released during exercise, thus stimulating the inflammatory responses. The aim of the present work was to evaluate the effect of regular exercise on the eHsp72-induced release of IL-1β, IL-6, and TNFα by macrophages from genetically obese Zucker rats (fa/fa) (ObZ), using lean Zucker (LZ) rats (Fa/fa) to provide reference values. ObZ presented a higher plasma concentration of eHsp72 than LZ, and exercise increased that concentration. In response to eHsp72, the macrophages from ObZ released less IL-1β and TNFα, but more IL-6, than macrophages from LZ. While eHsp72 stimulated the release of IL-1β, TNFα, and IL-6 in the macrophages from healthy LZ (with respect to the constitutive release), it inhibited the release of IL-1β and IL-6 in macrophages from ObZ. The habitual exercise improved the release of inflammatory cytokines by macrophages from ObZ in response to eHsp72 (it increased IL-1β and TNFα, and decreased IL-6), tending to values closer to those determined in healthy LZ. A deregulated macrophage inflammatory and stress response induced by eHsp72 underlies MS, and this is improved by habitual exercise. © Georg Thieme Verlag KG Stuttgart · New York.

Recombinant heat shock protein 27 (HSP27/HSPB1) protects against cadmium-induced oxidative stress and toxicity in human cervical cancer cells.

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Alvarez-Olmedo, Daiana G; Biaggio, Veronica S; Koumbadinga, Geremy A; Gómez, Nidia N; Shi, Chunhua; Ciocca, Daniel R; Batulan, Zarah; Fanelli, Mariel A; O'Brien, Edward R

2017-05-01

Cadmium (Cd) is a carcinogen with several well-described toxicological effects in humans, but its molecular mechanisms are still not fully understood. Overexpression of heat shock protein 27 (HSP27/HSPB1)-a multifunctional protein chaperone-has been shown to protect cells from oxidative damage and apoptosis triggered by Cd exposure. The aims of this work were to investigate the potential use of extracellular recombinant HSP27 to prevent/counteract Cd-induced cellular toxicity and to evaluate if peroxynitrite was involved in the development of Cd-induced toxicity. Here, we report that the harmful effects of Cd correlated with changes in oxidative stress markers: upregulation of reactive oxygen species, reduction in nitric oxide (NO) bioavailability, increment in lipid peroxidation, peroxynitrite (PN), and protein nitration; intracellular HSP27 was reduced. Treatments with Cd (100 μM) for 24 h or with the peroxynitrite donor, SIN-1, decreased HSP27 levels (~50%), suggesting that PN formation is responsible for the reduction of HSP27. Pre-treatments of the cells either with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) (a pharmacological inhibitor of NO synthase) or with recombinant HSP27 (rHSP27) attenuated the disruption of the cellular metabolism induced by Cd, increasing in a 55 and 52%, respectively, the cell viability measured by CCK-8. Cd induced necrotic cell death pathways, although apoptosis was also activated; pre-treatment with L-NAME or rHSP27 mitigated cell death. Our findings show for the first time a direct relationship between Cd-induced toxicity and PN production and a role for rHSP27 as a potential therapeutic agent that may counteract Cd toxicity.

Increased radiosensitivity and radiothermosensitivity of human pancreatic MIA PaCa-2 and U251 glioblastoma cell lines treated with the novel Hsp90 inhibitor NVP-HSP990

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Milanović, Dušan; Firat, Elke; Grosu, Anca Ligia; Niedermann, Gabriele

2013-01-01

Heat shock Protein 90 (Hsp90) is a molecular chaperone that folds, stabilizes, and functionally regulates many cellular proteins involved in oncogenic signaling and in the regulation of radiosensitivity. It is upregulated in response to stress such a heat. Hyperthermia is a potent radiosensitizer, but induction of Hsp90 may potentially limit its efficacy. Our aim was to investigate whether the new Hsp90 inhibitor NVP-HSP990 increases radiosensitivity, thermosensitivity and radiothermosensitivity of human tumor cell lines. U251 glioblastoma and MIA PaCa-2 pancreatic carcinoma cells were used. To determine clonogenic survival, colony forming assays were performed. Cell viability and proliferation were assesed by Trypan blue staining. Cell cycle and apoptosis analyses were performed by flow cytometry. DAPI staining was used to detect mitotic catastrophe. NVP-HSP990 increased the thermosensitivity, radiosensitivity and radio-thermosensitivity of both cell lines in clonogenic assays. 72 hours after irradiation with 4 Gy, a significant reduction in cell number associated with considerable G2/M acumulation and mitotic catastrophe as well as cell death by apoptosis/necrosis was observed. Treatment with NVP-HSP990 strongly sensitized U251 and MIA PaCa-2 cells to hyperthermia and ionizing radiation or combination thereof through augmentation of G2/M arrest, mitotic catastrophe and associated apoptosis

Heat shock protein 72: release and biological significance during exercise.

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Whitham, Martin; Fortes, Matthew Benjamin

2008-01-01

The cumulative stressors of exercise manifest themselves at a cellular level by threatening the protein homeostasis of the cell. In these conditions, Heat Shock Proteins (HSP) are synthesised to chaperone mis-folded and denatured proteins. As such, the intracellular HSP response is thought to aid cell survival in the face of otherwise lethal cellular stress. Recently, the inducible isoform of the 70 Kda heat shock protein family, Hsp72 has been detected in the extracellular environment. Furthermore, the release of this protein into the circulation has been shown to occur in response to a range of exercise bouts. The present review summarises the current research on the exercise Hsp72 response, the possible mediators and mechanisms of extracellular (e)Hsp72 release, and the possible biological significance of this systemic response. In particular, the possible role of eHsp72 in the modulation of immunity during exercise is discussed.

Níveis distintos de Hsp72 no miocárdio de ratas em resposta aos exercícios voluntário e forçado Niveles distintos de Hsp72 en el miocardio de ratas en respuesta a los ejercicios voluntario y forzado Different levels of Hsp72 in female rat myocardium in response to voluntary exercise and forced exercise

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Stéphano Freitas Soares Melo

2009-11-01

Full Text Available FUNDAMENTO: O exercício físico promove estresse hemodinâmico. OBJETIVO: Testar se programas de treinamento com corridas voluntária e forçada induzem níveis distintos de expressão de Hsp72 no miocárdio de ratas Wistar. MÉTODOS: Ratas Wistar foram alocadas em três grupos (n = 6, cada: treinadas com corrida voluntária (TCV, treinadas com corrida forçada (TCF e grupo controle (C. Os animais do TCV tiveram livre acesso à roda de corrida voluntária, enquanto os do TCF foram submetidos à corrida forçada em esteira (18 m/min, 0% inclinação, 60 m/min, 5 dias/sem durante oito semanas. Fragmentos dos ventrículos esquerdo (VE e direito (VD foram coletados para análise dos níveis de Hsp72. RESULTADOS: As ratas do grupo TCV correram, em média, 4,87 km, e as do TCF, 4,88 km por semana. Os animais dos grupos TCV e TCF ganharam menos peso (p 0,05 entre os grupos TCV, TCF e C (4,54 ± 0,79 mg/g vs 4,94 ± 0,89 mg/g vs 4,34 ± 0,87 mg/g, respectivamente. Ratas treinadas com corrida forçada apresentaram níveis de Hsp72 maiores (p FUNDAMENTO: El ejercicio físico promueve estrés hemodinámico. OBJETIVO: Probar si programas de entrenamiento con carreras voluntaria y forzada inducen niveles distintos de expresión de Hsp72 en el miocardio de ratas hembra Wistar. MÉTODOS: Ratas hembra Wistar fueron distribuidas en tres grupos (n = 6, cada uno: entrenadas con carrera voluntaria (ECV, entrenadas con carrera forzada (ECF y grupo control (C. Los animales del ECV tuvieron libre acceso a la rueda de carrera voluntaria, mientras que los del ECF fueron sometidos a carrera forzada en cinta sin fin (18 m/min, 0% inclinación, 60 m/min, 5 días/sem durante ocho semanas. Fragmentos de los ventrículos izquierdo (VI y derecho (VD se recolectaron para análisis de los niveles de Hsp72. RESULTADOS: Las ratas del grupo ECV corrieron, en promedio 4,87 km, y las del ECF, 4,88 km por semana. Los animales de los grupos ECV y ECF ganaron menos peso (p0,05 entre

The Effect of One Session Continuous and Intermittent Aerobic Exercise on Blood Responses of HSP72 , Cortisol and Creatine Kinase

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M. Amani

2013-10-01

Full Text Available Introduction & Objective: Heat shock proteins help the cells’ ability to keep their structures against different stresses. The purpose of this study was to investigate the effect of one ses-sion continuous and intermittent aerobic exercise on blood responses of HSP72, cortisol and creatine kinase (CK. Materials & Methods: This study is semi-experimental in which 21 male student athletes were divided in continuous group (n=7, intermittent group (n=7 and control group (n=7. Exer-cise protocol of continuous group included 1 hour running with 80% maximum heart rate in-tensity and that of intermittent group was 3 stages of 20 minute running with the same inten-sity as of continuous group . Blood sampling of basal, pre exercise, immediately after exer-cise and 90 minutes after exercise were gathered and the amounts of HSP72, cortisol and CK, were measured by ELISA, RIA and Enzymatic methods respectively. The data was analyzed with one way ANOVA and repeated measure analysis of variance at P?0.05 significance level. Results: HSP72 levels in the continuous group and intermittent group despite an increase in the average did not show a statistically significant difference. Changes between the groups were significant in immediately after exercise and 90 minutes after exercise (P.values respectively 0.017 and 0.002. CK changes in continuous group were significant but cortisol changes in different stages hadn’t significant difference Conclusion: Exercise with its role associated with cortisol and CK will stimulate HSP72 and continuous exercise will make further increase in HSP72 and CK increasing leads to a greater HSP72 response. (Sci J Hamadan Univ Med Sci 2013; 20 (3:223-231

Hsp90 inhibitor 17-AAG sensitizes Bcl-2 inhibitor (-)-gossypol by suppressing ERK-mediated protective autophagy and Mcl-1 accumulation in hepatocellular carcinoma cells.

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Wang, Bin; Chen, Linfeng; Ni, Zhenhong; Dai, Xufang; Qin, Liyan; Wu, Yaran; Li, Xinzhe; Xu, Liang; Lian, Jiqin; He, Fengtian

2014-11-01

Natural BH3-memitic (-)-gossypol shows promising antitumor efficacy in several kinds of cancer. However, our previous studies have demonstrated that protective autophagy decreases the drug sensitivities of Bcl-2 inhibitors in hepatocellular carcinoma (HCC) cells. In the present study, we are the first to report that Hsp90 inhibitor 17-AAG enhanced (-)-gossypol-induced apoptosis via suppressing (-)-gossypol-triggered protective autophagy and Mcl-1 accumulation. The suppression effect of 17-AAG on autophagy was mediated by inhibiting ERK-mediated Bcl-2 phosphorylation while was not related to Beclin1 or LC3 protein instability. Meanwhile, 17-AAG downregulated (-)-gossypol-triggered Mcl-1 accumulation by suppressing Mcl-1(Thr163) phosphorylation and promoting protein degradation. Collectively, our study indicates that Hsp90 plays an important role in tumor maintenance and inhibition of Hsp90 may become a new strategy for sensitizing Bcl-2-targeted chemotherapies in HCC cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Hsp20 Protects against Oxygen-Glucose Deprivation/Reperfusion-Induced Golgi Fragmentation and Apoptosis through Fas/FasL Pathway

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Bingwu Zhong

2015-01-01

Full Text Available Cerebral ischemia-reperfusion injury plays an important role in the development of tissue injury after acute ischemic stroke. Finding effective neuroprotective agents has become a priority in the treatment of ischemic stroke. The Golgi apparatus (GA is a pivotal organelle and its protection is an attractive target in the treatment of cerebral ischemia-reperfusion injury. Protective effects of Hsp20, a potential cytoprotective agent due to its chaperone-like activity and involvement in regulation of many vital processes, on GA were assessed in an ischemia-reperfusion injury model. Mouse neuroblastoma Neuro2a (N2a cells were subjected to oxygen-glucose deprivation/reperfusion (OGDR insult. OGDR induces Golgi fragmentation, apoptosis, and p115 cleavage in N2a cells. However, transfection with Hsp20 significantly attenuates OGDR-induced Golgi fragmentation and apoptosis. Hsp20 interacts with Bax, decreases FasL and Bax expression, and inhibits caspases 3 and p115 cleavage in N2a cells exposed to OGDR. Our data demonstrate that increased Hsp20 expression protects against OGDR-induced Golgi fragmentation and apoptosis, likely through interaction with Bax and subsequent amelioration of the OGDR-induced elevation in p115 cleavage via the Fas/FasL signaling pathway. This neuroprotective potential of Hsp20 against OGDR insult and the underlying mechanism will pave the way for its potential clinical application for cerebral ischemia-reperfusion related disorders.

Mesothelioma Cells Escape Heat Stress by Upregulating Hsp40/Hsp70 Expression via Mitogen-Activated Protein Kinases

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Michael Roth

2009-01-01

Full Text Available Therapy with hyperthermal chemotherapy in pleural diffuse malignant mesothelioma had limited benefits for patients. Here we investigated the effect of heat stress on heat shock proteins (HSP, which rescue tumour cells from apoptosis. In human mesothelioma and mesothelial cells heat stress (39–42°C induced the phosphorylation of two mitogen activated kinases (MAPK Erk1/2 and p38, and increased Hsp40, and Hsp70 expression. Mesothelioma cells expressed more Hsp40 and were less sensitive to heat stress compared to mesothelial cells. Inhibition of Erk1/2 MAPK by PD98059 or by Erk1 siRNA down-regulated heat stress-induced Hsp40 and Hsp70 expression and reduced mesothelioma cell survival. Inhibition of p38MAPK by SB203580 or siRNA reduced Hsp40, but not Hsp70, expression and also increased mesothelioma cell death. Thus hyperthermia combined with suppression of p38 MAPK or Hsp40 may represent a novel approach to improve mesothelioma therapy.

Oxygen glucose deprivation post-conditioning protects cortical neurons against oxygen-glucose deprivation injury: role of HSP70 and inhibition of apoptosis.

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Zhao, Jian-hua; Meng, Xian-li; Zhang, Jian; Li, Yong-li; Li, Yue-juan; Fan, Zhe-ming

2014-02-01

In the present study, we examined the effect of oxygen glucose deprivation (OGD) post-conditioning (PostC) on neural cell apoptosis in OGD-PostC model and the protective effect on primary cortical neurons against OGD injury in vitro. Four-h OGD was induced by OGD by using a specialized and humidified chamber. To initiate OGD, culture medium was replaced with de-oxygenated and glucose-free extracellular solution-Locke's medium. After OGD treatment for 4 h, cells were then allowed to recover for 6 h or 20 h. Then lactate dehydrogenase (LDH) release assay, Western blotting and flow cytometry were used to detect cell death, protein levels and apoptotic cells, respectively. For the PostC treatment, three cycles of 15-min OGD, followed by 15 min normal cultivation, were applied immediately after injurious 4-h OGD. Cells were then allowed to recover for 6 h or 20 h, and cell death was assessed by LDH release assay. Apoptotic cells were flow cytometrically evaluated after 4-h OGD, followed by re-oxygenation for 20 h (O4/R20). In addition, Western blotting was used to examine the expression of heat-shock protein 70 (HSP70), Bcl-2 and Bax. The ratio of Bcl-2 expression was (0.44±0.08)% and (0.76±0.10)%, and that of Bax expression was (0.51±0.05)% and (0.39±0.04)%, and that of HSP70 was (0.42±0.031)% and (0.72±0.045)% respectively in OGD group and PostC group. After O4/R6, the rate of neuron death in PostC group and OGD groups was (28.96±3.03)% and (37.02±4.47)%, respectively. Therefore, the PostC treatment could up-regulate the expression of HSP70 and Bcl-2, but down-regulate Bax expression. As compared with OGD group, OGD-induced neuron death and apoptosis were significantly decreased in PostC group (Pneuron death. This neuro-protective effect is likely achieved by anti-apoptotic mechanisms and is associated with over-expression of HSP70.

Anaplasma phagocytophilum MSP4 and HSP70 Proteins Are Involved in Interactions with Host Cells during Pathogen Infection

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Marinela Contreras

2017-07-01

Full Text Available Anaplasma phagocytophilum transmembrane and surface proteins play a role during infection and multiplication in host neutrophils and tick vector cells. Recently, A. phagocytophilum Major surface protein 4 (MSP4 and Heat shock protein 70 (HSP70 were shown to be localized on the bacterial membrane, with a possible role during pathogen infection in ticks. In this study, we hypothesized that A. phagocytophilum MSP4 and HSP70 have similar functions in tick-pathogen and host-pathogen interactions. To address this hypothesis, herein we characterized the role of these bacterial proteins in interaction and infection of vertebrate host cells. The results showed that A. phagocytophilum MSP4 and HSP70 are involved in host-pathogen interactions, with a role for HSP70 during pathogen infection. The analysis of the potential protective capacity of MSP4 and MSP4-HSP70 antigens in immunized sheep showed that MSP4-HSP70 was only partially protective against pathogen infection. This limited protection may be associated with several factors, including the recognition of non-protective epitopes by IgG in immunized lambs. Nevertheless, these antigens may be combined with other candidate protective antigens for the development of vaccines for the control of human and animal granulocytic anaplasmosis. Focusing on the characterization of host protective immune mechanisms and protein-protein interactions at the host-pathogen interface may lead to the discovery and design of new effective protective antigens.

Heat shock proteins (Hsp 70) response is not systematic to cell stress

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Hassen, Wafa; Ayed-Boussema, Imen; Bouslimi, Amel; Bacha, Hassen

2007-01-01

Ochratoxin A (OTA) is a mycotoxin routinely detected in improperly stored animal and human food supplies as well as in human sera worldwide. OTA has multiple toxic effects; however, the most prominent is nephrotoxicity. Thus, OTA is involved in the pathogenesis of human nephropathy in Balkan areas. In this study, we address the question of the appropriate functioning of the basal cellular defense mechanisms, after exposure to OTA, which, up to now, has not been investigated satisfactorily. In this context, we have monitored the effect of OTA on (i) the inhibition of cell viability, (ii) the oxidative damage using the GSH depletion, (iii) the inhibition of protein synthesis through the incorporation of [ 3 H] Leucine and (iv) the induction of Hsp 70 gene expression as a parameter of cytotoxicity, oxidative damage and particularly as a protective and adaptative response. This study was conducted using the Human Hep G2 hepatocytes and monkey kidney Vero cells under exposure conditions ranging from non-cytotoxic to sub-lethal. Our results clearly showed that OTA inhibits cell proliferation, strongly reduces protein synthesis and induces the decrease of GSH in concentration-dependent manner in both Hep G2 and Vero cells. However, although cytotoxicity and oxidative damage (main inducers of Hsp expression) occur, no change was observed in Hsp 70 level under the multiple tested conditions. Inhibition of protein synthesis could not explain the absence of Hsp 70 response since concentrations, which did not influence protein synthesis, also failed to display the expected Hsp 70 response. Our data are consistent with recently published reports where considerable differences were noticed in the ability of relevant toxicants to induce Hsp. These results raised doubt about the universal character of Hsp induction which seems to be more complex than originally envisioned. It could be concluded that Hsp 70 induction is not systematic to cell stress

A novel Hsp70 inhibitor prevents cell intoxication with the actin ADP-ribosylating Clostridium perfringens iota toxin

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Ernst, Katharina; Liebscher, Markus; Mathea, Sebastian; Granzhan, Anton; Schmid, Johannes; Popoff, Michel R.; Ihmels, Heiko; Barth, Holger; Schiene-Fischer, Cordelia

2016-01-01

Hsp70 family proteins are folding helper proteins involved in a wide variety of cellular pathways. Members of this family interact with key factors in signal transduction, transcription, cell-cycle control, and stress response. Here, we developed the first Hsp70 low molecular weight inhibitor specifically targeting the peptide binding site of human Hsp70. After demonstrating that the inhibitor modulates the Hsp70 function in the cell, we used the inhibitor to show for the first time that the stress-inducible chaperone Hsp70 functions as molecular component for entry of a bacterial protein toxin into mammalian cells. Pharmacological inhibition of Hsp70 protected cells from intoxication with the binary actin ADP-ribosylating iota toxin from Clostridium perfringens, the prototype of a family of enterotoxins from pathogenic Clostridia and inhibited translocation of its enzyme component across cell membranes into the cytosol. This finding offers a starting point for novel therapeutic strategies against certain bacterial toxins. PMID:26839186

NK cells of the oldest seniors represent constant and resistant to stimulation high expression of cellular protective proteins SIRT1 and HSP70.

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Kaszubowska, Lucyna; Foerster, Jerzy; Kaczor, Jan Jacek; Schetz, Daria; Ślebioda, Tomasz Jerzy; Kmieć, Zbigniew

2018-01-01

Natural killer cells (NK cells) are cytotoxic lymphocytes of innate immunity that reveal some immunoregulatory properties, however, their role in the process of ageing is not completely understood. The study aimed to analyze the expression of proteins involved in cellular stress response: sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in human NK cells with reference to the process of ageing. Non-stimulated and stimulated with IL-2, LPS or PMA with ionomycin cells originated from peripheral blood samples of: seniors aged over 85 ('the oldest'; n  = 25; 88.5 ± 0.5 years, mean ± SEM), seniors aged under 85 ('the old'; n  = 30; 75.6 ± 0.9 years) and the young ( n  = 31; 20.9 ± 0.3 years). The relationships between the levels of expression of cellular protective proteins in the studied population were also analyzed. The concentrations of carbonyl groups and 8-isoprostanes, markers of oxidative stress, in both stimulated and non-stimulated cultured NK cells were measured to assess the level of the oxidative stress in the cells. The oldest seniors varied from the other age groups by significantly higher expression of SIRT1 and HSP70 both in non-stimulated and stimulated NK cells. These cells also appeared to be resistant to further stimulations with IL-2, LPS or PMA with ionomycin. Highly positive correlations between SIRT1 and intracellular HSP70 in both stimulated and non-stimulated NK cells were observed. SOD2 presented low expression in non-stimulated cells, whereas its sensitivity to stimulation increased with age of donors. High positive correlations between SOD2 and surface HSP70 were observed. We found that the markers of oxidative stress in NK cells did not change with ageing. The oldest seniors revealed well developed adaptive stress response in NK cells with increased, constant levels of SIRT1 and intracellular HSP70. They presented also very high positive correlations between

Recombinant TgHSP70 Immunization Protects against Toxoplasma gondii Brain Cyst Formation by Enhancing Inducible Nitric Oxide Expression

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Neide M. Silva

2017-04-01

Full Text Available Toxoplasma gondii is known to cause congenital infection in humans and animals and severe disease in immunocompromised individuals; consequently development of vaccines against the parasite is highly necessary. Under stress conditions, T. gondii expresses the highly immunogenic heat shock protein 70 (TgHSP70. Here, we assessed the protective efficacy of rTgHSP70 immunization combined with Alum in oral ME-49 T. gondii infection and the mechanisms involved on it. It was observed that immunized mice with rTgHSP70 or rTgHSP70 adsorbed in Alum presented a significantly reduced number of cysts in the brain that was associated with increased iNOS+ cell numbers in the organ, irrespective the use of the adjuvant. Indeed, ex vivo experiments showed that peritoneal macrophages pre-stimulated with rTgHSP70 presented increased NO production and enhanced parasite killing, and the protein was able to directly stimulate B cells toward antibody producing profile. In addition, rTgHSP70 immunization leads to high specific antibody titters systemically and a mixed IgG1/IgG2a response, with predominance of IgG1 production. Nonetheless, it was observed that the pretreatment of the parasite with rTgHSP70 immune sera was not able to control T. gondii internalization and replication by NIH fibroblast neither peritoneal murine macrophages, nor anti-rTgHSP70 antibodies were able to kill T. gondii by complement-mediated lysis, suggesting that these mechanisms are not crucial to resistance. Interestingly, when in combination with Alum, rTgHSP70 immunization was able to reduce inflammation in the brain of infected mice and in parallel anti-rTgHSP70 immune complexes in the serum. In conclusion, immunization with rTgHSP70 induces massive amounts of iNOS expression and reduced brain parasitism, suggesting that iNOS expression and consequently NO production in the brain is a protective mechanism induced by TgHSP70 immunization, therefore rTgHSP70 can be a good candidate for

The expression of HSP27 is associated with poor clinical outcome in intrahepatic cholangiocarcinoma

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Romani, Antonello A; Crafa, Pellegrino; Desenzani, Silvia; Graiani, Gallia; Lagrasta, Costanza; Sianesi, Mario; Soliani, Paolo; Borghetti, Angelo F

2007-01-01

The heat shock proteins (HSPs) 27-kDa (HSP27) and 72-kDa (HSP72), are ubiquitous chaperone molecules inducible in cells exposed to different stress conditions. Increased level of HSPs are reported in several human cancers, and found to be associated with the resistance to some anticancer treatments and poor prognosis. However, there is no study of the relationship between HSPs expression and patient's prognosis in intrahepatic cholangiocarcinoma (IHCCA). In this exploratory retrospective study, we investigated the expressions of HSP27 and HSP72 as potential prognostic factors in IHCCA. Thirty-one paraffin-embedded samples were analyzed by immunohistochemical methods using HSP27 and HSP72 monoclonal antibodies. Proliferation rate was assessed in the same specimens by using monoclonal antibody against phosphorylated histone H3 (pHH3). Fisher's exact test was used to assess the hypothesis of independence between categorical variables in 2 × 2 tables. The ANOVA procedure was used to evaluate the association between ordinal and categorical variables. Estimates of the survival probability were calculated using the Kaplan-Meier method, and the log rank test was employed to test the null hypothesis of equality in overall survival among groups. The hazard ratio associated with HSP27 and HSP72 expression was estimated by Cox hazard-proportional regression. The expression of HSP27 was related to mitotic index, tumor greatest dimension, capsular and vascular invasion while the expression of HSP72 was only related to the presence of necrosis and the lymphoid infiltration. Kaplan-Maier analysis suggested that the expression of HSP27 significantly worsened the patients' median overall survival (11 ± 3.18 vs 55 ± 4.1 months, P-value = 0.0003). Moreover HSP27-positive patients exhibited the worst mean survival (7.0 ± 3.2 months) in the absence of concomitant HSP72 expression. The expression of HSP27, likely increasing cell proliferation, tumor mass, vascular and

Cell type-specific variations in the induction of hsp70 in human leukocytes by feverlike whole body hyperthermia.

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Oehler, R; Pusch, E; Zellner, M; Dungel, P; Hergovics, N; Homoncik, M; Eliasen, M M; Brabec, M; Roth, E

2001-10-01

Fever has been associated with shortened duration and improved survival in infectious disease. The mechanism of this beneficial response is still poorly understood. The heat-inducible 70-kDa heat shock protein (Hsp70) has been associated with protection of leukocytes against the cytotoxicity of inflammatory mediators and with improved survival of severe infections. This study characterizes the induction of Hsp70 by feverlike temperatures in human leukocytes in vitro and in vivo. Using flow cytometry, Hsp70 expression was determined in whole blood samples. This approach eliminated cell isolation procedures that would greatly affect the results. Heat treatment of whole blood in vitro for 2 hours at different temperatures revealed that Hsp70 expression depends on temperature and cell type; up to 41 degrees C, Hsp70 increased only slightly in lymphocytes and polymorphonuclear leukocytes. However, in monocytes a strong induction was already seen at 39 degrees C, and Hsp70 levels at 41 degrees C were 10-fold higher than in the 37 degrees C control. To be as close as possible to the physiological situation during fever, we immersed healthy volunteers in a hot water bath, inducing whole body hyperthermia (39 degrees C), and measured leukocyte Hsp70 expression. Hsp70 was induced in all leukocytes with comparable but less pronounced cell type-specific variations as observed in vitro. Thus, a systemic increase of body temperature as triggered by fever stimulates Hsp70 expression in peripheral leukocytes, especially in monocytes. This fever-induced Hsp70 expression may protect monocytes when confronted with cytotoxic inflammatory mediators, thereby improving the course of the disease.

Heat shock protein (Hsp) 40 mutants inhibit Hsp70 in mammalian cells

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Michels, AA; Kanon, B; Bensaude, O; Kampinga, HH

1999-01-01

Heat shock protein (Hsp) 70 and Hsp40 expressed in mammalian cells had been previously shown to cooperate in accelerating the reactivation of heat-denatured firefly luciferase (Michels, A. A., Kanon, B., Konings, A. W. T., Ohtsuka, K,, Bensaude, O., and Kampinga, H. H. (1997) J. Biol. Chem. 272,

Role of membrane Hsp70 in radiation sensitivity of tumor cells

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Murakami, Naoya; Kühnel, Annett; Schmid, Thomas E.; Ilicic, Katarina; Stangl, Stefan; Braun, Isabella S.; Gehrmann, Mathias; Molls, Michael; Itami, Jun; Multhoff, Gabriele

2015-01-01

The major stress-inducible heat shock protein 70 (Hsp70) is frequently overexpressed in the cytosol and integrated in the plasma membrane of tumor cells via lipid anchorage. Following stress such as non-lethal irradiation Hsp70 synthesis is up-regulated. Intracellular located Hsp70 is known to exert cytoprotective properties, however, less is known about membrane (m)Hsp70. Herein, we investigate the role of mHsp70 in the sensitivity towards irradiation in tumor sublines that differ in their cytosolic and/or mHsp70 levels. The isogenic human colon carcinoma sublines CX + with stable high and CX − with stable low expression of mHsp70 were generated by fluorescence activated cell sorting, the mouse mammary carcinoma sublines 4 T1 (4 T1 ctrl) and Hsp70 knock-down (4 T1 Hsp70 KD) were produced using the CRISPR/Cas9 system, and the Hsp70 down-regulation in human lung carcinoma sublines H1339 ctrl/H1339 HSF-1 KD and EPLC-272H ctrl/EPLC-272H HSF-1 KD was achieved by small interfering (si)RNA against Heat shock factor 1 (HSF-1). Cytosolic and mHsp70 was quantified by Western blot analysis/ELISA and flow cytometry; double strand breaks (DSBs) and apoptosis were measured by flow cytometry using antibodies against γH2AX and real-time PCR (RT-PCR) using primers and antibodies directed against apoptosis related genes; and radiation sensitivity was determined using clonogenic cell surviving assays. CX + /CX − tumor cells exhibited similar cytosolic but differed significantly in their mHsp70 levels, 4 T1 ctrl/4 T1 Hsp70 KD cells showed significant differences in their cytosolic and mHsp70 levels and H1339 ctrl/H1339 HSF-1 KD and EPLC-272H ctrl/EPLC-272H HSF-1 KD lung carcinoma cell sublines had similar mHsp70 but significantly different cytosolic Hsp70 levels. γH2AX was significantly up-regulated in irradiated CX − and 4 T1 Hsp70 KD with low basal mHsp70 levels, but not in their mHsp70 high expressing counterparts, irrespectively of their cytosolic Hsp70 content. After

The Hsp72 and Hsp90α mRNA Responses to Hot Downhill Running Are Reduced Following a Prior Bout of Hot Downhill Running, and Occur Concurrently within Leukocytes and the Vastus Lateralis

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James A. Tuttle

2017-07-01

Full Text Available The leukocyte heat shock response (HSR is used to determine individual's thermotolerance. The HSR and thermotolerance are enhanced following interventions such as preconditioning and/or acclimation/acclimatization. However, it is unclear whether the leukocyte HSR is an appropriate surrogate for the HSR in other tissues implicated within the pathophysiology of exertional heat illnesses (e.g., skeletal muscle, and whether an acute preconditioning strategy (e.g., downhill running can improve subsequent thermotolerance. Physically active, non-heat acclimated participants were split into two groups to investigate the benefits of hot downhill running as preconditioning strategy. A hot preconditioning group (HPC; n = 6 completed two trials (HPC1HOTDOWN and HPC2HOTDOWN of 30 min running at lactate threshold (LT on −10% gradient in 30°C and 50% relative humidity (RH separated by 7 d. A temperate preconditioning group (TPC; n = 5 completed 30 min running at LT on a −1% gradient in 20°C and 50% (TPC1TEMPFLAT and 7 d later completed 30 min running at LT on −10% gradient in 30°C and 50% RH (TPC2HOTDOWN. Venous blood samples and muscle biopsies (vastus lateralis; VL were obtained before, immediately after, 3, 24, and 48 h after each trial. Leukocyte and VL Hsp72, Hsp90α, and Grp78 mRNA relative expression was determined via RT-QPCR. Attenuated leukocyte and VL Hsp72 (2.8 to 1.8 fold and 5.9 to 2.4 fold; p < 0.05 and Hsp90α mRNA (2.9 to 2.4 fold and 5.2 to 2.4 fold; p < 0.05 responses accompanied reductions (p < 0.05 in physiological strain [exercising rectal temperature (−0.3°C and perceived muscle soreness (~ −14%] during HPC2HOTDOWN compared to HPC1HOTDOWN (i.e., a preconditioning effect. Both VL and leukocyte Hsp72 and Hsp90α mRNA increased (p < 0.05 simultaneously following downhill runs and demonstrated a strong relationship (p < 0.01 of similar magnitudes with one another. Hot downhill running is an effective preconditioning strategy

Imbalance of Hsp70 family variants fosters tau accumulation.

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Jinwal, Umesh K; Akoury, Elias; Abisambra, Jose F; O'Leary, John C; Thompson, Andrea D; Blair, Laura J; Jin, Ying; Bacon, Justin; Nordhues, Bryce A; Cockman, Matthew; Zhang, Juan; Li, Pengfei; Zhang, Bo; Borysov, Sergiy; Uversky, Vladimir N; Biernat, Jacek; Mandelkow, Eckhard; Gestwicki, Jason E; Zweckstetter, Markus; Dickey, Chad A

2013-04-01

Dysfunctional tau accumulation is a major contributing factor in tauopathies, and the heat-shock protein 70 (Hsp70) seems to play an important role in this accumulation. Several reports suggest that Hsp70 proteins can cause tau degradation to be accelerated or slowed, but how these opposing activities are controlled is unclear. Here we demonstrate that highly homologous variants in the Hsp70 family can have opposing effects on tau clearance kinetics. When overexpressed in a tetracycline (Tet)-based protein chase model, constitutive heat shock cognate 70 (Hsc70) and inducible Hsp72 slowed or accelerated tau clearance, respectively. Tau synergized with Hsc70, but not Hsp72, to promote microtubule assembly at nearly twice the rate of either Hsp70 homologue in reconstituted, ATP-regenerating Xenopus extracts supplemented with rhodamine-labeled tubulin and human recombinant Hsp72 and Hsc70. Nuclear magnetic resonance spectroscopy with human recombinant protein revealed that Hsp72 had greater affinity for tau than Hsc70 (I/I0 ratio difference of 0.3), but Hsc70 was 30 times more abundant than Hsp72 in human and mouse brain tissue. This indicates that the predominant Hsp70 variant in the brain is Hsc70, suggesting that the brain environment primarily supports slower tau clearance. Despite its capacity to clear tau, Hsp72 was not induced in the Alzheimer's disease brain, suggesting a mechanism for age-associated onset of the disease. Through the use of chimeras that blended the domains of Hsp72 and Hsc70, we determined that the reason for these differences between Hsc70 and Hsp72 with regard to tau clearance kinetics lies within their C-terminal domains, which are essential for their interactions with substrates and cochaperones. Hsp72 but not Hsc70 in the presence of tau was able to recruit the cochaperone ubiquitin ligase CHIP, which is known to facilitate the ubiquitination of tau, describing a possible mechanism of how the C-termini of these homologous Hsp70 variants

Detection of irradiation-induced, membrane heat shock protein 70 (Hsp70) in mouse tumors using Hsp70 Fab fragment

International Nuclear Information System (INIS)

Stangl, Stefan; Themelis, George; Friedrich, Lars; Ntziachristos, Vasilis; Sarantopoulos, Athanasios; Molls, Michael; Skerra, Arne; Multhoff, Gabriele

2011-01-01

Background and purpose: The major stress-inducible heat shock protein 70 (Hsp70) is frequently overexpressed in highly aggressive tumors, and elevated intracellular Hsp70 levels mediate protection against apoptosis. Following therapeutic intervention, such as ionizing irradiation, translocation of cytosolic Hsp70 to the plasma membrane is selectively increased in tumor cells and therefore, membrane Hsp70 might serve as a therapy-inducible, tumor-specific target structure. Materials and methods: Based on the IgG1 mouse monoclonal antibody (mAb) cmHsp70.1, we produced the Hsp70-specific recombinant Fab fragment (Hsp70 Fab), as an imaging tool for the detection of membrane Hsp70 positive tumor cells in vitro and in vivo. Results: The binding characteristics of Hsp70 Fab towards mouse colon (CT26) and pancreatic (1048) carcinoma cells at 4 deg. C were comparable to that of cmHsp70.1 mAb, as determined by flow cytometry. Following a temperature shift to 37 deg. C, Hsp70 Fab rapidly translocates into subcellular vesicles of mouse tumor cells. Furthermore, in tumor-bearing mice Cy5.5-conjugated Hsp70 Fab, but not unrelated IN-1 control Fab fragment (IN-1 ctrl Fab), gradually accumulates in CT26 tumors between 12 and 55 h after i.v. injection. Conclusions: In summary, the Hsp70 Fab provides an innovative, low immunogenic tool for imaging of membrane Hsp70 positive tumors, in vivo.

Plant Hsp90 Proteins Interact with B-Cells and Stimulate Their Proliferation

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Corigliano, Mariana G.; Maglioco, Andrea; Laguía Becher, Melina; Goldman, Alejandra; Martín, Valentina; Angel, Sergio O.; Clemente, Marina

2011-01-01

Background The molecular chaperone heat shock protein 90 (Hsp90) plays an important role in folding stabilization and activation of client proteins. Besides, Hsp90 of mammals and mammalian pathogens displays immunostimulatory properties. Here, we investigated the role of plant-derived Hsp90s as B-cell mitogens by measuring their proliferative responses in vitro. Methodology Plant cytosolic Hsp90 isoforms from Arabidopsis thaliana (AtHsp81.2) and Nicotiana benthamiana (NbHsp90.3) were expressed in E. coli. Over-expression of recombinant plant Hsp90s (rpHsp90s) was confirmed by SDS-PAGE and western blot using and anti-AtHsp81.2 polyclonal anti-body. Both recombinant proteins were purified by Ni-NTA affinity chromatography and their identity confirmed by MALDI-TOF-TOF. Recombinant AtHsp81.2 and NbHsp90.3 proteins induced prominent proliferative responses in spleen cells form BALB/c mice. Polymyxin-B, a potent inhibitor of lipopolysaccharide (LPS), did not eliminate the rpHsp90-induced proliferation. In addition, in vitro incubation of spleen cells with rpHsp90 led to the expansion of CD19-bearing populations, suggesting a direct effect of these proteins on B lymphocytes. This effect was confirmed by immunofluorescence analysis, where a direct binding of rpHsp90 to B- but not to T-cells was observed in cells from BALB/c and C3H/HeN mice. Finally, we examined the involvement of Toll Like Receptor 4 (TLR4) molecules in the rpHsp90s induction of B-cell proliferation. Spleen cells from C3H/HeJ mice, which carry a point mutation in the cytoplasmic region of TLR4, responded poorly to prAtHsp90. However, the interaction between rpHsp90 and B-cells from C3H/HeJ mice was not altered, suggesting that the mutation on TLR4 would be affecting the signal cascade but not the rpHsp90-TLR4 receptor interaction. Conclusions Our results show for the first time that spleen cell proliferation can be stimulated by a non-pathogen-derived Hsp90. Furthermore, our data provide a new example of

Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells.

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Ernst, Katharina; Schmid, Johannes; Beck, Matthias; Hägele, Marlen; Hohwieler, Meike; Hauff, Patricia; Ückert, Anna Katharina; Anastasia, Anna; Fauler, Michael; Jank, Thomas; Aktories, Klaus; Popoff, Michel R; Schiene-Fischer, Cordelia; Kleger, Alexander; Müller, Martin; Frick, Manfred; Barth, Holger

2017-06-02

Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.

PREFERENTIAL SECRETION OF INDUCIBLE HSP70 BY VITILIGO MELANOCYTES UNDER STRESS

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Mosenson, Jeffrey A.; Flood, Kelsey; Klarquist, Jared; Eby, Jonathan M.; Koshoffer, Amy; Boissy, Raymond E.; Overbeck, Andreas; C.Tung, Rebecca; Poole, I. Caroline Le

2014-01-01

SUMMARY Inducible HSP70 (HSP70i) chaperones peptides from stressed cells, protecting them from apoptosis. Upon extracellular release, HSP70i serves an adjuvant function, enhancing immune responses to bound peptides. We questioned whether HSP70i differentially protects control and vitiligo melanocytes from stress and subsequent immune responses. We compared expression of HSP70i in skin samples, evaluated the viability of primary vitiligo and control melanocytes exposed to bleaching phenols, and measured secreted HSP70i. We determined whether HSP70i traffics to melanosomes to contact immunogenic proteins by cell fractionation, western blotting, electron microscopy and confocal microscopy. Viability of vitiligo and control melanocytes was equally affected under stress. However, vitiligo melanocytes secreted increased amounts of HSP70i in response to MBEH, corroborating with aberrant HSP70i expression in patient skin. Intracellular HSP70i colocalized with melanosomes, and more so in response to MBEH in vitiligo melanocytes. Thus whereas either agent is cytotoxic to melanocytes, MBEH preferentially induces immune responses to melanocytes. PMID:24354861

Essential role of the NH2-terminal WD/EPF motif in the phosphorylation-activated protective function of mammalian Hsp27.

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Thériault, Jimmy R; Lambert, Herman; Chávez-Zobel, Aura T; Charest, Gabriel; Lavigne, Pierre; Landry, Jacques

2004-05-28

Hsp27 is expressed at high levels after mild heat shock and contributes to making cells extremely resistant to subsequent treatments. The activity of the protein is regulated at the transcriptional level, but also by phosphorylation, which occurs rapidly during stress and is responsible for causing the dissociation of large 700-kDa Hsp27 oligomers into dimers. We investigated the mechanism by which phosphorylation and oligomerization modulate the protective activity of Chinese hamster Hsp27. In contrast to oligomer dissociation, which only required Ser90 phosphorylation, activation of Hsp27 thermoprotective activity required the phosphorylation of both Ser90 and Ser15. Replacement of Ser90 by Ala90, which prevented the dissociation of the oligomer upon stress, did cause a severe defect in the protective activity. Dissociation was, however, not a sufficient condition to activate the protein because replacement of Ser15 by Ala15, which caused little effect in the oligomeric organization of the protein, also yielded an inactive protein. Analyzes of mutants with short deletions in the NH2 terminus identified the Hsp27 WD/EPF or PF-rich domain as essential for protection, maintenance of the oligomeric structure, and in vitro chaperone activity of the protein. In light of a three-dimensional model of Hsp27 based on the crystallographic structure of wheat Hsp16.9, we propose that the conserved WD/EPF motif of mammalian Hsp27 mediates important intramolecular interactions with hydrophic surfaces of the alpha-crystallin domain of the protein. These interactions are destabilized by Ser90 phosphorylation, making the motif free to interact with heterologous molecular targets upon the additional phosphorylation of the nearby Ser15.

Traditional Herbal Medicine, Rikkunshito, Induces HSP60 and Enhances Cytoprotection of Small Intestinal Mucosal Cells as a Nontoxic Chaperone Inducer

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Kumiko Tamaki

2012-01-01

Full Text Available Increasing incidence of small intestinal ulcers associated with nonsteroidal anti-inflammatory drugs (NSAIDs has become a topic with recent advances of endoscopic technology. However, the pathogenesis and therapy are not fully understood. The aim of this study is to examine the effect of Rikkunshito (TJ-43, a traditional herbal medicine, on expression of HSP60 and cytoprotective ability in small intestinal cell line (IEC-6. Effect of TJ-43 on HSP60 expression in IEC-6 cells was evaluated by immunoblot analysis. The effect of TJ-43 on cytoprotective abilities of IEC-6 cells against hydrogen peroxide or indomethacin was studied by MTT assay, LDH-release assay, caspase-8 activity, and TUNEL. HSP60 was significantly induced by TJ-43. Cell necrosis and apoptosis were significantly suppressed in IEC-6 cells pretreated by TJ-43 with overexpression of HSP60. Our results suggested that HSP60 induced by TJ-43 might play an important role in protecting small intestinal epithelial cells from apoptosis and necrosis in vitro.

A Novel Therapeutic Strategy for the Treatment of Glioma, Combining Chemical and Molecular Targeting of Hsp90α

International Nuclear Information System (INIS)

Mehta, Adi; Shervington, Leroy; Munje, Chinmay; Shervington, Amal

2011-01-01

Hsp90α's vital role in tumour survival and progression, together with its highly inducible expression profile in gliomas and its absence in normal tissue and cell lines validates it as a therapeutic target for glioma. Hsp90α was downregulated using the post-transcriptional RNAi strategy (sihsp90α) and a post-translational inhibitor, the benzoquinone antibiotic 17-AAG. Glioblastoma U87-MG and normal human astrocyte SVGp12 were treated with sihsp90α, 17-AAG and concurrent sihsp90α/17-AAG (combined treatment). Both Hsp90α gene silencing and the protein inhibitor approaches resulted in a dramatic reduction in cell viability. Results showed that sihsp90α, 17-AAG and a combination of sihsp90α/17-AAG, reduced cell viability by 27%, 75% and 88% (p < 0.001), respectively, after 72 h. hsp90α mRNA copy numbers were downregulated by 65%, 90% and 99% after 72 h treatment with sihsp90α, 17-AAG and sihsp90α/17-AAG, respectively. The relationship between Hsp90α protein expression and its client Akt kinase activity levels were monitored following treatment with sihsp90α, 17-AAG and sihsp90α/17-AAG. Akt kinase activity was downregulated as a direct consequence of Hsp90α inhibition. Both Hsp90α and Akt kinase levels were significantly downregulated after 72 h. Although, 17-AAG when used as a single agent reduces the Hsp90α protein and the Akt kinase levels, the efficacy demonstrated by combinatorial treatment was found to be far more effective. Combination treatment reduced the Hsp90α protein and Akt kinase levels to 4.3% and 43%, respectively, after 72 h. hsp90α mRNA expression detected in SVGp12 was negligible compared to U87-MG, also, the combination treatment did not compromise the normal cell viability. Taking into account the role of Hsp90α in tumour progression and the involvement of Akt kinase in cell signalling and the anti-apoptotic pathways in tumours, this double targets treatment infers a novel therapeutic strategy

Effects of HSP27 chaperone on THP-1 tumor cell apoptosis.

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Kaigorodova, E V; Ryazantseva, N V; Novitskii, V V; Maroshkina, A N; Belkina, M V

2012-11-01

The role of Hsp27 (heat shock protein 27) chaperone in regulation of THP-1 tumor cell apoptosis was studied. Realization of tumor cell apoptosis under conditions of in vitro culturing with Hsp27 specific inhibitor (KRIBB3) was evaluated by fluorescent microscopy with FITC-labeled annexin V and propidium iodide. Measurements of Bcl-2 family proteins (Bcl-2, Bax, Bad) in tumor cells incubated with Hsp27 inhibitor were carried out by Western blotting. Chaperone Hsp27 acted as apoptosis inhibitor in THP-1 tumor cells modulating the proportion of antiapoptotic (Bcl-2) and proapoptotic (Bax and Bad) proteins.

Altered Cross-linking of HSP27 by Zerumbone as a Novel Strategy for Overcoming HSP27- mediated Resistance

International Nuclear Information System (INIS)

Choi, Seo Hyun; Lee, Yoon Jin; Lee, Hae June; Lee, Yun Sil; Kim, Joon; Seo, Woo Duck; Nam, Joo Won; Lee, Yoo Jin; Seo, Eun Kyung

2010-01-01

HSPs have diverse roles in the regulation of signal transduction and in numerous aspects of cell growth and death. Indeed, HSP90, HSP70, and HSP27 have each been implicated in promoting cancer. Most HSP27 exists as large oligomeric complexes ranging from 100- 800 kDa, which are probably stabilized by complex interactions between dimeric building blocks. The functional properties of HSP27 are dependent on the quaternary structure of the protein. For example, HSP27 acts as a chaperone and binds to cytochrome c or Daxx as a dimer. Therefore, the oligomerization pattern of HPS27 is believed to have HSP27-mediated protective functions. In this study, zerumbone (ZER), the cytotoxic component isolated from Zingiber zerumbet Smith, induced cross-linking of HSP27 protein by its insertion between the disulfide bond of HSP27, and ZERmediated altered cross-linking of HSP27 modified normal HSP27 dimerization, which resulted in a sensitizing effect to tumors after treatment with radiation. Therefore, altered cross-linking by ZER may be a novel strategy for inhibition of HSP27-mediated resistance

Targeting HSP70 and GRP78 in canine osteosarcoma cells in combination with doxorubicin chemotherapy.

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Asling, Jonathan; Morrison, Jodi; Mutsaers, Anthony J

2016-11-01

Heat shock proteins (HSPs) are molecular chaperones subdivided into several families based on their molecular weight. Due to their cytoprotective roles, these proteins may help protect cancer cells against chemotherapy-induced cell death. Investigation into the biologic activity of HSPs in a variety of cancers including primary bone tumors, such as osteosarcoma (OSA), is of great interest. Both human and canine OSA tumor samples have aberrant production of HSP70. This study assessed the response of canine OSA cells to inhibition of HSP70 and GRP78 by the ATP-mimetic VER-155008 and whether this treatment strategy could sensitize cells to doxorubicin chemotherapy. Single-agent VER-155008 treatment decreased cellular viability and clonogenic survival and increased apoptosis in canine OSA cell lines. However, combination schedules with doxorubicin after pretreatment with VER-155008 did not improve inhibition of cellular viability, apoptosis, or clonogenic survival. Treatment with VER-155008 prior to chemotherapy resulted in an upregulation of target proteins HSP70 and GRP78 in addition to the co-chaperone proteins Herp, C/EBP homologous transcription protein (CHOP), and BAG-1. The increased GRP78 was more cytoplasmic in location compared to untreated cells. Single-agent treatment also revealed a dose-dependent reduction in activated and total Akt. Based on these results, targeting GRP78 and HSP70 may have biologic activity in canine osteosarcoma. Further studies are required to determine if and how this strategy may impact the response of osteosarcoma cells to chemotherapy.

[HSP90 Inhibitor 17-AAG Inhibits Multiple Myeloma Cell Proliferation by Down-regulating Wnt/β-Catenin Signaling Pathway].

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Chen, Kan-Kan; He, Zheng-Mei; Ding, Bang-He; Chen, Yue; Zhang, Li-Juan; Yu, Liang; Gao, Jian

2016-02-01

To investigate the inhibitory effect of HSP90 inhibitory 17-AAG on proliferation of multiple myeloma cells and its main mechanism. The multiple myeloma cells U266 were treated with 17-AAG of different concentrations (200, 400, 600 and 800 nmol/L) for 24, 48, and 72 hours respectively, then the proliferation rate, expression levels of β-catenin and C-MYC protein, as well as cell cycle of U266 cells were treated with 17-AAG and were detected by MTT method, Western blot and flow cytometry, respectively. The 17-AAG showed inhibitory effect on the proliferation of U266 cells in dose- and time-depetent manners (r = -0.518, P AAG displayed no inhibitory effect on proliferation of U266 cells (P > 0.05). The result of culturing U266 cells for 72 hours by 17-AAG of different concentrations showed that the more high of 17-AAG concentration, the more low level of β-catenin and C-MYC proteins (P AAG concentration, the more high of cell ratio in G1 phase (P AAG, the more long time of culture, the more high of cell ratio in G1 phase (P AAG can inhibit the proliferation of multiple myeloma cells, the down-regulation of Wnt/β-catenin signaling pathway and inhibition of HSP90 expression may be the main mechnisms of 17-AAG effect.

Inhibition of HSP27 alone or in combination with pAKT inhibition as therapeutic approaches to target SPARC-induced glioma cell survival

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Schultz Chad R

2012-04-01

Full Text Available Abstract Background The current treatment regimen for glioma patients is surgery, followed by radiation therapy plus temozolomide (TMZ, followed by 6 months of adjuvant TMZ. Despite this aggressive treatment regimen, the overall survival of all surgically treated GBM patients remains dismal, and additional or different therapies are required. Depending on the cancer type, SPARC has been proposed both as a therapeutic target and as a therapeutic agent. In glioma, SPARC promotes invasion via upregulation of the p38 MAPK/MAPKAPK2/HSP27 signaling pathway, and promotes tumor cell survival by upregulating pAKT. As HSP27 and AKT interact to regulate the activity of each other, we determined whether inhibition of HSP27 was better than targeting SPARC as a therapeutic approach to inhibit both SPARC-induced glioma cell invasion and survival. Results Our studies found the following. 1 SPARC increases the expression of tumor cell pro-survival and pro-death protein signaling in balance, and, as a net result, tumor cell survival remains unchanged. 2 Suppressing SPARC increases tumor cell survival, indicating it is not a good therapeutic target. 3 Suppressing HSP27 decreases tumor cell survival in all gliomas, but is more effective in SPARC-expressing tumor cells due to the removal of HSP27 inhibition of SPARC-induced pro-apoptotic signaling. 4 Suppressing total AKT1/2 paradoxically enhanced tumor cell survival, indicating that AKT1 or 2 are poor therapeutic targets. 5 However, inhibiting pAKT suppresses tumor cell survival. 6 Inhibiting both HSP27 and pAKT synergistically decreases tumor cell survival. 7 There appears to be a complex feedback system between SPARC, HSP27, and AKT. 8 This interaction is likely influenced by PTEN status. With respect to chemosensitization, we found the following. 1 SPARC enhances pro-apoptotic signaling in cells exposed to TMZ. 2 Despite this enhanced signaling, SPARC protects cells against TMZ. 3 This protection can be reduced

Efficient activation of T cells by human monocyte-derived dendritic cells (HMDCs pulsed with Coxiella burnetii outer membrane protein Com1 but not by HspB-pulsed HMDCs

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Wang Xile

2011-09-01

Full Text Available Abstract Background Coxiella burnetii is an obligate intracellular bacterium and the etiologic agent of Q fever; both coxiella outer membrane protein 1 (Com1 and heat shock protein B (HspB are its major immunodominant antigens. It is not clear whether Com1 and HspB have the ability to mount immune responses against C. burnetii infection. Results The recombinant proteins Com1 and HspB were applied to pulse human monocyte-derived dendritic cells (HMDCs, and the pulsed HMDCs were used to stimulate isogenic T cells. Com1-pulsed HMDCs expressed substantially higher levels of surface molecules (CD83, CD40, CD80, CD86, CD54, and CD58 and a higher level of interleukin-12 than HspB-pulsed HMDCs. Moreover, Com1-pulsed HMDCs induced high-level proliferation and activation of CD4+ and CD8+ cells, which expressed high levels of T-cell activation marker CD69 and inflammatory cytokines IFN-γ and TNF-α. In contrast, HspB-pulsed HMDCs were unable to induce efficient T-cell proliferation and activation. Conclusions Our results demonstrate that Com1-pulsed HMDCs are able to induce efficient T-cell proliferation and drive T cells toward Th1 and Tc1 polarization; however, HspB-pulsed HMDCs are unable to do so. Unlike HspB, Com1 is a protective antigen, which was demonstrated by the adoptive transfer of Com1-pulsed bone marrow dendritic cells into naive BALB/c mice.

10 CFR 72.10 - Employee protection.

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2010-01-01

... adverse action occurs because the employee has engaged in protected activities. An employee's engagement... 10 Energy 2 2010-01-01 2010-01-01 false Employee protection. 72.10 Section 72.10 Energy NUCLEAR... Employee protection. (a) Discrimination by a Commission licensee, certificate holder, an applicant for a...

Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells

International Nuclear Information System (INIS)

Banzet, N.; Richaud, C.; Deveaux, Y.; Kazmaier, M.; Gagnon, J.; Triantaphylides, C.

1998-01-01

Changes in gene expression, by application of H2O2, O2.- generating agents (methyl viologen, digitonin) and gamma irradiation to tomato suspension cultures, were investigated and compared to the well-described heat shock response. Two-dimensional gel protein mapping analyses gave the first indication that at least small heat shock proteins (smHSP) accumulated in response to application of H2O2 and gamma irradiation, but not to O2.- generating agents. While some proteins seemed to be induced specifically by each treatment, only part of the heat shock response was observed. On the basis of Northern hybridization experiments performed with four heterologous cDNA, corresponding to classes I-IV of pea smHSP, it could be concluded that significant amounts of class I and II smHSP mRNA are induced by H2O2 and by irradiation. Taken together, these results demonstrate that in plants some HSP genes are inducible by oxidative stresses, as in micro-organisms and other eukaryotic cells. HSP22, the main stress protein that accumulates following H2O2 action or gamma irradiation, was also purified. Sequence homology of amino terminal and internal sequences, and immunoreactivity with Chenopodium rubrum mitochondrial smHSP antibody, indicated that the protein belongs to the recently discovered class of plant mitochondrial smHSP. Heat shock or a mild H2O2 pretreatment was also shown to lead to plant cell protection against oxidative injury. Therefore, the synthesis of these stress proteins can be considered as an adaptive mechanism in which mitochondrial protection could be essential

The small-molecule kinase inhibitor D11 counteracts 17-AAG-mediated up-regulation of HSP70 in brain cancer cells.

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Schaefer, Susanne; Svenstrup, Tina H; Guerra, Barbara

2017-01-01

Many types of cancer express high levels of heat shock proteins (HSPs) that are molecular chaperones regulating protein folding and stability ensuring protection of cells from potentially lethal stress. HSPs in cancer cells promote survival, growth and spreading even in situations of growth factors deprivation by associating with oncogenic proteins responsible for cell transformation. Hence, it is not surprising that the identification of potent inhibitors of HSPs, notably HSP90, has been the primary research focus, in recent years. Exposure of cancer cells to HSP90 inhibitors, including 17-AAG, has been shown to cause resistance to chemotherapeutic treatment mostly attributable to induction of the heat shock response and increased cellular levels of pro-survival chaperones. In this study, we show that treatment of glioblastoma cells with 17-AAG leads to HSP90 inhibition indicated by loss of stability of the EGFR client protein, and significant increase in HSP70 expression. Conversely, co-treatment with the small-molecule kinase inhibitor D11 leads to suppression of the heat shock response and inhibition of HSF1 transcriptional activity. Beside HSP70, Western blot and differential mRNA expression analysis reveal that combination treatment causes strong down-regulation of the small chaperone protein HSP27. Finally, we demonstrate that incubation of cells with both agents leads to enhanced cytotoxicity and significantly high levels of LC3-II suggesting autophagy induction. Taken together, results reported here support the notion that including D11 in future treatment regimens based on HSP90 inhibition can potentially overcome acquired resistance induced by the heat shock response in brain cancer cells.

Hsp27, Hsp70, and metallothionein in MDCK and LLC-PK1 renal epithelial cells: effects of prolonged exposure to cadmium

International Nuclear Information System (INIS)

Bonham, Rita T.; Fine, Michael R.; Pollock, Fiona M.; Shelden, Eric A.

2003-01-01

Cadmium is a widely distributed industrial and environmental toxin. The principal target organ of chronic sublethal cadmium exposure is the kidney. In renal epithelial cells, acute high-dose cadmium exposure induces differential expression of proteins, including heat shock proteins. However, few studies have examined heat shock protein expression in cells after prolonged exposure to cadmium at sublethal concentrations. Here, we assayed total cell protein, neutral red uptake, cell death, and levels of metallothionein and heat shock proteins Hsp27 and inducible Hsp70 in cultures of MDCK and LLC-PK1 renal epithelial cells treated with cadmium for 3 days. Treatment with cadmium at concentrations equal to or greater than 10 μM (LLC-PK1) or 25 μM (MDCK) reduced measures of cell vitality and induced cell death. However, a concentration-dependent increase in Hsp27 was detected in both cell types treated with as little as 5 μM cadmium. Accumulation of Hsp70 was correlated only with cadmium treatment at concentrations also causing cell death. Metallothionein was maximally detected in cells treated with cadmium at concentrations that did not reduce cell vitality, and further increases were not detected at greater concentrations. These results reveal that heat shock proteins accumulate in renal epithelial cells during prolonged cadmium exposure, that cadmium induces differential expression of heat shock protein in epithelial cells, and that protein expression patterns in epithelial cells are specific to the cadmium concentration and degree of cellular injury. A potential role for Hsp27 in the cellular response to sublethal cadmium-induced injury is also implicated by our results

HSP90 is essential for Jak-STAT signaling in classical Hodgkin lymphoma cells

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Kube Dieter

2009-07-01

Full Text Available Abstract In classical Hodgkin lymphoma (cHL chemotherapeutic regimens are associated with stagnant rates of secondary malignancies requiring the development of new therapeutic strategies. We and others have shown that permanently activated Signal Transducer and Activator of Transcription (STAT molecules are essential for cHL cells. Recently an overexpression of heat-shock protein 90 (HSP90 in cHL cells has been shown and inhibition of HSP90 seems to affect cHL cell survival. Here we analysed the effects of HSP90 inhibition by geldanamycin derivative 17-AAG or RNA interference (RNAi on aberrant Jak-STAT signaling in cHL cells. Treatment of cHL cell lines with 17-AAG led to reduced cell proliferation and a complete inhibition of STAT1, -3, -5 and -6 tyrosine phosphorylation probably as a result of reduced protein expression of Janus kinases (Jaks. RNAi-mediated inhibition of HSP90 showed similar effects on Jak-STAT signaling in L428 cHL cells. These results suggest a central role of HSP90 in permanently activated Jak-STAT signaling in cHL cells. Therapeutics targeting HSP90 may be a promising strategy in cHL and other cancer entities associated with deregulated Jak-STAT pathway activation.

secHsp70 as a tool to approach amyloid-β42 and other extracellular amyloids.

Science.gov (United States)

De Mena, Lorena; Chhangani, Deepak; Fernandez-Funez, Pedro; Rincon-Limas, Diego E

2017-07-03

Self-association of amyloidogenic proteins is the main pathological trigger in a wide variety of neurodegenerative disorders. These aggregates are deposited inside or outside the cell due to hereditary mutations, environmental exposures or even normal aging. Cumulative evidence indicates that the heat shock chaperone Hsp70 possesses robust neuroprotection against various intracellular amyloids in Drosophila and mouse models. However, its protective role against extracellular amyloids was largely unknown as its presence outside the cells is very limited. Our recent manuscript in PNAS revealed that an engineered form of secreted Hsp70 (secHsp70) is highly protective against toxicity induced by extracellular deposition of the amyloid-β42 (Aβ42) peptide. In this Extra View article, we extend our analysis to other members of the heat shock protein family. We created PhiC31-based transgenic lines for human Hsp27, Hsp40, Hsp60 and Hsp70 and compared their activities in parallel against extracellular Aβ42. Strikingly, only secreted Hsp70 exhibits robust protection against Aβ42-triggered toxicity in the extracellular milieu. These observations indicate that the ability of secHsp70 to suppress Aβ42 insults is quite unique and suggest that targeted secretion of Hsp70 may represent a new therapeutic approach against Aβ42 and other extracellular amyloids. The potential applications of this engineered chaperone are discussed.

Protective effects of red grape (Vitis vinifera) juice through restoration of antioxidant defense, endocrine swing and Hsf1, Hsp72 levels in heat stress induced testicular dysregulation of Wister rat.

Science.gov (United States)

Halder, Soma; Sarkar, Mrinmoy; Dey, Sananda; Kumar Bhunia, Sujay; Ranjan Koley, Alok; Giri, Biplab

2018-01-01

Ability of red grape juice (RGJ), a known antioxidant, on testis of adult Wister rat to protect from oxidative stress induced damages by heat stress has been investigated in this study. Heat stress was induced maintaining body and testicular temperature at 43°C for 30min/day for 15 days using a hyperthermia induction chamber. Four groups of rats (n=6 per group) comprising of Group-I (control) -kept at 32°C, Group-II -exposed to heat stress alone, Group-III received RGJ (0.8ml/rat/day) alone and Group-IV -exposed to heat stress and received RGJ at same dose. Analysis of blood and testicular tissue exhibited significant reduction in serum testosterone, testicular superoxide dismutase, testicular catalase and testicular glutathione (all p rise in the level of serum corticosteroid, testicular lipid peroxidase and the apoptotic enzyme caspase-3 of testis (all p < 0.001) were observed along with substantial increase in testicular Hsp72 and Hsf-1, and decrease in 17β-HSD3 were noted in heat stressed rats compared to controls. In Group-IV rats, RGJ administration could restore these parameters to normal levels. The signs of retention were clear in Group-IV rats and found to be significantly different as compared to that of the Group-II rats. In testicular histology of rats exposed to heat stress alone revealed remarkable germ cell degeneration and tubular deformations which were prevented by RGJ treatment (Group-IV). The reduced number of sperm level in Group-II also restored in RGJ treatment (Group-IV). The above results indicate that consumption of RGJ may substantially protect testis from heat stress induce dysfunctions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cell stress promotes the association of phosphorylated HspB1 with F-actin.

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Joseph P Clarke

Full Text Available Previous studies have suggested that the small heat shock protein, HspB1, has a direct influence on the dynamics of cytoskeletal elements, in particular, filamentous actin (F-actin polymerization. In this study we have assessed the influence of HspB1 phosphorylation on its interaction(s with F-actin. We first determined the distribution of endogenous non-phosphorylated HspB1, phosphorylated HspB1 and F-actin in neuroendocrine PC12 cells by immunocytochemistry and confocal microscopy. We then investigated a potential direct interaction between HspB1 with F-actin by precipitating F-actin directly with biotinylated phalloidin followed by Western analyses; the reverse immunoprecipitation of HspB1 was also carried out. The phosphorylation influence of HspB1 in this interaction was investigated by using pharmacologic inhibition of p38 MAPK. In control cells, HspB1 interacts with F-actin as a predominantly non-phosphorylated protein, but subsequent to stress there is a redistribution of HspB1 to the cytoskeletal fraction and a significantly increased association of pHspB1 with F-actin. Our data demonstrate HspB1 is found in a complex with F-actin both in phosphorylated and non-phosphorylated forms, with an increased association of pHspB1 with F-actin after heat stress. Overall, our study combines both cellular and biochemical approaches to show cellular localization and direct demonstration of an interaction between endogenous HspB1 and F-actin using methodolgy that specifically isolates F-actin.

The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster.

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Jennifer L Bandura

Full Text Available The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite(+ reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C, suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis.

The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster.

Science.gov (United States)

Bandura, Jennifer L; Jiang, Huaqi; Nickerson, Derek W; Edgar, Bruce A

2013-01-01

The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite(+) reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C), suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis.

Effects of Lactobacillus johnsonii and Lactobacillus reuteri on gut barrier function and heat shock proteins in intestinal porcine epithelial cells.

Science.gov (United States)

Liu, Hao-Yu; Roos, Stefan; Jonsson, Hans; Ahl, David; Dicksved, Johan; Lindberg, Jan Erik; Lundh, Torbjörn

2015-04-01

Heat shock proteins (HSPs) are a set of highly conserved proteins that can serve as intestinal gate keepers in gut homeostasis. Here, effects of a probiotic, Lactobacillus rhamnosus GG (LGG), and two novel porcine isolates, Lactobacillus johnsonii strain P47-HY and Lactobacillus reuteri strain P43-HUV, on cytoprotective HSP expression and gut barrier function, were investigated in a porcine IPEC-J2 intestinal epithelial cell line model. The IPEC-J2 cells polarized on a permeable filter exhibited villus-like cell phenotype with development of apical microvilli. Western blot analysis detected HSP expression in IPEC-J2 and revealed that L. johnsonii and L. reuteri strains were able to significantly induce HSP27, despite high basal expression in IPEC-J2, whereas LGG did not. For HSP72, only the supernatant of L. reuteri induced the expression, which was comparable to the heat shock treatment, which indicated that HSP72 expression was more stimulus specific. The protective effect of lactobacilli was further studied in IPEC-J2 under an enterotoxigenic Escherichia coli (ETEC) challenge. ETEC caused intestinal barrier destruction, as reflected by loss of cell-cell contact, reduced IPEC-J2 cell viability and transepithelial electrical resistance, and disruption of tight junction protein zonula occludens-1. In contrast, the L. reuteri treatment substantially counteracted these detrimental effects and preserved the barrier function. L. johnsonii and LGG also achieved barrier protection, partly by directly inhibiting ETEC attachment. Together, the results indicate that specific strains of Lactobacillus can enhance gut barrier function through cytoprotective HSP induction and fortify the cell protection against ETEC challenge through tight junction protein modulation and direct interaction with pathogens. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Enhancement of Hsp70 synthesis protects common carp, Cyprinus carpio L., against lethal ammonia toxicity.

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Sung, Y Y; Roberts, R J; Bossier, P

2012-08-01

Exposure to TEX-OE®, a patented extract of the prickly pear cactus (Opuntia ficus indica) containing chaperone-stimulating factor, was shown to protect common carp, Cyprinus carpio L., fingerlings against acute ammonia stress. Survival was enhanced twofold from 50% to 95% after exposure to 5.92 mg L(-1) NH(3) , a level determined in the ammonia challenge bioassay as the 1-h LD50 concentration for this species. Survival of TEX-OE®-pre-exposed fish was enhanced by 20% over non-exposed controls during lethal ammonia challenge (14.21 mg L(-1)  NH(3) ). Increase in the levels of gill and muscle Hsp70 was evident in TEX-OE®-pre-exposed fish but not in the unexposed controls, indicating that application of TEX-OE® accelerated carp endogenous Hsp70 synthesis during ammonia perturbation. Protection against ammonia was correlated with Hsp70 accretion. © 2012 Blackwell Publishing Ltd.

Heat shock protein-27 protects human bronchial epithelial cells against oxidative stress–mediated apoptosis: possible implication in asthma

Science.gov (United States)

Merendino, Anna M.; Paul, Catherine; Vignola, Antonio M.; Costa, Maria A.; Melis, Mario; Chiappara, Giuseppina; Izzo, V.; Bousquet, J.; Arrigo, André-Patrick

2002-01-01

Inflammation of the human bronchial epithelium, as observed in asthmatics, is characterized by the selective death of the columnar epithelial cells, which desquamate from the basal cells. Tissue repair initiates from basal cells that resist inflammation. Here, we have evaluated the extent of apoptosis as well as the Hsp27 level of expression in epithelial cells from bronchial biopsy samples taken from normal and asthmatic subjects. Hsp27 is a chaperone whose expression protects against oxidative stress. We report that in asthmatic subjects the basal epithelium cells express a high level of Hsp27 but no apoptotic morphology. In contrast, apoptotic columnar cells are devoid of Hsp27 expression. Moreover, we observed a decreased resistance to hydrogen peroxide–induced apoptosis in human bronchial epithelial 16–HBE cells when they were genetically modified to express reduced levels of Hsp27. PMID:12482203

Systemic and mucosal immunization with Candida albicans hsp90 elicits hsp90-specific humoral response in vaginal mucosa which is further enhanced during experimental vaginal candidiasis.

Science.gov (United States)

Raska, Milan; Belakova, Jana; Horynova, Milada; Krupka, Michal; Novotny, Jiri; Sebestova, Martina; Weigl, Evzen

2008-08-01

The Candida albicans heat shock protein 90 kDa (hsp90-CA) is an important target for protective antibodies in disseminated candidiasis of experimental mice and humans. Hsp90-CA is present in the cell wall of Candida pseudohyphae or hyphae--typical pathogenic morphotypes in both mucosal and systemic Candida infections. However, the potential protective effects of hsp90-CA-specific antibodies in vaginal candidiasis has not yet been reported. In the present study we used various vaccine formulations (recombinant hsp90-CA protein and hsp90-CA-encoding DNA vaccine) and routes of administration (intradermal, intranasal, and intravenous) to induce both hsp90-CA-specific systemic and vaginal mucosa immune responses in experimental BALB/c mice. The results showed that intradermal recombinant hsp90-CA protein priming, followed by intranasal or intradermal recombinant hsp90-CA protein boosting induced significant increases in both serum and vaginal hsp90-CA-specific IgG and IgA antibodies compared to the control group, as well as enhanced hsp90-CA-specific splenocyte responses in vitro. In the intradermally boosted group, subsequent experimental vaginal Candida infection induced additional increases in the hsp90-CA specific IgG isotype, suggesting that Candida has the ability to induce a local hsp90-specific antibody (IgG) response during vulvovaginal candidiasis. Further work is required to elucidate the importance of immunity to highly conserved antigens during infection of the human female reproductive tract where a balance between immunity to and tolerance for commonly antigens such as hsp90 is necessary for the maintenance of fertility.

Recovery From Radiation-induced Bone Marrow Damage by HSP25 Through Tie2 Signaling

Energy Technology Data Exchange (ETDEWEB)

Lee, Hae-June [Division of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Kwon, Hee-Chung [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Chung, Hee-Yong [College of Medicine, Hanyang University, Seoul (Korea, Republic of); Lee, Yoon-Jin [Division of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Lee, Yun-Sil, E-mail: yslee0425@ewha.ac.kr [College of Pharmacy and Division of Life and Pharmaceutical Sciences, Ewha Woman' s University, Seoul (Korea, Republic of)

2012-09-01

Purpose: Whole-body radiation therapy can cause severe injury to the hematopoietic system, and therefore it is necessary to identify a novel strategy for overcoming this injury. Methods and Materials: Mice were irradiated with 4.5 Gy after heat shock protein 25 (HSP25) gene transfer using an adenoviral vector. Then, peripheral blood cell counts, histopathological analysis, and Western blotting on bone marrow (BM) cells were performed. The interaction of HSP25 with Tie2 was investigated with mouse OP9 and human BM-derived mesenchymal stem cells to determine the mechanism of HSP25 in the hematopoietic system. Results: HSP25 transfer increased BM regeneration and reduced apoptosis following whole-body exposure to ionizing radiation (IR). The decrease in Tie2 protein expression that followed irradiation of the BM was blocked by HSP25 transfer, and Tie2-positive cells were more abundant among the BM cells of HSP25-transferred mice, even after IR exposure. Following systemic RNA interference of Tie2 before IR, HSP25-mediated radioprotective effects were partially blocked in both mice and cell line systems. Stability of Tie2 was increased by HSP25, a response mediated by the interaction of HSP25 with Tie2. IR-induced tyrosine phosphorylation of Tie2 was augmented by HSP25 overexpression; downstream events in the Tie2 signaling pathway, including phosphorylation of AKT and EKR1/2, were also activated. Conclusions: HSP25 protects against radiation-induced BM damage by interacting with and stabilizing Tie2. This may be a novel strategy for HSP25-mediated radioprotection in BM.

Fine particulate matter potentiates type 2 diabetes development in high-fat diet-treated mice: stress response and extracellular to intracellular HSP70 ratio analysis.

Science.gov (United States)

Goettems-Fiorin, Pauline Brendler; Grochanke, Bethânia Salamoni; Baldissera, Fernanda Giesel; Dos Santos, Analu Bender; Homem de Bittencourt, Paulo Ivo; Ludwig, Mirna Stela; Rhoden, Claudia Ramos; Heck, Thiago Gomes

2016-12-01

Exposure to fine particulate matter (PM 2.5 ) air pollution is a risk factor for type 2 diabetes (T2DM). We argue whether the potentiating effect of PM 2.5 over the development of T2DM in high-fat diet (HFD)-fed mice would be related to modification in cell stress response, particularly in antioxidant defenses and 70-kDa heat shock proteins (HSP70) status. Male mice were fed standard chow or HFD for 12 weeks and then randomly exposed to daily nasotropic instillation of PM 2.5 for additional 12 weeks under the same diet schedule, divided into four groups (n = 14-15 each): Control, PM 2.5 , HFD, and HFD + PM 2.5 were evaluated biometric and metabolic profiles of mice, and cellular stress response (antioxidant defense and HSP70 status) of metabolic tissues. Extracellular to intracellular HSP70 ratio ([eHSP72]/[iHSP70]), viz. H-index, was then calculated. HFD + PM 2.5 mice presented a positive correlation between adiposity, increased body weight and glucose intolerance, and increased glucose and triacylglycerol plasma levels. Pancreas exhibited lower iHSP70 expression, accompanied by 3.7-fold increase in the plasma to pancreas [eHSP72]/[iHSP70] ratio. Exposure to PM 2.5 markedly potentiated metabolic dysfunction in HFD-treated mice and promoted relevant alteration in cell stress response assessed by [eHSP72]/[iHSP70], a relevant biomarker of chronic low-grade inflammatory state and T2DM risk.

Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity

DEFF Research Database (Denmark)

Jensen, Helle

frequently express Hsp70 on their cell surface, whereas the corresponding normal tissues do not. In addition, several clinically applied reagents, such as alkyl-lysophospholipides, chemotherapeutic agents, and anti-inflammatory reagents, have been found to enhance Hsp70 cell surface expression on cancer...

HSP27 Inhibits Homocysteine-Induced Endothelial Apoptosis by Modulation of ROS Production and Mitochondrial Caspase-Dependent Apoptotic Pathway

Directory of Open Access Journals (Sweden)

Xin Tian

2016-01-01

Full Text Available Objectives. Elevated plasma homocysteine (Hcy could lead to endothelial dysfunction and is viewed as an independent risk factor for atherosclerosis. Heat shock protein 27 (HSP27, a small heat shock protein, is reported to exert protective effect against atherosclerosis. This study aims to investigate the protective effect of HSP27 against Hcy-induced endothelial cell apoptosis in human umbilical vein endothelial cells (HUVECs and to determine the underlying mechanisms. Methods. Apoptosis, reactive oxygen species (ROS, and mitochondrial membrane potential (MMP of normal or HSP27-overexpressing HUVECs in the presence of Hcy were analyzed by flow cytometry. The mRNA and protein expression levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR and western blot. Results. We found that Hcy could induce cell apoptosis with corresponding decrease of nitric oxide (NO level, increase of endothelin-1 (ET-1, intracellular adhesion molecule-1 (ICAM-1, vascular cellular adhesion molecule-1 (VCAM-1, and monocyte chemoattractant protein-1 (MCP-1 levels, elevation of ROS, and dissipation of MMP. In addition, HSP27 could protect the cell against Hcy-induced apoptosis and inhibit the effect of Hcy on HUVECs. Furthermore, HSP27 could increase the ratio of Bcl-2/Bax and inhibit caspase-3 activity. Conclusions. Therefore, we concluded that HSP27 played a protective role against Hcy-induced endothelial apoptosis through modulation of ROS production and the mitochondrial caspase-dependent apoptotic pathway.

SGT, a Hsp90β binding partner, is accumulated in the nucleus during cell apoptosis

International Nuclear Information System (INIS)

Yin Hongyan; Wang Hanzhou; Zong Hongliang; Chen Xiaoning; Wang Yanlin; Yun Xiaojing; Wu Yihong; Wang Jiadong; Gu Jianxin

2006-01-01

In this study, we reported that small glutamine-rich TPR-containing protein (SGT) interacted with not only Hsp90α but also Hsp90β. Confocal analysis showed that treatment of cells with Hsp90-specific inhibitor geldanamycin (GA) disrupted the interaction of SGT with Hsp90β and this contributed to the increase of nuclear localization of SGT in HeLa cells. The increased nuclear localization of SGT was further confirmed by the Western blotting in GA-treated HeLa cells and H1299 cells. In our previous study, SGT was found to be a new pro-apoptotic factor, so we wondered whether the sub-cellular localization of SGT was related with cell apoptosis. By confocal analysis we found that the nuclear import of SGT was significantly increased in STS-induced apoptotic HeLa cells, which implied that the sub-cellular localization of SGT was closely associated with Hsp90β and apoptosis

Ubiquitin-fusion degradation pathway: A new strategy for inducing CD8 cells specific for mycobacterial HSP65

International Nuclear Information System (INIS)

Shen Jianying; Hisaeda, Hajime; Chou Bin; Yu Qingsheng; Tu Liping; Himeno, Kunisuke

2008-01-01

The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8 + T cells. In this study, we exploited UPS to induce CD8 + T cells specific for mycobacterial HSP65 (mHSP65), one of the leading vaccine candidates against infection with Mycobacterium tuberculosis. A chimeric DNA termed pU-HSP65 encoding a fusion protein between murine ubiquitin and mHSP65 was constructed, and C57BL/6 (B6) mice were immunized with the DNA using gene gun bombardment. Mice immunized with the chimeric DNA acquired potent resistance against challenge with the syngeneic B16F1 melanoma cells transfected with the mHSP65 gene (HSP65/B16F1), compared with those immunized with DNA encoding only mHSP65. Splenocytes from the former group of mice showed a higher grade of cytotoxic activity against HSP65/B16F1 cells and contained a larger number of granzyme B- or IFN-γ-producing CD8 + T cells compared with those from the latter group of mice

DNA double strand breaks and Hsp70 expression in proton irradiated living cells

International Nuclear Information System (INIS)

Fiedler, Anja; Reinert, Tilo; Tanner, Judith; Butz, Tilman

2007-01-01

DNA double strand breaks (DSBs) in living cells can be directly provoked by ionising radiation. DSBs can be visualized by immunostaining the phosphorylated histone γH2AX. Our concern was to test the feasibility of γH2AX staining for a direct visualization of single proton hits. If single protons produce detectable foci, DNA DSBs could be used as 'biological track detectors' for protons. Ionising radiation can also damage proteins indirectly by inducing free radicals. Heat shock proteins (Hsp) help to refold or even degrade the damaged proteins. The level of the most famous heat shock protein Hsp70 is increased by ionising radiation. We investigated the expression of γH2AX and Hsp70 after cross and line patterned irradiation with counted numbers of 2.25 MeV protons on primary human skin fibroblasts. The proton induced DSBs appear more delocalised than it was expected by the ion hit accuracy. Cooling the cells before the irradiation reduces the delocalisation of DNA DSBs, which is probably caused by the reduced diffusion of DNA damaging agents. Proton irradiation seems to provoke protein damages mainly in the cytoplasm indicated by cytoplasmic Hsp70 aggregates. On the contrary, in control heat shocked cells the Hsp70 was predominantly localized in the cell nucleus. However, the irradiated area could not be recognized, all cells on the Si 3 N 4 window showed a homogenous Hsp70 expression pattern

DNA double strand breaks and Hsp70 expression in proton irradiated living cells

Energy Technology Data Exchange (ETDEWEB)

Fiedler, Anja [Institute for Experimental Physics II, University of Leipzig (Germany) and Faculty of Biology, Pharmacy and Psychology, University of Leipzig (Germany)]. E-mail: afiedler@uni-leipzig.de; Reinert, Tilo [Institute for Experimental Physics II, University of Leipzig (Germany); Tanner, Judith [Clinic and Polyclinic for Radiation Oncology, University of Halle-Wittenberg (Germany); Butz, Tilman [Institute for Experimental Physics II, University of Leipzig (Germany)

2007-07-15

DNA double strand breaks (DSBs) in living cells can be directly provoked by ionising radiation. DSBs can be visualized by immunostaining the phosphorylated histone {gamma}H2AX. Our concern was to test the feasibility of {gamma}H2AX staining for a direct visualization of single proton hits. If single protons produce detectable foci, DNA DSBs could be used as 'biological track detectors' for protons. Ionising radiation can also damage proteins indirectly by inducing free radicals. Heat shock proteins (Hsp) help to refold or even degrade the damaged proteins. The level of the most famous heat shock protein Hsp70 is increased by ionising radiation. We investigated the expression of {gamma}H2AX and Hsp70 after cross and line patterned irradiation with counted numbers of 2.25 MeV protons on primary human skin fibroblasts. The proton induced DSBs appear more delocalised than it was expected by the ion hit accuracy. Cooling the cells before the irradiation reduces the delocalisation of DNA DSBs, which is probably caused by the reduced diffusion of DNA damaging agents. Proton irradiation seems to provoke protein damages mainly in the cytoplasm indicated by cytoplasmic Hsp70 aggregates. On the contrary, in control heat shocked cells the Hsp70 was predominantly localized in the cell nucleus. However, the irradiated area could not be recognized, all cells on the Si{sub 3}N{sub 4} window showed a homogenous Hsp70 expression pattern.

A Recombinant Trivalent Fusion Protein F1-LcrV-HSP70(II) Augments Humoral and Cellular Immune Responses and Imparts Full Protection against Yersinia pestis.

Science.gov (United States)

Verma, Shailendra K; Batra, Lalit; Tuteja, Urmil

2016-01-01

Plague is one of the most dangerous infections in humans caused by Yersinia pestis, a Gram-negative bacterium. Despite of an overwhelming research success, no ideal vaccine against plague is available yet. It is well established that F1/LcrV based vaccine requires a strong cellular immune response for complete protection against plague. In our earlier study, we demonstrated that HSP70(II) of Mycobacterium tuberculosis modulates the humoral and cellular immunity of F1/LcrV vaccine candidates individually as well as in combinations in a mouse model. Here, we made two recombinant constructs caf1-lcrV and caf1-lcrV-hsp70(II). The caf1 and lcrV genes of Y. pestis and hsp70 domain II of M. tuberculosis were amplified by polymerase chain reaction. Both the recombinant constructs caf1-lcrV and caf1-lcrV-hsp70(II) were cloned in pET28a vector and expressed in Escherichia coli. The recombinant fusion proteins F1-LcrV and F1-LcrV-HSP70(II) were purified using Ni-NTA columns and formulated with alum to evaluate the humoral and cell mediated immune responses in mice. The protective efficacies of F1-LcrV and F1-LcrV-HSP70(II) were determined following challenge of immunized mice with 100 LD50 of Y. pestis through intraperitoneal route. Significant differences were noticed in the titers of IgG and it's isotypes, i.e., IgG1, IgG2b, and IgG3 in anti- F1-LcrV-HSP70(II) sera in comparison to anti-F1-LcrV sera. Similarly, significant differences were also noticed in the expression levels of IL-2, IFN-γ and TNF-α in splenocytes of F1-LcrV-HSP(II) immunized mice in comparison to F1-LcrV. Both F1-LcrV and F1-LcrV-HSP70(II) provided 100% protection. Our research findings suggest that F1-LcrV fused with HSP70 domain II of M. tuberculosis significantly enhanced the humoral and cellular immune responses in mouse model.

Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

Energy Technology Data Exchange (ETDEWEB)

Rocha, C.D.; Trombone, A.P.F.; Lorenzi, J.C.C.; Almeida, L.P.; Gembre, A.F.; Padilha, E. [Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Ramos, S.G. [Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Silva, C.L.; Coelho-Castelo, A.A.M. [Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

2012-09-21

In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

Reduced glycogen availability is associated with an elevation in HSP72 in contracting human skeletal muscle

DEFF Research Database (Denmark)

Febbraio, Mark A; Steensberg, Adam; Walsh, Rory

2002-01-01

To test the hypothesis that a decrease in intramuscular glycogen availability may stimulate heat shock protein expression, seven men depleted one leg of muscle glycogen the day before performing 4-5 h of exhaustive, two-legged knee extensor exercise at 40 % of leg peak power output. Subjects...... and both femoral veins and blood was sampled from these catheters prior to exercise and at 1 h intervals during exercise and into recovery for the measurement of arterial-venous differences in serum HSP72. Plasma creatine kinase (CK) was also measured from arterial blood samples. Pre-exercise muscle...

Sensitization of multidrug-resistant human cancer cells to Hsp90 inhibitors by down-regulation of SIRT1

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Kim, Hak-Bong; Lee, Su-Hoon; Um, Jee-Hyun; Oh, Won Keun; Kim, Dong-Wan; Kang, Chi-Dug; Kim, Sun-Hee

2015-01-01

The effectiveness of Hsp90 inhibitors as anticancer agents was limited in multidrug-resistant (MDR) human cancer cells due to induction of heat shock proteins (Hsps) such as Hsp70/Hsp27 and P-glycoprotein (P-gp)-mediated efflux. In the present study, we showed that resistance to Hsp90 inhibitors of MDR human cancer cells could be overcome with SIRT1 inhibition. SIRT1 knock-down or SIRT1 inhibitors (amurensin G and EX527) effectively suppressed the resistance to Hsp90 inhibitors (17-AAG and AUY922) in several MDR variants of human lymphoblastic leukemia and human breast cancer cell lines. SIRT1 inhibition down-regulated the expression of heat shock factor 1 (HSF1) and subsequently Hsps and facilitated Hsp90 multichaperone complex disruption via hyperacetylation of Hsp90/Hsp70. These findings were followed by acceleration of ubiquitin ligase CHIP-mediated mutant p53 (mut p53) degradation and subsequent down-regulation of P-gp in 17-AAG-treated MDR cancer cells expressing P-gp and mut p53 after inhibition of SIRT1. Therefore, combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be a more effective therapeutic approach for Hsp90 inhibitor-resistant MDR cells via down-regulation of HSF1/Hsps, mut p53 and P-gp. PMID:26416354

Involvement of yeast HSP90 isoforms in response to stress and cell death induced by acetic acid.

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Alexandra Silva

Full Text Available Acetic acid-induced apoptosis in yeast is accompanied by an impairment of the general protein synthesis machinery, yet paradoxically also by the up-regulation of the two isoforms of the heat shock protein 90 (HSP90 chaperone family, Hsc82p and Hsp82p. Herein, we show that impairment of cap-dependent translation initiation induced by acetic acid is caused by the phosphorylation and inactivation of eIF2α by Gcn2p kinase. A microarray analysis of polysome-associated mRNAs engaged in translation in acetic acid challenged cells further revealed that HSP90 mRNAs are over-represented in this polysome fraction suggesting preferential translation of HSP90 upon acetic acid treatment. The relevance of HSP90 isoform translation during programmed cell death (PCD was unveiled using genetic and pharmacological abrogation of HSP90, which suggests opposing roles for HSP90 isoforms in cell survival and death. Hsc82p appears to promote survival and its deletion leads to necrotic cell death, while Hsp82p is a pro-death molecule involved in acetic acid-induced apoptosis. Therefore, HSP90 isoforms have distinct roles in the control of cell fate during PCD and their selective translation regulates cellular response to acetic acid stress.

Synthesis and Evaluation of the Tumor Cell Growth Inhibitory Potential of New Putative HSP90 Inhibitors.

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Bizarro, Ana; Sousa, Diana; Lima, Raquel T; Musso, Loana; Cincinelli, Raffaella; Zuco, Vantina; De Cesare, Michelandrea; Dallavalle, Sabrina; Vasconcelos, M Helena

2018-02-13

Heat shock protein 90 (HSP90) is a well-known target for cancer therapy. In a previous work, some of us have reported a series of 3-aryl-naphtho[2,3- d ]isoxazole-4,9-diones as inhibitors of HSP90. In the present work, various compounds with new chromenopyridinone and thiochromenopyridinone scaffolds were synthesized as potential HSP90 inhibitors. Their binding affinity to HSP90 was studied in vitro. Selected compounds ( 5 and 8 ) were further studied in various tumor cell lines regarding their potential to cause cell growth inhibition, alter the cell cycle profile, inhibit proliferation, and induce apoptosis. Their effect on HSP90 client protein levels was also confirmed in two cell lines. Finally, the antitumor activity of compound 8 was studied in A431 squamous cell carcinoma xenografts in nude mice. Our results indicated that treatment with compounds 5 and 8 decreased the proliferation of tumor cell lines and compound 8 induced apoptosis. In addition, these two compounds were able to downregulate selected proteins known as "clients" of HSP90. Finally, treatment of xenografted mice with compound 5 resulted in a considerable dose-dependent inhibition of tumor growth. Our results show that two new compounds with a chromenopyridinone and thiochromenopyridinone scaffold are promising putative HSP90 inhibitors causing tumor cell growth inhibition.

Hsp105 family proteins suppress staurosporine-induced apoptosis by inhibiting the translocation of Bax to mitochondria in HeLa cells

International Nuclear Information System (INIS)

Yamagishi, Nobuyuki; Ishihara, Keiichi; Saito, Youhei; Hatayama, Takumi

2006-01-01

Hsp105 (Hsp105α and Hsp105β), major heat shock proteins in mammalian cells, belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105α has completely different effects on stress-induced apoptosis depending on cell type. However, the molecular mechanisms by which Hsp105α regulates stress-induced apoptosis are not fully understood. Here, we established HeLa cells that overexpress either Hsp105α or Hsp105β by removing doxycycline and examined how Hsp105 modifies staurosporine (STS)-induced apoptosis in HeLa cells. Apoptotic features such as the externalization of phosphatidylserine on the plasma membrane and nuclear morphological changes were induced by the treatment with STS, and the STS-induced apoptosis was suppressed by overexpression of Hsp105α or Hsp105β. In addition, we found that overexpression of Hsp105α or Hsp105β suppressed the activation of caspase-3 and caspase-9 by preventing the release of cytochrome c from mitochondria. Furthermore, the translocation of Bax to mitochondria, which results in the release of cytochrome c from the mitochondria, was also suppressed by the overexpression of Hsp105α or Hsp105β. Thus, it is suggested that Hsp105 suppresses the stress-induced apoptosis at its initial step, the translocation of Bax to mitochondria in HeLa cells

An Impermeant Ganetespib Analog Inhibits Extracellular Hsp90-Mediated Cancer Cell Migration that Involves Lysyl Oxidase 2-like Protein

Energy Technology Data Exchange (ETDEWEB)

McCready, Jessica [Department of Natural Sciences, Assumption College, Worcester, MA 01609 (United States); Wong, Daniel S. [Department of Developmental Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Cell and Molecular Physiology Program, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA 02111 (United States); Burlison, Joseph A.; Ying, Weiwen [Synta Pharmaceuticals, Lexington, MA 02421 (United States); Jay, Daniel G., E-mail: daniel.jay@tufts.edu [Department of Developmental Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Cell and Molecular Physiology Program, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA 02111 (United States)

2014-04-30

Extracellular Hsp90 (eHsp90) activates a number of client proteins outside of cancer cells required for migration and invasion. Therefore, eHsp90 may serve as a novel target for anti-metastatic drugs as its inhibition using impermeant Hsp90 inhibitors would not affect the numerous vital intracellular Hsp90 functions in normal cells. While some eHsp90 clients are known, it is important to establish other proteins that act outside the cell to validate eHsp90 as a drug target to limit cancer spread. Using mass spectrometry we identified two precursor proteins Galectin 3 binding protein (G3BP) and Lysyl oxidase 2-like protein (LOXL2) that associate with eHsp90 in MDA-MB231 breast cancer cell conditioned media and confirmed that LOXL2 binds to eHsp90 in immunoprecipitates. We introduce a novel impermeant Hsp90 inhibitor STA-12-7191 derived from ganetespib and show that it is markedly less toxic to cells and can inhibit cancer cell migration in a dose dependent manner. We used STA-12-7191 to test if LOXL2 and G3BP are potential eHsp90 clients. We showed that while LOXL2 can increase wound healing and compensate for STA-12-7191-mediated inhibition of wound closure, addition of G3BP had no affect on this assay. These findings support of role for LOXL2 in eHsp90 stimulated cancer cell migration and provide preliminary evidence for the use of STA-12-7191 to inhibit eHsp90 to limit cancer invasion.

An Impermeant Ganetespib Analog Inhibits Extracellular Hsp90-Mediated Cancer Cell Migration that Involves Lysyl Oxidase 2-like Protein

International Nuclear Information System (INIS)

McCready, Jessica; Wong, Daniel S.; Burlison, Joseph A.; Ying, Weiwen; Jay, Daniel G.

2014-01-01

Extracellular Hsp90 (eHsp90) activates a number of client proteins outside of cancer cells required for migration and invasion. Therefore, eHsp90 may serve as a novel target for anti-metastatic drugs as its inhibition using impermeant Hsp90 inhibitors would not affect the numerous vital intracellular Hsp90 functions in normal cells. While some eHsp90 clients are known, it is important to establish other proteins that act outside the cell to validate eHsp90 as a drug target to limit cancer spread. Using mass spectrometry we identified two precursor proteins Galectin 3 binding protein (G3BP) and Lysyl oxidase 2-like protein (LOXL2) that associate with eHsp90 in MDA-MB231 breast cancer cell conditioned media and confirmed that LOXL2 binds to eHsp90 in immunoprecipitates. We introduce a novel impermeant Hsp90 inhibitor STA-12-7191 derived from ganetespib and show that it is markedly less toxic to cells and can inhibit cancer cell migration in a dose dependent manner. We used STA-12-7191 to test if LOXL2 and G3BP are potential eHsp90 clients. We showed that while LOXL2 can increase wound healing and compensate for STA-12-7191-mediated inhibition of wound closure, addition of G3BP had no affect on this assay. These findings support of role for LOXL2 in eHsp90 stimulated cancer cell migration and provide preliminary evidence for the use of STA-12-7191 to inhibit eHsp90 to limit cancer invasion

The regulatory mechanism of Hsp90α secretion from endothelial cells and its role in angiogenesis during wound healing

International Nuclear Information System (INIS)

Song, Xiaomin; Luo, Yongzhang

2010-01-01

Research highlights: → Growth factors such as bFGF, VEGF, PDGF and SDF-1 stimulate Hsp90α secretion from endothelial cells. → Secreted Hsp90α localizes on the leading edge of activated endothelial cells. → Secreted Hsp90α promotes angiogenesis in wound healing. -- Abstract: Heat shock protein 90α (Hsp90α) is a ubiquitously expressed molecular chaperone, which is essential for the maintenance of eukaryote homeostasis. Hsp90α can also be secreted extracellularly and is associated with several physiological and pathological processes including wound healing, cancer, infectious diseases and diabetes. Angiogenesis, defined as the sprouting of new blood vessels from pre-existing capillaries via endothelial cell proliferation and migration, commonly occurs in and contributes to the above mentioned processes. However, the secretion of Hsp90α from endothelial cells and also its function in angiogenesis are still unclear. Here we investigated the role of extracellular Hsp90α in angiogenesis using dermal endothelial cells in vitro and a wound healing model in vivo. We find that the secretion of Hsp90α but not Hsp90β is increased in activated endothelial cells with the induction of angiogenic factors and matrix proteins. Secreted Hsp90α localizes on the leading edge of endothelial cells and promotes their angiogenic activities, whereas Hsp90α neutralizing antibodies reverse the effect. Furthermore, using a mouse skin wound healing model in vivo, we demonstrate that extracellular Hsp90α localizes on blood vessels in granulation tissues of wounded skin and promotes angiogenesis during wound healing. Taken together, our study reveals that Hsp90α can be secreted by activated endothelial cells and is a positive regulator of angiogenesis, suggesting the potential application of Hsp90α as a stimulator for wound repair.

The regulatory mechanism of Hsp90{alpha} secretion from endothelial cells and its role in angiogenesis during wound healing

Energy Technology Data Exchange (ETDEWEB)

Song, Xiaomin [National Engineering Laboratory for Anti-tumor Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Beijing Key Laboratory for Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing 100084 (China); Luo, Yongzhang, E-mail: yluo@tsinghua.edu.cn [National Engineering Laboratory for Anti-tumor Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Beijing Key Laboratory for Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing 100084 (China)

2010-07-16

Research highlights: {yields} Growth factors such as bFGF, VEGF, PDGF and SDF-1 stimulate Hsp90{alpha} secretion from endothelial cells. {yields} Secreted Hsp90{alpha} localizes on the leading edge of activated endothelial cells. {yields} Secreted Hsp90{alpha} promotes angiogenesis in wound healing. -- Abstract: Heat shock protein 90{alpha} (Hsp90{alpha}) is a ubiquitously expressed molecular chaperone, which is essential for the maintenance of eukaryote homeostasis. Hsp90{alpha} can also be secreted extracellularly and is associated with several physiological and pathological processes including wound healing, cancer, infectious diseases and diabetes. Angiogenesis, defined as the sprouting of new blood vessels from pre-existing capillaries via endothelial cell proliferation and migration, commonly occurs in and contributes to the above mentioned processes. However, the secretion of Hsp90{alpha} from endothelial cells and also its function in angiogenesis are still unclear. Here we investigated the role of extracellular Hsp90{alpha} in angiogenesis using dermal endothelial cells in vitro and a wound healing model in vivo. We find that the secretion of Hsp90{alpha} but not Hsp90{beta} is increased in activated endothelial cells with the induction of angiogenic factors and matrix proteins. Secreted Hsp90{alpha} localizes on the leading edge of endothelial cells and promotes their angiogenic activities, whereas Hsp90{alpha} neutralizing antibodies reverse the effect. Furthermore, using a mouse skin wound healing model in vivo, we demonstrate that extracellular Hsp90{alpha} localizes on blood vessels in granulation tissues of wounded skin and promotes angiogenesis during wound healing. Taken together, our study reveals that Hsp90{alpha} can be secreted by activated endothelial cells and is a positive regulator of angiogenesis, suggesting the potential application of Hsp90{alpha} as a stimulator for wound repair.

Stress-induced localization of HSPA6 (HSP70B') and HSPA1A (HSP70-1) proteins to centrioles in human neuronal cells.

Science.gov (United States)

Khalouei, Sam; Chow, Ari M; Brown, Ian R

2014-05-01

The localization of yellow fluorescent protein (YFP)-tagged HSP70 proteins was employed to identify stress-sensitive sites in human neurons following temperature elevation. Stable lines of human SH-SY5Y neuronal cells were established that expressed YFP-tagged protein products of the human inducible HSP70 genes HSPA6 (HSP70B') and HSPA1A (HSP70-1). Following a brief period of thermal stress, YFP-tagged HSPA6 and HSPA1A rapidly appeared at centrioles in the cytoplasm of human neuronal cells, with HSPA6 demonstrating a more prolonged signal compared to HSPA1A. Each centriole is composed of a distal end and a proximal end, the latter linking the centriole doublet. The YFP-tagged HSP70 proteins targeted the proximal end of centrioles (identified by γ-tubulin marker) rather than the distal end (centrin marker). Centrioles play key roles in cellular polarity and migration during neuronal differentiation. The proximal end of the centriole, which is involved in centriole stabilization, may be stress-sensitive in post-mitotic, differentiating human neurons.

Intracellular dynamics of the Hsp90 co-chaperone p23 is dictated by Hsp90

International Nuclear Information System (INIS)

Picard, Didier

2006-01-01

p23 is a component of the Hsp90 molecular chaperone machine. It binds and stabilizes the ATP-bound dimeric form of Hsp90. Since Hsp90 binds protein substrates in the ATP conformation, p23 has been proposed to stabilize Hsp90-substrate complexes. In addition, p23 can also function as a molecular chaperone by itself and even possesses an unrelated enzymatic activity. Whether it fulfills the latter functions in cells while bound to Hsp90 remains unknown and is difficult to extrapolate from cell-free biochemical experiments. Using the 'fluorescence recovery after photobleaching' (FRAP) technology, I have examined the dynamics of human p23, expressed as a fusion protein with the green fluorescent protein (GFP), in living human HeLa cells. GFP-p23 is distributed throughout the cell, and its mobility is identical in the cytoplasm and in the nucleus. When the Hsp90 interaction is disrupted either with the Hsp90 inhibitor geldanamycin or by introduction of point mutations into p23, the mobility of p23 is greatly accelerated. Under these conditions, its intracellular movement may be diffusion-controlled. In contrast, when wild-type p23 is able to bind Hsp90, a more complex FRAP behavior is observed, suggesting that it is quantitatively bound in Hsp90 complexes undergoing a multitude of other interactions

Heat Shock Protein HSP27 Secretion by Ovarian Cancer Cells Is Linked to Intracellular Expression Levels, Occurs Independently of the Endoplasmic Reticulum Pathway and HSP27’s Phosphorylation Status, and Is Mediated by Exosome Liberation

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Matthias B. Stope

2017-01-01

Full Text Available The heat shock protein HSP27 has been correlated in ovarian cancer (OC patients with aggressiveness and chemoresistance and, therefore, represents a promising potential biomarker for OC diagnosis, prognosis, and treatment response. Notably, secretion of soluble HSP27 has been described by a few cell types and may take place as well in OC cells. Therefore, we studied HSP27 secretion mechanisms under diverse cellular conditions in an OC cell model system. Secretion of HSP27 was characterized after overexpression of HSP27 by transfected plasmids and after heat shock. Intra- and extracellular HSP27 amounts were assessed by Western blotting and ELISA. Protein secretion was blocked by brefeldin A and the impact of the HSP27 phosphorylation status was analyzed overexpressing HSP27 phosphomutants. The present study demonstrated that HSP27 secretion by OVCAR-3 and SK-OV-3 cells depends on intracellular HSP27 concentrations. Moreover, HSP27 secretion is independent of the endoplasmic reticulum secretory pathway and HSP27 phosphorylation. Notably, analysis of OC cell-born exosomes not only confirmed the concentration-dependent correlation of HSP27 expression and secretion but also demonstrated a concentration-dependent incorporation of HSP27 protein into exosomes. Thus, secreted HSP27 may become more important as an extracellular factor which controls the tumor microenvironment and might be a noninvasive biomarker.

HIF-1α-induced HSP70 regulates anabolic responses in articular chondrocytes under hypoxic conditions.

Science.gov (United States)

Tsuchida, Shinji; Arai, Yuji; Takahashi, Kenji A; Kishida, Tsunao; Terauchi, Ryu; Honjo, Kuniaki; Nakagawa, Shuji; Inoue, Hiroaki; Ikoma, Kazuya; Ueshima, Keiichiro; Matsuki, Tomohiro; Mazda, Osam; Kubo, Toshikazu

2014-08-01

We assessed whether heat shock protein 70 (HSP70) is involved in hypoxia inducible factor 1 alpha (HIF-1α)-dependent anabolic pathways in articular chondrocytes under hypoxic conditions. Primary rabbit chondrocytes were cultured under normoxia (20% oxygen condition) or hypoxia (1% oxygen condition). Alternatively, cells cultured under normoxia were treated with CoCl2 , which induces HIF-1α, to simulate hypoxia, or transfected with siRNAs targeting HIF-1α (si-HIF-1α) and HSP70 (si-HSP70) under hypoxia. HSP70 expression was enhanced by the increased expression of HIF-1α under hypoxia or simulated hypoxia, but not in the presence of si-HIF-1α. Hypoxia-induced overexpression of ECM genes was significantly suppressed by si-HIF-1α or si-HSP70. Cell viability positively correlated with hypoxia, but transfection with si-HIF-1α or si-HSP70 abrogated the chondroprotective effects of hypoxia. Although LDH release from sodium nitroprusside-treated cells and the proportion of TUNEL positive cells were decreased under hypoxia, transfection with si-HIF-1α or si-HSP70 almost completely blocked these effects. These findings indicated that HIF-1α-induced HSP70 overexpression increased the expression levels of ECM genes and cell viability, and protected chondrocytes from apoptosis. HIF-1α may regulate the anabolic effects of chondrocytes under hypoxic conditions by regulating HSP70 expression. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Long-Term Overexpression of Hsp70 Does Not Protect against Cardiac Dysfunction and Adverse Remodeling in a MURC Transgenic Mouse Model with Chronic Heart Failure and Atrial Fibrillation.

Science.gov (United States)

Bernardo, Bianca C; Sapra, Geeta; Patterson, Natalie L; Cemerlang, Nelly; Kiriazis, Helen; Ueyama, Tomomi; Febbraio, Mark A; McMullen, Julie R

2015-01-01

Previous animal studies had shown that increasing heat shock protein 70 (Hsp70) using a transgenic, gene therapy or pharmacological approach provided cardiac protection in models of acute cardiac stress. Furthermore, clinical studies had reported associations between Hsp70 levels and protection against atrial fibrillation (AF). AF is the most common cardiac arrhythmia presenting in cardiology clinics and is associated with increased rates of heart failure and stroke. Improved therapies for AF and heart failure are urgently required. Despite promising observations in animal studies which targeted Hsp70, we recently reported that increasing Hsp70 was unable to attenuate cardiac dysfunction and pathology in a mouse model which develops heart failure and intermittent AF. Given our somewhat unexpected finding and the extensive literature suggesting Hsp70 provides cardiac protection, it was considered important to assess whether Hsp70 could provide protection in another mouse model of heart failure and AF. The aim of the current study was to determine whether increasing Hsp70 could attenuate adverse cardiac remodeling, cardiac dysfunction and episodes of arrhythmia in a mouse model of heart failure and AF due to overexpression of Muscle-Restricted Coiled-Coil (MURC). Cardiac function and pathology were assessed in mice at approximately 12 months of age. We report here, that chronic overexpression of Hsp70 was unable to provide protection against cardiac dysfunction, conduction abnormalities, fibrosis or characteristic molecular markers of the failing heart. In summary, elevated Hsp70 may provide protection in acute cardiac stress settings, but appears insufficient to protect the heart under chronic cardiac disease conditions.

Long-Term Overexpression of Hsp70 Does Not Protect against Cardiac Dysfunction and Adverse Remodeling in a MURC Transgenic Mouse Model with Chronic Heart Failure and Atrial Fibrillation.

Directory of Open Access Journals (Sweden)

Bianca C Bernardo

Full Text Available Previous animal studies had shown that increasing heat shock protein 70 (Hsp70 using a transgenic, gene therapy or pharmacological approach provided cardiac protection in models of acute cardiac stress. Furthermore, clinical studies had reported associations between Hsp70 levels and protection against atrial fibrillation (AF. AF is the most common cardiac arrhythmia presenting in cardiology clinics and is associated with increased rates of heart failure and stroke. Improved therapies for AF and heart failure are urgently required. Despite promising observations in animal studies which targeted Hsp70, we recently reported that increasing Hsp70 was unable to attenuate cardiac dysfunction and pathology in a mouse model which develops heart failure and intermittent AF. Given our somewhat unexpected finding and the extensive literature suggesting Hsp70 provides cardiac protection, it was considered important to assess whether Hsp70 could provide protection in another mouse model of heart failure and AF. The aim of the current study was to determine whether increasing Hsp70 could attenuate adverse cardiac remodeling, cardiac dysfunction and episodes of arrhythmia in a mouse model of heart failure and AF due to overexpression of Muscle-Restricted Coiled-Coil (MURC. Cardiac function and pathology were assessed in mice at approximately 12 months of age. We report here, that chronic overexpression of Hsp70 was unable to provide protection against cardiac dysfunction, conduction abnormalities, fibrosis or characteristic molecular markers of the failing heart. In summary, elevated Hsp70 may provide protection in acute cardiac stress settings, but appears insufficient to protect the heart under chronic cardiac disease conditions.

Fisetin, a dietary flavonoid, induces apoptosis of cancer cells by inhibiting HSF1 activity through blocking its binding to the hsp70 promoter.

Science.gov (United States)

Kim, Joo Ae; Lee, Somyoung; Kim, Da-Eun; Kim, Moonil; Kwon, Byoung-Mog; Han, Dong Cho

2015-06-01

Heat shock factor 1 (HSF1) is a transcription factor for heat shock proteins (HSPs) expression that enhances the survival of cancer cells exposed to various stresses. HSF1 knockout suppresses carcinogen-induced cancer induction in mice. Therefore, HSF1 is a promising therapeutic and chemopreventive target. We performed cell-based screening with a natural compound collection and identified fisetin, a dietary flavonoid, as a HSF1 inhibitor. Fisetin abolished heat shock-induced luciferase activity with an IC50 of 14 μM in HCT-116 cancer cells. The treatment of HCT-116 with fisetin inhibited proliferation with a GI50 of 23 μM. When the cells were exposed to heat shock in the presence of fisetin, the induction of HSF1 target proteins, such as HSP70, HSP27 and BAG3 (Bcl-2-associated athanogene domain 3), were inhibited. HSP70/BAG3 complexes protect cancer cells from apoptosis by stabilizing anti-apoptotic Bcl-2 family proteins. The downregulation of HSP70/BAG3 by fisetin significantly reduced the amounts of Bcl-2, Bcl-xL and Mcl-1 proteins, subsequently inducing apoptotic cell death. Chromatin immunoprecipitation assays showed that fisetin inhibited HSF1 activity by blocking the binding of HSF1 to the hsp70 promoter. Intraperitoneal treatment of nude mice with fisetin at 30mg/kg resulted in a 35.7% (P < 0.001) inhibition of tumor growth. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Multifunctional selenium nanoparticles as carriers of HSP70 siRNA to induce apoptosis of HepG2 cells

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Li Y

2016-07-01

Full Text Available Yinghua Li,1 Zhengfang Lin,1 Mingqi Zhao,1 Tiantian Xu,1 Changbing Wang,1 Huimin Xia,1,* Hanzhong Wang,2,* Bing Zhu1,* 1Guangzhou Women and Children’s Medical Center, Guangzhou, Guangdong, 2State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People’s Republic of China *These authors contributed equally to this work Abstract: Small interfering RNA (siRNA as a new therapeutic modality holds promise for cancer treatment, but it is unable to cross cell membrane. To overcome this limitation, nanotechnology has been proposed for mediation of siRNA transfection. Selenium (Se is a vital dietary trace element for mammalian life and plays an essential role in the growth and functioning of humans. As a novel Se species, Se nanoparticles have attracted more and more attention for their higher anticancer efficacy. In the present study, siRNAs with polyethylenimine (PEI-modified Se nanoparticles (Se@PEI@siRNA have been demonstrated to enhance the apoptosis of HepG2 cells. Heat shock protein (HSP-70 is overexpressed in many types of human cancer and plays a significant role in several biological processes including the regulation of apoptosis. The objective of this study was to silence inducible HSP70 and promote the apoptosis of Se-induced HepG2 cells. Se@PEI@siRNA were successfully prepared and characterized by various microscopic methods. Se@PEI@siRNA showed satisfactory size distribution, high stability, and selectivity between cancer and normal cells. The cytotoxicity of Se@PEI@siRNA was lower for normal cells than tumor cells, indicating that these compounds may have fewer side effects. The gene-silencing efficiency of Se@PEI@siRNA was significantly much higher than Lipofectamine 2000@siRNA and resulted in a significantly reduced HSP70 mRNA and protein expression in cancer cells. When the expression of HSP70 was diminished, the function of cell protection was also removed and cancer cells became more

[The Role of Membrane-Bound Heat Shock Proteins Hsp90 in Migration of Tumor Cells in vitro and Involvement of Cell Surface Heparan Sulfate Proteoglycans in Protein Binding to Plasma Membrane].

Science.gov (United States)

Snigireva, A V; Vrublevskaya, V V; Skarga, Y Y; Morenkov, O S

2016-01-01

Heat shock protein Hsp90, detected in the extracellular space and on the membrane of cells, plays an important role in cell motility, migration, invasion and metastasis of tumor cells. At present, the functional role and molecular mechanisms of Hsp90 binding to plasma membrane are not elucidated. Using isoform-specific antibodies against Hsp90, Hsp9α and Hsp90β, we showed that membrane-bound Hsp90α and Hsp90β play a significant role in migration of human fibrosarcoma (HT1080) and glioblastoma (A-172) cells in vitro. Disorders of sulfonation of cell heparan sulfates, cleavage of cell heparan. sulfates by heparinase I/III as well as treatment of cells with heparin lead to an abrupt reduction in the expression level of Hsp90 isoforms. Furthermore, heparin significantly inhibits tumor cell migration. The results obtained demonstrate that two isoforms of membrane-bound Hsp90 are involved in migration of tumor cells in vitro and that cell surface heparan sulfate proteoglycans play a pivotal role in the "anchoring" of Hsp90α and Hsp90β to the plasma membrane.

Identification of the key structural motifs involved in HspB8/HspB6-Bag3 interaction

NARCIS (Netherlands)

Fuchs, Margit; Poirier, Dominic J.; Seguin, Samuel J.; Lambert, Herman; Carra, Serena; Charette, Steve J.; Landry, Jacques

2010-01-01

The molecular chaperone HspB8 [Hsp (heat-shock protein) B8] is member of the B-group of Hsps. These proteins bind to unfolded or misfolded proteins and protect them from aggregation. HspB8 has been reported to form a stable molecular complex with the chaperone cohort protein Bag3 (Bcl-2-associated

SPRINT-INTERVAL TRAINING INDUCES HEAT SHOCK PROTEIN 72 IN RAT SKELETAL MUSCLES

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Yuji Ogura

2006-06-01

Full Text Available Previous studies have demonstrated that endurance exercise training increases the level of heat shock proteins (HSPs in skeletal muscles. However, little attention has been drawn to the effects of high intensity-short duration exercise, or sprint- interval training (SIT on HSP72 level in rat skeletal muscles. This study performed to test the hypothesis that the SIT would induce the HSP72 in fast and slow skeletal muscles of rats. Young male Wistar rats (8 weeks old were randomly assigned to a control (CON or a SIT group (n = 8/group. Animals in the SIT group were trained (1 min/sprint, 6~10 sets/day and 5~6 days/week on a treadmill for 9 weeks. After the training period, HSP72 levels in the plantaris (fast and soleus (slow muscles were analyzed by Western blotting method. Enzyme activities (hexokinase, phosphofructokinase and citrate synthase and histochemical properties (muscle fiber type compositions and cross sectional area in both muscles were also determined. The SIT resulted in significantly (p < 0.05 higher levels of HSP72 in both the plantaris and soleus muscles compared to the CON group, with the plantaris producing a greater HSP72 increase than the soleus (plantaris; 550 ± 116%, soleus; 26 ± 8%, p < 0.05. Further, there were bioenergetic improvements, fast-to-slow shift of muscle fiber composition and hypertrophy in the type IIA fiber only in the plantaris muscle. These findings indicate that the SIT program increases HSP72 level of the rat hindlimb muscles, and the SIT-induced accumulation of HSP72 differs between fast and slow muscles

Involvement of p27CIP/KIP in HSP25 or HSP70 Mediated Adaptive Response by Low Dose Radiation

International Nuclear Information System (INIS)

Seo, Hang Rhan; Lee, Yoon Jin; Lee, Su Jae; Bae, Sang woo; Lee, Yun Sil

2005-01-01

Adaptive responses that reduce the harmful effects of subsequent exposure to high-dose radiation have demonstrated in chromosome aberration, cell survival, sister chromatid exchanges, micronucleus induction, mutation and neoplastic transformation. The mechanisms and conditions for the adaptive response to radiation have not been clarified, although the continuous production of free radicals from radiation and other sources has stimulated cells to evolve a repair system for chromosome breaks. An alteration of the DNA molecule triggers the repair system, and frequent activation may increase the general repair capacity, irrespective of the cause of the damage. Besides, cell cycle regulation systems, antioxidant defense systems, molecular chaperone or stress-response systems. Our previous data showed that when cells were preirradiated with 1cGy, they showed the adaptive response. A reduction of apoptosis by low-dose preirradiation is another potential mechanism for this effect. We previously demonstrated that mouse RIF cells, which did not induce HSP25 and HSP70 did not exhibit a adaptive response after 1cGy preirradiation. whereas the thermoresistant TR cells, which expressed inducible HSP25 and HSP70 showed a response. Moreover, when HSP70 and HSP25 were transfected to RIF cells, the cells acquired adaptive response. In this study, to elucidate the mechanisms in induction of adaptiveresponse, we compared cell cycle distribution by low dose radiation after HSP25 or HSP70 transfected cells and p27CIP/KIP is responsible for the different induction of adaptive response

Imaging of Hsp70-positive tumors with cmHsp70.1 antibody-conjugated gold nanoparticles

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Gehrmann MK

2015-09-01

Full Text Available Mathias K Gehrmann,1 Melanie A Kimm,2 Stefan Stangl,1 Thomas E Schmid,1 Peter B Noël,2 Ernst J Rummeny,2 Gabriele Multhoff11Department of Radiation Oncology, 2Department of Diagnostic and Interventional Radiology, Klinikum rechts der Isar, Technische Universität München, Munich, GermanyAbstract: Real-time imaging of small tumors is still one of the challenges in cancer diagnosis, prognosis, and monitoring of clinical outcome. Targeting novel biomarkers that are selectively expressed on a large variety of different tumors but not normal cells has the potential to improve the imaging capacity of existing methods such as computed tomography. Herein, we present a novel technique using cmHsp70.1 monoclonal antibody-conjugated spherical gold nanoparticles for quantification of the targeted uptake of gold nanoparticles into membrane Hsp70-positive tumor cells. Upon binding, cmHsp70.1-conjugated gold nanoparticles but not nanoparticles coupled to an isotype-matched IgG1 antibody or empty nanoparticles are rapidly taken up by highly malignant Hsp70 membrane-positive mouse tumor cells. After 24 hours, the cmHsp70.1-conjugated gold nanoparticles are found to be enriched in the perinuclear region. Specificity for membrane Hsp70 was shown by using an Hsp70 knockout tumor cell system. Toxic side effects of the cmHsp70.1-conjugated nanoparticles are not observed at a concentration of 1–10 µg/mL. Experiments are ongoing to evaluate whether cmHsp70.1 antibody-conjugated gold nanoparticles are suitable for the detection of membrane-Hsp70-positive tumors in vivo.Keywords: heat shock protein 70, tumor biomarker, theranostics, multimodal CT, multispectral CT, k-edge

Hsp90 inhibitors reduce influenza virus replication in cell culture

International Nuclear Information System (INIS)

Chase, Geoffrey; Deng, Tao; Fodor, Ervin; Leung, B.W.; Mayer, Daniel; Schwemmle, Martin; Brownlee, George

2008-01-01

The viral RNA polymerase complex of influenza A virus consists of three subunits PB1, PB2 and PA. Recently, the cellular chaperone Hsp90 was shown to play a role in nuclear import and assembly of the trimeric polymerase complex by binding to PB1 and PB2. Here we show that Hsp90 inhibitors, geldanamycin or its derivative 17-AAG, delay the growth of influenza virus in cell culture resulting in a 1-2 log reduction in viral titre early in infection. We suggest that this is caused by the reduced half-life of PB1 and PB2 and inhibition of nuclear import of PB1 and PA which lead to reduction in viral RNP assembly. Hsp90 inhibitors may represent a new class of antiviral compounds against influenza viruses

[The impact of melafen on the expression of chloroplastic chaperone protein HSP70B and photosynthetic pigments in cells of Chlamydomonas reinhardtii].

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Ermokhina, O V; Belkina, G G; Oleskina, Iu P; Fattakhov, S G; Iurina, N P

2009-01-01

The effects of growth regulator of the new generation-melamine salt of bis(oxymethyl)phosphine acid (melafen)--on culture growth, pigment and protein content, and the induction of protective chloroplastic chaperone HSP70B in Chlamydomonas reinhardtii CW15 cells were studied. Melafen exhibited 10-30% growth inhibition at 10(-9)-10(-2)% concentration. At 10(-9)-10(-4)% of melafen electrophoretic concentration, the pattern of cellular proteins was similar to the control. The alterations in protein content of algae cells were detected only at 10(-2)% concentration. The content of chlorophyll and carotenoids in melafen-treated cells was 17-40% lower than in the control. Melafen at 10(-9)-109-2)% concentration inhibited HSP70B induction by 39-43% compared to untreated cells. The potential mechanism of melafen effect might involve its influence on nuclear gene expression.

Phloroglucinol-Mediated Hsp70 Production in Crustaceans: Protection against Vibrio parahaemolyticus in Artemia franciscana and Macrobrachium rosenbergii

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Kumar, Vikash; Baruah, Kartik; Nguyen, Dung Viet; Smagghe, Guy; Vossen, Els; Bossier, Peter

2018-01-01

The halophilic aquatic bacterium, Vibrio parahaemolyticus, is an important aquatic pathogen, also capable of causing acute hepatopancreatic necrosis disease (AHPND) in shrimp resulting in significant economic losses. Therefore, there is an urgent need to develop anti-infective strategies to control AHPND. The gnotobiotic Artemia model is used to establish whether a phenolic compound phloroglucinol is effective against the AHPND strain V. parahaemolyticus MO904. We found that pretreatment with phloroglucinol, at an optimum concentration (30 µM), protects axenic brine shrimp larvae against V. parahaemolyticus infection and induced heat shock protein 70 (Hsp70) production (twofolds or more) as compared with the control. We further demonstrated that the Vibrio-protective effect of phloroglucinol was caused by its prooxidant effect and is linked to the induction of Hsp70. In addition, RNA interference confirms that phloroglucinol-induced Hsp70 mediates the survival of brine shrimp larvae against V. parahaemolyticus infection. The study was validated in xenic Artemia model and in a Macrobrachium rosenbergii system. Pretreatment of xenic brine shrimp larvae (30 µM) and Macrobrachium larvae (5 µM) with phloroglucinol increases the survival of xenic brine shrimp and Macrobrachium larvae against subsequent V. parahaemolyticus challenge. Taken together, our study provides substantial evidence that the prooxidant activity of phloroglucinol induces Hsp70 production protecting brine shrimp, A. franciscana, and freshwater shrimp, M. rosenbergii, against the AHPND V. parahaemolyticus strain MO904. Probably, phloroglucinol treatment might become part of a holistic strategy to control AHPND in shrimp.

Phloroglucinol-Mediated Hsp70 Production in Crustaceans: Protection against Vibrio parahaemolyticus in Artemia franciscana and Macrobrachium rosenbergii

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Vikash Kumar

2018-05-01

Full Text Available The halophilic aquatic bacterium, Vibrio parahaemolyticus, is an important aquatic pathogen, also capable of causing acute hepatopancreatic necrosis disease (AHPND in shrimp resulting in significant economic losses. Therefore, there is an urgent need to develop anti-infective strategies to control AHPND. The gnotobiotic Artemia model is used to establish whether a phenolic compound phloroglucinol is effective against the AHPND strain V. parahaemolyticus MO904. We found that pretreatment with phloroglucinol, at an optimum concentration (30 µM, protects axenic brine shrimp larvae against V. parahaemolyticus infection and induced heat shock protein 70 (Hsp70 production (twofolds or more as compared with the control. We further demonstrated that the Vibrio-protective effect of phloroglucinol was caused by its prooxidant effect and is linked to the induction of Hsp70. In addition, RNA interference confirms that phloroglucinol-induced Hsp70 mediates the survival of brine shrimp larvae against V. parahaemolyticus infection. The study was validated in xenic Artemia model and in a Macrobrachium rosenbergii system. Pretreatment of xenic brine shrimp larvae (30 µM and Macrobrachium larvae (5 µM with phloroglucinol increases the survival of xenic brine shrimp and Macrobrachium larvae against subsequent V. parahaemolyticus challenge. Taken together, our study provides substantial evidence that the prooxidant activity of phloroglucinol induces Hsp70 production protecting brine shrimp, A. franciscana, and freshwater shrimp, M. rosenbergii, against the AHPND V. parahaemolyticus strain MO904. Probably, phloroglucinol treatment might become part of a holistic strategy to control AHPND in shrimp.

Whey protein hydrolysate enhances HSP90 but does not alter HSP60 and HSP25 in skeletal muscle of rats.

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Carolina Soares Moura

Full Text Available Whey protein hydrolysate (WPH intake has shown to increase HSP70 expression. The aim of the present study was to investigate whether WPH intake would also influences HSP90, HSP60 and HSP25 expression, as well as associated parameters. Forty-eight male Wistar rats were divided into sedentary (unstressed and exercised (stressed groups, and were fed with three different sources of protein: whey protein (WP, whey protein hydrolysate (WPH and casein (CAS as a control, based on the AIN93G diet for 3 weeks. WPH intake increased HSP90 expression in both sedentary and exercised animals compared to WP or CAS, however no alteration was found from exercise or diet to HSP60 or HSP25. Co-chaperone Aha1 and p-HSF1 were also increased in the exercised animals fed with WPH in comparison with WP or CAS, consistent with enhanced HSP90 expression. VEGF and p-AKT were increased in the WPH exercised group. No alteration was found in BCKDH, PI3-Kinase (p85, GFAT, OGT or PGC for diet or exercise. The antioxidant system GPx, catalase and SOD showed different responses to diet and exercise. The data indicate that WPH intake enhanced factors related to cell survival, such as HSP90 and VEGF, but does not alter HSP60 or HSP25 in rat skeletal muscle.

The expression of HSP60 and HSP10 in large bowel carcinomas with lymph node metastase

International Nuclear Information System (INIS)

Cappello, Francesco; David, Sabrina; Rappa, Francesca; Bucchieri, Fabio; Marasà, Lorenzo; Bartolotta, Tommaso E; Farina, Felicia; Zummo, Giovanni

2005-01-01

The involvement of Heat Shock Proteins (HSP) in cancer development and progression is a widely debated topic. The objective of the present study was to evaluate the presence and expression of HSP60 and HSP10 in a series of large bowel carcinomas and locoregional lymph nodes with and without metastases. 82 Astler and Coller's stage C2 colorectal cancers, of which 48 well-differentiated and 34 poorly-differentiated, were selected along with 661 lymph nodes, including 372 with metastases and 289 with reactive hyperplasia only, from the same tumours. Primitive tumours and both metastatic and reactive lymph nodes were studied; specifically, three different compartments of the lymph nodes, secondary follicle, paracortex and medullary sinus, were also analysed. An immunohistochemical research for HSP60 and HSP10 was performed and the semiquantitative results were analysed by statistical analysis to determine the correlation between HSPs expression and 1) tumour grading; 2) degree of inflammation; 3) number of lymph nodes involved; 4) lymph node compartment hyperplasia. Moreover, western blotting was performed on a smaller group of samples to confirm the immunohistochemical results. Our data show that the expression of HSP60, in both primary tumour and lymph node metastasis, is correlated with the tumoral grade, while the HSP10 expression is not. Nevertheless, the levels of HSP10 are commonly higher than the levels of HSP60. In addition, statistical analyses do not show any correlation between the degree of inflammation and the immunopositivity for both HSP60 and HSP10. Moreover, we find a significant correlation between the presence of lymph node metastases and the positivity for both HSP60 and HSP10. In particular, metastatic lymph nodes show a higher percentage of cells positive for both HSP60 and HSP10 in the secondary follicles, and for HSP10 in the medullary sinuses, when compared with hyperplastic lymph nodes. HSP60 and HSP10 may have diagnostic and prognostic

Production and immune response of recombinant Hsp60 and Hsp70 from the salmon pathogen Piscirickettsia salmonis

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VIVIAN WILHELM

2005-01-01

Full Text Available We have isolated and sequenced the genes encoding the heat shock proteins 60 (Hsp60 and 70 (Hsp70 of the salmon pathogen Piscirickettsia salmonis. The sequence analysis revealed the expected two open reading frames that encode proteins with calculated molecular weights of 60,060 and 70,400. The proteins exhibit a 70-80% homology with other known prokaryotic Hsp60 and Hsp70 sequences. The coding regions have been expressed in E. coli as thioredoxin fusion proteins. Both recombinant proteins were shown to elicit a humoral response when injected intraperitoneally in Atlantic salmon and also conferred protection to fish challenged with P. salmonis. The present data will facilitate further studies on the involvement of heat shock proteins in protective immunity of fish to infection by P. salmonis and their potential use in recombinants vaccines against this intracellular pathogen.

The HSP90 Inhibitor Ganetespib Radiosensitizes Human Lung Adenocarcinoma Cells

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Roberto Gomez-Casal

2015-05-01

Full Text Available The molecular chaperone HSP90 is involved in stabilization and function of multiple client proteins, many of which represent important oncogenic drivers in NSCLC. Utilization of HSP90 inhibitors as radiosensitizing agents is a promising approach. The antitumor activity of ganetespib, HSP90 inhibitor, was evaluated in human lung adenocarcinoma (AC cells for its ability to potentiate the effects of IR treatment in both in vitro and in vivo. The cytotoxic effects of ganetespib included; G2/M cell cycle arrest, inhibition of DNA repair, apoptosis induction, and promotion of senescence. All of these antitumor effects were both concentration- and time-dependent. Both pretreatment and post-radiation treatment with ganetespib at low nanomolar concentrations induced radiosensitization in lung AC cells in vitro. Ganetespib may impart radiosensitization through multiple mechanisms: such as down regulation of the PI3K/Akt pathway; diminished DNA repair capacity and promotion of cellular senescence. In vivo, ganetespib reduced growth of T2821 tumor xenografts in mice and sensitized tumors to IR. Tumor irradiation led to dramatic upregulation of β-catenin expression in tumor tissues, an effect that was mitigated in T2821 xenografts when ganetespib was combined with IR treatments. These data highlight the promise of combining ganetespib with IR therapies in the treatment of AC lung tumors.

Glutamine's protection against cellular injury is dependent on heat shock factor-1.

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Morrison, Angela L; Dinges, Martin; Singleton, Kristen D; Odoms, Kelli; Wong, Hector R; Wischmeyer, Paul E

2006-06-01

Glutamine (GLN) has been shown to protect cells, tissues, and whole organisms from stress and injury. Enhanced expression of heat shock protein (HSP) has been hypothesized to be responsible for this protection. To date, there are no clear mechanistic data confirming this relationship. This study tested the hypothesis that GLN-mediated activation of the HSP pathway via heat shock factor-1 (HSF-1) is responsible for cellular protection. Wild-type HSF-1 (HSF-1(+/+)) and knockout (HSF-1(-/-)) mouse fibroblasts were used in all experiments. Cells were treated with GLN concentrations ranging from 0 to 16 mM and exposed to heat stress injury in a concurrent treatment model. Cell viability was assayed with phenazine methosulfate plus tetrazolium salt, HSP-70, HSP-25, and nuclear HSF-1 expression via Western blot analysis, and HSF-1/heat shock element (HSE) binding via EMSA. GLN significantly attenuated heat-stress induced cell death in HSF-1(+/+) cells in a dose-dependent manner; however, the survival benefit of GLN was lost in HSF-1(-/-) cells. GLN led to a dose-dependent increase in HSP-70 and HSP-25 expression after heat stress. No inducible HSP expression was observed in HSF-1(-/-) cells. GLN increased unphosphorylated HSF-1 in the nucleus before heat stress. This was accompanied by a GLN-mediated increase in HSF-1/HSE binding and nuclear content of phosphorylated HSF-1 after heat stress. This is the first demonstration that GLN-mediated cellular protection after heat-stress injury is related to HSF-1 expression and cellular capacity to activate an HSP response. Furthermore, the mechanism of GLN-mediated protection against injury appears to involve an increase in nuclear HSF-1 content before stress and increased HSF-1 promoter binding and phosphorylation.

Effect of vibrational stress and spaceflight on regulation of heat shock proteins hsp70 and hsp27 in human lymphocytes (Jurkat)

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Cubano, L. A.; Lewis, M. L.

2001-01-01

Heat shock protein levels are increased in cells as a result of exposure to stress. To determine whether heat shock protein regulation could be used to evaluate stress in cells during spaceflight, the response of Jurkat cells to spaceflight and simulated space shuttle launch vibration was investigated by evaluating hsp70 and hsp27 gene expression. Gene expression was assessed by reverse transcription-polymerase chain reaction using mRNA extracted from vibrated, nonvibrated, space-flown, and ground control cells. Results indicate that mechanical stresses of vibration and low gravity do not up-regulate the mRNA for hsp70, although the gene encoding hsp27 is up-regulated by spaceflight but not by vibration. In ground controls, the mRNA for hsp70 and hsp27 increased with time in culture. We conclude that hsp70 gene expression is a useful indicator of stress related to culture density but is not an indicator of the stresses of launch vibration or microgravity. Up-regulation of hsp27 gene expression in microgravity is a new finding.

Nickel, lead, and cadmium induce differential cellular responses in sea urchin embryos by activating the synthesis of different HSP70s

International Nuclear Information System (INIS)

Geraci, Fabiana; Pinsino, Annalisa; Turturici, Guiseppina; Savona, Rosalia; Giudice, Giovanni; Sconzo, Gabriella

2004-01-01

Treatment with heavy metals, such as nickel, lead or cadmium, elicits different cellular stress responses according to the metal used and the length of treatment. In Paracentrotus lividus embryos the inducible forms of HSP70 (HSP70/72) are different in molecular mass from the constitutively expressed HSP75, and they can be used as markers of cellular stress. Even a short treatment with each metal induces the synthesis of HSP70/72 which remain stable for at least 20 h and differ little in their isoelectric points. Continuous treatment from fertilization with nickel or lead produces late irregular pluteus embryos, with peak HSP70/72 synthesis at blastula followed by the arrest of synthesis by pluteus. On the contrary, the same treatment with cadmium induces continuous HSP70/72 synthesis and produces irregular gastrula embryos which then degenerate. Moreover, a long treatment induces over control embryos a slight increase in the amount of constitutive HSP75 during development while lead treatment depresses constitutive HSP75 at early stages and doubles its quantity at late stages

Exercise induces the release of heat shock protein 72 from the human brain in vivo

OpenAIRE

Lancaster, G. I.; Møller, K.; Nielsen, B.; Secher, N. H.; Febbraio, M. A.; Nybo, L.

2004-01-01

The present study tested the hypothesis that in response to physical stress the human brain has the capacity to release heat shock protein 72 (Hsp72) in vivo. Therefore, 6 humans (males) cycled for 180 minutes at 60% of their maximal oxygen uptake, and the cerebral Hsp72 response was determined on the basis of the internal jugular venous to arterial difference and global cerebral blood flow. At rest, there was a net balance of Hsp72 across the brain, but after 180 minutes of exercise, we were...

Hsp90 inhibition differentially destabilises MAP kinase and TGF-beta signalling components in cancer cells revealed by kinase-targeted chemoproteomics

International Nuclear Information System (INIS)

Haupt, Armin; Dahl, Andreas; Lappe, Michael; Lehrach, Hans; Gonzalez, Cayetano; Drewes, Gerard; Lange, Bodo MH; Joberty, Gerard; Bantscheff, Marcus; Fröhlich, Holger; Stehr, Henning; Schweiger, Michal R; Fischer, Axel; Kerick, Martin; Boerno, Stefan T

2012-01-01

The heat shock protein 90 (Hsp90) is required for the stability of many signalling kinases. As a target for cancer therapy it allows the simultaneous inhibition of several signalling pathways. However, its inhibition in healthy cells could also lead to severe side effects. This is the first comprehensive analysis of the response to Hsp90 inhibition at the kinome level. We quantitatively profiled the effects of Hsp90 inhibition by geldanamycin on the kinome of one primary (Hs68) and three tumour cell lines (SW480, U2OS, A549) by affinity proteomics based on immobilized broad spectrum kinase inhibitors ('kinobeads'). To identify affected pathways we used the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway classification. We combined Hsp90 and proteasome inhibition to identify Hsp90 substrates in Hs68 and SW480 cells. The mutational status of kinases from the used cell lines was determined using next-generation sequencing. A mutation of Hsp90 candidate client RIPK2 was mapped onto its structure. We measured relative abundances of > 140 protein kinases from the four cell lines in response to geldanamycin treatment and identified many new potential Hsp90 substrates. These kinases represent diverse families and cellular functions, with a strong representation of pathways involved in tumour progression like the BMP, MAPK and TGF-beta signalling cascades. Co-treatment with the proteasome inhibitor MG132 enabled us to classify 64 kinases as true Hsp90 clients. Finally, mutations in 7 kinases correlate with an altered response to Hsp90 inhibition. Structural modelling of the candidate client RIPK2 suggests an impact of the mutation on a proposed Hsp90 binding domain. We propose a high confidence list of Hsp90 kinase clients, which provides new opportunities for targeted and combinatorial cancer treatment and diagnostic applications

Systematic Proteomic Identification of the Heat Shock Proteins (Hsp) that Interact with Estrogen Receptor Alpha (ERα) and Biochemical Characterization of the ERα-Hsp70 Interaction.

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Dhamad, Ahmed E; Zhou, Zhenqi; Zhou, Jianhong; Du, Yuchun

2016-01-01

Heat shock proteins (Hsps) are known to associate with estrogen receptors (ER) and regulate ER-mediated cell proliferation. Historically, the studies in this area have focused on Hsp90. However, some critical aspects of the Hsp-ERα interactions remain unclear. For example, we do not know which Hsps are the major or minor ERα interactants and whether or not different Hsp isoforms associate equally with ERα. In the present study, through a quantitative proteomic method we found that 21 Hsps and 3 Hsp cochaperones were associated with ERα in human 293T cells that were cultured in a medium containing necessary elements for cell proliferation. Four Hsp70s (Hsp70-1, Hsc70, Grp75, and Grp78) were the most abundant Hsps identified to associate with ERα, followed by two Hsp90s (Hsp90α and Hsp90β) and three Hsp110s (Hsp105, HspA4, and HspA4L). Hsp90α was found to be 2-3 times more abundant than Hsp90β in the ERα-containing complexes. Among the reported Hsp cochaperones, we detected prostaglandin E synthase 3 (p23), peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP51), and E3 ubiquitin-protein ligase CHIP (CHIP). Studies with the two most abundant ERα-associated Hsps, Hsp70-1 and Hsc70, using human breast cancer MCF7 cells demonstrate that the two Hsps interacted with ERα in both the cytoplasm and nucleus when the cells were cultured in a medium supplemented with fetal bovine serum and phenol red. Interestingly, the ERα-Hsp70-1/Hsc70 interactions were detected only in the cytoplasm but not in the nucleus under hormone starvation conditions, and stimulation of the starved cells with 17β-estradiol (E2) did not change this. In addition, E2-treatment weakened the ERα-Hsc70 interaction but had no effect on the ERα-Hsp70-1 interaction. Further studies showed that significant portions of Hsp70-1 and Hsc70 were associated with transcriptionally active chromatin and inactive chromatin, and the two Hsps interacted with ERα in both forms of the chromatins in MCF7 cells.

Cathepsin B is involved in the heat shock induced cardiomyocytes apoptosis as well as the anti-apoptosis effect of HSP-70.

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Hsu, Shu-Fen; Hsu, Chuan-Chih; Cheng, Bor-Chih; Lin, Cheng-Hsien

2014-11-01

Cathepsin B is one of the major lysosomal cysteine proteases that plays an important role in apoptosis. Herein, we investigated whether Cathepsin B is involved in cardiomyocyte apoptosis caused by hyperthermic injury (HI) and heat shock protein (HSP)-70 protects these cells from HI-induced apoptosis mediated by Cathepsin. HI was produced in H9C2 cells by putting them in a circulating 43 °C water bath for 120 min, whereas preinduction of HSP-70 was produced in H9C2 cells by mild heat preconditioning (or putting them in 42 °C water bath for 30 min) 8 h before the start of HI. It was found that HI caused both cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. E-64-c, in addition to reducing Cathepsin B activity, significantly attenuated HI-induced cardiomyocyte apoptosis (evidenced by increased apoptotic cell numbers, increased tuncated Bid (t-Bid), increased cytochrome C, increased caspase-9/-3, and decreased Bcl-2/Bax) in H9C2 cells. In addition, preinduction of HSP-70 by mild heat preconditioning or inhibition of HSP-70 by Tripolide significantly attenuated or exacerbated respectively both the cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. Furthermore, the beneficial effects of pre-induction of HSP-70 by mild heat production in reducing both cardiomyocyte apoptosis and increased Cathepsin B activity caused by HI can be significantly reduced by Triptolide preconditioning. These results indicate that Cathepsin B is involved in HI-induced cardiomyocyte apoptosis in H9C2 cells and HSP-70 protects these cells from HI-induced cardiomyocyte apoptosis through Cathepsin B pathways.

Downregulation of the Hsp90 system causes defects in muscle cells of Caenorhabditis elegans.

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Andreas M Gaiser

Full Text Available The ATP-dependent molecular chaperone Hsp90 is required for the activation of a variety of client proteins involved in various cellular processes. Despite the abundance of known client proteins, functions of Hsp90 in the organismal context are not fully explored. In Caenorhabditis elegans, Hsp90 (DAF-21 has been implicated in the regulation of the stress-resistant dauer state, in chemosensing and in gonad formation. In a C. elegans strain carrying a DAF-21 mutation with a lower ATP turnover, we observed motility defects. Similarly, a reduction of DAF-21 levels in wild type nematodes leads to reduced motility and induction of the muscular stress response. Furthermore, aggregates of the myosin MYO-3 are visible in muscle cells, if DAF-21 is depleted, implying a role of Hsp90 in the maintenance of muscle cell functionality. Similar defects can also be observed upon knockdown of the Hsp90-cochaperone UNC-45. In life nematodes YFP-DAF-21 localizes to the I-band and the M-line of the muscular ultrastructure, but the protein is not stably attached there. The Hsp90-cofactor UNC-45-CFP contrarily can be found in all bands of the nematode muscle ultrastructure and stably associates with the UNC-54 containing A-band. Thus, despite the physical interaction between DAF-21 and UNC-45, apparently the two proteins are not always localized to the same muscular structures. While UNC-45 can stably bind to myofilaments in the muscular ultrastructure, Hsp90 (DAF-21 appears to participate in the maintenance of muscle structures as a transiently associated diffusible factor.

Early Response Roles for Prolactin Cortisol and Circulating and Cellular Levels of Heat Shock Proteins 72 and 90α in Severe Sepsis and SIRS

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K. Vardas

2014-01-01

Full Text Available Objective. To evaluate the early heat shock protein (HSP and hormonal stress response of intensive care unit (ICU patients with severe sepsis/septic shock (SS or systemic inflammatory response syndrome (SIRS compared to healthy subjects (H. Methods. Patients with early (first 48 hrs SS (n=29 or SIRS (n=29 admitted to a university ICU and 16 H were enrolled in the study. Serum prolactin, cortisol, and plasma ACTH were determined using immunoassay analyzers. ELISA was used to evaluate extracellular HSPs (eHSP90α, eHSP72 and interleukins. Mean fluorescence intensity (MFI values for intracellular HSPs (iHSP72, iHSP90α were measured using 4-colour flow-cytometry. Results. Prolactin, cortisol, and eHSP90α levels were significantly increased in SS patients compared to SIRS and H (P<0.003. ACTH and eHSP72 were significantly higher in SS and SIRS compared to H (P<0.005. SS monocytes expressed lower iHSP72 MFI levels compared to H (P=0.03. Prolactin was related with SAPS III and APACHE II scores and cortisol with eHSP90α, IL-6, and lactate (P<0.05. In SS and SIRS eHSP90α was related with eHSP72, IL-6, and IL-10. Conclusion. Prolactin, apart from cortisol, may have a role in the acute stress response in severe sepsis. In this early-onset inflammatory process, cortisol relates to eHSP90α, monocytes suppress iHSP72, and plasma eHSP72 increases.

Epigallocatechin-3-gallate suppresses the expression of HSP70 and HSP90 and exhibits anti-tumor activity in vitro and in vivo

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Tran, Phan LCHB; Kim, Soo-A; Choi, Hong Seok; Yoon, Jung-Hoon; Ahn, Sang-Gun

2010-01-01

Epigallocatechin-3-gallate (EGCG), one of the major catechins in green tea, is a potential chemopreventive agent for various cancers. The aim of this study was to examine the effect of EGCG on the expression of heat shock proteins (HSPs) and tumor suppression. Cell colony formation was evaluated by a soft agar assay. Transcriptional activity of HSP70 and HSP90 was determined by luciferase reporter assay. An EGCG-HSPs complex was prepared using EGCG attached to the cyanogen bromide (CNBr)-activated Sepharose 4B. In vivo effect of EGCG on tumor growth was examined in a xenograft model. Treatment with EGCG decreased cell proliferation and colony formation of MCF-7 human breast cancer cells. EGCG specifically inhibited the expression of HSP70 and HSP90 by inhibiting the promoter activity of HSP70 and HSP90. Pretreatment with EGCG increased the stress sensitivity of MCF-7 cells upon heat shock (44°C for 1 h) or oxidative stress (H 2 O 2 , 500 μM for 24 h). Moreover, treatment with EGCG (10 mg/kg) in a xenograft model resulted in delayed tumor incidence and reduced tumor size, as well as the inhibition of HSP70 and HSP90 expression. Overall, these findings demonstrate that HSP70 and HSP90 are potent molecular targets of EGCG and suggest EGCG as a drug candidate for the treatment of human cancer

Targeting HSP90 and monoclonal protein trafficking modulates the unfolded protein response, chaperone regulation and apoptosis in myeloma cells

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Born, E J; Hartman, S V; Holstein, S A

2013-01-01

Multiple myeloma is characterized by the production of substantial quantities of monoclonal protein. We have previously demonstrated that select inhibitors of the isoprenoid biosynthetic pathway (IBP) induce apoptosis of myeloma cells via inhibition of Rab geranylgeranylation, leading to disruption of monoclonal protein trafficking and induction of the unfolded protein response (UPR) pathway. Heat-shock protein 90 (HSP90) inhibitors disrupt protein folding and are currently under clinical investigation in myeloma. The effects of combining IBP and HSP90 inhibitors on cell death, monoclonal protein trafficking, the UPR and chaperone regulation were investigated in monoclonal protein-producing cells. An enhanced induction of cell death was observed following treatment with IBP and HSP90 inhibitors, which occurred through both ER stress and non-ER stress pathways. The HSP90 inhibitor 17-AAG abrogated the effects of the IBP inhibitors on intracellular monoclonal protein levels and localization as well as induction of the UPR in myeloma cells. Disparate effects on chaperone expression were observed in myeloma vs amyloid light chain cells. Here we demonstrate that the novel strategy of targeting MP trafficking in concert with HSP90 enhances myeloma cell death via a complex modulation of ER stress, UPR, and cell death pathways

Modification of HSP proteins and Ca2+ are responsible for the NO-derived peroxynitrite mediated neurological damage in PC12 cell.

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Wen, Jun; Li, Hua; Zhang, Yudan; Li, Xia; Liu, Fang

2015-01-01

Peroxynitrite as one crucial metabolite of NO-derived agents has been well multi-investigated to inspect its potential role and sought to define its concrete mechanism underlying the memory loss and impaired cognition involved in pathological processes. In this investigation, the cell viability was assessed by the MTT assay. The neurotoxicity of peroxynitrite was analyzed by using immunohistochemical measurements in cultured PC12 cells to explore the underlying mechanisms. The generation of ROS was evaluated by a fluorometry assay by a fluorometry assay. Apoptosis was assayed by annexin V-FITC and PI staining with flow cytometry. [Ca2+]i was examined by using the microspectrofluorometer. Hsp70 was detected by western blot assay. The results revealed that PC12 cells were inhibited by peroxynitrite both in a dose-dependent and time-dependent manner. The level of ROS in PC12 cells exposed to SIN-1 was increased in a dose-dependent manner. The result indicated that the SIN-1 induced apoptosis of PC12 cells in a dose-dependent manner. Quercetin inhibited the viability of PC12 cells in a concentration-dependent manner. [Ca2+]i was increased gradually when cells treated with quercetin alone and also increased with treatment of dantrolene-containing. Hsp70 was significantly decreased in SIN-1-treated group compared with that of control group (P<0.01). In conclusion, Ca2+ homeostasis and chaperone Hsp70 were critically involved in peroxynitrite induced nitrosative stress as protective. Peroxynitrite acts as the pathological agent in learning and memory defects in CNS disorders associated with challenge.

Chaperone roles for TMAO and HSP70 during hyposmotic stress in the spiny dogfish shark (Squalus acanthias).

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MacLellan, Robyn J; Tunnah, Louise; Barnett, David; Wright, Patricia A; MacCormack, Tyson; Currie, Suzanne

2015-10-01

Salinity decreases are experienced by many marine elasmobranchs. To understand how these fishes cope with hyposmotic stress on a cellular level, we used the spiny dogfish shark (Squalus acanthias) as a model to test whether a reciprocal relationship exists between the cell's two primary protein protection mechanisms, the chemical (e.g., trimethylamine oxide, TMAO) and molecular (e.g., heat shock protein 70, HSP70) chaperone systems. This relationship is interesting given that many elasmobranchs are expected to gain water and lose osmolytes, chemical chaperones, and ions as they osmoconform to new, lowered salinity. Dogfish were cannulated for repeated blood sampling and exposed to 70% seawater (SW) for 48 h. These hyposmotic conditions had no effect on red blood cell (RBC) and white muscle TMAO concentrations, and did not result in HSP70 induction or signs of protein damage (i.e., increased ubiquitin), suggesting that TMAO levels were sufficiently protective in these tissues. However, in the gill, we observed a significant decrease in TMAO concentration and a significant induction of HSP70 as well as signs of protein damage. In the face of this cellular stress response, gill Na(+)/K(+)-ATPase (NKA) activity significantly increased during hyposmotic conditions, as expected. We suggest that this functional preservation in the gill is partly the result of HSP70 induction with lowered salinity. We conclude a reciprocal relationship between TMAO and HSP70 in the gills of dogfish as a result of in vivo hyposmotic stress. When osmotically induced protein damage surpasses the protective capacity of remaining TMAO, HSP70 is induced to preserve tissue and organismal function.

Requirement of Hsp105 in CoCl{sub 2}-induced HIF-1α accumulation and transcriptional activation

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Mikami, Hiroki; Saito, Youhei, E-mail: ysaito@mb.kyoto-phu.ac.jp; Okamoto, Namiko; Kakihana, Ayana; Kuga, Takahisa; Nakayama, Yuji, E-mail: nakayama@mb.kyoto-phu.ac.jp

2017-03-15

The mammalian stress protein Hsp105α protects cells from stress conditions. Several studies have indicated that Hsp105α is overexpressed in many types of solid tumors, which contain hypoxic microenvironments. However, the role of Hsp105α in hypoxic tumors remains largely unknown. We herein demonstrated the involvement of Hsp105α in HIF-1 functions induced by the hypoxia-mimetic agent CoCl{sub 2}. While Hsp105α is mainly localized in the cytoplasm under normal conditions, a treatment with CoCl{sub 2} induces the nuclear localization of Hsp105α, which correlated with HIF-1α expression levels. The overexpression of degradation-resistant HIF-1α enhances the nuclear localization of Hsp105α without the CoCl{sub 2} treatment. The CoCl{sub 2}-dependent transcriptional activation of HIF-1, which is measured using a reporter gene containing a HIF-responsive element, is reduced by the knockdown of Hsp105α. Furthermore, the CoCl{sub 2}-induced accumulation of HIF-1α is enhanced by heat shock, which results in the nuclear localization of Hsp105, and is suppressed by the knockdown of Hsp105. Hsp105 associates with HIF-1α in CoCl{sub 2}-treated cells. These results suggest that Hsp105α plays an important role in the functions of HIF-1 under hypoxic conditions, in which Hsp105α enhances the accumulation and transcriptional activity of HIF-1 through the HIF-1α-mediated nuclear localization of Hsp105α. - Highlights: • Hsp105α is required for the CoCl{sub 2}-induced transcriptional activation and accumulation of HIF-1. • Hsp105α localizes to the nucleus and interacts with HIF-1α in CoCl{sub 2}-treated cells. • Hsp105 enhances the CoCl{sub 2}-induced accumulation of HIF-1α under heat shock conditions.

Novel Hsp90 inhibitor NVP-AUY922 radiosensitizes prostate cancer cells.

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Gandhi, Nishant; Wild, Aaron T; Chettiar, Sivarajan T; Aziz, Khaled; Kato, Yoshinori; Gajula, Rajendra P; Williams, Russell D; Cades, Jessica A; Annadanam, Anvesh; Song, Danny; Zhang, Yonggang; Hales, Russell K; Herman, Joseph M; Armour, Elwood; DeWeese, Theodore L; Schaeffer, Edward M; Tran, Phuoc T

2013-04-01

Outcomes for poor-risk localized prostate cancers treated with radiation are still insufficient. Targeting the "non-oncogene" addiction or stress response machinery is an appealing strategy for cancer therapeutics. Heat-shock-protein-90 (Hsp90), an integral member of this machinery, is a molecular chaperone required for energy-driven stabilization and selective degradation of misfolded "client" proteins, that is commonly overexpressed in tumor cells. Hsp90 client proteins include critical components of pathways implicated in prostate cancer cell survival and radioresistance, such as androgen receptor signaling and the PI3K-Akt-mTOR pathway. We examined the effects of a novel non-geldanamycin Hsp90 inhibitor, AUY922, combined with radiation (RT) on two prostate cancer cell lines, Myc-CaP and PC3, using in vitro assays for clonogenic survival, apoptosis, cell cycle distribution, γ-H2AX foci kinetics and client protein expression in pathways important for prostate cancer survival and radioresistance. We then evaluated tumor growth delay and effects of the combined treatment (RT-AUY922) on the PI3K-Akt-mTOR and AR pathways in a hind-flank tumor graft model. We observed that AUY922 caused supra-additive radiosensitization in both cell lines at low nanomolar doses with enhancement ratios between 1.4-1.7 (p < 0.01). RT-AUY922 increased apoptotic cell death compared with either therapy alone, induced G 2-M arrest and produced marked changes in client protein expression. These results were confirmed in vivo, where RT-AUY922 combination therapy produced supra-additive tumor growth delay compared with either therapy by itself in Myc-CaP and PC3 tumor grafts (both p < 0.0001). Our data suggest that combined RT-AUY922 therapy exhibits promising activity against prostate cancer cells, which should be investigated in clinical studies.

Small heat shock proteins mediate cell-autonomous and -nonautonomous protection in a Drosophila model for environmental-stress-induced degeneration.

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Kawasaki, Fumiko; Koonce, Noelle L; Guo, Linda; Fatima, Shahroz; Qiu, Catherine; Moon, Mackenzie T; Zheng, Yunzhen; Ordway, Richard W

2016-09-01

Cell and tissue degeneration, and the development of degenerative diseases, are influenced by genetic and environmental factors that affect protein misfolding and proteotoxicity. To better understand the role of the environment in degeneration, we developed a genetic model for heat shock (HS)-stress-induced degeneration in Drosophila This model exhibits a unique combination of features that enhance genetic analysis of degeneration and protection mechanisms involving environmental stress. These include cell-type-specific failure of proteostasis and degeneration in response to global stress, cell-nonautonomous interactions within a simple and accessible network of susceptible cell types, and precise temporal control over the induction of degeneration. In wild-type flies, HS stress causes selective loss of the flight ability and degeneration of three susceptible cell types comprising the flight motor: muscle, motor neurons and associated glia. Other motor behaviors persist and, accordingly, the corresponding cell types controlling leg motor function are resistant to degeneration. Flight motor degeneration was preceded by a failure of muscle proteostasis characterized by diffuse ubiquitinated protein aggregates. Moreover, muscle-specific overexpression of a small heat shock protein (HSP), HSP23, promoted proteostasis and protected muscle from HS stress. Notably, neurons and glia were protected as well, indicating that a small HSP can mediate cell-nonautonomous protection. Cell-autonomous protection of muscle was characterized by a distinct distribution of ubiquitinated proteins, including perinuclear localization and clearance of protein aggregates associated with the perinuclear microtubule network. This network was severely disrupted in wild-type preparations prior to degeneration, suggesting that it serves an important role in muscle proteostasis and protection. Finally, studies of resistant leg muscles revealed that they sustain proteostasis and the microtubule

Development and characterization of a novel C-terminal inhibitor of Hsp90 in androgen dependent and independent prostate cancer cells

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Eskew, Jeffery D; Rajewski, Roger A; Blagg, Brian SJ; Manjarrez, Jacob R; Matts, Robert L; Holzbeierlein, Jeffrey M; Vielhauer, George A; Sadikot, Takrima; Morales, Pedro; Duren, Alicia; Dunwiddie, Irene; Swink, Megan; Zhang, Xiaoying; Hembruff, Stacey; Donnelly, Alison

2011-01-01

The molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells. PC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography) to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies. KU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model. Overall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer

Development and characterization of a novel C-terminal inhibitor of Hsp90 in androgen dependent and independent prostate cancer cells

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Eskew Jeffery D

2011-10-01

Full Text Available Abstract Background The molecular chaperone, heat shock protein 90 (Hsp90 has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells. Methods PC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies. Results KU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model. Conclusions Overall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer.

Repercussion of mitochondria deformity induced by anti-Hsp90 drug 17AAG in human tumor cells

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Vishal, Chaturvedi

2011-06-07

Inhibiting Hsp90 chaperone roles using 17AAG induces cytostasis or apoptosis in tumor cells through destabilization of several mutated cancer promoting proteins. Although mitochondria are central in deciding the fate of cells, 17AAG induced effects on tumor cell mitochondria were largely unknown. Here, we show that Hsp90 inhibition with 17AAG first affects mitochondrial integrity in different human tumor cells, neuroblastoma, cervical cancer and glial cells. Using human neuroblastoma tumor cells, we found the early effects associated with a change in mitochondrial membrane potential, elongation and engorgement of mitochondria because of an increased matrix vacuolization. These effects are specific to Hsp90 inhibition as other chemotherapeutic drugs did not induce similar mitochondrial deformity. Further, the effects are independent of oxidative damage and cytoarchitecture destabilization since cytoskeletal disruptors and mitochondrial metabolic inhibitors also do not induce similar deformity induced by 17AAG. The 1D PAGE LC MS/ MS mitochondrial proteome analysis of 17AAG treated human neuroblastoma cells showed a loss of 61% proteins from membrane, metabolic, chaperone and ribonucleoprotein families. About 31 unmapped protein IDs were identified from proteolytic processing map using Swiss-Prot accession number, and converted to the matching gene name searching the ExPASy proteomics server. Our studies display that Hsp90 inhibition effects at first embark on mitochondria of tumor cells and compromise mitochondrial integrity. the author(s), publisher and licensee Libertas Academica Ltd.

Tumor imaging and targeting potential of an Hsp70-derived 14-mer peptide.

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Mathias Gehrmann

Full Text Available We have previously used a unique mouse monoclonal antibody cmHsp70.1 to demonstrate the selective presence of a membrane-bound form of Hsp70 (memHsp70 on a variety of leukemia cells and on single cell suspensions derived from solid tumors of different entities, but not on non-transformed cells or cells from corresponding 'healthy' tissue. This antibody can be used to image tumors in vivo and target them for antibody-dependent cellular cytotoxicity. Tumor-specific expression of memHsp70 therefore has the potential to be exploited for theranostic purposes. Given the advantages of peptides as imaging and targeting agents, this study assessed whether a 14-mer tumor penetrating peptide (TPP; TKDNNLLGRFELSG, the sequence of which is derived from the oligomerization domain of Hsp70 which is expressed on the cell surface of tumor cells, can also be used for targeting membrane Hsp70 positive (memHsp70+ tumor cells, in vitro.The specificity of carboxy-fluorescein (CF- labeled TPP (TPP to Hsp70 was proven in an Hsp70 knockout mammary tumor cell system. TPP specifically binds to different memHsp70+ mouse and human tumor cell lines and is rapidly taken up via endosomes. Two to four-fold higher levels of CF-labeled TPP were detected in MCF7 (82% memHsp70+ and MDA-MB-231 (75% memHsp70+ cells compared to T47D cells (29% memHsp70+ that exhibit a lower Hsp70 membrane positivity. After 90 min incubation, TPP co-localized with mitochondrial membranes in memHsp70+ tumors. Although there was no evidence that any given vesicle population was specifically localized, fluorophore-labeled cmHsp70.1 antibody and TPP preferentially accumulated in the proximity of the adherent surface of cultured cells. These findings suggest a potential association between membrane Hsp70 expression and cytoskeletal elements that are involved in adherence, the establishment of intercellular synapses and/or membrane reorganization.This study demonstrates the specific binding and rapid

HDAC6 inhibition enhances 17-AAG--mediated abrogation of hsp90 chaperone function in human leukemia cells.

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Rao, Rekha; Fiskus, Warren; Yang, Yonghua; Lee, Pearl; Joshi, Rajeshree; Fernandez, Pravina; Mandawat, Aditya; Atadja, Peter; Bradner, James E; Bhalla, Kapil

2008-09-01

Histone deacetylase 6 (HDAC6) is a heat shock protein 90 (hsp90) deacetylase. Treatment with pan-HDAC inhibitors or depletion of HDAC6 by siRNA induces hyperacetylation and inhibits ATP binding and chaperone function of hsp90. Treatment with 17-allylamino-demothoxy geldanamycin (17-AAG) also inhibits ATP binding and chaperone function of hsp90, resulting in polyubiquitylation and proteasomal degradation of hsp90 client proteins. In this study, we determined the effect of hsp90 hyperacetylation on the anti-hsp90 and antileukemia activity of 17-AAG. Hyperacetylation of hsp90 increased its binding to 17-AAG, as well as enhanced 17-AAG-mediated attenuation of ATP and the cochaperone p23 binding to hsp90. Notably, treatment with 17-AAG alone also reduced HDAC6 binding to hsp90 and induced hyperacetylation of hsp90. This promoted the proteasomal degradation of HDAC6. Cotreatment with 17-AAG and siRNA to HDAC6 induced more inhibition of hsp90 chaperone function and depletion of BCR-ABL and c-Raf than treatment with either agent alone. In addition, cotreatment with 17-AAG and tubacin augmented the loss of survival of K562 cells and viability of primary acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) samples. These findings demonstrate that HDAC6 is an hsp90 client protein and hyperacetylation of hsp90 augments the anti-hsp90 and antileukemia effects of 17-AAG.

Hsp40 gene therapy exerts therapeutic effects on polyglutamine disease mice via a non-cell autonomous mechanism.

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H Akiko Popiel

Full Text Available The polyglutamine (polyQ diseases such as Huntington's disease (HD, are neurodegenerative diseases caused by proteins with an expanded polyQ stretch, which misfold and aggregate, and eventually accumulate as inclusion bodies within neurons. Molecules that inhibit polyQ protein misfolding/aggregation, such as Polyglutamine Binding Peptide 1 (QBP1 and molecular chaperones, have been shown to exert therapeutic effects in vivo by crossing of transgenic animals. Towards developing a therapy using these aggregation inhibitors, we here investigated the effect of viral vector-mediated gene therapy using QBP1 and molecular chaperones on polyQ disease model mice. We found that injection of adeno-associated virus type 5 (AAV5 expressing QBP1 or Hsp40 into the striatum both dramatically suppresses inclusion body formation in the HD mouse R6/2. AAV5-Hsp40 injection also ameliorated the motor impairment and extended the lifespan of R6/2 mice. Unexpectedly, we found even in virus non-infected cells that AAV5-Hsp40 appreciably suppresses inclusion body formation, suggesting a non-cell autonomous therapeutic effect. We further show that Hsp40 inhibits secretion of the polyQ protein from cultured cells, implying that it inhibits the recently suggested cell-cell transmission of the polyQ protein. Our results demonstrate for the first time the therapeutic effect of Hsp40 gene therapy on the neurological phenotypes of polyQ disease mice.

Targets Fishing and Identification of Calenduloside E as Hsp90AB1: Design, Synthesis, and Evaluation of Clickable Activity-Based Probe

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Wang, Shan; Tian, Yu; Zhang, Jing-Yi; Xu, Hui-Bo; Zhou, Ping; Wang, Min; Lu, Sen-Bao; Luo, Yun; Wang, Min; Sun, Gui-Bo; Xu, Xu-Dong; Sun, Xiao-Bo

2018-01-01

Calenduloside E (CE), a natural triterpenoid compound isolated from Aralia elata, can protect against ox-LDL-induced human umbilical vein endothelial cell (HUVEC) injury in our previous reports. However, the exact targets and mechanisms of CE remain elusive. For the sake of resolving this question, we designed and synthesized a clickable activity-based probe (CE-P), which could be utilized to fish the functional targets in HUVECs using a gel-based strategy. Based on the previous studies of the structure-activity relationship (SAR), we introduced an alkyne moiety at the C-28 carboxylic group of CE, which kept the protective and anti-apoptosis activity. Via proteomic approach, one of the potential proteins bound to CE-P was identified as Hsp90AB1, and further verification was performed by pure recombinant Hsp90AB1 and competitive assay. These results demonstrated that CE could bind to Hsp90AB1. We also found that CE could reverse the Hsp90AB1 decrease after ox-LDL treatment. To make our results more convincing, we performed SPR analysis and the affinity kinetic assay showed that CE/CE-P could bind to Hsp90AB1 in a dose-dependent manner. Taken together, our research showed CE could probably bind to Hsp90AB1 to protect the cell injury, which might provide the basis for the further exploration of its cardiovascular protective mechanisms. For the sake of resolving this question, we designed and synthesized a clickable activity-based probe (CE-P), which could be utilized to fish the functional targets in HUVECs using a gel-based strategy. PMID:29875664

The Double-Edged Sword: Conserved Functions of Extracellular Hsp90 in Wound Healing and Cancer

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Hance, Michael W.; Nolan, Krystal D.; Isaacs, Jennifer S., E-mail: isaacsj@musc.edu [Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Hollings Cancer Center, Charleston, SC 29412 (United States)

2014-05-06

Heat shock proteins (Hsps) represent a diverse group of chaperones that play a vital role in the protection of cells against numerous environmental stresses. Although our understanding of chaperone biology has deepened over the last decade, the “atypical” extracellular functions of Hsps have remained somewhat enigmatic and comparatively understudied. The heat shock protein 90 (Hsp90) chaperone is a prototypic model for an Hsp family member exhibiting a duality of intracellular and extracellular functions. Intracellular Hsp90 is best known as a master regulator of protein folding. Cancers are particularly adept at exploiting this function of Hsp90, providing the impetus for the robust clinical development of small molecule Hsp90 inhibitors. However, in addition to its maintenance of protein homeostasis, Hsp90 has also been identified as an extracellular protein. Although early reports ascribed immunoregulatory functions to extracellular Hsp90 (eHsp90), recent studies have illuminated expanded functions for eHsp90 in wound healing and cancer. While the intended physiological role of eHsp90 remains enigmatic, its evolutionarily conserved functions in wound healing are easily co-opted during malignancy, a pathology sharing many properties of wounded tissue. This review will highlight the emerging functions of eHsp90 and shed light on its seemingly dichotomous roles as a benevolent facilitator of wound healing and as a sinister effector of tumor progression.

Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin inhibits the proliferation of ARPE-19 cells

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Wang Lin

2010-04-01

Full Text Available Abstract Background The antiproliferative effect of the Hsp90 inhibitor 17-AAG (17-allylamino-17-demethoxygeldanamycin on human retinal pigment epithelial cells is investigated. Methods MTT and flow cytometry were used to study the antiproliferative effects of the 17-AAG treatment of ARPE-19 cells. 2D gel electrophoresis (2-DE and mass spectrometry were applied to detect the altered expression of proteins, which was verified by real-time PCR. Gene Ontology analysis and Ingenuity Pathway Analysis (IPA were utilized to analyze the signaling pathways, cellular location, function, and network connections of the identified proteins. And SOD assay was employed to confirm the analysis. Results 17-AAG suppressed the proliferation of ARPE-19 cells by inducing cell cycle arrest and apoptosis. Proteomic analysis revealed that the expression of 94 proteins was altered by a factor of more than 1.5 following exposure to 17-AAG. Of these 94, 87 proteins were identified. Real-time PCR results indicated that Hsp90 and Hsp70, which were not identified by proteomic analysis, were both upregulated upon 17-AAG treatment. IPA revealed that most of the proteins have functions that are related to oxidative stress, as verified by SOD assay, while canonical pathway analysis revealed glycolysis/gluconeogenesis. Conclusions 17-AAG suppressed the proliferation of ARPE-19 cells by inducing cell cycle arrest and apoptosis, and possibly by oxidative stress.

HSP90 inhibitor 17-AAG selectively eradicates lymphoma stem cells.

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Newman, Bryan; Liu, Yan; Lee, Hsiu-Fang; Sun, Duxin; Wang, Yin

2012-09-01

Cancer stem cells (CSC; also called tumor-initiating cells) comprise tumor cell subpopulations that preserve the properties of quiescence, self-renewal, and differentiation of normal stem cells. In addition, CSCs are therapeutically important because of their key contributions toward drug resistance. The hypoxia-inducible transcription factor HIF1α is critical for CSC maintenance in mouse lymphoma. In this study, we showed that low concentrations of the HSP90 inhibitor 17-AAG eliminate lymphoma CSCs in vitro and in vivo by disrupting the transcriptional function of HIF1α, a client protein of HSP90. 17-AAG preferentially induced apoptosis and eliminated the colony formation capacity of mouse lymphoma CSCs and human acute myeloid leukemia (AML) CSCs. However, low concentrations of 17-AAG failed to eliminate highly proliferative lymphoma and AML cells (non-CSCs), in which the AKT-GSK3 signaling pathway is constitutively active. The heat shock transcription factor HSF1 is highly expressed in non-CSCs, but it was weakly expressed in lymphoma CSCs. However, siRNA-mediated attenuation of HSF1 abrogated the colony formation ability of both lymphoma and AML CSCs. This study supports the use of 17-AAG as a CSC targeting agent and, in addition, shows that HSF1 is an important target for elimination of both CSCs and non-CSCs in cancer. ©2012 AACR.

Proteomic profiling reveals that resveratrol inhibits HSP27 expression and sensitizes breast cancer cells to doxorubicin therapy.

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José Díaz-Chávez

Full Text Available The use of chemopreventive natural compounds represents a promising strategy in the search for novel therapeutic agents in cancer. Resveratrol (3,4',5-trans-trihydroxystilbilene is a dietary polyphenol found in fruits, vegetables and medicinal plants that exhibits chemopreventive and antitumor effects. In this study, we searched for modulated proteins with preventive or therapeutic potential in MCF-7 breast cancer cells exposed to resveratrol. Using two-dimensional electrophoresis we found significant changes (FC >2.0; p≤0.05 in the expression of 16 proteins in resveratrol-treated MCF-7 cells. Six down-regulated proteins were identified by tandem mass spectrometry (ESI-MS/MS as heat shock protein 27 (HSP27, translationally-controlled tumor protein, peroxiredoxin-6, stress-induced-phosphoprotein-1, pyridoxine-5'-phosphate oxidase-1 and hypoxanthine-guanine phosphoribosyl transferase; whereas one up-regulated protein was identified as triosephosphate isomerase. Particularly, HSP27 overexpression has been associated to apoptosis inhibition and resistance of human cancer cells to therapy. Consistently, we demonstrated that resveratrol induces apoptosis in MCF-7 cells. Apoptosis was associated with a significant increase in mitochondrial permeability transition, cytochrome c release in cytoplasm, and caspases -3 and -9 independent cell death. Then, we evaluated the chemosensitization effect of increasing concentrations of resveratrol in combination with doxorubicin anti-neoplastic agent in vitro. We found that resveratrol effectively sensitize MCF-7 cells to cytotoxic therapy. Next, we evaluated the relevance of HSP27 targeted inhibition in therapy effectiveness. Results evidenced that HSP27 inhibition using RNA interference enhances the cytotoxicity of doxorubicin. In conclusion, our data indicate that resveratrol may improve the therapeutic effects of doxorubicin in part by cell death induction. We propose that potential modulation of HSP27

Proteomic profiling reveals that resveratrol inhibits HSP27 expression and sensitizes breast cancer cells to doxorubicin therapy.

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Díaz-Chávez, José; Fonseca-Sánchez, Miguel A; Arechaga-Ocampo, Elena; Flores-Pérez, Ali; Palacios-Rodríguez, Yadira; Domínguez-Gómez, Guadalupe; Marchat, Laurence A; Fuentes-Mera, Lizeth; Mendoza-Hernández, Guillermo; Gariglio, Patricio; López-Camarillo, César

2013-01-01

The use of chemopreventive natural compounds represents a promising strategy in the search for novel therapeutic agents in cancer. Resveratrol (3,4',5-trans-trihydroxystilbilene) is a dietary polyphenol found in fruits, vegetables and medicinal plants that exhibits chemopreventive and antitumor effects. In this study, we searched for modulated proteins with preventive or therapeutic potential in MCF-7 breast cancer cells exposed to resveratrol. Using two-dimensional electrophoresis we found significant changes (FC >2.0; p≤0.05) in the expression of 16 proteins in resveratrol-treated MCF-7 cells. Six down-regulated proteins were identified by tandem mass spectrometry (ESI-MS/MS) as heat shock protein 27 (HSP27), translationally-controlled tumor protein, peroxiredoxin-6, stress-induced-phosphoprotein-1, pyridoxine-5'-phosphate oxidase-1 and hypoxanthine-guanine phosphoribosyl transferase; whereas one up-regulated protein was identified as triosephosphate isomerase. Particularly, HSP27 overexpression has been associated to apoptosis inhibition and resistance of human cancer cells to therapy. Consistently, we demonstrated that resveratrol induces apoptosis in MCF-7 cells. Apoptosis was associated with a significant increase in mitochondrial permeability transition, cytochrome c release in cytoplasm, and caspases -3 and -9 independent cell death. Then, we evaluated the chemosensitization effect of increasing concentrations of resveratrol in combination with doxorubicin anti-neoplastic agent in vitro. We found that resveratrol effectively sensitize MCF-7 cells to cytotoxic therapy. Next, we evaluated the relevance of HSP27 targeted inhibition in therapy effectiveness. Results evidenced that HSP27 inhibition using RNA interference enhances the cytotoxicity of doxorubicin. In conclusion, our data indicate that resveratrol may improve the therapeutic effects of doxorubicin in part by cell death induction. We propose that potential modulation of HSP27 levels using natural

The Leishmania HSP20 Is Antigenic during Natural Infections, but, as DNA Vaccine, It does not Protect BALB/c Mice against Experimental L. amazonensis Infection

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Ana M. Montalvo-Álvarez

2008-01-01

Full Text Available Protozoa of the genus Leishmania are causative agents of leishmaniasis, an important health problem in both human and veterinary medicine. Here, we describe a new heat shock protein (HSP in Leishmania, belonging to the small HSP (sHSP family in kinetoplastids. The protein is highly conserved in different Leishmania species, showing instead significant divergence with sHSP's from other organisms. The humoral response elicited against this protein during Leishmania infection has been investigated in natural infected humans and dogs, and in experimentally infected hamsters. Leishmania HSP20 is a prominent antigen for canine hosts; on the contrary, the protein seems to be a poor antigen for human immune system. Time-course analysis of appearance of anti-HSP20 antibodies in golden hamsters indicated that these antibodies are produced at late stages of the infection, when clinical symptoms of disease are patent. Finally, the protective efficacy of HSP20 was assessed in mice using a DNA vaccine approach prior to challenge with Leishmania amazonensis.

Hsp27, Hsp70 and mismatch repair proteins hMLH1 and hMSH2 expression in peripheral blood lymphocytes from healthy subjects and cancer patients.

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Nadin, Silvina Beatriz; Vargas-Roig, Laura M; Drago, Gisela; Ibarra, Jorge; Ciocca, Daniel R

2007-07-08

Mismatch repair (MMR) deficiency and higher expression levels of heat shock proteins (Hsps) have been implicated with drug resistance to topoisomerase II poisons (doxorubicin) and to platinum compounds (cisplatin). This study was designed to determine individual influences of doxorubicin and cisplatin treatment on the expression of Hsp27, Hsp70, hMLH1 and hMSH2 proteins and in the DNA damage status in peripheral blood lymphocytes (PBLs). In addition, we studied whether these proteins and the DNA damage correlated with the survival of cancer patients. PBLs from 10 healthy donors and 25 cancer patients (before and after three cycles of chemotherapy) were exposed to in vitro treatments: C (control), HS (heat shock at 42 degrees C), Do or Pt (doxorubicin or cisplatin alone), and HS+Do or HS+Pt (heat shock+doxorubicin or heat shock+cisplatin). PBLs were collected at time 0 (T0: immediately after drug treatment) and after 24h of repair (T24). Hsp27, Hsp70, hMLH1 and hMSH2 were studied by immunocytochemistry and the DNA damage by alkaline comet assay. Immunofluorescence studies and confocal microscopy revealed that hMLH1 and hMSH2 colocalized with Hsp27 and Hsp72 (inducible form of Hsp70). hMLH1 and hMSH2 were significantly induced by Pt and HS+Pt at T24 in cancer patients, but only modestly influenced by Do. Cancer patients presented higher basal expression of total and nuclear Hsp27 and Hsp70 than controls, and these proteins were also increased by HS, Do and HS+Do. The Hsp70 induction by Pt and HS+Pt was noted in cancer patients, especially nuclear Hsp70. In cancer patients, basal DNA damage was slightly higher than in healthy persons; and after Pt and HS+Pt treatments, DNA migration and number of apoptotic cells were higher than controls. Hsps accomplished a cytoprotective function in pre-chemotherapy PBLs (HS before Do or Pt), but not in post-chemotherapy samples. In Pt-treated patients the ratio N/C (nuclear/cytoplasmic) of Hsp27 was related to disease free survival

Proteotoxicity is not the reason for the dependence of cancer cells on the major chaperone Hsp70.

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Colvin, Teresa A; Gabai, Vladimir L; Sherman, Michael Y

2014-01-01

Several years ago a hypothesis was proposed that the survival of cancer cells depend on elevated expression of molecular chaperones because these cells are prone to proteotoxic stress. A critical prediction of this hypothesis is that depletion of chaperones in cancer cells should lead to proteotoxicity. Here, using the major chaperone Hsp70 as example, we demonstrate that its depletion does not trigger proteotoxic stress, thus refuting the model. Accordingly, other functions of chaperones, e.g., their role in cell signaling, might define the requirements for chaperones in cancer cells, which is critical for rational targeting Hsp70 in cancer treatment.

17-AAG, an Hsp90 inhibitor, causes kinetochore defects: a novel mechanism by which 17-AAG inhibits cell proliferation.

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Niikura, Y; Ohta, S; Vandenbeldt, K J; Abdulle, R; McEwen, B F; Kitagawa, K

2006-07-13

The Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG), which is currently in clinical trials, is thought to exert antitumor activity by simultaneously targeting several oncogenic signaling pathways. Here we report a novel mechanism by which 17-AAG inhibits cell proliferation, and we provide the first evidence that HSP90 is required for the assembly of kinetochore protein complexes in humans. 17-AAG caused delocalization of several kinetochore proteins including CENP-I and CENP-H but excluding CENP-B and CENP-C. Consistently, 17-AAG induced a mitotic arrest that depends on the spindle checkpoint and induced misalignment of chromosomes and aneuploidy. We found that HSP90 associates with SGT1 (suppressor of G2 allele of skp1; SUGT1) in human cells and that depletion of SGT1 sensitizes HeLa cells to 17-AAG. Overexpression of SGT1 restored the localization of specific kinetochore proteins and chromosome alignment in cells treated with 17-AAG. Biochemical and genetic results suggest that HSP90, through its interaction with SGT1 (SUGT1), is required for kinetochore assembly. Furthermore, time-course experiments revealed that transient treatment with 17-AAG between late S and G2/M phases causes substantial delocalization of CENP-H and CENP-I, a finding that strongly suggests that HSP90 participates in kinetochore assembly in a cell cycle-dependent manner.

Identification of cytosolic peroxisome proliferator binding protein as a member of the heat shock protein HSP70 family.

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Alvares, K; Carrillo, A; Yuan, P M; Kawano, H; Morimoto, R I; Reddy, J K

1990-01-01

Clofibrate and many of its structural analogues induce proliferation of peroxisomes in the hepatic parenchymal cells of rodents and certain nonrodent species including primates. This induction is tissue specific, occurring mainly in the liver parenchymal cells and to a lesser extent in the kidney cortical epithelium. The induction of peroxisomes is associated with a predictable pleiotropic response, characterized by hepatomegaly, and increased activities and mRNA levels of certain peroxisomal enzymes. Using affinity chromatography, we had previously isolated a protein that binds to clofibric acid. We now show that this protein is homologous with the heat shock protein HSP70 family by analysis of amino acid sequences of isolated peptides from trypsin-treated clofibric acid binding protein and by cross-reactivity with a monoclonal antibody raised against the conserved region of the 70-kDa heat shock proteins. The clofibric acid-Sepharose column could bind HSP70 proteins isolated from various species, which could then be eluted with either clofibric acid or ATP. Conversely, when a rat liver cytosol containing multiple members of the HSP70 family was passed through an ATP-agarose column, and eluted with clofibric acid, only P72 (HSC70) was eluted. These results suggest that clofibric acid, a peroxisome proliferator, preferentially interacts with P72 at or near the ATP binding site. Images PMID:2371272

Hsp70 and ceramide release by diode laser-treated mouse skin cells in vivo

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Sokolovskii, G. S.; Onikienko, S. B.; Zemlyanoi, A. V.; Soboleva, K. K.; Pikhtin, N. A.; Tarasov, I. S.; Guzova, I. V.; Margulis, B. A.

2014-12-01

We report experimental study of generation of extracellular heat shock proteins (Hsp70) and ceramides under pulsed irradiation by quantum-well laser diodes. Our results are of great promise for applications in practical medicine such as protection against biopathogenes and abiotic stress factor challenges.

S-nitrosylation of TRIM72 at cysteine 144 is critical for protection against oxidation-induced protein degradation and cell death.

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Kohr, Mark J; Evangelista, Alicia M; Ferlito, Marcella; Steenbergen, Charles; Murphy, Elizabeth

2014-04-01

Oxidative stress and membrane damage following myocardial ischemia/reperfusion injury are important contributors to cardiomyocyte death and the loss of myocardial function. Our previous study identified cysteine 144 (C144) of tripartite motif-containing protein 72 (TRIM72) as a potential site for S-nitrosylation (SNO). TRIM72 is a cardioprotective membrane repair protein that can be both activated and targeted for degradation by different oxidative modifications. Consistent with the potential regulation of TRIM72 by various oxidative modifications, we found that SNO levels increased at C144 of TRIM72 with ischemic preconditioning. Therefore, to investigate the role of C144 in the regulation of TRIM72 function, we mutated C144 of TRIM72 to a serine residue (TRIM72(C144S)), and expressed either TRIM72(WT) or TRIM72(C144S) in HEK-293 cells, which lack endogenous TRIM72, in order to examine the effect of this mutation on the functional stability of TRIM72 and on cell survival. We hypothesized that SNO of TRIM72 stabilizes the protein, thus allowing for membrane repair and enhanced cell survival. Upon treatment with hydrogen peroxide (H2O2), we found that TRIM72(WT) levels were decreased, but not TRIM72(C144S) and this correlated with increased H2O2-induced cell death in TRIM72(WT) cells. Additionally, we found that treatment with the cardioprotective S-nitrosylating agent S-nitrosoglutathione (GSNO), was able to preserve TRIM72(WT) protein levels and enhance TRIM72(WT)-mediated cell survival, but had no effect on TRIM72(C144S) levels. Consistent with our hypothesis, GSNO was also found to increase SNO levels and inhibit H2O2-induced irreversible oxidation for TRIM72(WT) without affecting TRIM72(C144S). In further support of our hypothesis, GSNO blocked the ischemia/reperfusion-induced decrease in TRIM72 levels and reduced infarct size in a Langendorff-perfused heart model. The results of these studies have important implications for cardioprotection and suggest that

Hsp90 orchestrates transcriptional regulation by Hsf1 and cell wall remodelling by MAPK signalling during thermal adaptation in a pathogenic yeast.

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Michelle D Leach

2012-12-01

Full Text Available Thermal adaptation is essential in all organisms. In yeasts, the heat shock response is commanded by the heat shock transcription factor Hsf1. Here we have integrated unbiased genetic screens with directed molecular dissection to demonstrate that multiple signalling cascades contribute to thermal adaptation in the pathogenic yeast Candida albicans. We show that the molecular chaperone heat shock protein 90 (Hsp90 interacts with and down-regulates Hsf1 thereby modulating short term thermal adaptation. In the longer term, thermal adaptation depends on key MAP kinase signalling pathways that are associated with cell wall remodelling: the Hog1, Mkc1 and Cek1 pathways. We demonstrate that these pathways are differentially activated and display cross talk during heat shock. As a result ambient temperature significantly affects the resistance of C. albicans cells to cell wall stresses (Calcofluor White and Congo Red, but not osmotic stress (NaCl. We also show that the inactivation of MAP kinase signalling disrupts this cross talk between thermal and cell wall adaptation. Critically, Hsp90 coordinates this cross talk. Genetic and pharmacological inhibition of Hsp90 disrupts the Hsf1-Hsp90 regulatory circuit thereby disturbing HSP gene regulation and reducing the resistance of C. albicans to proteotoxic stresses. Hsp90 depletion also affects cell wall biogenesis by impairing the activation of its client proteins Mkc1 and Hog1, as well as Cek1, which we implicate as a new Hsp90 client in this study. Therefore Hsp90 modulates the short term Hsf1-mediated activation of the classic heat shock response, coordinating this response with long term thermal adaptation via Mkc1- Hog1- and Cek1-mediated cell wall remodelling.

Active participation of Hsp90 in the biogenesis of the trimeric reovirus cell attachment protein sigma1.

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Gilmore, R; Coffey, M C; Lee, P W

1998-06-12

The reovirus cell attachment protein, sigma1, is a lollipop-shaped homotrimer with an N-terminal fibrous tail and a C-terminal globular head. Biogenesis of this protein involves two trimerization events: N-terminal trimerization, which occurs cotranslationally and is Hsp70/ATP-independent, and C-terminal trimerization, which occurs posttranslationally and is Hsp70/ATP-dependent. To determine if Hsp90 also plays a role in sigma1 biogenesis, we analyzed sigma1 synthesized in rabbit reticulocyte lysate. Coprecipitation experiments using anti-Hsp90 antibodies revealed that Hsp90 was associated with immature sigma1 trimers (hydra-like intermediates with assembled N termini and unassembled C termini) but not with mature trimers. The use of truncated sigma1 further demonstrated that only the C-terminal half of sigma1 associated with Hsp90. In the presence of the Hsp90 binding drug geldanamycin, N-terminal trimerization proceeded normally, but C-terminal trimerization was blocked. Geldanamycin did not inhibit the association of Hsp90 with sigma 1 but prevented the subsequent release of Hsp90 from the immature sigma1 complex. We also examined the status of p23, an Hsp90-associated cochaperone. Like Hsp90, p23 only associated with immature sigma1 trimers, and this association was mapped to the C-terminal half of sigma1. However, unlike Hsp90, p23 was released from the sigma1 complex upon the addition of geldanamycin. These results highlight an all-or-none concept of chaperone involvement in different oligomerization domains within a single protein and suggest a possible common usage of chaperones in the regulation of general protein folding and of steroid receptor activation.

Efficacy of the HSP90 inhibitor 17-AAG in human glioma cell lines and tumorigenic glioma stem cells.

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Sauvageot, Claire Marie-Elisabeth; Weatherbee, Jessica Leigh; Kesari, Santosh; Winters, Susan Elizabeth; Barnes, Jessica; Dellagatta, Jamie; Ramakrishna, Naren Raj; Stiles, Charles Dean; Kung, Andrew Li-Jen; Kieran, Mark W; Wen, Patrick Yung Chih

2009-04-01

Glioblastoma multiforme (GBM) arises from genetic and signaling abnormalities in components of signal transduction pathways involved in proliferation, survival, and the cell cycle axis. Studies to date with single-agent targeted molecular therapy have revealed only modest effects in attenuating the growth of these tumors, suggesting that targeting multiple aberrant pathways may be more beneficial. Heat-shock protein 90 (HSP90) is a molecular chaperone that is involved in the conformational maturation of a defined group of client proteins, many of which are deregulated in GBM. 17-allylamino-17-demethoxygeldanamycin (17-AAG) is a well-characterized HSP90 inhibitor that should be able to target many of the aberrant signal transduction pathways in GBM. We assessed the ability of 17-AAG to inhibit the growth of glioma cell lines and glioma stem cells both in vitro and in vivo and assessed its ability to synergize with radiation and/or temozolomide, the standard therapies for GBM. Our results reveal that 17-AAG is able to inhibit the growth of both human glioma cell lines and glioma stem cells in vitro and is able to target the appropriate proteins within these cells. In addition, 17-AAG can inhibit the growth of intracranial tumors and can synergize with radiation both in tissue culture and in intracranial tumors. This compound was not found to synergize with temozolomide in any of our models of gliomas. Our results suggest that HSP90 inhibitors like 17-AAG may have therapeutic potential in GBM, either as a single agent or in combination with radiation.

Expression dynamics of HSP70 during chronic heat stress in Tharparkar cattle.

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Bharati, Jaya; Dangi, S S; Chouhan, V S; Mishra, S R; Bharti, M K; Verma, V; Shankar, O; Yadav, V P; Das, K; Paul, A; Bag, S; Maurya, V P; Singh, G; Kumar, P; Sarkar, M

2017-06-01

Six male Tharparkar cattle aged 2-3 years were selected for the study. The animals were acclimatized in the psychrometric chamber at thermoneutral zone (TNZ) for 15 days and then exposed to 42 °C temperature up to 23 days followed by 12 days of recovery period. Physiological responses were estimated, and peripheral blood mononuclear cells (PBMCs) were isolated at TNZ on day 1, day 5, and day 12; after 6 h of heat stress exposure on day 16 to day 20, day 25, day 30, day 32, day 34, day 36, and day 38; and a recovery period on day 45 and day 50. The PBMCs were cultured to study the effect of thermal challenge on HSP70 messenger RNA (mRNA) expression pattern at different temperature-time combinations. The mRNA and protein expression of HSP70 in PBMCs along with serum extracellular HSP70 (eHSP70) was increased (P cattle and the biphasic expression pattern may be providing a second window of protection during chronic heat stress.

The Complex Function of Hsp70 in Metastatic Cancer

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Juhasz, Kata; Lipp, Anna-Maria; Nimmervoll, Benedikt; Sonnleitner, Alois; Hesse, Jan; Haselgruebler, Thomas; Balogi, Zsolt, E-mail: zsolt.balogi@cbl.at [Center for Advanced Bioanalysis GmbH, Gruberstr. 40-42, A-4020 Linz (Austria)

2013-12-20

Elevated expression of the inducible heat shock protein 70 (Hsp70) is known to correlate with poor prognosis in many cancers. Hsp70 confers survival advantage as well as resistance to chemotherapeutic agents, and promotes tumor cell invasion. At the same time, tumor-derived extracellular Hsp70 has been recognized as a “chaperokine”, activating antitumor immunity. In this review we discuss localization dependent functions of Hsp70 in the context of invasive cancer. Understanding the molecular principles of metastasis formation steps, as well as interactions of the tumor cells with the microenvironment and the immune system is essential for fighting metastatic cancer. Although Hsp70 has been implicated in different steps of the metastatic process, the exact mechanisms of its action remain to be explored. Known and potential functions of Hsp70 in controlling or modulating of invasion and metastasis are discussed.

Wild-type EGFR Is Stabilized by Direct Interaction with HSP90 in Cancer Cells and Tumors

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Aarif Ahsan

2012-08-01

Full Text Available The epidermal growth factor receptor (EGFR has been targeted for inhibition using tyrosine kinase inhibitors and monoclonal antibodies, with improvement in outcome in subsets of patients with head and neck, lung, and colorectal carcinomas. We have previously found that EGFR stability plays a key role in cell survival after chemotherapy and radiotherapy. Heat shock protein 90 (HSP90 is known to stabilize mutant EGFR and ErbB2, but its role in cancers with wild-type (WT WT-EGFR is unclear. In this report, we demonstrate that fully mature, membrane-bound WT-EGFR interacts with HSP90 independent of ErbB2. Further, the HSP90 inhibitors geldanamycin (GA and AT13387 cause a decrease in WT-EGFR in cultured head and neck cancer cells. This decrease results from a significantly reduced half-life of WT-EGFR. WT-EGFR was also lost in head and neck xenograft specimens after treatment with AT13387 under conditions that inhibited tumor growth and prolonged survival of the mice. Our findings demonstrate that WT-EGFR is a client protein of HSP90 and that their interaction is critical for maintaining both the stability of the receptor as well as the growth of EGFR-dependent cancers. Furthermore, these findings support the search for specific agents that disrupt HSP90's ability to act as an EGFR chaperone.

HSP20 phosphorylation and airway smooth muscle relaxation

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Mariam Ba

2009-06-01

Full Text Available Mariam Ba1, Cherie A Singer1, Manoj Tyagi2, Colleen Brophy3, Josh E Baker4, Christine Cremo4, Andrew Halayko5, William T Gerthoffer21Department of Pharmacology, University of Nevada School of Medicine, Reno, NV, USA; 2Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, AL, USA; 3Harrington Department of Biochemistry, Arizona State University, Tempe, AZ, USA; 4Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV, USA; 5Departments of Physiology and Internal Medicine, University of Manitoba, Winnipeg, MB, CanadaAbstract: HSP20 (HSPB6 is a small heat shock protein expressed in smooth muscles that is hypothesized to inhibit contraction when phosphorylated by cAMP-dependent protein kinase. To investigate this hypothesis in airway smooth muscle (ASM we showed that HSP20 was constitutively expressed as well as being inducible in cultured hASM cells by treatment with 1 µM isoproterenol or 10 µM salmeterol. In contrast, a mixture of proinflammatory mediators (interleukin-1β, tumor necrosis factor α, and interferon γ inhibited expression of HSP20 by about 50% in 48 hours. To determine whether phosphorylation of HSP20 is sufficient to induce relaxation, canine tracheal smooth muscle was treated with a cell permeant phosphopeptide that mimics the phosphorylation of HSP20. The HSP20 phosphopeptide antagonized carbacholinduced contraction by 60% with no change in myosin light chain phosphorylation. Recombinant full length HSP20 inhibited skeletal actin binding to smooth muscle myosin subfragment 1 (S1, and recombinant cell permeant TAT-HSP20 S16D mutant reduced F-actin filaments in cultured hASM cells. Carbachol stimulation of canine tracheal smooth muscle tissue caused redistribution of HSP20 from large macromolecular complexes (200–500 kDa to smaller complexes (<60 kDa. The results are consistent with HSP20 expression and macromolecular structure being dynamically regulated in airway

Hsp70-Bag3 interactions regulate cancer-related signaling networks.

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Colvin, Teresa A; Gabai, Vladimir L; Gong, Jianlin; Calderwood, Stuart K; Li, Hu; Gummuluru, Suryaram; Matchuk, Olga N; Smirnova, Svetlana G; Orlova, Nina V; Zamulaeva, Irina A; Garcia-Marcos, Mikel; Li, Xiaokai; Young, Z T; Rauch, Jennifer N; Gestwicki, Jason E; Takayama, Shinichi; Sherman, Michael Y

2014-09-01

Bag3, a nucleotide exchange factor of the heat shock protein Hsp70, has been implicated in cell signaling. Here, we report that Bag3 interacts with the SH3 domain of Src, thereby mediating the effects of Hsp70 on Src signaling. Using several complementary approaches, we established that the Hsp70-Bag3 module is a broad-acting regulator of cancer cell signaling by modulating the activity of the transcription factors NF-κB, FoxM1, Hif1α, the translation regulator HuR, and the cell-cycle regulators p21 and survivin. We also identified a small-molecule inhibitor, YM-1, that disrupts the Hsp70-Bag3 interaction. YM-1 mirrored the effects of Hsp70 depletion on these signaling pathways, and in vivo administration of this drug was sufficient to suppress tumor growth in mice. Overall, our results defined Bag3 as a critical factor in Hsp70-modulated signaling and offered a preclinical proof-of-concept that the Hsp70-Bag3 complex may offer an appealing anticancer target. ©2014 American Association for Cancer Research.

The p53/HSP70 inhibitor, 2-phenylethynesulfonamide, causes oxidative stress, unfolded protein response and apoptosis in rainbow trout cells

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Zeng, Fanxing; Tee, Catherine; Liu, Michelle [Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1 (Canada); Sherry, James P. [Aquatic Contaminants Research Division, Environment Canada, Burlington, Ontario L7R 4A6 (Canada); Dixon, Brian; Duncker, Bernard P. [Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1 (Canada); Bols, Niels C., E-mail: ncbols@uwaterloo.ca [Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1 (Canada)

2014-01-15

Highlights: •2-Phenylethynesulfonamide (PES) is an inhibitor of p53 and HSP 70 in mammals. •In the fish epithelial cell line, RTgill-W1, PES enhanced ROS generation and was cytotoxic. •RTgill-W1 death was by apoptosis and blocked by the anti-oxidant N-acetylcysteine. •This is the first report linking PES-induced cell death to ROS. •With this background PES should be useful for studying fish cell survival pathways. -- Abstract: The effect of 2-phenylethynesulfonamide (PES), which is a p53 and HSP70 inhibitor in mammalian cells, was studied on the rainbow trout (Oncorhynchus mykiss) gill epithelial cell line, RTgill-W1, in order to evaluate PES as a tool for understanding the cellular survival pathways operating in fish. As judged by three viability assays, fish cells were killed by 24 h exposures to PES, but cell death was blocked by the anti-oxidant N-acetylcysteine (NAC). Cell death had several hallmarks of apoptosis: DNA laddering, nuclear fragmentation, Annexin V staining, mitochondrial membrane potential decline, and caspases activation. Reactive oxygen species (ROS) production peaked in several hours after the addition of PES and before cell death. HSP70 and BiP levels were higher in cultures treated with PES for 24 h, but this was blocked by NAC. As well, PES treatment caused HSP70, BiP and p53 to accumulate in the detergent-insoluble fraction, and this too was prevented by NAC. Of several possible scenarios to explain the results, the following one is the simplest. PES enhances the generation of ROS, possibly by inhibiting the anti-oxidant actions of p53 and HSP70. ER stress arises from the ROS and from PES inhibiting the chaperone activities of HSP70. The ER stress in turn initiates the unfolded protein response (UPR), but this fails to restore ER homeostasis so proteins aggregate and cells die. Despite these multiple actions, PES should be useful for studying fish cellular survival pathways.

RNAi knockdown of Hop (Hsp70/Hsp90 organising protein) decreases invasion via MMP-2 down regulation.

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Walsh, Naomi

2011-07-28

We previously identified Hop as over expressed in invasive pancreatic cancer cell lines and malignant tissues of pancreatic cancer patients, suggesting an important role for Hop in the biology of invasive pancreatic cancer. Hop is a co-chaperone protein that binds to both Hsp70\\/Hsp90. We hypothesised that by targeting Hop, signalling pathways modulating invasion and client protein stabilisation involving Hsp90-dependent complexes may be altered. In this study, we show that Hop knockdown by small interfering (si)RNA reduces the invasion of pancreatic cancer cells, resulting in decreased expression of the downstream target gene, matrix metalloproteinases-2 (MMP-2). Hop in conditioned media co-immunoprecipitates with MMP-2, implicating a possible extracellular function for Hop. Knockdown of Hop expression also reduced expression levels of Hsp90 client proteins, HER2, Bcr-Abl, c-MET and v-Src. Furthermore, Hop is strongly expressed in high grade PanINs compared to lower PanIN grades, displaying differential localisation in invasive ductal pancreatic cancer, indicating that the localisation of Hop is an important factor in pancreatic tumours. Our data suggests that the attenuation of Hop expression inactivates key signal transduction proteins which may decrease the invasiveness of pancreatic cancer cells possibly through the modulation of Hsp90 activity. Therefore, targeting Hop in pancreatic cancer may constitute a viable strategy for targeted cancer therapy.

Model of Oxygen and Glucose Deprivation in PC12 Cells and Detection of HSP70 Protein

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He, Jinting; Yang, Le; Shao, Yankun

2018-01-01

Objective: PC12 cell was used to set up a ischemia model by OGD and detected HSP70 protein. Methods: Use of PC12 cells induced by NGF stimulation into nerve cells, oxygen and glucose deprivation to build the nerve cells of oxygen and glucose deprivation model; using Western blot analysis of PC12 cells into neuron-like cells and oxygen-glucose deprivation model established. Results: The application of a final concentration of 50 ng / ml of NGF in DMEM complete mediumPC12 cells showed a typical neuronal morphology with the increase in cell culture time. NGF culture time showed a positive correlation, the establishment of oxygen and glucose deprivation (OGD) training environment, the OGD after nerve element appears different degrees of damage, OGD can effectively induce the expression of HSP70. Conclusion: PC12 cell transformed into cells by NGF; the cell model of OGD was established.

The Malarial Exported PFA0660w Is an Hsp40 Co-Chaperone of PfHsp70-x.

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Michael O Daniyan

Full Text Available Plasmodium falciparum, the human pathogen responsible for the most dangerous malaria infection, survives and develops in mature erythrocytes through the export of proteins needed for remodelling of the host cell. Molecular chaperones of the heat shock protein (Hsp family are prominent members of the exportome, including a number of Hsp40s and a Hsp70. PFA0660w, a type II Hsp40, has been shown to be exported and possibly form a complex with PfHsp70-x in the infected erythrocyte cytosol. However, the chaperone properties of PFA0660w and its interaction with human and parasite Hsp70s are yet to be investigated. Recombinant PFA0660w was found to exist as a monomer in solution, and was able to significantly stimulate the ATPase activity of PfHsp70-x but not that of a second plasmodial Hsp70 (PfHsp70-1 or a human Hsp70 (HSPA1A, indicating a potential specific functional partnership with PfHsp70-x. Protein binding studies in the presence and absence of ATP suggested that the interaction of PFA0660w with PfHsp70-x most likely represented a co-chaperone/chaperone interaction. Also, PFA0660w alone produced a concentration-dependent suppression of rhodanese aggregation, demonstrating its chaperone properties. Overall, we have provided the first biochemical evidence for the possible role of PFA0660w as a chaperone and as co-chaperone of PfHsp70-x. We propose that these chaperones boost the chaperone power of the infected erythrocyte, enabling successful protein trafficking and folding, and thereby making a fundamental contribution to the pathology of malaria.

[Apoptosis-modulating effects of heat shock proteins: the influence of Hsp27 chaperone on TBA Bcl-2 family proteins in Jurkat cell line].

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Riazantseva, N V; Kaĭgorodova, E V; Maroshkina, A N; Belkina, M V; Novitskiĭ, V V

2012-01-01

The in vitro phosphorylated and non-phosphorylated Hsp27 forms concentrations and Bcl-2 proteins affected by Hsp27 inhibition were studied in Jurkat-line tumor cells and healthy donor mononuclear lymphocytes by Western blotting technique. The Hsp27 inhibition causes the increase of intracellular Bax protein concentration and the decrease of Bcl-2 level leading to an increase of apoptotic changes in Jurkat line cells.

Down-regulation of cellular FLICE-inhibitory protein (Long Form contributes to apoptosis induced by Hsp90 inhibition in human lung cancer cells

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Wang Qilin

2012-12-01

Full Text Available Abstract Background Cellular FLICE-Inhibitory Protein (long form, c-FLIPL is a critical negative regulator of death receptor-mediated apoptosis. Overexpression of c-FLIPL has been reported in many cancer cell lines and is associated with chemoresistance. In contrast, down-regulation of c-FLIP may drive cancer cells into cellular apoptosis. This study aims to demonstrate that inhibition of the heat shock protein 90 (Hsp90 either by inhibitors geldanamycin/17-N-Allylamino-17-demethoxygeldanamycin (GA/17-AAG or siRNA technique in human lung cancer cells induces c-FLIPL degradation and cellular apoptosis through C-terminus of Hsp70-interacting protein (CHIP-mediated mechanisms. Methods Calu-1 and H157 cell lines (including H157-c-FLIPL overexpressing c-FLIPL and control cell H157-lacZ were treated with 17-AAG and the cell lysates were prepared to detect the given proteins by Western Blot and the cell survival was assayed by SRB assay. CHIP and Hsp90 α/β proteins were knocked down by siRNA technique. CHIP and c-FLIPL plasmids were transfected into cells and immunoprecipitation experiments were performed to testify the interactions between c-FLIPL, CHIP and Hsp90. Results c-FLIPL down-regulation induced by 17-AAG can be reversed with the proteasome inhibitor MG132, which suggested that c-FLIPL degradation is mediated by a ubiquitin-proteasome system. Inhibition of Hsp90α/β reduced c-FLIPL level, whereas knocking down CHIP expression with siRNA technique inhibited c-FLIPL degradation. Furthermore, c-FLIPL and CHIP were co-precipitated in the IP complexes. In addition, overexpression of c-FLIPL can rescue cancer cells from apoptosis. When 17-AAG was combined with an anti-cancer agent celecoxib(CCB, c-FLIPL level declined further and there was a higher degree of caspase activation. Conclusion We have elucidated c-FLIPL degradation contributes to apoptosis induced by Hsp90 inhibition, suggesting c-FLIP and Hsp90 may be the promising combined targets

Small but Crucial: The Novel Small Heat Shock Protein Hsp21 Mediates Stress Adaptation and Virulence in Candida albicans

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Mayer, François L.; Wilson, Duncan; Jacobsen, Ilse D.; Miramón, Pedro; Slesiona, Silvia; Bohovych, Iryna M.; Brown, Alistair J. P.; Hube, Bernhard

2012-01-01

Small heat shock proteins (sHsps) have multiple cellular functions. However, the biological function of sHsps in pathogenic microorganisms is largely unknown. In the present study we identified and characterized the novel sHsp Hsp21 of the human fungal pathogen Candida albicans. Using a reverse genetics approach we demonstrate the importance of Hsp21 for resistance of C. albicans to specific stresses, including thermal and oxidative stress. Furthermore, a hsp21Δ/Δ mutant was defective in invasive growth and formed significantly shorter filaments compared to the wild type under various filament-inducing conditions. Although adhesion to and invasion into human-derived endothelial and oral epithelial cells was unaltered, the hsp21Δ/Δ mutant exhibited a strongly reduced capacity to damage both cell lines. Furthermore, Hsp21 was required for resisting killing by human neutrophils. Measurements of intracellular levels of stress protective molecules demonstrated that Hsp21 is involved in both glycerol and glycogen regulation and plays a major role in trehalose homeostasis in response to elevated temperatures. Mutants defective in trehalose and, to a lesser extent, glycerol synthesis phenocopied HSP21 deletion in terms of increased susceptibility to environmental stress, strongly impaired capacity to damage epithelial cells and increased sensitivity to the killing activities of human primary neutrophils. Via systematic analysis of the three main C. albicans stress-responsive kinases (Mkc1, Cek1, Hog1) under a range of stressors, we demonstrate Hsp21-dependent phosphorylation of Cek1 in response to elevated temperatures. Finally, the hsp21Δ/Δ mutant displayed strongly attenuated virulence in two in vivo infection models. Taken together, Hsp21 mediates adaptation to specific stresses via fine-tuning homeostasis of compatible solutes and activation of the Cek1 pathway, and is crucial for multiple stages of C. albicans pathogenicity. Hsp21 therefore represents the first

[HSP90 inhibitor 17-AAG plays an important role in JAK3/STAT5 signaling pathways in HTLV-1 infection cell line HUT-102].

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Yang, Q Q; Tan, H; Fu, Z P; Ma, Q; Song, J L

2017-08-14

Objective: To analyze whether heat-shock protein 90 (HSP90) be involved in a permanently abnormal activated JAK/STAT signaling in ATL cells in vitro. Methods: The effect of 17-AAG on proliferation of ATL cell lines HUT-102 was assessed using CCK8 at different time points. Cell apoptosis was measured by flow cytometry. The specific proteins HSP90, STAT5, p-STAT5 and JAK3 were detected by Western blotting. Results: Overexpression of HSP90 in HUT-102 cell lines was disclosed ( P AAG led to reduced cell proliferation, but there was no significant change in terms of cell proliferation when the concentration of 17-AAG between 2 000-8 000 nmol/L ( P >0.05) . 17-AAG induced cell apoptosis in different time-points and concentrations. 17-AAG don't affect the expression of JAK3 gene. Conclusion: This study indicated that JAK3 as HSP90 client protein was aberrantly activated in HTLV-1-infected T-cell lines, leading to constitutive activation of p-STAT5 in JAK/STAT signal pathway, which demonstrated that HSP90-inhibitors 17-AAG inhibited the growth of HTLV-1-infected T-cell lines by reducing cell proliferation and inducing cell apoptosis.

Hsp40s specify functions of Hsp104 and Hsp90 protein chaperone machines.

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Michael Reidy

2014-10-01

Full Text Available Hsp100 family chaperones of microorganisms and plants cooperate with the Hsp70/Hsp40/NEF system to resolubilize and reactivate stress-denatured proteins. In yeast this machinery also promotes propagation of prions by fragmenting prion polymers. We previously showed the bacterial Hsp100 machinery cooperates with the yeast Hsp40 Ydj1 to support yeast thermotolerance and with the yeast Hsp40 Sis1 to propagate [PSI+] prions. Here we find these Hsp40s similarly directed specific activities of the yeast Hsp104-based machinery. By assessing the ability of Ydj1-Sis1 hybrid proteins to complement Ydj1 and Sis1 functions we show their C-terminal substrate-binding domains determined distinctions in these and other cellular functions of Ydj1 and Sis1. We find propagation of [URE3] prions was acutely sensitive to alterations in Sis1 activity, while that of [PIN+] prions was less sensitive than [URE3], but more sensitive than [PSI+]. These findings support the ideas that overexpressing Ydj1 cures [URE3] by competing with Sis1 for interaction with the Hsp104-based disaggregation machine, and that different prions rely differently on activity of this machinery, which can explain the various ways they respond to alterations in chaperone function.

Pulmonary heat shock protein expression after exposure to a metabolically activated Clara cell toxicant: relationship to protein adduct formation

International Nuclear Information System (INIS)

Williams, Kurt J.; Cruikshank, Michael K.; Plopper, Charles G.

2003-01-01

Heat shock proteins/stress proteins (Hsps) participate in regulation of protein synthesis and degradation and serve as general cytoprotectants, yet their role in lethal Clara cell injury is not clear. To define the pattern of Hsp expression in acute lethal Clara cell injury, mice were treated with the Clara cell-specific toxicant naphthalene (NA), and patterns of expression compared to electrophilic protein adduction and previously established organellar degradation and gluathione (GSH) depletion. In sites of lethal injury (distal bronchiole), prior to organellar degradation (1 h post-NA), protein adduction is detectable and ubiquitin, Hsp 25, Hsp 72, and heme-oxygenase 1 (HO-1) are increased. Maximal Hsp expression, protein adduction, and GSH depletion occur simultaneous (by 2-3 h) with early organelle disruption. Hsp expression is higher later (6-24 h), only in exfoliating cells. In airway sites (proximal bronchiole) with nonlethal Clara cell injury elevation of Hsp 25, 72, and HO-1 expression follows significant GSH depletion (greater than 50% 2 h post-NA). This data build upon our previous studies and we conclude that (1) in lethal (terminal bronchiole) and nonlethal (proximal bronchiole) Clara cell injury, Hsp induction is associated with the loss of GSH and increased protein adduction, and (2) in these same sites, organelle disruption is not a prerequisite for Hsp induction

Genome-wide identification of heat shock proteins (Hsps) and Hsp interactors in rice: Hsp70s as a case study.

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Wang, Yongfei; Lin, Shoukai; Song, Qi; Li, Kuan; Tao, Huan; Huang, Jian; Chen, Xinhai; Que, Shufu; He, Huaqin

2014-05-07

Heat shock proteins (Hsps) perform a fundamental role in protecting plants against abiotic stresses. Although researchers have made great efforts on the functional analysis of individual family members, Hsps have not been fully characterized in rice (Oryza sativa L.) and little is known about their interactors. In this study, we combined orthology-based approach with expression association data to screen rice Hsps for the expression patterns of which strongly correlated with that of heat responsive probe-sets. Twenty-seven Hsp candidates were identified, including 12 small Hsps, six Hsp70s, three Hsp60s, three Hsp90s, and three clpB/Hsp100s. Then, using a combination of interolog and expression profile-based methods, we inferred 430 interactors of Hsp70s in rice, and validated the interactions by co-localization and function-based methods. Subsequent analysis showed 13 interacting domains and 28 target motifs were over-represented in Hsp70s interactors. Twenty-four GO terms of biological processes and five GO terms of molecular functions were enriched in the positive interactors, whose expression levels were positively associated with Hsp70s. Hsp70s interaction network implied that Hsp70s were involved in macromolecular translocation, carbohydrate metabolism, innate immunity, photosystem II repair and regulation of kinase activities. Twenty-seven Hsps in rice were identified and 430 interactors of Hsp70s were inferred and validated, then the interacting network of Hsp70s was induced and the function of Hsp70s was analyzed. Furthermore, two databases named Rice Heat Shock Proteins (RiceHsps) and Rice Gene Expression Profile (RGEP), and one online tool named Protein-Protein Interaction Predictor (PPIP), were constructed and could be accessed at http://bioinformatics.fafu.edu.cn/.

The Hsc/Hsp70 co-chaperone network controls antigen aggregation and presentation during maturation of professional antigen presenting cells.

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Nadja Kettern

Full Text Available The maturation of mouse macrophages and dendritic cells involves the transient deposition of ubiquitylated proteins in the form of dendritic cell aggresome-like induced structures (DALIS. Transient DALIS formation was used here as a paradigm to study how mammalian cells influence the formation and disassembly of protein aggregates through alterations of their proteostasis machinery. Co-chaperones that modulate the interplay of Hsc70 and Hsp70 with the ubiquitin-proteasome system (UPS and the autophagosome-lysosome pathway emerged as key regulators of this process. The chaperone-associated ubiquitin ligase CHIP and the ubiquitin-domain protein BAG-1 are essential for DALIS formation in mouse macrophages and bone-marrow derived dendritic cells (BMDCs. CHIP also cooperates with BAG-3 and the autophagic ubiquitin adaptor p62 in the clearance of DALIS through chaperone-assisted selective autophagy (CASA. On the other hand, the co-chaperone HspBP1 inhibits the activity of CHIP and thereby attenuates antigen sequestration. Through a modulation of DALIS formation CHIP, BAG-1 and HspBP1 alter MHC class I mediated antigen presentation in mouse BMDCs. Our data show that the Hsc/Hsp70 co-chaperone network controls transient protein aggregation during maturation of professional antigen presenting cells and in this way regulates the immune response. Similar mechanisms may modulate the formation of aggresomes and aggresome-like induced structures (ALIS in other mammalian cell types.

17-Allylamino-17-demethoxygeldanamycin induces downregulation of critical Hsp90 protein clients and results in cell cycle arrest and apoptosis of human urinary bladder cancer cells

International Nuclear Information System (INIS)

Karkoulis, Panagiotis K; Stravopodis, Dimitrios J; Margaritis, Lukas H; Voutsinas, Gerassimos E

2010-01-01

17-Allylamino-17-demethoxygeldanamycin (17-AAG), a benzoquinone ansamycin antibiotic, specifically targets heat shock protein 90 (Hsp90) and interferes with its function as a molecular chaperone that maintains the structural and functional integrity of various protein clients involved in cellular signaling. In this study, we have investigated the effect of 17-AAG on the regulation of Hsp90-dependent signaling pathways directly implicated in cell cycle progression, survival and motility of human urinary bladder cancer cell lines. We have used MTT-based assays, FACS analysis, Western blotting, semi-quantitative RT-PCR, immunocytochemistry and scratch-wound assay in RT4, RT112 and T24 human urinary bladder cancer cell lines. We have demonstrated that, upon 17-AAG treatment, bladder cancer cells are arrested in the G1 phase of the cell cycle and eventually undergo apoptotic cell death in a dose-dependent manner. Furthermore, 17-AAG administration was shown to induce a pronounced downregulation of multiple Hsp90 protein clients and other downstream effectors, such as IGF-IR, Akt, IKK-α, IKK-β, FOXO1, ERK1/2 and c-Met, resulting in sequestration-mediated inactivation of NF-κB, reduced cell proliferation and decline of cell motility. In total, we have clearly evinced a dose-dependent and cell type-specific effect of 17-AAG on cell cycle progression, survival and motility of human bladder cancer cells, due to downregulation of multiple Hsp90 clients and subsequent disruption of signaling integrity

Acquired resistance to HSP90 inhibitor 17-AAG and increased metastatic potential are associated with MUC1 expression in colon carcinoma cells.

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Liu, Xin; Ban, Li-Li; Luo, Gang; Li, Zhi-Yao; Li, Yun-Feng; Zhou, Yong-Chun; Wang, Xi-Cai; Jin, Cong-Guo; Ye, Jia-Gui; Ma, Ding-Ding; Xie, Qing; Huang, You-Guang

2016-06-01

Heat shock protein 90 (HSP90) is a molecular chaperone required for the stability and function of many proteins. The chaperoning of oncoproteins by HSP90 enhances the survival, growth, and invasive potential of cancer cells. HSP90 inhibitors are promising new anticancer agents, in which the benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin (17-AAG) is currently in clinical evaluation. However, the implications of acquired resistance to this class of drug remain largely unexplored. In the present study, we have generated isogenic human colon cancer cell lines that are resistant to 17-AAG by continued culturing in the compound. Cross-resistance was found with another HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin. The resistant cells showed obvious morphology changes with a metastatic phenotype and significant increases in migration and adhesion to collagens. Western blotting analysis of epithelial-mesenchymal transition molecular markers found that expression of E-cadherin downregulated, whereas expression of N-cadherin and β-catenin upregulated in the resistant cells. Mucin 1 (MUC1) has been reported to mediate metastasis as well as chemical resistance in many cancers. Here, we found that MUC1 expression was significantly elevated in the acquired drug resistance cells. 17-AAG treatment could decrease MUC1 more in parental cells than in acquired 17-AAG-resistant cells. Further study found that knockdown of MUC1 expression by small interfering RNA could obviously re-sensitize the resistant cells to 17-AAG treatment, and decrease the cell migration and adhesion. These were coupled with a downregulation in N-cadherin and β-catenin. The results indicate that HSP90 inhibitor therapies in colon carcinomas could generate resistance and increase metastatic potential that might mediated by upregulation of MUC1 expression. Findings from this study further our understanding of the potential clinical effects of HSP90-directed therapies in

Chlamydia trachomatis Infection and Anti-Hsp60 Immunity: The Two Sides of the Coin

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Cappello, Francesco; Conway de Macario, Everly; Di Felice, Valentina; Zummo, Giovanni; Macario, Alberto J. L.

2009-01-01

Chlamydia trachomatis (CT) infection is one of the most common causes of reproductive tract diseases and infertility. CT-Hsp60 is synthesized during infection and is released in the bloodstream. As a consequence, immune cells will produce anti-CT-Hsp60 antibodies. Hsp60, a ubiquitous and evolutionarily conserved chaperonin, is normally sequestered inside the cell, particularly into mitochondria. However, upon cell stress, as well as during carcinogenesis, the chaperonin becomes exposed on the cell surface (sf-Hsp60) and/or is secreted from cells into the extracellular space and circulation. Reports in the literature on circulating Hsp and anti-Hsp antibodies are in many cases short on details about Hsp60 concentrations, and about the specificity spectra of the antibodies, their titers, and their true, direct, pathogenetic effects. Thus, more studies are still needed to obtain a definitive picture on these matters. Nevertheless, the information already available indicates that the concurrence of persistent CT infection and appearance of sf-Hsp60 can promote an autoimmune aggression towards stressed cells and the development of diseases such as autoimmune arthritis, multiple sclerosis, atherosclerosis, vasculitis, diabetes, and thyroiditis, among others. At the same time, immunocomplexes composed of anti-CT-Hsp60 antibodies and circulating Hsp60 (both CT and human) may form deposits in several anatomical locations, e.g., at the glomerular basal membrane. The opposite side of the coin is that pre-tumor and tumor cells with sf-Hsp60 can be destroyed with participation of the anti-Hsp60 antibody, thus stopping cancer progression before it is even noticed by the patient or physician. PMID:19714222

Chlamydia trachomatis infection and anti-Hsp60 immunity: the two sides of the coin.

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Francesco Cappello

2009-08-01

Full Text Available Chlamydia trachomatis (CT infection is one of the most common causes of reproductive tract diseases and infertility. CT-Hsp60 is synthesized during infection and is released in the bloodstream. As a consequence, immune cells will produce anti-CT-Hsp60 antibodies. Hsp60, a ubiquitous and evolutionarily conserved chaperonin, is normally sequestered inside the cell, particularly into mitochondria. However, upon cell stress, as well as during carcinogenesis, the chaperonin becomes exposed on the cell surface (sf-Hsp60 and/or is secreted from cells into the extracellular space and circulation. Reports in the literature on circulating Hsp and anti-Hsp antibodies are in many cases short on details about Hsp60 concentrations, and about the specificity spectra of the antibodies, their titers, and their true, direct, pathogenetic effects. Thus, more studies are still needed to obtain a definitive picture on these matters. Nevertheless, the information already available indicates that the concurrence of persistent CT infection and appearance of sf-Hsp60 can promote an autoimmune aggression towards stressed cells and the development of diseases such as autoimmune arthritis, multiple sclerosis, atherosclerosis, vasculitis, diabetes, and thyroiditis, among others. At the same time, immunocomplexes composed of anti-CT-Hsp60 antibodies and circulating Hsp60 (both CT and human may form deposits in several anatomical locations, e.g., at the glomerular basal membrane. The opposite side of the coin is that pre-tumor and tumor cells with sf-Hsp60 can be destroyed with participation of the anti-Hsp60 antibody, thus stopping cancer progression before it is even noticed by the patient or physician.

Expression analysis of HSP70 in the testis of Octopus tankahkeei under thermal stress.

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Long, Ling-Li; Han, Ying-Li; Sheng, Zhang; Du, Chen; Wang, You-Fa; Zhu, Jun-Quan

2015-09-01

The gene encoding heat shock protein 70 (HSP70) was identified in Octopus tankahkeei by homologous cloning and rapid amplification of cDNA ends (RACE). The full-length cDNA (2471 bp) consists of a 5'-untranslated region (UTR) (89 bp), a 3'-UTR (426 bp), and an open reading frame (1956 bp) that encodes 651 amino acid residues with a predicted molecular mass of 71.8 kDa and an isoelectric point of 5.34. Based on the amino acid sequence analysis and multiple sequence alignment, this cDNA is a member of cytoplasmic hsp70 subfamily of the hsp70 family and was designated as ot-hsp70. Tissue expression analysis showed that HSP70 expression is highest in the testes when all examined organs were compared. Immunohistochemistry analysis, together with hematoxylin-eosin staining, revealed that the HSP70 protein was expressed in all spermatogenic cells, but not in fibroblasts. In addition, O. tankahkeei were heat challenged by exposure to 32 °C seawater for 2 h, then returned to 13 °C for various recovery time (0-24 h). Relative expression of ot-hsp70 mRNA in the testes was measured at different time points post-challenge by quantitative real-time PCR. A clear time-dependent mRNA expression of ot-hsp70 after thermal stress indicates that the HSP70 gene is inducible. Ultrastructural changes of the heat-stressed testis were observed by transmission electron microscopy. We suggest that HSP70 plays an important role in spermatogenesis and testis protection against thermal stress in O. tankahkeei. Copyright © 2015 Elsevier Inc. All rights reserved.

The relationship between heat shock protein 72 expression in skeletal muscle and insulin sensitivity is dependent on adiposity

DEFF Research Database (Denmark)

Henstridge, Darren C; Forbes, Josephine M; Penfold, Sally A

2010-01-01

Decreased gene expression of heat shock protein 72 (HSP72) in skeletal muscle is associated with insulin resistance in humans. We aimed to determine whether HSP72 protein expression in insulin-sensitive tissues is related to criterion standard measures of adiposity and insulin resistance in a young...... healthy human population free of hyperglycemia. Healthy participants (N = 17; age, 30 ± 3 years) underwent measurement of body composition (dual-energy x-ray absorptiometry), a maximum aerobic capacity test (VO(2max)), an oral glucose tolerance test, and a hyperinsulinemic-euglycemic clamp (M) to access...... insulin sensitivity. Skeletal muscle and subcutaneous adipose tissue biopsies were obtained by percutaneous needle biopsy. HSP72 protein expression in skeletal muscle was inversely related to percentage body fat (r = -0.54, P

Gene expression of Hsp70, Hsp90 and Hsp110 families in normal palate and cleft palate during mouse embryogenesis.

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Zhu, Yongfei; Ren, Chuanlu; Wan, Xuying; Zhu, Yuping; Zhu, Jiangbo; Zhou, Hongyuan; Zhang, Tianbao

2013-11-01

Most previous studies focused on a small number of heat shock proteins (Hsps) and their relationships with embryogenesis, and the actual roles of these Hsps in normal and abnormal embryonic development remain unclear. It was found in the present systemic study that except for Grp170, whose expression was not detectable at GD18, all 19 Hsps of Hsp70, Hsp90 and Hsp110 families were expressed in the normal development of embryonic palate tissue in mice, but their expression patterns varied with different Hsps, presenting as a correlation with the developmental phases. In the treatment group by all-trans retinoic acid (atRA), the messenger RNA (mRNA) abundance of HspA1A, HspA1L, HspA8, HspA9, HspA12A, HspA12B, HspA13, HspA14, Hsp90AA1, Hsp90AB1, Grp94, Trap1, Hsp105, Hsp110 and Grp170 was higher in the palates at GD11 (the beginning of palate development), the mRNA abundance of HspA1A, HspA12A and HspA12B was higher at GD18 (before birth) and an mRNA expression peak of HspA1L, HspA8, HspA9, Hsp90AA1, Grp94, Hsp110 and Grp170 was observed at GD17. The mRNA abundance of most genes in atRA-induced cleft palates of the treatment group was different from that of the control group. Grp78, HspA14 and Hsp105 were closely associated with the normal palate development and cleft palate in mouse embryo, possibly as palate development-related genes. Except Grp170, the other genes may be closely associated with the development of mouse palates through participating in the stress response process and/or the antiapoptosis process.

Identification and characterization of cytosolic Hansenula polymorpha proteins belonging to the Hsp70 protein family

NARCIS (Netherlands)

Titorenko, Vladimir I.; Evers, Melchior E.; Diesel, Andre; Samyn, Bart; Beeumen, Josef van; Roggenkamp, Rainer; Kiel, Jan A.K.W.; Klei, Ida J. van der; Veenhuis, Marten

We have isolated two members of the Hsp70 protein family from the yeast Hansenula polymorpha using affinity chromatography. Both proteins were located in the cytoplasm. One of these, designated Hsp72, was inducible in nature (e.g. by heat shock). The second protein (designated Hsc74) was

Recruitment of phosphorylated small heat shock protein Hsp27 to nuclear speckles without stress

International Nuclear Information System (INIS)

Bryantsev, A.L.; Chechenova, M.B.; Shelden, E.A.

2007-01-01

During stress, the mammalian small heat shock protein Hsp27 enters cell nuclei. The present study examines the requirements for entry of Hsp27 into nuclei of normal rat kidney (NRK) renal epithelial cells, and for its interactions with specific nuclear structures. We find that phosphorylation of Hsp27 is necessary for the efficient entry into nuclei during heat shock but not sufficient for efficient nuclear entry under control conditions. We further report that Hsp27 is recruited to an RNAse sensitive fraction of SC35 positive nuclear speckles, but not other intranuclear structures, in response to heat shock. Intriguingly, Hsp27 phosphorylation, in the absence of stress, is sufficient for recruitment to speckles found in post-anaphase stage mitotic cells. Additionally, pseudophosphorylated Hsp27 fused to a nuclear localization peptide (NLS) is recruited to nuclear speckles in unstressed interphase cells, but wildtype and nonphosphorylatable Hsp27 NLS fusion proteins are not. The expression of NLS-Hsp27 mutants does not enhance colony forming abilities of cells subjected to severe heat shock, but does regulate nuclear speckle morphology. These data demonstrate that phosphorylation, but not stress, mediates Hsp27 recruitment to an RNAse soluble fraction of nuclear speckles and support a site-specific role for Hsp27 within the nucleus

Ganetespib, an HSP90 inhibitor, kills Epstein-Barr virus (EBV)-infected B and T cells and reduces the percentage of EBV-infected cells in the blood.

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Shatzer, Amber; Ali, Mir A; Chavez, Mayra; Dowdell, Kennichi; Lee, Min-Jung; Tomita, Yusuke; El-Hariry, Iman; Trepel, Jane B; Proia, David A; Cohen, Jeffrey I

2017-04-01

HSP90 inhibitors have been shown to kill Epstein-Barr virus (EBV)-infected cells by reducing the level of EBV EBNA-1 and/or LMP1. We treated virus-infected cells with ganetespib, an HSP90 inhibitor currently being evaluated in multiple clinical trials for cancer and found that the drug killed EBV-positive B and T cells and reduced the level of both EBV EBNA-1 and LMP1. Treatment of cells with ganetespib also reduced the level of pAkt. Ganetespib delayed the onset of EBV-positive lymphomas and prolonged survival in SCID mice inoculated with one EBV-transformed B-cell line, but not another B-cell line. The former cell line showed lower levels of EBNA-1 after treatment with ganetespib in vitro. Treatment of a patient with T-cell chronic active EBV with ganetespib reduced the percentage of EBV-positive cells in the peripheral blood. These data indicate that HSP90 inhibitors may have a role in the therapy of certain EBV-associated diseases.

HDJC9, a novel human type C DnaJ/HSP40 member interacts with and cochaperones HSP70 through the J domain

International Nuclear Information System (INIS)

Han Chaofeng; Chen Taoyong; Li Nan; Yang Mingjin; Wan Tao; Cao Xuetao

2007-01-01

HSP40s are a subfamily of heat shock proteins (HSPs) and play important roles in regulation of cell proliferation, survival and apoptosis by serving as chaperones for HSP70s. Up to date hundreds of HSP40 proteins derived from various species ranging from Escherichia coli to homo sapiens have been identified. Here we report the cloning and characterization of a novel human type C DnaJ homologue, HDJC9, containing a typical N-terminal J domain. HDJC9 is upregulated at both mRNA and protein levels upon various stress and mitogenic stimulations. HDJC9 is mainly localized in cell nuclei under normal culture conditions while it is transported into cytoplasm and plasma membrane upon heat shock stress through a non-classical and lipid-dependent pathway. HDJC9 can interact with HSP70s and activate the ATPase activity of HSP70s, both of which are dependent on the J domain. Our data suggest that HDJC9 is a novel cochaperone for HSP70s

Hsp90: A New Player in DNA Repair?

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Rosa Pennisi

2015-10-01

Full Text Available Heat shock protein 90 (Hsp90 is an evolutionary conserved molecular chaperone that, together with Hsp70 and co-chaperones makes up the Hsp90 chaperone machinery, stabilizing and activating more than 200 proteins, involved in protein homeostasis (i.e., proteostasis, transcriptional regulation, chromatin remodeling, and DNA repair. Cells respond to DNA damage by activating complex DNA damage response (DDR pathways that include: (i cell cycle arrest; (ii transcriptional and post-translational activation of a subset of genes, including those associated with DNA repair; and (iii triggering of programmed cell death. The efficacy of the DDR pathways is influenced by the nuclear levels of DNA repair proteins, which are regulated by balancing between protein synthesis and degradation as well as by nuclear import and export. The inability to respond properly to either DNA damage or to DNA repair leads to genetic instability, which in turn may enhance the rate of cancer development. Multiple components of the DNA double strand breaks repair machinery, including BRCA1, BRCA2, CHK1, DNA-PKcs, FANCA, and the MRE11/RAD50/NBN complex, have been described to be client proteins of Hsp90, which acts as a regulator of the diverse DDR pathways. Inhibition of Hsp90 actions leads to the altered localization and stabilization of DDR proteins after DNA damage and may represent a cell-specific and tumor-selective radiosensibilizer. Here, the role of Hsp90-dependent molecular mechanisms involved in cancer onset and in the maintenance of the genome integrity is discussed and highlighted.

The HSP90 inhibitor 17-AAG synergizes with doxorubicin and U0126 in anaplastic large cell lymphoma irrespective of ALK expression.

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Georgakis, Georgios V; Li, Yang; Rassidakis, Georgios Z; Medeiros, L Jeffrey; Younes, Anas

2006-12-01

Heat shock protein 90 (HSP90) chaperones and maintains the molecular integrity of a variety of signal transduction proteins, including the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) oncogenic protein, a genetic abnormality that is frequently observed in anaplastic large cell lymphoma (ALCL) cells. Here we demonstrate that HSP90 is overexpressed in primary and cultured ALK-positive and ALK-negative ALCL cells, and we evaluate the potential role of the small molecule inhibitor of HSP90, 17-allylamino-17-demethoxygeldanamycin (17-AAG) in treating ALCL. The antiproliferative effect of 17-AAG-cultured cells was determined by MTS assay. Apoptosis and cell-cycle arrest were determined by Annexin-V/propidium iodide and propidium iodide staining, respectively, and fluorescein-activated cell sorting analysis. Expression of HSP90 was evaluated by immunohistochemistry, and molecular changes were determined by Western blot. Treatment of cultured ALCL cells with 17-AAG induced cell-cycle arrest and apoptosis, irrespective of ALK expression. At the molecular level, 17-AAG induced degradation of ALK and Akt proteins, dephosphorylated extracellular signal-regulated kinase, and degraded the cell-cycle regulatory protein cyclin D1 and its cyclin-dependent kinases, CDK4 and CDK6, but had a differential effect on p27 and p53 proteins. Inhibition of extracellular signal-regulated kinase phosphorylation by the mitogen activated protein kinase inhibitor U0126 induced cell death in all ALCL cell lines, and sublethal concentration 17-AAG showed synergistic antiproliferative effects when combined with U0126 or doxorubicin. Our data demonstrate that targeting HSP90 function by 17-AAG may offer a novel therapeutic strategy for ALCL, either as single-agent activity or by combining 17-AAG with conventional or targeted therapeutic schemes.

MEK inhibition potentiates the activity of Hsp90 inhibitor 17-AAG against pancreatic cancer cells.

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Zhang, Tao; Li, Yanyan; Zhu, Zhenkun; Gu, Mancang; Newman, Bryan; Sun, Duxin

2010-10-04

The Ras/Raf/MEK/ERK signaling has been implicated in uncontrolled cell proliferation and tumor progression in pancreatic cancer. The purpose of this study is to evaluate the antitumor activity of MEK inhibitor U0126 in combination with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) in pancreatic cancer cells. Western blotting showed that 17-AAG caused a 2- to 3-fold transient activation of MEK/ERK signaling in pancreatic cancer cells. The activation sustained for 6 h before phospho-ERK (p-ERK) destabilization. The selective MEK inhibitor U0126 completely abolished 17-AAG induced ERK1/2 activation and resulted in more than 80% of phospho-ERK degradation after only 15 min treatment. Moreover, U0126 had complementary effect on 17-AAG regulated oncogenic and cell cycle related proteins. Although 17-AAG downregulated cyclin D1, cyclin E, CDK4 and CDK6, it led to cyclin A and CDK2 accumulation, which was reversed by the addition of U0126. Antiproliferation assay showed that combination of U0126 and 17-AAG resulted in synergistic cytotoxic effect. More importantly, 17-AAG alone only exhibited moderate inhibition of cell migration in vitro, while addition of U0126 dramatically enhanced the inhibitory effect by 2- to 5-fold. Taken together, these data demonstrate that MEK inhibitor U0126 potentiates the activity of Hsp90 inhibitor 17-AAG against pancreatic cancer cells. The combination of Hsp90 and MEK inhibition could provide a promising avenue for the treatment of pancreatic cancer.

In contrast to conventional inactivated influenza vaccines, 4xM2e.HSP70c fusion protein fully protected mice against lethal dose of H1, H3 and H9 influenza A isolates circulating in Iran

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Ebrahimi, Seyyed Mahmoud, E-mail: smebrahimi@shirazu.ac.ir [Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, P.O. Box 14155-3651,Tehran (Iran, Islamic Republic of); Research Center of Virus and Vaccine, Baqiyatallah University of Medical Science, P.O.Box 14155-3651, Tehran (Iran, Islamic Republic of); Dabaghian, Mehran [Department of Pathobiology, University of Tehran, Faculty of Veterinary Medicine, P.O. Box 14155-6453, Tehran (Iran, Islamic Republic of); Tebianian, Majid [Department of Biotechnology, Razi Vaccine and Serum Research Institute (RVSRI), P.O. Box 31975/148, Karaj, Tehran (Iran, Islamic Republic of); Zabeh Jazi, Mohammad Hossein [Department of Pathobiology, University of Tehran, Faculty of Veterinary Medicine, P.O. Box 14155-6453, Tehran (Iran, Islamic Republic of)

2012-08-15

Ideal vaccines against influenza viruses should elicit not only a humoral response, but also a cellular response. Mycobacterium tuberculosis HSP70 (mHSP70) have been found to promote immunogenic APCs function, elicit a strong cytotoxic T lymphocyte (CTL) response, and prevent the induction of tolerance. Moreover, it showed linkage of antigens to the C-terminus of mHSP70 (mHSP70c) can represent them as vaccines resulted in more potent, protective antigen specific responses in the absence of adjuvants or complex formulations. Hence, recombinant fusion protein comprising C-terminus of mHSP70 genetically fused to four tandem repeats of the ectodomain of the conserved influenza matrix protein M2 (M2e) was expressed in Escherichia coli, purified under denaturing condition, refolding, and then confirmed by SDS-PAGE, respectively. The recombinant fusion protein, 4xM2e.HSP70c, retained its immunogenicity and displayed the protective epitope of M2e by ELISA and FITC assays. A prime-boost administration of 4xM2e.HSP70c formulated in F105 buffer by intramuscular route in mice (Balb/C) provided full protection against lethal dose of mouse-adapted H1N1, H3N2, or H9N2 influenza A isolates from Iran compared to 0-33.34% survival rate of challenged unimmunized and immunized mice with the currently in use conventional vaccines designated as control groups. However, protection induced by immunization with 4xM2e.HSP70c failed to prevent weight loss in challenged mice; they experienced significantly lower weight loss, clinical symptoms and higher lung viral clearance in comparison with protective effects of conventional influenza vaccines in challenged mice. These data demonstrate that C-terminal domain of mHSP70 can be a superior candidate to deliver the adjuvant function in M2e-based influenza A vaccine in order to provide significant protection against multiple influenza A virus strains.

In contrast to conventional inactivated influenza vaccines, 4xM2e.HSP70c fusion protein fully protected mice against lethal dose of H1, H3 and H9 influenza A isolates circulating in Iran

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Ebrahimi, Seyyed Mahmoud; Dabaghian, Mehran; Tebianian, Majid; Zabeh Jazi, Mohammad Hossein

2012-01-01

Ideal vaccines against influenza viruses should elicit not only a humoral response, but also a cellular response. Mycobacterium tuberculosis HSP70 (mHSP70) have been found to promote immunogenic APCs function, elicit a strong cytotoxic T lymphocyte (CTL) response, and prevent the induction of tolerance. Moreover, it showed linkage of antigens to the C-terminus of mHSP70 (mHSP70c) can represent them as vaccines resulted in more potent, protective antigen specific responses in the absence of adjuvants or complex formulations. Hence, recombinant fusion protein comprising C-terminus of mHSP70 genetically fused to four tandem repeats of the ectodomain of the conserved influenza matrix protein M2 (M2e) was expressed in Escherichia coli, purified under denaturing condition, refolding, and then confirmed by SDS–PAGE, respectively. The recombinant fusion protein, 4xM2e.HSP70c, retained its immunogenicity and displayed the protective epitope of M2e by ELISA and FITC assays. A prime-boost administration of 4xM2e.HSP70c formulated in F105 buffer by intramuscular route in mice (Balb/C) provided full protection against lethal dose of mouse-adapted H1N1, H3N2, or H9N2 influenza A isolates from Iran compared to 0–33.34% survival rate of challenged unimmunized and immunized mice with the currently in use conventional vaccines designated as control groups. However, protection induced by immunization with 4xM2e.HSP70c failed to prevent weight loss in challenged mice; they experienced significantly lower weight loss, clinical symptoms and higher lung viral clearance in comparison with protective effects of conventional influenza vaccines in challenged mice. These data demonstrate that C-terminal domain of mHSP70 can be a superior candidate to deliver the adjuvant function in M2e-based influenza A vaccine in order to provide significant protection against multiple influenza A virus strains.

Differences in the curing of [PSI+] prion by various methods of Hsp104 inactivation.

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Yang-Nim Park

Full Text Available [PSI(+] yeast, containing the misfolded amyloid conformation of Sup35 prion, is cured by inactivation of Hsp104. There has been controversy as to whether inactivation of Hsp104 by guanidine treatment or by overexpression of the dominant negative Hsp104 mutant, Hsp104-2KT, cures [PSI(+] by the same mechanism- inhibition of the severing of the prion seeds. Using live cell imaging of Sup35-GFP, overexpression of Hsp104-2KT caused the foci to increase in size, then decrease in number, and finally disappear when the cells were cured, similar to that observed in cells cured by depletion of Hsp104. In contrast, guanidine initially caused an increase in foci size but then the foci disappeared before the cells were cured. By starving the yeast to make the foci visible in cells grown with guanidine, the number of cells with foci was found to correlate exactly with the number of [PSI(+] cells, regardless of the curing method. Therefore, the fluorescent foci are the prion seeds required for maintenance of [PSI(+] and inactivation of Hsp104 cures [PSI(+] by preventing severing of the prion seeds. During curing with guanidine, the reduction in seed size is an Hsp104-dependent effect that cannot be explained by limited severing of the seeds. Instead, in the presence of guanidine, Hsp104 retains an activity that trims or reduces the size of the prion seeds by releasing Sup35 molecules that are unable to form new prion seeds. This Hsp104 activity may also occur in propagating yeast.

Repercussion of mitochondria deformity induced by anti-Hsp90 drug 17AAG in human tumor cells

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Vishal, Chaturvedi; Kumar, Jonnala Ujwal; Veera Brahmendra Swamy, Cherukuvada; Nandini, Rangaraj; Srinivas, Gunda; Kumaresan, Rathinam; Shashi, Singh; Sreedhar, Amere Subbarao

2011-01-01

Inhibiting Hsp90 chaperone roles using 17AAG induces cytostasis or apoptosis in tumor cells through destabilization of several mutated cancer promoting proteins. Although mitochondria are central in deciding the fate of cells, 17AAG induced effects

The role of heat shock protein (HSP as inhibitor apoptosis in hypoxic conditions of bone marrow stem cell culture

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Sri Wigati Mardi Mulyani

2014-03-01

Full Text Available Background: The concept of stem cell therapy is one of the new hope as a medical therapy on salivary gland defect. However, the lack of viability of the transplanted stem cells survival rate led to the decrease of effectiveness of stem cell therapy. The underlying assumption in the decrease of viability and function of stem cells is an increase of apoptosis incidence. It suggests that the microenvironment in the area of damaged tissues is not conducive to support stem cell viability. One of the microenvironment is the hypoxia condition. Several scientific journals revealed that the administration of hypoxic cell culture can result in stress cells but on the other hand the stress condition of the cells also stimulates heat shock protein 27 (HSP 27 as antiapoptosis through inhibition of caspase 9. Purpose: The purpose of this study was to examine the role of heat shock protein 27 as inhibitor apoptosis in hypoxic conditions of bone marrow stem cell culture. Methods: Stem cell culture was performed in hypoxic conditions (O2 1% and measured the resistance to apoptosis through HSP 27 and caspase 9 expression of bone marrow mesenchymal stem cells by using immunoflorecence and real time PCR. Results: The result of study showed that preconditioning hypoxia could inhibit apoptosis through increasing HSP 27 and decreasing level of caspase 9. Conclusion: The study suggested that hypoxic precondition could reduce apoptosis by increasing amount of heat shock protein 27 and decreasing caspase 9.Latar belakang: Konsep terapi stem cell merupakan salah satu harapan baru sebagai terapi medis kelainan kelenjar ludah. Namun, rendahnya viabilitas stem cell yang ditransplantasikan menyebabkan penurunan efektivitas terapi. Asumsi yang mendasari rendahnya viabilitas dan fungsi stem cell adalah tingginya kejadian apoptosis. Hal ini menunjukkan bahwa lingkungan mikro di daerah jaringan yang rusak tidak kondusif untuk mendukung viabilitas stem cell. Salah satu lingkungan

Pharmacological targeting of HSP90 with 17-AAG induces apoptosis of myogenic cells through activation of the intrinsic pathway.

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Wagatsuma, Akira; Takayama, Yuzo; Hoshino, Takayuki; Shiozuka, Masataka; Yamada, Shigeru; Matsuda, Ryoichi; Mabuchi, Kunihiko

2017-12-16

We have shown that pharmacological inhibition of HSP90 ATPase activity induces apoptosis of myoblasts during their differentiation. However, the signaling pathways remain not fully characterized. We report that pharmacological targeting of HSP90 with 17-AAG activates the intrinsic pathway including caspase-dependent and caspase-independent pathways. 17-AAG induces the typical apoptotic phenotypes including PARP cleavage, chromatin condensation, and nuclear fragmentation with mitochondrial release of cytochrome c, Smac/DIABLO, procaspase-9 processing, and caspase-3 activation. AIF and EndoG redistribute from the mitochondria into the cytosol and are partially translocated to the nucleus in 17-AAG-treated cells. These results suggest that caspase-dependent and caspase-independent pathways should be considered in apoptosis of myogenic cells induced by inhibition of HSP90 ATPase activity.

Dietary Nutrients and Bioactive Substances Modulate Heat Shock Protein (HSP) Expression: A Review.

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Moura, Carolina Soares; Lollo, Pablo Christiano Barboza; Morato, Priscila Neder; Amaya-Farfan, Jaime

2018-05-28

Interest in the heat shock proteins (HSPs), as a natural physiological toolkit of living organisms, has ranged from their chaperone function in nascent proteins to the remedial role following cell stress. As part of the defence system, HSPs guarantee cell tolerance against a variety of stressors, including exercise, oxidative stress, hyper and hypothermia, hyper and hypoxia and improper diets. For the past couple of decades, research on functional foods has revealed a number of substances likely to trigger cell protection through mechanisms that involve the induction of HSP expression. This review will summarize the occurrence of the most easily inducible HSPs and describe the effects of dietary proteins, peptides, amino acids, probiotics, high-fat diets and other food-derived substances reported to induce HSP response in animals and humans studies. Future research may clarify the mechanisms and explore the usefulness of this natural alternative of defense and the modulating mechanism of each substance.

HSP10 selective preference for myeloid and megakaryocytic precursors in normal human bone marrow

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F Cappello

2009-06-01

Full Text Available Heat shock proteins (HSPs constitute a heterogeneous family of proteins involved in cell homeostasis. During cell life they are involved in harmful insults, as well as in immune and inflammatory reactions. It is known that they regulate gene expression, and cell proliferation, differentiation and death. HSP60 is a mitochondrial chaperonin, highly preserved during evolution, responsible of protein folding. Its function is strictly dependent on HSP10 in both prokaryotic and eukaryotic elements. We investigated the presence and the expression of HSP60 and HSP10 in a series of 20 normal human bone marrow specimens (NHBM by the means of immunohistochemistry. NHBM showed no expression of HSP60, probably due to its being below the detectable threshold, as already demonstrated in other normal human tissues. By contrast, HSP10 showed a selective positivity for myeloid and megakaryocytic lineages. The positivity was restricted to precursor cells, while mature elements were constantly negative.We postulate that HSP10 plays a role in bone marrow cell differentiation other than being a mitochondrial co-chaperonin. The present data emphasize the role of HSP10 during cellular homeostasis and encourage further investigations in this field.

4-Phenylbutyrate stimulates Hsp70 expression through the Elp2 component of elongator and STAT-3 in cystic fibrosis epithelial cells.

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Suaud, Laurence; Miller, Katelyn; Panichelli, Ashley E; Randell, Rachel L; Marando, Catherine M; Rubenstein, Ronald C

2011-12-30

Sodium 4-phenylbutyrate (4PBA) corrects trafficking of ΔF508-CFTR in Cystic Fibrosis (CF) epithelia, which is hypothesized to, at least in part, result from increased expression of Hsp70 (stress-induced 70 kDa heat shock protein). To identify other 4PBA-regulated proteins that may promote correction of ΔF508 trafficking, we performed differential display RT-PCR on mRNA from IB3-1 CF bronchiolar epithelial cells treated for 0-24 h with 1 mM 4PBA. In this screen, a STAT-3 (signal transducer and activator of transcription-3)-interacting protein, StIP-1 that regulates STAT-3 activation had transiently increased expression. StIP-1 is identical to Elongator protein 2 (Elp2), a component of the Elongator complex that regulates RNA polymerase II. Previous studies have suggested that Elongator regulates Hsp70 mRNA transcription, and that the Hsp70 promoter contains functional STAT-3-binding sites. We therefore tested the hypothesis that 4PBA increases Hsp70 expression by an Elongator- and STAT-3-dependent mechanism. 4PBA treatment of IB3-1 CF bronchiolar epithelial cells caused transiently increased expression of Hsp70 protein, as well as Elp2 protein and mRNA. Elp2 depletion by transfection of small interfering RNAs, reduced both Elp2 and Hsp70 protein expression. 4PBA also caused transient activation of STAT-3, and increased abundance of nuclear proteins that bind to the STAT-3-responsive element of the Hsp70 promoter. Luciferase reporter assays demonstrated that both Elp2 overexpression and 4PBA increase Hsp70 promoter activity, while Elp2 depletion blocked the ability of 4PBA to stimulate Hsp70 promoter activity. Together, these data suggest that Elp2 and STAT-3 mediate, at least in part, the stimulation of Hsp70 expression by 4PBA.

Overexpression of GmHsp90s, a heat shock protein 90 (Hsp90 gene family cloning from soybean, decrease damage of abiotic stresses in Arabidopsis thaliana.

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Jinyan Xu

Full Text Available Hsp90 is one of the most conserved and abundant molecular chaperones and is an essential component of the protective stress response; however, its roles in abiotic stress responses in soybean (Glycine max remain obscure. Here, 12 GmHsp90 genes from soybean were identified and found to be expressed and to function differentially under abiotic stresses. The 12 GmHsp90 genes were isolated and named GmHsp90A1-GmHsp90A6, GmHsp90B1, GmHsp90B2, GmHsp90C1.1, GmHsp90C1.2, GmHsp90C2.1 and GmHsp90C2.2 based on their characteristics and high homology to other Hsp90s according to a new nomenclature system. Quantitative real-time PCR expression data revealed that all the genes exhibited higher transcript levels in leaves and could be strongly induced under heat, osmotic and salt stress but not cold stress. Overexpression of five typical genes (GmHsp90A2, GmHsp90A4, GmHsp90B1, GmHsp90C1.1 and GmHsp90C2.1 in Arabidopsis thaliana provided useful evidences that GmHsp90 genes can decrease damage of abiotic stresses. In addition, an abnormal accumulation of proline was detected in some transgenic Arabidopsis plants suggested overexpressing GmHsp90s may affect the synthesis and response system of proline. Our work represents a systematic determination of soybean genes encoding Hsp90s, and provides useful evidence that GmHsp90 genes function differently in response to abiotic stresses and may affect the synthesis and response system of proline.

Interaction of HSP20 with a viral RdRp changes its sub-cellular localization and distribution pattern in plants.

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Li, Jing; Xiang, Cong-Ying; Yang, Jian; Chen, Jian-Ping; Zhang, Heng-Mu

2015-09-11

Small heat shock proteins (sHSPs) perform a fundamental role in protecting cells against a wide array of stresses but their biological function during viral infection remains unknown. Rice stripe virus (RSV) causes a severe disease of rice in Eastern Asia. OsHSP20 and its homologue (NbHSP20) were used as baits in yeast two-hybrid (YTH) assays to screen an RSV cDNA library and were found to interact with the viral RNA-dependent RNA polymerase (RdRp) of RSV. Interactions were confirmed by pull-down and BiFC assays. Further analysis showed that the N-terminus (residues 1-296) of the RdRp was crucial for the interaction between the HSP20s and viral RdRp and responsible for the alteration of the sub-cellular localization and distribution pattern of HSP20s in protoplasts of rice and epidermal cells of Nicotiana benthamiana. This is the first report that a plant virus or a viral protein alters the expression pattern or sub-cellular distribution of sHSPs.

Effects of Thermal Stress on the mRNA Expression of SOD, HSP90, and HSP70 in the Spotted Sea Bass ( Lateolabrax maculatus)

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Shin, Moon-Kyeong; Park, Ho-Ra; Yeo, Won-Jun; Han, Kyung-Nam

2018-03-01

The aim of this study was to elucidate the molecular mechanisms underlying the thermal stress response in the spotted sea bass ( Lateolabrax maculatus). Spotted sea basses were exposed to 4 different water temperatures (20, 22, 24, and 28°C) in increasing increments of 2°C/h from 18°C (control) for different time periods (0, 6, 12, 24, 48, 72, and 96 h). Subsequently, 3 tissues (liver, muscle, and gill) were isolated, and the levels of SOD, HSP90, and HSP70 mRNA were assessed. SOD mRNA expression was maintained at baseline levels of control fish at all water temperatures in the liver, while muscle and gill tissue showed an increase followed by a decrease over each certain time with higher water temperature. HSP90 mRNA expression increased in the liver at ≤ 24°C over time, but maintained baseline expression at 28°C. In muscle, HSP90 mRNA expression gradually increased at all water temperatures, but increased and then decreased at ≥ 24°C in gill tissue. HSP70 mRNA expression exhibited an increase and then a decrease in liver tissue at 28°C, but mainly showed similar expression patterns to HSP90 in all tissues. These results suggest the activity of a defense mechanism using SOD, HSP90, and HSP70 in the spotted sea bass upon rapid increases in water temperature, where the expression of these genes indicated differences between tissues in the extent of the defense mechanisms. Also, these results indicate that high water temperature and long-term thermal stress exposure can inhibit physiological defense mechanisms.

Neurotherapeutic activity of the recombinant heat shock protein Hsp70 in a model of focal cerebral ischemia in rats

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Shevtsov MA

2014-05-01

Full Text Available Maxim A Shevtsov,1,2 Boris P Nikolaev,3 Ludmila Y Yakovleva,3 Anatolii V Dobrodumov,4 Anastasiy S Dayneko,5 Alexey A Shmonin,5,6 Timur D Vlasov,5 Elena V Melnikova,5 Alexander D Vilisov,4,5 Irina V Guzhova,1 Alexander M Ischenko,3 Anastasiya L Mikhrina,7 Oleg V Galibin,5 Igor V Yakovenko,2 Boris A Margulis1 1Institute of Cytology of the Russian Academy of Sciences (RAS, St Petersburg, Russia; 2AL Polenov Russian Research Scientific Institute of Neurosurgery, St Petersburg, Russia; 3Research Institute of Highly Pure Biopreparations, St Petersburg, Russia; 4Institute of Macromolecular Compounds of the Russian Academy of Sciences (RAS, St Petersburg, Russia; 5First St Petersburg IP Pavlov State Medical University, St Petersburg, Russia; 6Federal Almazov Medical Research Centre, St Petersburg, Russia; 7IM Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences (RAS, St Petersburg, Russia Abstract: Recombinant 70 kDa heat shock protein (Hsp70 is an antiapoptotic protein that has a cell protective activity in stress stimuli and thus could be a useful therapeutic agent in the management of patients with acute ischemic stroke. The neuroprotective and neurotherapeutic activity of recombinant Hsp70 was explored in a model of experimental stroke in rats. Ischemia was produced by the occlusion of the middle cerebral artery for 45 minutes. To assess its neuroprotective capacity, Hsp70, at various concentrations, was intravenously injected 20 minutes prior to ischemia. Forty-eight hours after ischemia, rats were sacrificed and brain tissue sections were stained with 2% triphenyl tetrazolium chloride. Preliminary treatment with Hsp70 significantly reduced the ischemic zone (optimal response at 2.5 mg/kg. To assess Hsp70’s neurotherapeutic activity, we intravenously administered Hsp70 via the tail vein 2 hours after reperfusion (2 hours and 45 minutes after ischemia. Rats were then kept alive for 72 hours. The

Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells

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Rezende, Rafael M.; Oliveira, Rafael P.; Medeiros, Samara R.; Gomes-Santos, Ana C.; Alves, Andrea C.; Loli, Flávia G.; Guimarães, Mauro A.F.; Amaral, Sylvia S.; da Cunha, André P.; Weiner, Howard L.; Azevedo, Vasco; Miyoshi, Anderson; Faria, Ana M.C.

2013-01-01

Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L. lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-β - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice. PMID:22939403

Hsp70 in the atrial neuroendocrine units of the snail, Achatina fulica.

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Martynova, M G; Bystrova, O A; Shabelnikov, S V; Margulis, B A; Prokofjeva, D S

2007-04-01

Heat shock proteins (Hsps) are evolutionary conserved peptides well known as molecular chaperones and stress proteins. Elevated levels of extracellular Hsps in blood plasma have been observed during the stress responses and some diseases. Information on the cellular sources of extracellular Hsps and mechanisms regulating their release is still scanty. Here we showed the presence and localization of Hsp70 in the neuroendocrine system in the atrium of the snail, Achatina fulica. The occurrence of the peptide in snail atrium lysate was detected by Western blot analysis. Immunoperoxidase and immunogold staining demonstrated that Hsp70-immunoreactivity is mainly confined to the peculiar atrial neuroendocrine units which are formed by nerve fibers tightly contacted with large granular cells. Immunolabelling intensity differed in morphologically distinct types of secretory granules in the granular cells. The pictures of exocytosis of Hsp70-immunolabeled granules from the granular cells were observed. In nerve bundles, axon profiles with Hsp70-immunoreactive and those with non-immunoreactive neurosecretory granules were found. In addition, Hsp70-like material was also revealed in the granules of glia-interstitial cells that accompanied nerve fibers. Our findings provide an immuno-morphological basis for a role of Hsp70 in the functioning of the neuroendocrine system in the snail heart, and show that the atrial granular cells are a probable source of extracellular Hsp70 in the snail hemolymph.

Quercetin suppresses drug-resistant spheres via the p38 MAPK-Hsp27 apoptotic pathway in oral cancer cells.

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Su-Feng Chen

Full Text Available BACKGROUND: Treatment failure in oral squamous cell carcinoma (OSCC leading to local recurrence(s and metastases is mainly due to drug resistance. Cancer stem cells (CSCs are thought be responsible for the development of drug resistance. However, the correlations between CSCs, drug resistance, and new strategy against drug resistance in OSCC remain elusive. METHODS: A drug-resistant sphere (DRSP model was generated by using a nonadhesive culture system to induce drug-resistant cells from SCC25 oral cancer cells. A comparative analysis was performed between the parent control cells and DRSPs with a related treatment strategy focusing on the expression of epithelial-mesenchymal transition (EMT-associated markers, drug-resistance-related genes, and CSC properties in vitro, as well as tumorigenicity and the regimen for tumor regression in vivo. RESULTS: Our data show the presence of a phenomenon of EMT with gradual cellular transition from an epithelioid to mesenchymal-like spheroid morphology during induction of drug resistance. The characterization of DRSPs revealed the upregulation of the drug-resistance-related genes ABCG2 and MDR-1 and of CSC-representative markers, suggesting that DRSPs have greater resistance to cisplatin (Cis and stronger CSC properties compared with the control. Moreover, overexpression of phosphorylated heat-shock protein 27 (p-Hsp27 via the activation of p38 MAPK signaling was observed in DRSPs. Knockdown of Hsp27 decreased Cis resistance and induced apoptosis in DRSPs. Furthermore, an inhibitor of Hsp27, quercetin (Qu, suppressed p-Hsp27 expression, with alterations of the EMT signature, leading to the promotion of apoptosis in DRSPs. A xenographic study also confirmed the increase of tumorigenicity in DRSPs. The combination of Qu and Cis can reduce tumor growth and decrease drug resistance in OSCC. CONCLUSIONS: The p38 MAPK-Hsp27 axis plays an important role in CSCs-mediated drug resistance in OSCC. Targeting this axis

Cytotoxicity and Hsp 70 induction in Hep G2 cells in response to zearalenone and cytoprotection by sub-lethal heat shock

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Hassen, Wafa; Golli, Emna El; Baudrimont, Isabelle; Mobio, A. Theophile; Ladjimi, M. Moncef; Creppy, E. Edmond; Bacha, Hassen

2005-01-01

Zearalenone (ZEN) is a mycotoxin with several adverse effects in laboratory and domestic animals. The mechanism of ZEN toxicity that involves mainly binding to oestrogen receptors and inhibition of macromolecules synthesis is not fully understood. Using human hepatocytes Hep G2 cells as a model, the aim of this work was (i) to investigate the ability of ZEN to induce heat shock proteins Hsp 70 and (ii) to find out the mechanisms of ZEN cytotoxicity by examining cell proliferation and protein synthesis. Our study demonstrated that ZEN induces Hsp 70 expression in a time and dose-dependant manner; this induction occurs at non-cytotoxic concentrations, it could be therefore considered as a biomarker of toxicity. A cytoprotective effect of Hsp 70 was elicited when Hep G2 cells were exposed to Sub-Lethal heat shock prior to ZEN treatment and evidenced by a reduced ZEN cytolethality. This cytoprotection suggests that Hsp 70 may constitute an important cellular defence mechanism. Finally, our data show that ZEN is cytotoxic in Hep G2 cells by inhibiting cell proliferation and total protein synthesis and pointed out oxidative damage as possible pathway involved in ZEN toxicity; however, other investigations are needed to further confirm Zen induced oxidative stress

The targeted inhibition of mitochondrial Hsp90 overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis

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Yan, Chunlan [Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Mitochondria Hub Regulation Center, Busan, 602-714 (Korea, Republic of); Department of Physiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058 (China); Oh, Joon Seok; Yoo, Seung Hee; Lee, Jee Suk [Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Mitochondria Hub Regulation Center, Busan, 602-714 (Korea, Republic of); Yoon, Young Geol [Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Mitochondria Hub Regulation Center, Busan, 602-714 (Korea, Republic of); Department of Biomedical Science, Institute for Biomedical and Health Sciences, Jungwon University, Chungbuk, 367-805 (Korea, Republic of); Oh, Yoo Jin; Jang, Min Seok [Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Mitochondria Hub Regulation Center, Busan, 602-714 (Korea, Republic of); Lee, Sang Yeob [Department of Rheumatology, Dong-A University College of Medicine, Busan, 602-714 (Korea, Republic of); Yang, Jun [Department of Toxicology, Hangzhou Normal University School of Public Health, Hangzhou, Zhejiang, 310036 China (China); Lee, Sang Hwa [Department of Microbiology and, Dong-A University College of Medicine, Busan, 602-714 (Korea, Republic of); Kim, Hye Young [Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Mitochondria Hub Regulation Center, Busan, 602-714 (Korea, Republic of); Yoo, Young Hyun, E-mail: yhyoo@dau.ac.kr [Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Mitochondria Hub Regulation Center, Busan, 602-714 (Korea, Republic of)

2013-01-01

Previous studies have reported that a Gamitrinib variant containing triphenylphosphonium (G-TPP) binds to mitochondrial Hsp90 and rapidly inhibits its activity, thus inducing the apoptotic pathway in the cells. Accordingly, G-TPP shows a potential as a promising drug for the treatment of cancer. A cell can die from different types of cell death such as apoptosis, necrosis, necroptosis, and autophagic cell death. In this study, we further investigated the mechanisms and modes of cell death in the G-TPP-treated Hep3B and U937 cell lines. We discovered that G-TPP kills the U937 cells through the apoptotic pathway and the overexpression of Bcl-2 significantly inhibits U937 cell death to G-TPP. We further discovered that G-TPP kills the Hep3B cells by activating necroptosis in combination with the partial activation of caspase-dependent apoptosis. Importantly, G-TPP overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis. We also observed that G-TPP induces compensatory autophagy in the Hep3B cell line. We further found that whereas there is a Bcl-2-Beclin 1 interaction in response to G-TPP, silencing the beclin 1 gene failed to block LC3-II accumulation in the Hep3B cells, indicating that G-TPP triggers Beclin 1-independent protective autophagy in Hep3B cells. Taken together, these data reveal that G-TPP induces cell death through a combination of death pathways, including necroptosis and apoptosis, and overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis. These findings are important for the therapeutic exploitation of necroptosis as an alternative cell death program to bypass the resistance to apoptosis. Highlights: ► G-TPP binds to mitochondrial Hsp90. ► G-TPP induces apoptosis in U937 human leukemia cancer cells. ► G-TPP induces combination of death pathways in Hep3B cell. ► G-TPP overcomes the resistance conferred by Bcl-2 in Hep3B cells via necroptosis. ► G-TPP triggers Beclin 1-independent

Heat Shock Protein 90 (Hsp90 Expression and Breast Cancer

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Christos A. Papadimitriou

2012-09-01

Full Text Available Hsp90 is an abundant protein in mammalian cells. It forms several discrete complexes, each containing distinct groups of co-chaperones that assist protein folding and refolding during stress, protein transport and degradation. It interacts with a variety of proteins that play key roles in breast neoplasia including estrogen receptors, tumor suppressor p53 protein, angiogenesis transcription factor HIF-1alpha, antiapoptotic kinase Akt, Raf-1 MAP kinase and a variety of receptor tyrosine kinases of the erbB family. Elevated Hsp90 expression has been documented in breast ductal carcinomas contributing to the proliferative activity of breast cancer cells; whilst a significantly decreased Hsp90 expression has been shown in infiltrative lobular carcinomas and lobular neoplasia. Hsp90 overexpression has been proposed as a component of a mechanism through which breast cancer cells become resistant to various stress stimuli. Therefore, pharmacological inhibition of HSPs can provide therapeutic opportunities in the field of cancer treatment. 17-allylamino,17-demethoxygeldanamycin is the first Hsp90 inhibitor that has clinically been investigated in phase II trial, yielding promising results in patients with HER2-overexpressing metastatic breast cancer, whilst other Hsp90 inhibitors (retaspimycin HCL, NVP-AUY922, NVP-BEP800, CNF2024/BIIB021, SNX-5422, STA-9090, etc. are currently under evaluation.

Heat shock protein 90β stabilizes focal adhesion kinase and enhances cell migration and invasion in breast cancer cells

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Xiong, Xiangyang; Wang, Yao; Liu, Chengmei; Lu, Quqin; Liu, Tao; Chen, Guoan; Rao, Hai; Luo, Shiwen

2014-01-01

Focal adhesion kinase (FAK) acts as a regulator of cellular signaling and may promote cell spreading, motility, invasion and survival in malignancy. Elevated expression and activity of FAK frequently correlate with tumor cell metastasis and poor prognosis in breast cancer. However, the mechanisms by which the turnover of FAK is regulated remain elusive. Here we report that heat shock protein 90β (HSP90β) interacts with FAK and the middle domain (amino acids 233–620) of HSP90β is mainly responsible for this interaction. Furthermore, we found that HSP90β regulates FAK stability since HSP90β inhibitor 17-AAG triggers FAK ubiquitylation and subsequent proteasome-dependent degradation. Moreover, disrupted FAK-HSP90β interaction induced by 17-AAG contributes to attenuation of tumor cell growth, migration, and invasion. Together, our results reveal how HSP90β regulates FAK stability and identifies a potential therapeutic strategy to breast cancer. - Highlights: • HSP90β protects FAK from degradation by the ubiquitin-proteasome pathway. • Inhibition of HSP90β or FAK attenuates tumorigenesis of breast cancer cells. • Genetic repression of HSP90β or FAK inhibits tumor cell migration and proliferation. • Inhibition of HSP90β or FAK interferes cell invasion and cytoskeleton

Heat shock protein 90β stabilizes focal adhesion kinase and enhances cell migration and invasion in breast cancer cells

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Xiong, Xiangyang [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China); Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Nanchang University, Nanchang, Jiangxi 330006 (China); State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047 (China); Wang, Yao [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China); Liu, Chengmei [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047 (China); Lu, Quqin [Department of Biostatistics and Epidemiology, School of Public Health, Nanchang University, Nanchang, Jiangxi 330006 (China); Liu, Tao [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China); Chen, Guoan [Department of Hematology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006 (China); Rao, Hai [Department of Molecular Medicine, University of Texas Health Science Center, San Antonio, TX 78229 (United States); Luo, Shiwen, E-mail: shiwenluo@ncu.edu.cn [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China)

2014-08-01

Focal adhesion kinase (FAK) acts as a regulator of cellular signaling and may promote cell spreading, motility, invasion and survival in malignancy. Elevated expression and activity of FAK frequently correlate with tumor cell metastasis and poor prognosis in breast cancer. However, the mechanisms by which the turnover of FAK is regulated remain elusive. Here we report that heat shock protein 90β (HSP90β) interacts with FAK and the middle domain (amino acids 233–620) of HSP90β is mainly responsible for this interaction. Furthermore, we found that HSP90β regulates FAK stability since HSP90β inhibitor 17-AAG triggers FAK ubiquitylation and subsequent proteasome-dependent degradation. Moreover, disrupted FAK-HSP90β interaction induced by 17-AAG contributes to attenuation of tumor cell growth, migration, and invasion. Together, our results reveal how HSP90β regulates FAK stability and identifies a potential therapeutic strategy to breast cancer. - Highlights: • HSP90β protects FAK from degradation by the ubiquitin-proteasome pathway. • Inhibition of HSP90β or FAK attenuates tumorigenesis of breast cancer cells. • Genetic repression of HSP90β or FAK inhibits tumor cell migration and proliferation. • Inhibition of HSP90β or FAK interferes cell invasion and cytoskeleton.

Interference with the HSF1/HSP70/BAG3 Pathway Primes Glioma Cells to Matrix Detachment and BH3 Mimetic-Induced Apoptosis.

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Antonietti, Patrick; Linder, Benedikt; Hehlgans, Stephanie; Mildenberger, Iris C; Burger, Michael C; Fulda, Simone; Steinbach, Joachim P; Gessler, Florian; Rödel, Franz; Mittelbronn, Michel; Kögel, Donat

2017-01-01

Malignant gliomas exhibit a high intrinsic resistance against stimuli triggering apoptotic cell death. HSF1 acts as transcription factor upstream of HSP70 and the HSP70 co-chaperone BAG3 that is overexpressed in glioblastoma. To specifically target this resistance mechanism, we applied the selective HSF1 inhibitor KRIBB11 and the HSP70/BAG3 interaction inhibitor YM-1 in combination with the pan-Bcl-2 inhibitor AT-101. Here, we demonstrate that lentiviral BAG3 silencing significantly enhances AT-101-induced cell death and reactivates effector caspase-mediated apoptosis in U251 glioma cells with high BAG3 expression, whereas these sensitizing effects were less pronounced in U343 cells expressing lower BAG3 levels. KRIBB11 decreased protein levels of HSP70, BAG3, and the antiapoptotic Bcl-2 protein Mcl-1, and both KRIBB11 and YM-1 elicited significantly increased mitochondrial dysfunction, effector caspase activity, and apoptotic cell death after combined treatment with AT-101 and ABT-737. Depletion of BAG3 also led to a pronounced loss of cell-matrix adhesion, FAK phosphorylation, and in vivo tumor growth in an orthotopic mouse glioma model. Furthermore, it reduced the plating efficiency of U251 cells in three-dimensional clonogenic assays and limited clonogenic survival after short-term treatment with AT-101. Collectively, our data suggest that the HSF1/HSP70/BAG3 pathway plays a pivotal role for overexpression of prosurvival Bcl-2 proteins and cell death resistance of glioma. They also support the hypothesis that interference with BAG3 function is an effective novel approach to prime glioma cells to anoikis. Mol Cancer Ther; 16(1); 156-68. ©2016 AACR. ©2016 American Association for Cancer Research.

Targeting Hsp27/eIF4E interaction with phenazine compound: a promising alternative for castration-resistant prostate cancer treatment.

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Hajer, Ziouziou; Claudia, Andrieu; Erik, Laurini; Sara, Karaki; Maurizio, Fermeglia; Ridha, Oueslati; David, Taieb; Michel, Camplo; Olivier, Siri; Sabrina, Pricl; Maria, Katsogiannou; Palma, Rocchi

2017-09-29

The actual strategy to improve current therapies in advanced prostate cancer involves targeting genes activated by androgen withdrawal, either to delay or prevent the emergence of the castration-refractory phenotype. However, these genes are often implicated in several physiological processes, and long-term inhibition of survival proteins might be accompanied with cytotoxic effects. To avoid this problem, an alternative therapeutic strategy relies on the identification and use of compounds that disrupt specific protein-protein interactions involved in androgen withdrawal. Specifically, the interaction of the chaperone protein Hsp27 with the initiation factor eIF4E leads to the protection of protein synthesis initiation process and enhances cell survival during cell stress induced by castration or chemotherapy. Thus, in this work we aimed at i) identifying the interaction site of the Hsp27/eIF4E complex and ii) interfere with the relevant protein/protein association mechanism involved in castration-resistant progression of prostate cancer. By a combination of experimental and modeling techniques, we proved that eIF4E interacts with the C-terminal part of Hsp27, preferentially when Hsp27 is phosphorylated. We also observed that the loss of this interaction increased cell chemo-and hormone-sensitivity. In order to find a potential inhibitor of Hsp27/eIF4E interaction, BRET assays in combination with molecular simulations identified the phenazine derivative 14 as the compound able to efficiently interfere with this protein/protein interaction, thereby inhibiting cell viability and increasing cell death in chemo- and castration-resistant prostate cancer models in vitro and in vivo .

Integration of gene dosage and gene expression in non-small cell lung cancer, identification of HSP90 as potential target.

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Mariëlle I Gallegos Ruiz

Full Text Available BACKGROUND: Lung cancer causes approximately 1.2 million deaths per year worldwide, and non-small cell lung cancer (NSCLC represents 85% of all lung cancers. Understanding the molecular events in non-small cell lung cancer (NSCLC is essential to improve early diagnosis and treatment for this disease. METHODOLOGY AND PRINCIPAL FINDINGS: In an attempt to identify novel NSCLC related genes, we performed a genome-wide screening of chromosomal copy number changes affecting gene expression using microarray based comparative genomic hybridization and gene expression arrays on 32 radically resected tumor samples from stage I and II NSCLC patients. An integrative analysis tool was applied to determine whether chromosomal copy number affects gene expression. We identified a deletion on 14q32.2-33 as a common alteration in NSCLC (44%, which significantly influenced gene expression for HSP90, residing on 14q32. This deletion was correlated with better overall survival (P = 0.008, survival was also longer in patients whose tumors had low expression levels of HSP90. We extended the analysis to three independent validation sets of NSCLC patients, and confirmed low HSP90 expression to be related with longer overall survival (P = 0.003, P = 0.07 and P = 0.04. Furthermore, in vitro treatment with an HSP90 inhibitor had potent antiproliferative activity in NSCLC cell lines. CONCLUSIONS: We suggest that targeting HSP90 will have clinical impact for NSCLC patients.

Human-derived physiological heat shock protein 27 complex protects brain after focal cerebral ischemia in mice.

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Shinichiro Teramoto

Full Text Available Although challenging, neuroprotective therapies for ischemic stroke remain an interesting strategy for countering ischemic injury and suppressing brain tissue damage. Among potential neuroprotective molecules, heat shock protein 27 (HSP27 is a strong cell death suppressor. To assess the neuroprotective effects of HSP27 in a mouse model of transient middle cerebral artery occlusion, we purified a "physiological" HSP27 (hHSP27 from normal human lymphocytes. hHSP27 differed from recombinant HSP27 in that it formed dimeric, tetrameric, and multimeric complexes, was phosphorylated, and contained small amounts of αβ-crystallin and HSP20. Mice received intravenous injections of hHSP27 following focal cerebral ischemia. Infarct volume, neurological deficit scores, physiological parameters, and immunohistochemical analyses were evaluated 24 h after reperfusion. Intravenous injections of hHSP27 1 h after reperfusion significantly reduced infarct size and improved neurological deficits. Injected hHSP27 was localized in neurons on the ischemic side of the brain. hHSP27 suppressed neuronal cell death resulting from cytochrome c-mediated caspase activation, oxidative stress, and inflammatory responses. Recombinant HSP27 (rHSP27, which was artificially expressed and purified from Escherichia coli, and dephosphorylated hHSP27 did not have brain protective effects, suggesting that the phosphorylation of hHSP27 may be important for neuroprotection after ischemic insults. The present study suggests that hHSP27 with posttranslational modifications provided neuroprotection against ischemia/reperfusion injury and that the protection was mediated through the inhibition of apoptosis, oxidative stress, and inflammation. Intravenously injected human HSP27 should be explored for the treatment of acute ischemic strokes.

Cloning of three heat shock protein genes (HSP70, HSP90α and HSP90β) and their expressions in response to thermal stress in loach (Misgurnus anguillicaudatus) fed with different levels of vitamin C.

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Yan, Jie; Liang, Xiao; Zhang, Yin; Li, Yang; Cao, Xiaojuan; Gao, Jian

2017-07-01

Heat shock protein 70 (HSP70) and 90 (HSP90) are the most broadly studied proteins in HSP families. They play key roles in cells as molecular chaperones, in response to stress conditions such as thermal stress. In this study, full-length cDNA sequences of HSP70, HSP90α and HSP90β from loach Misgurnus anguillicaudatus were cloned. The full-length cDNA of HSP70 in loach was 2332bp encoding 644 amino acids, while HSP90α and HSP90β were 2586bp and 2678bp in length, encoding 729 and 727 amino acids, respectively. The deduced amino acid sequences of HSP70 in loach shared the highest identity with those of Megalobrama amblycephala and Cyprinus carpio. The deduced amino acid sequences of HSP90α and HSP90β in loach both shared the highest identity with those of M. amblycephala. Their mRNA tissue expression results showed that the maximum expressions of HSP70, HSP90α and HSP90β were respectively present in the intestine, brain and kidney of loach. Quantitative real-time PCR was employed to analyze the temporal expressions of HSP70, HSP90α and HSP90β in livers of loaches fed with different levels of vitamin C under thermal stress. Expression levels of the three HSP genes in loach fed the diet without vitamin C supplemented at 0 h of thermal stress were significantly lower than those at 2 h, 6 h, 12 h and 24 h of thermal stress. It indicated that expressions of the three HSP genes were sensitive to thermal stress in loach. The three HSP genes in loaches fed with 1000 mg/kg vitamin C expressed significantly lower than other vitamin C groups at many time points of thermal stress, suggesting 1000 mg/kg dietary vitamin C might decrease the body damages caused by the thermal stress. This study will be of value for further studies into thermal stress tolerance in loach. Copyright © 2017 Elsevier Ltd. All rights reserved.

Decreased Hsp90 expression in infiltrative lobular carcinoma: an immunohistochemical study

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Zagouri, Flora; Patsouris, Effstratios; Zografos, George; Sergentanis, Theodoros; Nonni, Afrodite; Papadimitriou, Christos; Pazaiti, Anastasia; Michalopoulos, Nikolaos V; Safioleas, Panagiotis; Lazaris, Andreas; Theodoropoulos, George

2010-01-01

Elevated Hsp90 expression has been documented in breast ductal carcinomas, whereas decreased Hsp90 expression has been reported in precursor lobular lesions. This study aims to assess Hsp90 expression in infiltrative lobular carcinomas of the breast. Tissue specimens were taken from 32 patients with infiltrative lobular carcinoma. Immunohistochemical assessment of Hsp90 was performed both in the lesion and the adjacent normal breast ducts and lobules; the latter serving as control. Concerning Hsp90 assessment: i) the percentage of positive cells and ii) the intensity were separately analyzed. Subsequently, the Allred score was adopted and calculated. The intensity was treated as an ordinal variable-score (0: negative, low: 1, moderate: 2, high: 3). Statistical analysis followed. All infiltrative lobular carcinoma foci mainly presented with a positive cytoplasmic immunoreaction for Hsp90. Compared to the adjacent normal ducts and lobules, infiltrative lobular carcinoma exhibited a statistically significant decrease in Hsp90 expression, both in terms of Hsp90 positive cells (%) and Allred score (74.2 ± 11.2 vs. 59.1 ± 14.2 p = 0.0001; 7.00 ± 0.95 vs. 6.22 ± 1.01, p = 0.007, Wilcoxon matched-pairs signed-ranks test). Concerning the intensity of Hsp90 immunostaining only a marginal decrease was noted (2.16 ± 0.68 vs. 1.84 ± 0.63, p = 0.087, Wilcoxon matched-pairs signed-ranks test). ILC lesions seem to exhibit decreased Hsp90 expression, a finding contrary to what might have been expected, given that high Hsp90 expression is a trait of invasive ductal carcinomas

Albumin stimulates renal tubular inflammation through an HSP70-TLR4 axis in mice with early diabetic nephropathy

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Jheng, Huei-Fen; Tsai, Pei-Jane; Chuang, Yi-Lun; Shen, Yi-Ting; Tai, Ting-An; Chen, Wen-Chung; Chou, Chuan-Kai; Ho, Li-Chun; Tang, Ming-Jer; Lai, Kuei-Tai A.; Sung, Junne-Ming; Tsai, Yau-Sheng

2015-01-01

ABSTRACT Increased urinary albumin excretion is not simply an aftermath of glomerular injury, but is also involved in the progression of diabetic nephropathy (DN). Whereas Toll-like receptors (TLRs) are incriminated in the renal inflammation of DN, whether and how albumin is involved in the TLR-related renal inflammatory response remains to be clarified. Here, we showed that both TLR2 and TLR4, one of their putative endogenous ligands [heat shock protein 70 (HSP70)] and nuclear factor-κB promoter activity were markedly elevated in the kidneys of diabetic mice. A deficiency of TLR4 but not of TLR2 alleviated albuminuria, tubulointerstitial fibrosis and inflammation induced by diabetes. The protection against renal injury in diabetic Tlr4−/− mice was associated with reduced tubular injuries and preserved cubilin levels, rather than amelioration of glomerular lesions. In vitro studies revealed that albumin, a stronger inducer than high glucose (HG), induced the release of HSP70 from proximal tubular cells. HSP70 blockade ameliorated albumin-induced inflammatory mediators. HSP70 triggered the production of inflammatory mediators in a TLR4-dependent manner. Moreover, HSP70 inhibition in vivo ameliorated diabetes-induced albuminuria, inflammatory response and tubular injury. Finally, we found that individuals with DN had higher levels of TLR4 and HSP70 in the dilated tubules than non-diabetic controls. Thus, activation of the HSP70-TLR4 axis, stimulated at least in part by albumin, in the tubular cell is a newly identified mechanism associated with induction of tubulointerstitial inflammation and aggravation of pre-existing microalbuminuria in the progression of DN. PMID:26398934

Hsp90: Friends, clients and natural foes.

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Verma, Sharad; Goyal, Sukriti; Jamal, Salma; Singh, Aditi; Grover, Abhinav

2016-08-01

Hsp90, a homodimeric ATPase, is responsible for the correct folding of a number of newly synthesized polypeptides in addition to the correct folding of denatured/misfolded client proteins. It requires several co-chaperones and other partner proteins for chaperone activity. Due to the involvement of Hsp90-dependent client proteins in a variety of oncogenic signaling pathways, Hsp90 inhibition has emerged as one of the leading strategies for anticancer chemotherapeutics. Most of Hsp90 inhibitors blocks the N terminal ATP binding pocket and prevents the conformational changes which are essential for the loading of co-chaperones and client proteins. Several other inhibitors have also been reported which disrupt chaperone cycle in ways other than binding to N terminal ATP binding pocket. The Hsp90 inhibition is associated with heat shock response, mediated by HSF-1, to overcome the loss of Hsp90 and sustain cell survival. This review is an attempt to give an over view of all the important players of chaperone cycle. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

MiR-29b inhibits collagen maturation in hepatic stellate cells through down-regulating the expression of HSP47 and lysyl oxidase

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Zhang, Yifei; Ghazwani, Mohammed; Li, Jiang [Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Sun, Ming; Stolz, Donna B. [Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261 (United States); He, Fengtian [Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Third Military Medical University, Chongqing 400038 (China); Fan, Jie [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Xie, Wen [Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Li, Song, E-mail: sol4@pitt.edu [Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261 (United States)

2014-04-18

Highlights: • Enhanced HSP47 and LOX expression is associated with decreased miR-29b level in liver fibrosis. • miR-29b down-regulates HSP47 and LOX expression. • The suppression of HSP47 and LOX by miR-29b is mediated by putative sites at their 3′-UTRs. • miR-29b inhibits extracellular LOX activity and collagen maturation. - Abstract: Altered expression of miR-29b is implicated in the pathogenesis and progression of liver fibrosis. We and others previously demonstrated that miR-29b down-regulates the expression of several extracellular-matrix (ECM) genes including Col 1A1, Col 3A1 and Elastin via directly targeting their 3′-UTRs. However, whether or not miR-29b plays a role in the post-translational regulation of ECM biosynthesis has not been reported. Heat shock protein 47 (HSP47) and lysyl oxidase (LOX) are known to be essential for ECM maturation. In this study we have demonstrated that expression of HSP47 and LOX was significantly up-regulated in culture-activated primary rat hepatic stellate cells (HSCs), TGF-β stimulated LX-2 cells and liver tissue of CCl{sub 4}-treated mice, which was accompanied by a decrease of miR-29b level. In addition, over-expression of miR-29b in LX-2 cells resulted in significant inhibition on HSP47 and LOX expression. Mechanistically, miR-29b inhibited the expression of a reporter gene that contains the respective full-length 3′-UTR from HSP47 and LOX gene, and this inhibitory effect was abolished by the deletion of a putative miR-29b targeting sequence from the 3′-UTRs. Transfection of LX-2 cells with miR-29b led to abnormal collagen structure as shown by electron-microscopy, presumably through down-regulation of the expression of molecules involved in ECM maturation including HSP47 and LOX. These results demonstrated that miR-29b is involved in regulating the post-translational processing of ECM and fibril formation.

MiR-29b inhibits collagen maturation in hepatic stellate cells through down-regulating the expression of HSP47 and lysyl oxidase

International Nuclear Information System (INIS)

Zhang, Yifei; Ghazwani, Mohammed; Li, Jiang; Sun, Ming; Stolz, Donna B.; He, Fengtian; Fan, Jie; Xie, Wen; Li, Song

2014-01-01

Highlights: • Enhanced HSP47 and LOX expression is associated with decreased miR-29b level in liver fibrosis. • miR-29b down-regulates HSP47 and LOX expression. • The suppression of HSP47 and LOX by miR-29b is mediated by putative sites at their 3′-UTRs. • miR-29b inhibits extracellular LOX activity and collagen maturation. - Abstract: Altered expression of miR-29b is implicated in the pathogenesis and progression of liver fibrosis. We and others previously demonstrated that miR-29b down-regulates the expression of several extracellular-matrix (ECM) genes including Col 1A1, Col 3A1 and Elastin via directly targeting their 3′-UTRs. However, whether or not miR-29b plays a role in the post-translational regulation of ECM biosynthesis has not been reported. Heat shock protein 47 (HSP47) and lysyl oxidase (LOX) are known to be essential for ECM maturation. In this study we have demonstrated that expression of HSP47 and LOX was significantly up-regulated in culture-activated primary rat hepatic stellate cells (HSCs), TGF-β stimulated LX-2 cells and liver tissue of CCl 4 -treated mice, which was accompanied by a decrease of miR-29b level. In addition, over-expression of miR-29b in LX-2 cells resulted in significant inhibition on HSP47 and LOX expression. Mechanistically, miR-29b inhibited the expression of a reporter gene that contains the respective full-length 3′-UTR from HSP47 and LOX gene, and this inhibitory effect was abolished by the deletion of a putative miR-29b targeting sequence from the 3′-UTRs. Transfection of LX-2 cells with miR-29b led to abnormal collagen structure as shown by electron-microscopy, presumably through down-regulation of the expression of molecules involved in ECM maturation including HSP47 and LOX. These results demonstrated that miR-29b is involved in regulating the post-translational processing of ECM and fibril formation

TAS-116, a novel Hsp90 inhibitor, selectively enhances radio-sensitivity of human cancer cells to X-rays and carbon ion radiation

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Lee, Younghyun; Sunada, Shigeaki; Hirakawa, Hirokazu; Fujimori, Akira; Nickoloff, Jac A.; Okayasu, Ryuichi

2016-01-01

Hsp90 inhibitors have been investigated as cancer therapeutics in mono-therapy and to augment radiotherapy, however serious adverse effects of early generation Hsp90 inhibitors limited their development. TAS-116 is a novel Hsp90 inhibitor with lower adverse effects than other Hsp90 inhibitors, and here we investigated the radio-sensitizing effects of TAS-116 in low LET X-ray, and high LET carbon ion irradiated human cancer cells and mouse tumor xenografts. TAS-116 decreased cell survival of both X-ray and carbon ion-irradiated human cancer cell lines (HeLa and H1299 cells), and similar to other Hsp90 inhibitors, it did not affect radiosensitivity of non-cancerous human fibroblasts. TAS-116 increased the number of radiation-induced γ-H2AX foci, and delayed the repair of DNA double-strand breaks (DSBs). TAS-116 reduced the expression of proteins that mediate repair of DSBs by homologous recombination (RAD51) and non-homologous end joining (Ku, DNA-PKcs), and suppressed formation of RAD51 foci and phosphorylation/activation of DNA-PKcs. TAS-116 also decreased expression of the cdc25 cell cycle progression marker, markedly increasing G2/M arrest. Combined treatment of mouse tumor xenografts with carbon ions and TAS-116 showed promising delay in tumor growth compared to either individual treatment. These results demonstrate that TAS-116 radio-sensitizes human cancer cells to both X rays and carbon ions by inhibiting the two major DSB repair pathways, and these effects were accompanied by marked cell cycle arrest. The promising results of combination TAS-116 + carbon ion radiation therapy of tumor xenografts justify further exploration of TAS-116 as an adjunct to radiotherapy using low or high LET radiation. PMID:28062703

In Vivo Detection of HSP90 Identifies Breast Cancers with Aggressive Behavior.

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Osada, Takuya; Kaneko, Kensuke; Gwin, William R; Morse, Michael A; Hobeika, Amy; Pogue, Brian W; Hartman, Zachary C; Hughes, Philip F; Haystead, Timothy; Lyerly, H Kim

2017-12-15

Purpose: Hsp90, a chaperone to numerous molecular pathways in malignant cells, is elevated in aggressive breast cancers. We hypothesized that identifying breast cells with elevated Hsp90 activity in situ could result in early detection of aggressive breast cancers. Experimental Design: We exploited the uptake of an Hsp90 inhibitor by malignant cells to create an imaging probe (HS131) of Hsp90 activity by linking it to a near-infrared (nIR) dye. HS131 uptake into cells correlated with cell membrane expression of Hsp90 and was used to image molecular subtypes of murine and human breast cancers in vitro and in murine models. Results: HS131 imaging was both sensitive and specific in detecting the murine 4T1 breast cancer cell line, as well as subclones with differing metastatic potential. Highly metastatic subclones (4T07) had high HS131 uptake, but subclones with lower metastatic potential (67NR, 168FARN) had low HS131 uptake. We generated isogenic cell lines to demonstrate that overexpression of a variety of specific oncogenes resulted in high HS131 uptake and retention. Finally, we demonstrated that HS131 could be used to detect spontaneous tumors in MMTV-neu mice, as well as primary and metastatic human breast cancer xenografts. HS131 could image invasive lobular breast cancer, a histologic subtype of breast cancer which is often undetectable by mammography. Conclusions: An HSP90-targeting nIR probe is sensitive and specific in imaging all molecular subtypes of murine and human breast cancer, with higher uptake in aggressive and highly metastatic clones. Clinical studies with Hsp90-targeting nIR probes will be initiated shortly. Clin Cancer Res; 23(24); 7531-42. ©2017 AACR . ©2017 American Association for Cancer Research.

Spatio-temporal regulation of Hsp90-ligand complex leads to immune activation.

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Yasuaki eTamura

2016-05-01

Full Text Available Hsp90 is the most abundant cytosolic HSP and is known to act as a molecular chaperone. We found that an Hsp90-cancer antigen peptide complex was efficiently cross-presented by human monocyte-derived dendritic cells and induced peptide-specific cytotoxic T lymphocytes. Furthermore, we observed that the internalized Hsp90-peptide complex was strictly sorted to the Rab5+, EEA1+ static early endosome and the Hsp90-chaperoned peptide was processed and bound to MHC class I molecules through a endosome-recycling pathway. We also found that extracellular Hsp90 complexed with CpG-A or self-DNA stimulates production of a large amount of IFN-α from pDCs via static early endosome targeting. Thus, extracellular Hsp90 can target the antigen or nucleic acid to a static early endosome by spatio-temporal regulation. Moreover, we showed that Hsp90 associates with and delivers TLR7/9 from the ER to early endosomes for ligand recognition. Hsp90 inhibitor, geldanamycin derivative inhibited the Hsp90 association with TLR7/9, resulting in inhibition IFN-α production, leading to improvement of SLE symptoms. Interstingly, we observed that serum Hsp90 is clearly increased in patients with active SLE compared with that in patients with inactive disease. Serum Hsp90 detected in SLE patients binds to self-DNA and/or anti-DNA Ab, thus leading to stimulation of pDCs to produce IFN-α. Thus, Hsp90 plays a crucial role in the pathogenesis of SLE and that an Hsp90 inhibitor will therefore provide a new therapeutic approach to SLE and other nucleic acid-related autoimmune diseases. We will discuss how spatio-temporal regulation of Hsp90-ligand complexes within antigen-presenting cells affects the innate immunity and adaptive immunity.

Mobile phone base station-emitted radiation does not induce phosphorylation of Hsp27.

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Hirose, H; Sakuma, N; Kaji, N; Nakayama, K; Inoue, K; Sekijima, M; Nojima, T; Miyakoshi, J

2007-02-01

An in vitro study focusing on the effects of low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields act to induce phosphorylation and overexpression of heat shock protein hsp27. First, we evaluated the responses of human cells to microwave exposure at a specific absorption rate (SAR) of 80 mW/kg, which corresponds to the limit of the average whole-body SAR for general public exposure defined as a basic restriction in the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. Second, we investigated whether continuous wave (CW) and Wideband Code Division Multiple Access (W-CDMA) modulated signal RF fields at 2.1425 GHz induced activation or gene expression of hsp27 and other heat shock proteins (hsps). Human glioblastoma A172 cells were exposed to W-CDMA radiation at SARs of 80 and 800 mW/kg for 2-48 h, and CW radiation at 80 mW/kg for 24 h. Human IMR-90 fibroblasts from fetal lungs were exposed to W-CDMA at 80 and 800 mW/kg for 2 or 28 h, and CW at 80 mW/kg for 28 h. Under the RF field exposure conditions described above, no significant differences in the expression levels of phosphorylated hsp27 at serine 82 (hsp27[pS82]) were observed between the test groups exposed to W-CDMA or CW signal and the sham-exposed negative controls, as evaluated immediately after the exposure periods by bead-based multiplex assays. Moreover, no noticeable differences in the gene expression of hsps were observed between the test groups and the negative controls by DNA Chip analysis. Our results confirm that exposure to low-level RF field up to 800 mW/kg does not induce phosphorylation of hsp27 or expression of hsp gene family.

Decreased Hsp90 expression in infiltrative lobular carcinoma: an immunohistochemical study

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Zagouri Flora

2010-08-01

Full Text Available Abstract Background Elevated Hsp90 expression has been documented in breast ductal carcinomas, whereas decreased Hsp90 expression has been reported in precursor lobular lesions. This study aims to assess Hsp90 expression in infiltrative lobular carcinomas of the breast. Methods Tissue specimens were taken from 32 patients with infiltrative lobular carcinoma. Immunohistochemical assessment of Hsp90 was performed both in the lesion and the adjacent normal breast ducts and lobules; the latter serving as control. Concerning Hsp90 assessment: i the percentage of positive cells and ii the intensity were separately analyzed. Subsequently, the Allred score was adopted and calculated. The intensity was treated as an ordinal variable-score (0: negative, low: 1, moderate: 2, high: 3. Statistical analysis followed. Results All infiltrative lobular carcinoma foci mainly presented with a positive cytoplasmic immunoreaction for Hsp90. Compared to the adjacent normal ducts and lobules, infiltrative lobular carcinoma exhibited a statistically significant decrease in Hsp90 expression, both in terms of Hsp90 positive cells (% and Allred score (74.2 ± 11.2 vs. 59.1 ± 14.2 p = 0.0001; 7.00 ± 0.95 vs. 6.22 ± 1.01, p = 0.007, Wilcoxon matched-pairs signed-ranks test. Concerning the intensity of Hsp90 immunostaining only a marginal decrease was noted (2.16 ± 0.68 vs. 1.84 ± 0.63, p = 0.087, Wilcoxon matched-pairs signed-ranks test. Conclusion ILC lesions seem to exhibit decreased Hsp90 expression, a finding contrary to what might have been expected, given that high Hsp90 expression is a trait of invasive ductal carcinomas.

BAG3 Expression in Glioblastoma Cells Promotes Accumulation of Ubiquitinated Clients in an Hsp70-dependent Manner*

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Gentilella, Antonio; Khalili, Kamel

2011-01-01

Disposal of damaged proteins and protein aggregates is a prerequisite for the maintenance of cellular homeostasis and impairment of this disposal can lead to a broad range of pathological conditions, most notably in brain-associated disorders including Parkinson and Alzheimer diseases, and cancer. In this respect, the Protein Quality Control (PQC) pathway plays a central role in the clearance of damaged proteins. The Hsc/Hsp70-co-chaperone BAG3 has been described as a new and critical component of the PQC in several cellular contexts. For example, the expression of BAG3 in the rodent brain correlates with the engagement of protein degradation machineries in response to proteotoxic stress. Nevertheless, little is known about the molecular events assisted by BAG3. Here we show that ectopic expression of BAG3 in glioblastoma cells leads to the activation of an HSF1-driven stress response, as attested by transcriptional activation of BAG3 and Hsp70. BAG3 overexpression determines an accumulation of ubiquitinated proteins and this event requires the N-terminal region, WW domain of BAG3 and the association of BAG3 with Hsp70. The ubiquitination mainly occurs on BAG3-client proteins and the inhibition of proteasomal activity results in a further accumulation of ubiquitinated clients. At the cellular level, overexpression of BAG3 in glioblastoma cell lines, but not in non-glial cells, results in a remarkable decrease in colony formation capacity and this effect is reverted when the binding of BAG3 to Hsp70 is impaired. These observations provide the first evidence for an involvement of BAG3 in the ubiquitination and turnover of its partners. PMID:21233200

Plasmodium falciparum Hsp70-z, an Hsp110 homologue, exhibits independent chaperone activity and interacts with Hsp70-1 in a nucleotide-dependent fashion.

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Zininga, Tawanda; Achilonu, Ikechukwu; Hoppe, Heinrich; Prinsloo, Earl; Dirr, Heini W; Shonhai, Addmore

2016-05-01

The role of molecular chaperones, among them heat shock proteins (Hsps), in the development of malaria parasites has been well documented. Hsp70s are molecular chaperones that facilitate protein folding. Hsp70 proteins are composed of an N-terminal nucleotide binding domain (NBD), which confers them with ATPase activity and a C-terminal substrate binding domain (SBD). In the ADP-bound state, Hsp70 possesses high affinity for substrate and releases the folded substrate when it is bound to ATP. The two domains are connected by a conserved linker segment. Hsp110 proteins possess an extended lid segment, a feature that distinguishes them from canonical Hsp70s. Plasmodium falciparum Hsp70-z (PfHsp70-z) is a member of the Hsp110 family of Hsp70-like proteins. PfHsp70-z is essential for survival of malaria parasites and is thought to play an important role as a molecular chaperone and nucleotide exchange factor of its cytosolic canonical Hsp70 counterpart, PfHsp70-1. Unlike PfHsp70-1 whose functions are fairly well established, the structure-function features of PfHsp70-z remain to be fully elucidated. In the current study, we established that PfHsp70-z possesses independent chaperone activity. In fact, PfHsp70-z appears to be marginally more effective in suppressing protein aggregation than its cytosol-localized partner, PfHsp70-1. Furthermore, based on coimmunoaffinity chromatography and surface plasmon resonance analyses, PfHsp70-z associated with PfHsp70-1 in a nucleotide-dependent fashion. Our findings suggest that besides serving as a molecular chaperone, PfHsp70-z could facilitate the nucleotide exchange function of PfHsp70-1. These dual functions explain why it is essential for parasite survival.

In vitro study comparing the efficacy of the water-soluble HSP90 inhibitors, 17-AEPGA and 17-DMAG, with that of the non‑water-soluble HSP90 inhibitor, 17-AAG, in breast cancer cell lines.

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Ghadban, Tarik; Jessen, André; Reeh, Matthias; Dibbern, Judith L; Mahner, Sven; Mueller, Volkmar; Wellner, Ulrich F; Güngör, Cenap; Izbicki, Jakob R; Vashist, Yogesh K

2016-10-01

Heat shock protein (HSP)90 has emerged as an important target in cancer therapeutics. Diverse HSP90 inhibitors are under evaluation. The aim of the present study was to investigate the growth inhibitory effects of the newly developed water-soluble HSP90 inhibitors, 17-[2-(Pyrrolidin-1-yl)ethyl]amino-17-demethoxygeldanamycin (17-AEPGA) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), compared to that of the non-water-soluble HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG). The anti-proliferative effects of the 3 drugs on the human breast cancer cell lines, MCF-7, SKBR-3 and MDA-MB-231, were examined in vitro. In addition, tumor progression factors, including human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor 1 (EGFR1) and insulin-like growth factor type 1 receptor (IGF1R), as well as apoptotic markers were analysed. We found a time- and dose-dependent effect in all the tested cell lines. The effects of 17-AEPGA and 17-DMAG were equal or superior to those of 17-AAG. The 50% growth inhibition concentration was AAG.

The dimethylthiourea-induced attenuation of cisplatin nephrotoxicity is associated with the augmented induction of heat shock proteins

International Nuclear Information System (INIS)

Tsuji, Takayuki; Kato, Akihiko; Yasuda, Hideo; Miyaji, Takehiko; Luo, Jinghui; Sakao, Yukitoshi; Ito, Hideaki; Fujigaki, Yoshihide; Hishida, Akira

2009-01-01

Dimethylthiourea (DMTU), a potent hydroxyl radical scavenger, affords protection against cisplatin (CDDP)-induced acute renal failure (ARF). Since the suppression of oxidative stress and the enhancement of heat shock proteins (HSPs) are both reported to protect against CDDP-induced renal damage, we tested whether increased HSP expression is involved in the underlying mechanisms of the DMTU-induced renal protection. We examined the effect of DMTU treatment on the expression of HSPs in the kidney until day 5 following a single injection of CDDP (5 mg/kg BW). DMTU significantly inhibited the CDDP-induced increments of serum creatinine, the number of 8-hydroxyl-2'-deoxyguanosine (8-OHdG)- and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL)-positive tubular cells, and tubular damage score (p < 0.05). CDDP significantly increased renal abundances of HO-1, HSP60, HSP72 and HSP90 at days 1, 3, and 5. DMTU significantly augmented only the expression of HSP60 expression mainly in the cytoplasm of the proximal tubular cells at days 1 and 3 in CDDP-induced ARF. DMTU also inhibited the CDDP-induced increment of Bax, a pro-apoptotic protein, in the fraction of organelles/membranes at day 3. The findings suggest that DMTU may afford protection against CDDP-induced ARF, partially through the early induction of cytoplasmic HSP60, thereby preventing the Bax-mediated apoptosis in renal tubular cells

Hsp27 regulates Akt activation and polymorphonuclear leukocyte apoptosis by scaffolding MK2 to Akt signal complex.

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Wu, Rui; Kausar, Hina; Johnson, Paul; Montoya-Durango, Diego E; Merchant, Michael; Rane, Madhavi J

2007-07-27

We have shown previously that Akt exists in a signal complex with p38 MAPK, MAPK-activated protein kinase-2 (MK2), and heat shock protein 27 (Hsp27) and MK2 phosphorylates Akt on Ser-473. Additionally, dissociation of Hsp27 from Akt, prior to Akt activation, induced polymorphonuclear leukocyte (PMN) apoptosis. However, the role of Hsp27 in regulating Akt activation was not examined. This study tested the hypothesis that Hsp27 regulates Akt activation and promotes cell survival by scaffolding MK2 to the Akt signal complex. Here we show that loss of Akt/Hsp27 interaction by anti-Hsp27 antibody treatment resulted in loss of Akt/MK2 interaction, loss of Akt-Ser-473 phosphorylation, and induced PMN apoptosis. Transfection of myristoylated Akt (AktCA) in HK-11 cells induced Akt-Ser-473 phosphorylation, activation, and Hsp27-Ser-82 phosphorylation. Cotransfection of AktCA with Hsp27 short interfering RNA, but not scrambled short interfering RNA, silenced Hsp27 expression, without altering Akt expression in HK-11 cells. Silencing Hsp27 expression inhibited Akt/MK2 interaction, inhibited Akt phosphorylation and Akt activation, and induced HK-11 cell death. Deletion mutagenesis studies identified acidic linker region (amino acids 117-128) on Akt as an Hsp27 binding region. Deletion of amino acids 117-128 on Akt resulted in loss of its interaction with Hsp27 and MK2 but not with Hsp90 as demonstrated by immunoprecipitation and glutathione S-transferase pulldown studies. Co-transfection studies demonstrated that constitutively active MK2 (MK2EE) phosphorylated Aktwt (wild type) on Ser-473 but failed to phosphorylate Akt(Delta117-128) mutant in transfixed cells. These studies collectively define a novel role of Hsp27 in regulating Akt activation and cellular apoptosis by mediating interaction between Akt and its upstream activator MK2.

HSP90 Inhibitors, Geldanamycin and Radicicol, Enhance Fisetin-Induced Cytotoxicity via Induction of Apoptosis in Human Colonic Cancer Cells

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Ming-Shun Wu

2013-01-01

Full Text Available We revealed the cytotoxic effect of the flavonoid, fisetin (FIS, on human COLO205 colon cancer cells in the presence and absence of the HSP90 inhibitors, geldanamycin (GA and radicicol (RAD. Compared to FIS treatment alone of COLO205 cells, GA and RAD significantly enhanced FIS-induced cytotoxicity, increased expression of cleaved caspase-3 and the PAPR protein, and produced a greater density of DNA ladder formation. GA and RAD also reduced the MMPs with induction of caspase-9 protein cleavage in FIS-treated COLO205 cells. Increased caspase-3 and -9 activities were detected in COLO205 cells treated with FIS+GA or FIS+RAD, and the intensity of DNA ladder formation induced by FIS+GA was reduced by adding the caspase-3 inhibitor, DEVD-FMK. A decrease in Bcl-2 but not Bcl-XL or Bax protein by FIS+GA or FIS+RAD was identified in COLO205 cells by Western blotting. A reduction in p53 protein with increased ubiquitin-tagged proteins was observed in COLO205 cells treated with FIS+GA or FIS+RAD. Furthermore, GA and RAD reduced the stability of the p53 protein in COLO205 cells under FIS stimulation. The evidence supports HSP90 inhibitors possibly sensitizing human colon cancer cells to FIS-induced apoptosis, and treating colon cancer by combining HSP90 inhibitors with FIS deserves further in vivo study.

Hsp70-GlcNAc-binding activity is released by stress, proteasome inhibition, and protein misfolding

International Nuclear Information System (INIS)

Guinez, Celine; Mir, Anne-Marie; Leroy, Yves; Cacan, Rene; Michalski, Jean-Claude; Lefebvre, Tony

2007-01-01

Numerous recent works strengthen the idea that the nuclear and cytosolic-specific O-GlcNAc glycosylation protects cells against injuries. We have first investigated O-GlcNAc level and Hsp70-GlcNAc-binding activity (HGBA) behaviour after exposure of HeLa and HepG 2 cells to a wide variety of stresses. O-GlcNAc and HGBA responses were different according to the stress and according to the cell. HGBA was released for almost all stresses, while O-GlcNAc level was modified either upwards or downwards, depending to the stress. Against all expectations, we demonstrated that energy charge did not significantly vary with stress whereas UDP-GlcNAc pools were more dramatically affected even if differences in UDP-GlcNAc contents were not correlated with O-GlcNAc variations suggesting that O-GlcNAc transferase is itself finely regulated during cell injury. Finally, HGBA could be triggered by proteasome inhibition and by L-azetidine-2-carboxylic acid (a proline analogue) incorporation demonstrating that protein misfolding is one of the key-activator of this Hsp70 property

Hsp90 selectively modulates phenotype in vertebrate development.

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Patricia L Yeyati

2007-03-01

Full Text Available Compromised heat shock protein 90 (Hsp90 function reveals cryptic phenotypes in flies and plants. These observations were interpreted to suggest that this molecular stress-response chaperone has a capacity to buffer underlying genetic variation. Conversely, the protective role of Hsp90 could account for the variable penetrance or severity of some heritable developmental malformations in vertebrates. Using zebrafish as a model, we defined Hsp90 inhibitor levels that did not induce a heat shock response or perturb phenotype in wild-type strains. Under these conditions the severity of the recessive eye phenotype in sunrise, caused by a pax6b mutation, was increased, while in dreumes, caused by a sufu mutation, it was decreased. In another strain, a previously unobserved spectrum of severe structural eye malformations, reminiscent of anophthalmia, microphthalmia, and nanophthalmia complex in humans, was uncovered by this limited inhibition of Hsp90 function. Inbreeding of offspring from selected unaffected carrier parents led to significantly elevated malformation frequencies and revealed the oligogenic nature of this phenotype. Unlike in Drosophila, Hsp90 inhibition can decrease developmental stability in zebrafish, as indicated by increased asymmetric presentation of anophthalmia, microphthalmia, and nanophthalmia and sunrise phenotypes. Analysis of the sunrise pax6b mutation suggests a molecular mechanism for the buffering of mutations by Hsp90. The zebrafish studies imply that mild perturbation of Hsp90 function at critical developmental stages may underpin the variable penetrance and expressivity of many developmental anomalies where the interaction between genotype and environment plays a major role.

Changes in HSP gene and protein expression in natural scrapie with brain damage

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2011-01-01

Heat shock proteins (Hsp) perform cytoprotective functions such as apoptosis regulation and inflammatory response control. These proteins can also be secreted to the extracellular medium, acting as inflammatory mediators, and their chaperone activity permits correct folding of proteins and avoids the aggregation of anomalous isoforms. Several studies have proposed the implication of Hsp in prion diseases. We analysed the gene expression and protein distribution of different members of the Hsp27, Hsp70, and Hsp90 families in the central nervous system of sheep naturally infected with scrapie. Different expression profiles were observed in the areas analysed. Whereas changes in transcript levels were not observed in the cerebellum or medulla oblongata, a significant decrease in HSP27 and HSP90 was detected in the prefrontal cortex. In contrast, HSP73 was over-expressed in diencephalons of scrapie animals. Western blotting did not reveal significant differences in Hsp90 and Hsp70 protein expression between scrapie and control animals. Expression rates identified by real-time RT-PCR and western blotting were compared with the extent of classical scrapie lesions using stepwise regression. Changes in Hsp gene and protein expression were associated with prion protein deposition, gliosis and spongiosis rather than with apoptosis. Finally, immunohistochemistry revealed intense Hsp70 and Hsp90 immunolabelling in Purkinje cells of scrapie sheep. In contrast, controls displayed little or no staining in these cells. The observed differences in gene expression and protein distribution suggest that the heat shock proteins analysed play a role in the natural form of the disease. PMID:21314976

Changes in HSP gene and protein expression in natural scrapie with brain damage

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Serrano Carmen

2011-01-01

Full Text Available Abstract Heat shock proteins (Hsp perform cytoprotective functions such as apoptosis regulation and inflammatory response control. These proteins can also be secreted to the extracellular medium, acting as inflammatory mediators, and their chaperone activity permits correct folding of proteins and avoids the aggregation of anomalous isoforms. Several studies have proposed the implication of Hsp in prion diseases. We analysed the gene expression and protein distribution of different members of the Hsp27, Hsp70, and Hsp90 families in the central nervous system of sheep naturally infected with scrapie. Different expression profiles were observed in the areas analysed. Whereas changes in transcript levels were not observed in the cerebellum or medulla oblongata, a significant decrease in HSP27 and HSP90 was detected in the prefrontal cortex. In contrast, HSP73 was over-expressed in diencephalons of scrapie animals. Western blotting did not reveal significant differences in Hsp90 and Hsp70 protein expression between scrapie and control animals. Expression rates identified by real-time RT-PCR and western blotting were compared with the extent of classical scrapie lesions using stepwise regression. Changes in Hsp gene and protein expression were associated with prion protein deposition, gliosis and spongiosis rather than with apoptosis. Finally, immunohistochemistry revealed intense Hsp70 and Hsp90 immunolabelling in Purkinje cells of scrapie sheep. In contrast, controls displayed little or no staining in these cells. The observed differences in gene expression and protein distribution suggest that the heat shock proteins analysed play a role in the natural form of the disease.

A role for anti-HSP60 antibodies in arthritis

DEFF Research Database (Denmark)

Carlsen, Thomas Gelsing; Bennike, Tue; Christiansen, Gunna

2013-01-01

As a result of the high sequence similarity between HSP60 proteins, found in both prokaryotic and eukaryotic cells, it has been suggested, but never concluded, that anti-HSP60 antibodies could be of importance in the pathology of arthritis diseases explained by a concept named molecular mimicry...

Partial Protection of Mice against Trypanosoma cruzi after Immunizing with the TcY 72 Antigenic Preparation

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Yara M Gomes

1999-03-01

Full Text Available A 72 kDa Trypanosoma cruzi glycoprotein recognized by the 164C11 monoclonal antibody (IgM isotype was purified by preparative electrophoresis. The antigenic preparation obtained, named TcY 72, was used to immunize C57Bl/10 mice. The following results were observed after immunization: (1 induction of higher titres of IgG than IgM antibodies, as evaluated by indirect immunofluorescence; (2 significant DTH after injection of epimastigotes in mice footpads; (3 peak parasitemia in immunized mice was significantly reduced and animals were negative by 13 days post-infection, although the mice still succumb to infection; (4 the phenotypic analysis of spleen cell populations showed a decrease in the CD4/CD8 ratio in immunized mice. Taken as a whole, these findings indicate that TcY 72 is immunogenic and potentially important for protective immunity.

Comparative analysis of novel and conventional Hsp90 inhibitors on HIF activity and angiogenic potential in clear cell renal cell carcinoma: implications for clinical evaluation

International Nuclear Information System (INIS)

Bohonowych, Jessica ES; Peng, Shuping; Gopal, Udhayakumar; Hance, Michael W; Wing, Shane B; Argraves, Kelley M; Lundgren, Karen; Isaacs, Jennifer S

2011-01-01

Perturbing Hsp90 chaperone function targets hypoxia inducible factor (HIF) function in a von Hippel-Lindau (VHL) independent manner, and represents an approach to combat the contribution of HIF to cell renal carcinoma (CCRCC) progression. However, clinical trials with the prototypic Hsp90 inhibitor 17-AAG have been unsuccessful in halting the progression of advanced CCRCC. Here we evaluated a novel next generation small molecule Hsp90 inhibitor, EC154, against HIF isoforms and HIF-driven molecular and functional endpoints. The effects of EC154 were compared to those of the prototypic Hsp90 inhibitor 17-AAG and the histone deacetylase (HDAC) inhibitor LBH589. The findings indicate that EC154 is a potent inhibitor of HIF, effective at doses 10-fold lower than 17-AAG. While EC154, 17-AAG and the histone deacetylase (HDAC) inhibitor LBH589 impaired HIF transcriptional activity, CCRCC cell motility, and angiogenesis; these effects did not correlate with their ability to diminish HIF protein expression. Further, our results illustrate the complexity of HIF targeting, in that although these agents suppressed HIF transcripts with differential dynamics, these effects were not predictive of drug efficacy in other relevant assays. We provide evidence for EC154 targeting of HIF in CCRCC and for LBH589 acting as a suppressor of both HIF-1 and HIF-2 activity. We also demonstrate that 17-AAG and EC154, but not LBH589, can restore endothelial barrier function, highlighting a potentially new clinical application for Hsp90 inhibitors. Finally, given the discordance between HIF activity and protein expression, we conclude that HIF expression is not a reliable surrogate for HIF activity. Taken together, our findings emphasize the need to incorporate an integrated approach in evaluating Hsp90 inhibitors within the context of HIF suppression

Comparative analysis of novel and conventional Hsp90 inhibitors on HIF activity and angiogenic potential in clear cell renal cell carcinoma: implications for clinical evaluation

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Bohonowych Jessica ES

2011-12-01

Full Text Available Abstract Background Perturbing Hsp90 chaperone function targets hypoxia inducible factor (HIF function in a von Hippel-Lindau (VHL independent manner, and represents an approach to combat the contribution of HIF to cell renal carcinoma (CCRCC progression. However, clinical trials with the prototypic Hsp90 inhibitor 17-AAG have been unsuccessful in halting the progression of advanced CCRCC. Methods Here we evaluated a novel next generation small molecule Hsp90 inhibitor, EC154, against HIF isoforms and HIF-driven molecular and functional endpoints. The effects of EC154 were compared to those of the prototypic Hsp90 inhibitor 17-AAG and the histone deacetylase (HDAC inhibitor LBH589. Results The findings indicate that EC154 is a potent inhibitor of HIF, effective at doses 10-fold lower than 17-AAG. While EC154, 17-AAG and the histone deacetylase (HDAC inhibitor LBH589 impaired HIF transcriptional activity, CCRCC cell motility, and angiogenesis; these effects did not correlate with their ability to diminish HIF protein expression. Further, our results illustrate the complexity of HIF targeting, in that although these agents suppressed HIF transcripts with differential dynamics, these effects were not predictive of drug efficacy in other relevant assays. Conclusions We provide evidence for EC154 targeting of HIF in CCRCC and for LBH589 acting as a suppressor of both HIF-1 and HIF-2 activity. We also demonstrate that 17-AAG and EC154, but not LBH589, can restore endothelial barrier function, highlighting a potentially new clinical application for Hsp90 inhibitors. Finally, given the discordance between HIF activity and protein expression, we conclude that HIF expression is not a reliable surrogate for HIF activity. Taken together, our findings emphasize the need to incorporate an integrated approach in evaluating Hsp90 inhibitors within the context of HIF suppression.

Hsp47 and cyclophilin B traverse the endoplasmic reticulum with procollagen into pre-Golgi intermediate vesicles. A role for Hsp47 and cyclophilin B in the export of procollagen from the endoplasmic reticulum.

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Smith, T; Ferreira, L R; Hebert, C; Norris, K; Sauk, J J

1995-08-04

Hsp47 and cyclophilin B (CyPB) are residents of the endoplasmic reticulum (ER). Both of these proteins are closely associated with polysome-associated alpha 1(I) procollagen chains. Hsp47 possesses chaperone properties early during the translation of procollagen while the cis/trans-isomerase properties of CyPB facilitate procollagen folding. In this report, we further investigate the interaction of these proteins with procollagen I during export from the ER. To inhibit vesicular budding and retain procollagen within the ER, cells were treated with the heterotrimeric G protein inhibitor mastoparan or calphostin C, a specific inhibitor of diacylglycerol/phorbol ester binding proteins. To arrest procollagen in pre-Golgi intermediate vesicles, cells were treated with guanosine 5'-3-O-(thio)triphosphate. Pulse-chase experiments of cells labeled with [35S]methionine followed by immunoprecipitation during the chase period with anti-procollagen, anti-Hsp47, and anti-CyPB antibodies were performed to reveal the relationship between Hsp47/CyPB/procollagen I. The distribution of procollagen, Hsp47, and CyPB to the ER and/or pre-Golgi vesicles was verified by immunofluorescence. Hsp47 and CyPB remained associated with procollagen retained within the ER. Hsp47 and CyPB were also associated with procollagen exported from the ER into pre-Golgi intermediate vesicles. Treatment of cells with cyclosporin A diminished the levels of CyPB bound to procollagen and diminished the rate of Hsp47 released from procollagen and the rate of procollagen secretion, suggesting that Hsp47 release from procollagen may be driven by helix formation. Also, these studies suggest that Hsp47 may resemble protein disulfide isomerase and possess both chaperone and anti-chaperone properties. During translation, high levels of Hsp47 are seen to limit protein aggregation and facilitate chain registration. Later, Hsp47 and/or CyPB and protein disulfide isomerase act as anti-chaperones and provide the basis for

RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells

International Nuclear Information System (INIS)

Gilmour, D.S.; Lis, J.T.

1986-01-01

By using a protein-DNA cross-linking method, we examined the in vivo distribution of RNA polymerase II on the hsp70 heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end of the hsp70 gene, predominantly between nucleotides -12 and +65 relative to the start of transcription. This association of RNA polymerase II was apparent whether the cross-linking was performed by a 10-min UV irradiation of chilled cells with mercury vapor lamps or by a 40-microsecond irradiation of cells with a high-energy xenon flash lamp. We hypothesize that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerase II may be critical in the mechanism of transcription activation

Heat shock protein 10 (Hsp10) in immune-related diseases: one coin, two sides

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Jia, Haibo; Halilou, Amadou I.; Hu, Liang; Cai, Wenqian; Liu, Jing; Huang, Bo

2011-01-01

Heat shock protein 10 (Hsp10) in eukaryotes, originally identified as a mitochondrial chaperone, now is also known to be present in cytosol, cell surface, extracellular space and peripheral blood. Functionally besides participating in mitochondrial protein folding in association with Hsp60, Hsp10 appears to be related to pregnancy, cancer and autoimmune inhibition. Hsp10 can be released to peripheral blood at very early time point of pregnancy and given another name called early pregnancy factor (EPF), which seems to play a critical role in developing a pregnant niche. In malignant disorders, Hsp10 is usually abnormally expressed in the cytosol of malignant cells and further released to extracellular space, resulting in tumor-promoting effect from various aspects. Furthermore, distinct from other heat shock protein members, whose soluble form is recognized as danger signal by immune cells and triggers immune responses, Hsp10 after release, however, is designed to be an inhibitory signal by limiting immune response. This review discusses how Hsp10 participates in various physiological and pathological processes from basic protein molecule folding to pregnancy, cancer and autoimmune diseases, and emphasizes how important the location is for the function exertion of a molecule. PMID:21969171

Over-expression of Arabidopsis DnaJ (Hsp40) contributes to NaCl ...

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Over-expression of Arabidopsis DnaJ (Hsp40) contributes to NaCl-stress tolerance. Z Zhichang, Z Wanrong, Y Jinping, Z Jianjun, LZL Xufeng, Y Yang. Abstract. DnaJ (Hsp40), a heat shock protein, is a molecular chaperones responsive to various environmental stress. To analyze the protective role of DnaJ, we obtained ...

Quantitative analysis of the interplay between hsc70 and its co-chaperone HspBP1

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Hicham Mahboubi

2015-12-01

Full Text Available Background. Chaperones and their co-factors are components of a cellular network; they collaborate to maintain proteostasis under normal and harmful conditions. In particular, hsp70 family members and their co-chaperones are essential to repair damaged proteins. Co-chaperones are present in different subcellular compartments, where they modulate chaperone activities.Methods and Results. Our studies assessed the relationship between hsc70 and its co-factor HspBP1 in human cancer cells. HspBP1 promotes nucleotide exchange on hsc70, but has also chaperone-independent functions. We characterized the interplay between hsc70 and HspBP1 by quantitative confocal microscopy combined with automated image analyses and statistical evaluation. Stress and the recovery from insult changed significantly the subcellular distribution of hsc70, but had little effect on HspBP1. Single-cell measurements and regression analysis revealed that the links between the chaperone and its co-factor relied on (i the physiological state of the cell and (ii the subcellular compartment. As such, we identified a linear relationship and strong correlation between hsc70 and HspBP1 distribution in control and heat-shocked cells; this correlation changed in a compartment-specific fashion during the recovery from stress. Furthermore, we uncovered significant stress-induced changes in the colocalization between hsc70 and HspBP1 in the nucleus and cytoplasm.Discussion. Our quantitative approach defined novel properties of the co-chaperone HspBP1 as they relate to its interplay with hsc70. We propose that changes in cell physiology promote chaperone redistribution and thereby stimulate chaperone-independent functions of HspBP1.

Crosstalk between p38, Hsp25 and Akt in spinal motor neurons after sciatic nerve injury

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Murashov, A. K.; Ul Haq, I.; Hill, C.; Park, E.; Smith, M.; Wang, X.; Wang, X.; Goldberg, D. J.; Wolgemuth, D. J.

2001-01-01

The p38 stress-activated protein kinase pathway is involved in regulation of phosphorylation of Hsp25, which in turn regulates actin filament dynamic in non-neuronal cells. We report that p38, Hsp25 and Akt signaling pathways were specifically activated in spinal motor neurons after sciatic nerve axotomy. The activation of the p38 kinase was required for induction of Hsp25 expression. Furthermore, Hsp25 formed a complex with Akt, a member of PI-3 kinase pathway that prevents neuronal cell death. Together, our observations implicate Hsp25 as a central player in a complex system of signaling that may both promote regeneration of nerve fibers and prevent neuronal cell death in the injured spinal cord.

Apigenin inhibits proliferation and induces apoptosis in human multiple myeloma cells through targeting the trinity of CK2, Cdc37 and Hsp90

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Xu Yuan-Ji

2011-08-01

Full Text Available Abstract Background Multiple myeloma (MM is a B-cell malignancy that is largely incurable and is characterized by the accumulation of malignant plasma cells in the bone marrow. Apigenin, a common flavonoid, has been reported to suppress proliferation in a wide variety of solid tumors and hematological cancers; however its mechanism is not well understood and its effect on MM cells has not been determined. Results In this study, we investigated the effects of apigenin on MM cell lines and on primary MM cells. Cell viability assays demonstrated that apigenin exhibited cytotoxicity against both MM cell lines and primary MM cells but not against normal peripheral blood mononuclear cells. Together, kinase assays, immunoprecipitation and western blot analysis showed that apigenin inhibited CK2 kinase activity, decreased phosphorylation of Cdc37, disassociated the Hsp90/Cdc37/client complex and induced the degradation of multiple kinase clients, including RIP1, Src, Raf-1, Cdk4 and AKT. By depleting these kinases, apigenin suppressed both constitutive and inducible activation of STAT3, ERK, AKT and NF-κB. The treatment also downregulated the expression of the antiapoptotic proteins Mcl-1, Bcl-2, Bcl-xL, XIAP and Survivin, which ultimately induced apoptosis in MM cells. In addition, apigenin had a greater effects in depleting Hsp90 clients when used in combination with the Hsp90 inhibitor geldanamycin and the histone deacetylase inhibitor vorinostat. Conclusions Our results suggest that the primary mechanisms by which apigenin kill MM cells is by targeting the trinity of CK2-Cdc37-Hsp90, and this observation reveals the therapeutic potential of apigenin in treating multiple myeloma.

Heat shock protein 70 inhibits shrinkage-induced programmed cell death via mechanisms independent of effects on cell volume-regulatory membrane transport proteins

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Nylandsted, J; Jäättelä, M; Hoffmann, E K

2004-01-01

Cell shrinkage is a ubiquitous feature of programmed cell death (PCD), but whether it is an obligatory signalling event in PCD is unclear. Heat shock protein 70 (Hsp70) potently counteracts PCD in many cells, by mechanisms that are incompletely understood. In the present investigation, we found...... that severe hypertonic stress greatly diminished the viability of murine fibrosarcoma cells (WEHI-902) and immortalized murine embryonic fibroblasts (iMEFs). This effect was attenuated markedly by Hsp70 over-expression. To determine whether the protective effect of Hsp70 was mediated via an effect on volume...... regulatory ion transport, we compared regulatory volume decrease (RVD) and increase (RVI) in control WEHI-902 cells and after increasing Hsp70 levels by heat shock or over-expression (WEHI-912). Hsp70 levels affected neither RVD, RVI nor the relative contributions of the Na(+)/H(+)-exchanger (NHE1) and Na...

Identification of the plant compound geraniin as a novel Hsp90 inhibitor.

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Antonio Vassallo

Full Text Available Besides its function in normal cellular growth, the molecular chaperone heat shock protein 90 (Hsp90 binds to a large number of client proteins required for promoting cancer cell growth and/or survival. In an effort to discover new small molecules able to inhibit the Hsp90 ATPase and chaperoning activities, we screened, by a surface plasmon resonance assay, a small library including different plant polyphenols. The ellagitannin geraniin, was identified as the most promising molecule, showing a binding affinity to Hsp90α similar to that of 17-(allylamino-17-demethoxygeldanamycin (17AGG. Geraniin was able to inhibit in vitro the Hsp90α ATPase activity in a dose-dependent manner, with an inhibitory efficiency comparable to that measured for 17-AAG. In addition, this compound compromised the chaperone activity of Hsp90α, monitored by the citrate synthase thermal induced aggregation assay. Geraniin decreased the viability of HeLa and Jurkat cell lines and caused an arrest in G2/M phase. We also proved that following exposure to different concentrations of geraniin, the level of expression of the client proteins c-Raf, pAkt, and EGFR was strongly down-regulated in both the cell lines. These results, along with the finding that geraniin did not exert any appreciable cytotoxicity on normal cells, encourage further studies on this compound as a promising chemical scaffold for the design of new Hsp90 inhibitors.

Expression of Hsp27 and Hsp70 and vacuolization in the pituitary glands in cases of fatal hypothermia.

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Doberentz, Elke; Markwerth, Philipp; Wagner, Rebecca; Madea, Burkhard

2017-09-01

Hypothermia causes systemic cellular stress. The pituitary gland is an endocrine gland and plays an important role in thermoregulation. When the core body temperature drops, the pituitary gland is activated by stimulation of hypothalamic hormones. In this study, we investigated morphological alterations of the pituitary gland in cases of fatal hypothermia. Several morphological alterations of the anterior lobe of the pituitary gland, such as hemorrhage, vacuolization, and hyperemia, have been previously described in fatal hypothermia. However, the diagnostic value of these findings is controversial. We compared 11 cases of fatal hypothermia with 10 cases lacking antemortem hypothermic influences. In the presence of thermal cellular stress, the expression of heat shock proteins increases to protect cellular structures. Therefore, we immunohistochemically analyzed Hsp27 and Hsp70. Hsp27 expression was detected in 27.3% of the cases of fatal hypothermia and in 10.0% of the control cases, whereas Hsp70 expression was not detected in any case. Additionally, Sudan staining was performed to quantify fatty degeneration. A positive reaction was found in 45.5% of the study group and in 10.0% of the control group. This indicates that fatty degeneration might be a valuable marker when other macroscopic signs of hypothermia are absent.

Administration of Mycobacterium leprae rHsp65 aggravates experimental autoimmune uveitis in mice.

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Eliana B Marengo

Full Text Available The 60 kDa heat shock protein family, Hsp60, constitutes an abundant and highly conserved class of molecules that are highly expressed in chronic-inflammatory and autoimmune processes. Experimental autoimmune uveitis [EAU] is a T cell mediated intraocular inflammatory disease that resembles human uveitis. Mycobacterial and homologous Hsp60 peptides induces uveitis in rats, however their participation in aggravating the disease is poorly known. We here evaluate the effects of the Mycobacterium leprae Hsp65 in the development/progression of EAU and the autoimmune response against the eye through the induction of the endogenous disequilibrium by enhancing the entropy of the immunobiological system with the addition of homologous Hsp. B10.RIII mice were immunized subcutaneously with interphotoreceptor retinoid-binding protein [IRBP], followed by intraperitoneally inoculation of M. leprae recombinant Hsp65 [rHsp65]. We evaluated the proliferative response, cytokine production and the percentage of CD4(+IL-17(+, CD4(+IFN-gamma(+ and CD4(+Foxp3(+ cells ex vivo, by flow cytometry. Disease severity was determined by eye histological examination and serum levels of anti-IRBP and anti-Hsp60/65 measured by ELISA. EAU scores increased in the Hsp65 group and were associated with an expansion of CD4(+IFN-gamma(+ and CD4(+IL-17(+ T cells, corroborating with higher levels of IFN-gamma. Our data indicate that rHsp65 is one of the managers with a significant impact over the immune response during autoimmunity, skewing it to a pathogenic state, promoting both Th1 and Th17 commitment. It seems comprehensible that the specificity and primary function of Hsp60 molecules can be considered as a potential pathogenic factor acting as a whistleblower announcing chronic-inflammatory diseases progression.

Hsp90α regulates ATM and NBN functions in sensing and repair of DNA double-strand breaks.

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Pennisi, Rosa; Antoccia, Antonio; Leone, Stefano; Ascenzi, Paolo; di Masi, Alessandra

2017-08-01

The molecular chaperone heat shock protein 90 (Hsp90α) regulates cell proteostasis and mitigates the harmful effects of endogenous and exogenous stressors on the proteome. Indeed, the inhibition of Hsp90α ATPase activity affects the cellular response to ionizing radiation (IR). Although the interplay between Hsp90α and several DNA damage response (DDR) proteins has been reported, its role in the DDR is still unclear. Here, we show that ataxia-telangiectasia-mutated kinase (ATM) and nibrin (NBN), but not 53BP1, RAD50, and MRE11, are Hsp90α clients as the Hsp90α inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) induces ATM and NBN polyubiquitination and proteosomal degradation in normal fibroblasts and lymphoblastoid cell lines. Hsp90α-ATM and Hsp90α-NBN complexes are present in unstressed and irradiated cells, allowing the maintenance of ATM and NBN stability that is required for the MRE11/RAD50/NBN complex-dependent ATM activation and the ATM-dependent phosphorylation of both NBN and Hsp90α in response to IR-induced DNA double-strand breaks (DSBs). Hsp90α forms a complex also with ph-Ser1981-ATM following IR. Upon phosphorylation, NBN dissociates from Hsp90α and translocates at the DSBs, while phThr5/7-Hsp90α is not recruited at the damaged sites. The inhibition of Hsp90α affects nuclear localization of MRE11 and RAD50, impairs DDR signaling (e.g., BRCA1 and CHK2 phosphorylation), and slows down DSBs repair. Hsp90α inhibition does not affect DNA-dependent protein kinase (DNA-PK) activity, which possibly phosphorylates Hsp90α and H2AX after IR. Notably, Hsp90α inhibition causes H2AX phosphorylation in proliferating cells, this possibly indicating replication stress events. Overall, present data shed light on the regulatory role of Hsp90α on the DDR, controlling ATM and NBN stability and influencing the DSBs signaling and repair. © 2017 Federation of European Biochemical Societies.

Heat shock proteins and chronic fatigue in primary Sjögren's syndrome.

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Bårdsen, Kjetil; Nilsen, Mari Mæland; Kvaløy, Jan Terje; Norheim, Katrine Brække; Jonsson, Grete; Omdal, Roald

2016-04-01

Fatigue occurs frequently in patients with cancer, neurological diseases and chronic inflammatory diseases, but the biological mechanisms that lead to and regulate fatigue are largely unknown. When the innate immune system is activated, heat shock proteins (HSPs) are produced to protect cells. Some extracellular HSPs appear to recognize cellular targets in the brain, and we hypothesize that fatigue may be generated by specific HSPs signalling through neuronal or glial cells in the central nervous system. From a cohort of patients with primary Sjögren's syndrome, 20 patients with high and 20 patients with low fatigue were selected. Fatigue was evaluated with a fatigue visual analogue scale. Plasma concentrations of HSP32, HSP60, HSP72 and HSP90α were measured and analysed to determine if there were associations with the level of fatigue. Plasma concentrations of HSP90α were significantly higher in patients with high fatigue compared with those with low fatigue, and there was a tendency to higher concentrations of HSP72 in patients with high fatigue compared with patients with low fatigue. There were no differences in concentrations of HSP32 and HSP60 between the high- and low-fatigue groups. Thus, extracellular HSPs, particularly HSP90α, may signal fatigue in chronic inflammation. This supports the hypothesis that fatigue is generated by cellular defence mechanisms. © The Author(s) 2016.

Selection Transforms the Landscape of Genetic Variation Interacting with Hsp90.

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Geiler-Samerotte, Kerry A; Zhu, Yuan O; Goulet, Benjamin E; Hall, David W; Siegal, Mark L

2016-10-01

The protein-folding chaperone Hsp90 has been proposed to buffer the phenotypic effects of mutations. The potential for Hsp90 and other putative buffers to increase robustness to mutation has had major impact on disease models, quantitative genetics, and evolutionary theory. But Hsp90 sometimes contradicts expectations for a buffer by potentiating rapid phenotypic changes that would otherwise not occur. Here, we quantify Hsp90's ability to buffer or potentiate (i.e., diminish or enhance) the effects of genetic variation on single-cell morphological features in budding yeast. We corroborate reports that Hsp90 tends to buffer the effects of standing genetic variation in natural populations. However, we demonstrate that Hsp90 tends to have the opposite effect on genetic variation that has experienced reduced selection pressure. Specifically, Hsp90 tends to enhance, rather than diminish, the effects of spontaneous mutations and recombinations. This result implies that Hsp90 does not make phenotypes more robust to the effects of genetic perturbation. Instead, natural selection preferentially allows buffered alleles to persist and thereby creates the false impression that Hsp90 confers greater robustness.

Protein synthesis in TE 671/RD (human rabdomiosarcoma) cells treated with thapsigargin and hyperthermia: impairment of HSP 70 induction.

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Delpino, A; Piselli, P; Mangano, G

1995-01-01

In this study we considered the quantitative and qualitative changes of protein synthetic activity occurring in TE 671/RD cells treated with thapsigargin (TG), with hyperthermia (HT) or with a combination of both these agents. In cells treated with TG (100 nM, continuous exposure), the overall protein synthetic activity was initially inhibited but subsequently recovered to about 60% of the initial level. Chronic TG exposure was also able to induce the expression of GRP 78. The rate of synthesis of GRP 78, after a lag period of about 2 h, increased gradually to reach a maximum (9-fold induction) after 6 h of TG-treatment and was then maintained at that level up to 18 h. A weak induction of GRP 94 was observed following 6-8 h of continuous exposure to TG. In cells treated with HT (43 degrees C for 30 min), a typical heat shock response was observed: in particular, the relative rate of synthesis of HSP 70 (the major heat-inducible mammalian heat shock protein) was increased 10-fold over the constitutive level. The heat-promoted induction of HSP 70 was significantly reduced by concomitant or previous exposure to TG. When TG and HT were administred simultaneously, the increase in HSP 70 synthesis was only 4.7-fold over the control level, while in cells pre-treated for 1 h with TG before the hyperthermic challenge the rate of HSP 70 synthesis was only stimulated 2-fold. In both these conditions, by contrast, it was apparent that HT did not affect the TG-promoted induction of GRP 78. The correlations between the TG-induced mobilization of cytosolic Ca2+ and the effects on protein synthesis are discussed.

Vaccination with recombinant heat shock protein 60 from Histoplasma capsulatum protects mice against pulmonary histoplasmosis.

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Gomez, F J; Allendoerfer, R; Deepe, G S

1995-07-01

HIS-62 is a glycoprotein that has been isolated from the cell wall and cell membrane fraction of the pathogenic fungus Histoplasma capsulatum. It is a target of the cellular immune response to this fungus, and it protects mice against a lethal intravenous inoculum of H. capsulatum yeast cells. In this study, we cloned the gene encoding this antigen to reveal its biological nature and studied the immunological activity of recombinant antigen. The amino acid sequences of the NH2 terminus and internal peptides were obtained by Edman degradation. Degenerate oligonucleotides were used to isolate a gene fragment of HIS-62 by PCR. One 680-bp segment that corresponded to the known peptide sequence was amplified from H. capsulatum DNA. This DNA was used to screen a genomic library, and the full-length gene was isolated and sequenced. The deduced amino acid sequence of the gene demonstrated approximately 70 and approximately 50% identity to heat shock protein 60 (hsp 60) from Saccharomyces cerevisiae and hsp 60 from Escherichia coli, respectively. A cDNA was synthesized by reverse transcription PCR and was expressed in E. coli. Recombinant protein reacted with a monospecific polyclonal rabbit antiserum raised against native HIS-62, with monoclonal HIS-62-reactive T cells, and with splenocytes from mice immunized with viable yeast cells. Moreover, vaccination with the recombinant protein conferred protection in mice against a lethal intranasal inoculation with yeast cells. Thus, HIS-62 is a member of the hsp 60 family, and the recombinant hsp 60 is protective against pulmonary histoplasmosis in mice.

The role of HSP70 in mediating age-dependent mortality in sepsis

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McConnell, Kevin W.; Fox, Amy C.; Clark, Andrew T.; Chang, Nai-Yuan Nicholas; Dominguez, Jessica A.; Farris, Alton B.; Buchman, Timothy G.; Hunt, Clayton R.; Coopersmith, Craig M.

2011-01-01

Sepsis is primarily a disease of the aged, with increased incidence and mortality occurring in aged hosts. Heat shock protein (HSP) 70 plays an important role in both healthy aging and the stress response to injury. The purpose of this study was to determine the role of HSP70 in mediating mortality and the host inflammatory response in aged septic hosts. Sepsis was induced in both young (6–12week old) and aged (16–17 month old) HSP70−/− and wild type (WT) mice to determine if HSP70 modulated outcome in an age-dependent fashion. Young HSP70−/− and WT mice subjected to cecal ligation and puncture (CLP), Pseudomonas aeruginosa pneumonia or Streptococcus pneumoniae pneumonia had no differences in mortality, suggesting HSP70 does not mediate survival in young septic hosts. In contrast, mortality was higher in aged HSP70−/− mice than aged WT mice subjected to CLP (p=0.01), suggesting HSP70 mediates mortality in sepsis in an age-dependent fashion. Compared to WT mice, aged septic HSP70−/− mice had increased gut epithelial apoptosis and pulmonary inflammation. In addition, HSP70−/−mice had increased systemic levels of TNF-α, IL-6, IL-10 and IL-1β compared to WT mice. These data demonstrate that HSP70 is a key determinant of mortality in aged but not young hosts in sepsis. HSP70 may play a protective role in an age-dependent response to sepsis by preventing excessive gut apoptosis and both pulmonary and systemic inflammation. PMID:21296977

Localization of Hsp27 in the Rat Submandibular Gland Following the Application of Various Surgical Treatments

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Mizobe, Kenichi; Kawabe, Yoshihiro; Bando, Yasuhiko; Sakiyama, Koji; Araki, Hisao; Amano, Osamu

2014-01-01

Salivary glands repair and regenerate following various types of injuries and surgical procedures. However, the tissue responses induced in the contralateral glands have yet to be elucidated in detail. Hsp27, a member of the heat-shock protein (Hsp) family, is strongly expressed in physiological environments, particularly during development. Hsp27 was previously shown to play a role in the regulation of acinar cell proliferation and differentiation in the rat submandibular gland. The present study performed the following surgical treatments on the right submandibular glands of adult rats: 1) duct ligation followed by unligation after one week; 2) partial sialoadenectomy; and 3) total sialoadenectomy. Immunohistochemistry for Hsp27 and Ki67 was performed in the experimental and normal contralateral glands, and localization was histologically and morphometrically analyzed. The results obtained revealed the localization of Hsp27 to the intercalated duct in the submandibular glands of non-treated rats. The expression of Hsp27 was strongly induced in both the uninjured contralateral control glands as well as treated glands of experimental rats regardless of the surgical procedure performed. The number of Hsp27-immunopositive cells increased rapidly following surgery, and subsequently returned to the same level as that in non-treated rats after 4 weeks. However, no marked changes were observed in the number of Ki67-immunopositive proliferating cells. Therefore, the change in the number of Hsp27-immunopositive cells may have contributed to compensatory hypertrophy. The results of the present study indicate that the expression of Hsp27 in the intercalated duct in the submandibular gland may play a role in the differentiation of acinar cells

Heterologous Expression of the Carrot Hsp17.7 gene Increased Growth, Cell Viability, and Protein Solubility in Transformed Yeast (Saccharomyces cerevisiae) under Heat, Cold, Acid, and Osmotic Stress Conditions.

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Ko, Eunhye; Kim, Minhye; Park, Yunho; Ahn, Yeh-Jin

2017-08-01

In industrial fermentation of yeast (Saccharomyces cerevisiae), culture conditions are often modified from the optimal growth conditions of the cells to maintain large-scale cultures and/or to increase recombinant protein production. However, altered growth conditions can be stressful to yeast cells resulting in reduced cell growth and viability. In this study, a small heat shock protein gene from carrot (Daucus carota L.), Hsp17.7, was inserted into the yeast genome via homologous recombination to increase tolerance to stress conditions that can occur during industrial culture. A DNA construct, Translational elongation factor gene promoter-carrot Hsp17.7 gene-Phosphoribosyl-anthranilate isomerase gene (an auxotrophic marker), was generated by a series of PCRs and introduced into the chromosome IV of the yeast genome. Immunoblot analysis showed that carrot Hsp17.7 accumulated in the transformed yeast cell lines. Growth rates and cell viability of these cell lines were higher than control cell lines under heat, cold, acid, and hyperosmotic stress conditions. Soluble protein levels were higher in the transgenic cell lines than control cell lines under heat and cold conditions, suggesting the molecular chaperone function of the recombinant Hsp17.7. This study showed that a recombinant DNA construct containing a HSP gene from carrot was successfully expressed in yeast by homologous recombination and increased tolerances to abiotic stress conditions.

Function of SSA subfamily of Hsp70 within and across species varies widely in complementing Saccharomyces cerevisiae cell growth and prion propagation.

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Deepak Sharma

2009-08-01

Full Text Available The cytosol of most eukaryotic cells contains multiple highly conserved Hsp70 orthologs that differ mainly by their spatio-temporal expression patterns. Hsp70s play essential roles in protein folding, transport or degradation, and are major players of cellular quality control processes. However, while several reports suggest that specialized functions of Hsp70 orthologs were selected through evolution, few studies addressed systematically this issue.We compared the ability of Ssa1p-Ssa4p from Saccharomyces cerevisiae and Ssa5p-Ssa8p from the evolutionary distant yeast Yarrowia lipolytica to perform Hsp70-dependent tasks when expressed as the sole Hsp70 for S. cerevisiae in vivo. We show that Hsp70 isoforms (i supported yeast viability yet with markedly different growth rates, (ii influenced the propagation and stability of the [PSI(+] and [URE3] prions, but iii did not significantly affect the proteasomal degradation rate of CFTR. Additionally, we show that individual Hsp70 orthologs did not induce the formation of different prion strains, but rather influenced the aggregation properties of Sup35 in vivo. Finally, we show that [URE3] curing by the overexpression of Ydj1p is Hsp70-isoform dependent.Despite very high homology and overlapping functions, the different Hsp70 orthologs have evolved to possess distinct activities that are required to cope with different types of substrates or stress situations. Yeast prions provide a very sensitive model to uncover this functional specialization and to explore the intricate network of chaperone/co-chaperone/substrates interactions.

HSP60, a protein downregulated by IGFBP7 in colorectal carcinoma

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Lin Jie

2010-04-01

Full Text Available Abstract Background In our previous study, it was well defined that IGFBP7 was an important tumor suppressor gene in colorectal cancer (CRC. We aimed to uncover the downstream molecules responsible for IGFBP7's behaviour in this study. Methods Differentially expressed protein profiles between PcDNA3.1(IGFBP7-transfected RKO cells and the empty vector transfected controls were generated by two-dimensional gel electrophoresis (2-DE and mass spectrometry (MS identification. The selected differentially expressed protein induced by IGFBP7 was confirmed by western blot and ELISA. The biological behaviour of the protein was explored by cell growth assay and colony formation assay. Results Six unique proteins were found differentially expressed in PcDNA3.1(IGFBP7-transfected RKO cells, including albumin (ALB, 60 kDa heat shock protein(HSP60, Actin cytoplasmic 1 or 2, pyruvate kinase muscle 2(PKM2, beta subunit of phenylalanyl-tRNA synthetase(FARSB and hypothetical protein. The downregulation of HSP60 by IGFBP7 was confirmed by western blot and ELISA. Recombinant human HSP60 protein could increase the proliferation rate and the colony formation ability of PcDNA3.1(IGFBP7-RKO cells. Conclusion HSP60 was an important downstream molecule of IGFBP7. The downregulation of HSP60 induced by IGFBP7 may be, at least in part, responsible for IGFBP7's tumor suppressive biological behaviour in CRC.

HSP27 phosphorylation modulates TRAIL-induced activation of Src-Akt/ERK signaling through interaction with β-arrestin2.

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Qi, Shimei; Xin, Yinqiang; Qi, Zhilin; Xu, Yimiao; Diao, Ying; Lan, Lei; Luo, Lan; Yin, Zhimin

2014-03-01

Heat shock protein 27 (HSP27) regulates critical cellular functions such as development, differentiation, cell growth and apoptosis. A variety of stimuli induce the phosphorylation of HSP27, which affects its cellular functions. However, most previous studies focused on the role of HSP27 protein itself in apoptosis, the particular role of its phosphorylation state in signaling transduction remains largely unclear. In the present study, we reported that HSP27 phosphorylation modulated TRAIL-triggered pro-survival signaling transduction. In HeLa cells, suppression of HSP27 phosphorylation by specific inhibitor KRIBB3 or MAPKAPK2 (MK2) knockdown and by overexpression of non-phosphorylatable HSP27(3A) mutant demonstrated that hindered HSP27 phosphorylation enhanced the TRAIL-induced apoptosis. In addition, reduced HSP27 phosphorylation by KRIBB3 treatment or MK2 knockdown attenuated the TRAIL-induced activation of Akt and ERK survival signaling through suppressing the phosphorylation of Src. By overexpression of HSP27(15A) or HSP27(78/82A) phosphorylation mutant, we further showed that phosphorylation of HSP27 at serine 78/82 residues was essential to TRAIL-triggered Src-Akt/ERK signaling transduction. Co-immunoprecipitation and confocal microscopy showed that HSP27 interacted with Src and scaffolding protein β-arrestin2 in response of TRAIL stimulation and suppression of HSP27 phosphorylation apparently disrupted the TRAIL-induced interaction of HSP27 and Src or interaction of HSP27 and β-arrestin2. We further demonstrated that β-arrestin2 mediated HSP27 action on TRAIL-induced Src activation, which was achieved by recruiting signaling complex of HSP27/β-arrestin2/Src in response to TRAIL. Taken together, our study revealed that HSP27 phosphorylation modulates TRAIL-triggered activation of Src-Akt/ERK pro-survival signaling via interacting with β-arrestin2 in HeLa cells. Copyright © 2013 Elsevier Inc. All rights reserved.

17-AAG, a Hsp90 inhibitor, attenuates the hypoxia-induced expression of SDF-1alpha and ILK in mouse RPE cells.

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Wang, Ye Qing; Zhang, Xiao Mei; Wang, Xiao Dan; Wang, Bin Jie; Wang, Wei

2010-03-01

The aim of this study was to investigate the changes of SDF-1alpha and ILK expression in mouse retinal pigment epithelium (RPE) cells in response to hypoxia, and the effect of 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (Hsp90) inhibitor, on the hypoxia-induced expression of SDF-1alpha and ILK. RPE cells were cultured with 200 micromol/L cobalt chloride (CoCl(2)) for different times (1, 3, 6, 12, 24, 72 h) to imitate chemical hypoxia. Pretreatment of 17-AAG was 1 h prior to hypoxic insult. Cellular viability after 17-AAG treatment was assessed by MTT assay, and the changes of SDF-1alpha and ILK expression were examined by RT-PCR and Western blot. Up-regulation of SDF-1alpha and ILK expression in response to hypoxia was observed. One hour pretreatment of 17-AAG could remarkably decreased the hypoxia-induced SDF-1alpha and ILK expression in vitro. Our results indicated that SDF-1alpha and ILK involved in the hypoxic response of RPE cells, and 1 h pretreatment of 17-AAG had an inhibitive effect on the hypoxia-induced SDF-1alpha and ILK expression.

Hsp70 stabilizes lysosomes and reverts Niemann-Pick disease-associated lysosomal pathology

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Kirkegaard, Thomas; Roth, Anke G; Petersen, Nikolaj H T

2010-01-01

Heat shock protein 70 (Hsp70) is an evolutionarily highly conserved molecular chaperone that promotes the survival of stressed cells by inhibiting lysosomal membrane permeabilization, a hallmark of stress-induced cell death. Clues to its molecular mechanism of action may lay in the recently...... reported stress- and cancer-associated translocation of a small portion of Hsp70 to the lysosomal compartment. Here we show that Hsp70 stabilizes lysosomes by binding to an endolysosomal anionic phospholipid bis(monoacylglycero)phosphate (BMP), an essential co-factor for lysosomal sphingomyelin metabolism......-is also associated with a marked decrease in lysosomal stability, and this phenotype can be effectively corrected by treatment with recombinant Hsp70. Taken together, these data open exciting possibilities for the development of new treatments for lysosomal storage disorders and cancer with compounds...

Inhibition of homologous recombination repair in irradiated tumor cells pretreated with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin

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Noguchi, Miho; Yu, Dong; Hirayama, Ryoichi; Ninomiya, Yasuharu; Sekine, Emiko; Kubota, Nobuo; Ando, Koichi; Okayasu, Ryuichi

2006-01-01

In order to investigate the mechanism of radio-sensitization by an Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), we studied repair of DNA double strand breaks (DSBs) in irradiated human cells pre-treated with 17-AAG. DSBs are thought to be the critical target for radiation-induced cell death. Two human tumor cell lines DU145 and SQ-5 which showed clear radio-sensitization by 17-AAG revealed a significant inhibition of DSB repair, while normal human cells which did not show radio-sensitization by the drug indicated no change in the DSB repair kinetics with 17-AAG. We further demonstrated that BRCA2 was a novel client protein for Hsp90, and 17-AAG caused the degradation of BRCA2 and in turn altered the behavior of Rad51, a critical protein for homologous recombination (HR) pathway of DSB repair. Our data demonstrate for the first time that 17-AAG inhibits the HR repair process and could provide a new therapeutic strategy to selectively result in higher tumor cell killing

SNX-25a, a novel Hsp90 inhibitor, inhibited human cancer growth more potently than 17-AAG.

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Wang, Shaoxiang; Wang, Xiao; Du, Zhan; Liu, Yuting; Huang, Dane; Zheng, Kai; Liu, Kaisheng; Zhang, Yi; Zhong, Xueyun; Wang, Yifei

2014-07-18

17-Allylamino-17-demethoxygeldanamycin (17-AAG), a typical Hsp90 inhibitor derived from geldanamycin (GA), has entered Phase III clinical trials for cancer therapy. However, it has several significant limitations such as poor solubility, limited bioavailability and unacceptable hepatotoxicity. In this study, the anticancer activity and mechanism of SNX-25a, a novel Hsp90 inhibitor, was investigated comparing with that of 17-AAG. We showed that SNX-25a triggered growth inhibition more sensitively than 17-AAG against many human cancer cells, including K562, SW-620, A375, Hep-2, MCF-7, HepG2, HeLa, and A549 cell lines, especially at low concentrations (AAG, SNX-25a was more potent in arresting the cell cycle at G2 phase, and displayed more potent effects on human cancer cell apoptosis and Hsp90 client proteins. It also exhibited a stronger binding affinity to Hsp90 than 17-AAG using molecular docking. Considering the superiority effects on Hsp90 affinity, cell growth, cell cycle, apoptosis, and Hsp90 client proteins, SNX-25a is supposed as a potential anticancer agent that needs to be explored in detail. Copyright © 2014 Elsevier Inc. All rights reserved.

Blockade of MK-801-induced heat shock protein 72/73 in rat brain by antipsychotic and monoaminergic agents targeting D2, 5-HT1A, 5-HT2A and α1-adrenergic receptors.

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Romón, Tamara; Planas, Anna M; Adell, Albert

2014-02-01

Noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists can produce positive and negative symptomatology as well as impairment of cognitive function that closely resemble those present in schizophrenia. In rats, these drugs induce a behavioral syndrome (characterized by hyperlocomotion and stereotypies), an enhanced glutamatergic transmission in the medial prefrontal cortex, and damage to retrosplenial cortical neurons in adult rats, which was measured as the induction of the stress protein 72/73 kDa heat shock protein (Hsp72/73). In the present work, we have examined the existence of possible differences among different antipsychotic drugs in their capacity to block immunolabeling of Hsp72/73 in the retrosplenial cortex of the rat induced by the potent NMDA receptor antagonist, MK- 801. In addition, the effects of selective monoaminergic agents were also studied to delineate the particular receptors responsible for the actions of antipsychotic drugs. Pretreatment with clozapine, chlorpromazine, olanzapine, ziprasidone--and to a lesser extent haloperidol-reduced the formation of Hsp72/73 protein in the rat retrosplenial cortex after the administration of MK-801. In addition, antagonism at dopamine D2 (raclopride), 5-HT2 (M100907) and α1- adrenoceptors (prazosin) as well as agonism at 5-HT1A receptors (BAY x 3702) also diminished the MK-801-induced number of cells labeled with Hsp72/73. Each of these effects may contribute to antipsychotic action. The results suggest that the efficacy of atypical antipsychotic drugs in the clinic may result from a combined effect on 5-HT2, 5-HT1A and α1-adrenergic receptors added to the classical dopamine D2 receptor antagonism.

Mode of cell death induced by the HSP90 inhibitor 17-AAG (tanespimycin) is dependent on the expression of pro-apoptotic BAX.

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Powers, Marissa V; Valenti, Melanie; Miranda, Susana; Maloney, Alison; Eccles, Suzanne A; Thomas, George; Clarke, Paul A; Workman, Paul

2013-11-01

Inhibitors of the molecular chaperone heat shock protein 90 (HSP90) are of considerable current interest as targeted cancer therapeutic agents because of the ability to destabilize multiple oncogenic client proteins. Despite their resulting pleiotropic effects on multiple oncogenic pathways and hallmark traits of cancer, resistance to HSP90 inhibitors is possible and their ability to induce apoptosis is less than might be expected. Using an isogenic model for BAX knockout in HCT116 human colon carcinoma cells, we demonstrate the induction of BAX-dependent apoptosis at pharmacologically relevant concentrations of the HSP90 inhibitor 17-AAG both in vitro and in tumor xenografts in vivo. Removal of BAX expression by homologous recombination reduces apoptosis in vitro and in vivo but allows a lower level of cell death via a predominantly necrotic mechanism. Despite reducing apoptosis, the loss of BAX does not alter the overall sensitivity to 17-AAG in vitro or in vivo. The results indicate that 17-AAG acts predominantly to cause a cytostatic antiproliferative effect rather than cell death and further suggest that BAX status may not alter the overall clinical response to HSP90 inhibitors. Other agents may be required in combination to enhance tumor-selective killing by these promising drugs. In addition, there are implications for the use of apoptotic endpoints in the assessment of the activity of molecularly targeted agents.

Class I and II Small Heat Shock Proteins Together with HSP101 Protect Protein Translation Factors during Heat Stress.

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McLoughlin, Fionn; Basha, Eman; Fowler, Mary E; Kim, Minsoo; Bordowitz, Juliana; Katiyar-Agarwal, Surekha; Vierling, Elizabeth

2016-10-01

The ubiquitous small heat shock proteins (sHSPs) are well documented to act in vitro as molecular chaperones to prevent the irreversible aggregation of heat-sensitive proteins. However, the in vivo activities of sHSPs remain unclear. To investigate the two most abundant classes of plant cytosolic sHSPs (class I [CI] and class II [CII]), RNA interference (RNAi) and overexpression lines were created in Arabidopsis (Arabidopsis thaliana) and shown to have reduced and enhanced tolerance, respectively, to extreme heat stress. Affinity purification of CI and CII sHSPs from heat-stressed seedlings recovered eukaryotic translation elongation factor (eEF) 1B (α-, β-, and γ-subunits) and eukaryotic translation initiation factor 4A (three isoforms), although the association with CI sHSPs was stronger and additional proteins involved in translation were recovered with CI sHSPs. eEF1B subunits became partially insoluble during heat stress and, in the CI and CII RNAi lines, showed reduced recovery to the soluble cell fraction after heat stress, which was also dependent on HSP101. Furthermore, after heat stress, CI sHSPs showed increased retention in the insoluble fraction in the CII RNAi line and vice versa. Immunolocalization revealed that both CI and CII sHSPs were present in cytosolic foci, some of which colocalized with HSP101 and with eEF1Bγ and eEF1Bβ. Thus, CI and CII sHSPs have both unique and overlapping functions and act either directly or indirectly to protect specific translation factors in cytosolic stress granules. © 2016 American Society of Plant Biologists. All Rights Reserved.

Plasmodial Hsp70s are functionally adapted to the malaria parasite life cycle

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Jude M Przyborski

2015-06-01

Full Text Available The human malaria parasite, Plasmodium falciparum, encodes a minimal complement of six heat shock protein 70s (PfHSP70s, some of which are highly expressed and are thought to play an important role in the survival and pathology of the parasite. In addition to canonical features of molecular chaperones, these HSP70s possess properties that reflect functional adaptation to a parasitic life style, including resistance to thermal insult during fever periods and host-parasite interactions. The parasite even exports an HSP70 to the host cell where it is likely to be involved in host cell modification. This review focuses on the features of the PfHSP70s, particularly with respect to their adaptation to the malaria parasite life cycle.

Hsp104 Overexpression Cures Saccharomyces cerevisiae [PSI+] by Causing Dissolution of the Prion Seeds

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Park, Yang-Nim; Zhao, Xiaohong; Yim, Yang-In; Todor, Horia; Ellerbrock, Robyn; Reidy, Michael; Eisenberg, Evan; Masison, Daniel C.

2014-01-01

The [PSI+] yeast prion is formed when Sup35 misfolds into amyloid aggregates. [PSI+], like other yeast prions, is dependent on the molecular chaperone Hsp104, which severs the prion seeds so that they pass on as the yeast cells divide. Surprisingly, however, overexpression of Hsp104 also cures [PSI+]. Several models have been proposed to explain this effect: inhibition of severing, asymmetric segregation of the seeds between mother and daughter cells, and dissolution of the prion seeds. First, we found that neither the kinetics of curing nor the heterogeneity in the distribution of the green fluorescent protein (GFP)-labeled Sup35 foci in partially cured yeast cells is compatible with Hsp104 overexpression curing [PSI+] by inhibiting severing. Second, we ruled out the asymmetric segregation model by showing that the extent of curing was essentially the same in mother and daughter cells and that the fluorescent foci did not distribute asymmetrically, but rather, there was marked loss of foci in both mother and daughter cells. These results suggest that Hsp104 overexpression cures [PSI+] by dissolution of the prion seeds in a two-step process. First, trimming of the prion seeds by Hsp104 reduces their size, and second, their amyloid core is eliminated, most likely by proteolysis. PMID:24632242

Hsp104 overexpression cures Saccharomyces cerevisiae [PSI+] by causing dissolution of the prion seeds.

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Park, Yang-Nim; Zhao, Xiaohong; Yim, Yang-In; Todor, Horia; Ellerbrock, Robyn; Reidy, Michael; Eisenberg, Evan; Masison, Daniel C; Greene, Lois E

2014-05-01

The [PSI(+)] yeast prion is formed when Sup35 misfolds into amyloid aggregates. [PSI(+)], like other yeast prions, is dependent on the molecular chaperone Hsp104, which severs the prion seeds so that they pass on as the yeast cells divide. Surprisingly, however, overexpression of Hsp104 also cures [PSI(+)]. Several models have been proposed to explain this effect: inhibition of severing, asymmetric segregation of the seeds between mother and daughter cells, and dissolution of the prion seeds. First, we found that neither the kinetics of curing nor the heterogeneity in the distribution of the green fluorescent protein (GFP)-labeled Sup35 foci in partially cured yeast cells is compatible with Hsp104 overexpression curing [PSI(+)] by inhibiting severing. Second, we ruled out the asymmetric segregation model by showing that the extent of curing was essentially the same in mother and daughter cells and that the fluorescent foci did not distribute asymmetrically, but rather, there was marked loss of foci in both mother and daughter cells. These results suggest that Hsp104 overexpression cures [PSI(+)] by dissolution of the prion seeds in a two-step process. First, trimming of the prion seeds by Hsp104 reduces their size, and second, their amyloid core is eliminated, most likely by proteolysis.

Essential oil from Cymbopogon flexuosus as the potential inhibitor for HSP90.

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Gaonkar, Roopa; Shiralgi, Yallappa; Lakkappa, Dhananjaya B; Hegde, Gurumurthy

2018-01-01

The essential oil of Cymbopogon flexuosus or lemongrass oil (LO) is reported to have antibacterial, antifungal and anticancerous effects. HSP90 is one of the major chaperones responsible for the proper folding of cancer proteins. In this paper we show that the essential oil of C. flexuosus significantly suppresses the HSP90 gene expression. The cytotoxicity of the compounds was tested by MTT assay and the gene expression studies were carried out using HEK-293 and MCF-7 cells. Also we tested the efficacy of the major component of this essential oil viz. citral and geraniol in inhibiting the HSP90 expression. The oil was found to be more cytotoxic to MCF-7 cells with different IC 50 values for the oil (69.33 μg/mL), citral (140.7 μg/mL) and geraniol (117 μg/mL). The fold change of expression was calculated by RT-qPCR using ΔΔCt (2 ^-ΔΔCt ) method and it was 0.1 and 0.03 in MCF-7 cells at 80 μg/mL and 160 μg/mL of LO. Western blot results showed suppression of HSP90 protein expression and HSP90 - ATPase activity inhibition was also observed using LO. This study shows the anticancer mechanism exhibited by the essential oil of C. flexuosus is by the inhibition of the important chaperone protein HSP90.

Influence of In Vitro IL-2 or IL-15 Alone or in Combination with Hsp 70 Derived 14-Mer Peptide (TKD on the Expression of NK Cell Activatory and Inhibitory Receptors on Peripheral Blood T Cells, B Cells and NKT Cells.

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Ilona Hromadnikova

Full Text Available Previous studies from Multhoff and colleagues reported that plasma membrane Hsp70 acts as a tumour-specific recognition structure for activated NK cells, and that the incubation of NK cells with Hsp70 and/or a 14-mer peptide derived from the N-terminal sequence of Hsp70 (TKDNNLLGRFELSG, TKD, aa 450-463 plus a low dose of IL-2 triggers NK cell proliferation and migration, and their capacity to kill cancer cells expressing membrane Hsp70. Herein, we have used flow cytometry to determine the influence of in vitro stimulation of peripheral blood mononuclear cells from healthy individuals with IL-2 or IL-15, either alone or in combination with TKD peptide on the cell surface expression of CD94, NK cell activatory receptors (CD16, NK2D, NKG2C, NKp30, NKp44, NKp46, NKp80, KIR2DL4, DNAM-1 and LAMP1 and NK cell inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2 and NKR-P1A by CD3+CD56+ (NKT, CD3+CD4+, CD3+CD8+ and CD19+ populations. NKG2D, DNAM-1, LAMP1 and NKR-P1A expression was upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD in NKT, CD8+ T cells and B cells. CD94 was upregulated in NKT and CD8+ T cells. Concurrently, an increase in a number of CD8+ T cells expressing LIR1/ILT-2 and CD4+ T cells positive for NKR-P1A was observed. The proportion of CD8+ T cells that expressed NKG2D was higher after IL-2/TKD treatment, when compared with IL-2 treatment alone. In comparison with IL-15 alone, IL-15/TKD treatment increased the proportion of NKT cells that were positive for CD94, LAMP1 and NKRP-1A. The more potent effect of IL-15/TKD on cell surface expression of NKG2D, LIR1/ILT-2 and NKRP-1A was observed in B cells compared with IL-15 alone. However, this increase was not of statistical significance. IL-2/TKD induced significant upregulation of LAMP1 in CD8+ T cells compared with IL-2 alone. Besides NK cells, other immunocompetent cells present within the fraction of peripheral blood mononuclear cells were influenced by

Influence of In Vitro IL-2 or IL-15 Alone or in Combination with Hsp 70 Derived 14-Mer Peptide (TKD) on the Expression of NK Cell Activatory and Inhibitory Receptors on Peripheral Blood T Cells, B Cells and NKT Cells

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Hromadnikova, Ilona; Li, Shuang; Kotlabova, Katerina; Dickinson, Anne M.

2016-01-01

Previous studies from Multhoff and colleagues reported that plasma membrane Hsp70 acts as a tumour-specific recognition structure for activated NK cells, and that the incubation of NK cells with Hsp70 and/or a 14-mer peptide derived from the N-terminal sequence of Hsp70 (TKDNNLLGRFELSG, TKD, aa 450–463) plus a low dose of IL-2 triggers NK cell proliferation and migration, and their capacity to kill cancer cells expressing membrane Hsp70. Herein, we have used flow cytometry to determine the influence of in vitro stimulation of peripheral blood mononuclear cells from healthy individuals with IL-2 or IL-15, either alone or in combination with TKD peptide on the cell surface expression of CD94, NK cell activatory receptors (CD16, NK2D, NKG2C, NKp30, NKp44, NKp46, NKp80, KIR2DL4, DNAM-1 and LAMP1) and NK cell inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2 and NKR-P1A) by CD3+CD56+ (NKT), CD3+CD4+, CD3+CD8+ and CD19+ populations. NKG2D, DNAM-1, LAMP1 and NKR-P1A expression was upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD in NKT, CD8+ T cells and B cells. CD94 was upregulated in NKT and CD8+ T cells. Concurrently, an increase in a number of CD8+ T cells expressing LIR1/ILT-2 and CD4+ T cells positive for NKR-P1A was observed. The proportion of CD8+ T cells that expressed NKG2D was higher after IL-2/TKD treatment, when compared with IL-2 treatment alone. In comparison with IL-15 alone, IL-15/TKD treatment increased the proportion of NKT cells that were positive for CD94, LAMP1 and NKRP-1A. The more potent effect of IL-15/TKD on cell surface expression of NKG2D, LIR1/ILT-2 and NKRP-1A was observed in B cells compared with IL-15 alone. However, this increase was not of statistical significance. IL-2/TKD induced significant upregulation of LAMP1 in CD8+ T cells compared with IL-2 alone. Besides NK cells, other immunocompetent cells present within the fraction of peripheral blood mononuclear cells were influenced by the treatment

HECTD3 Mediates an HSP90-Dependent Degradation Pathway for Protein Kinase Clients

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Zhaobo Li

2017-06-01

Full Text Available Inhibition of the ATPase cycle of the HSP90 chaperone promotes ubiquitylation and proteasomal degradation of its client proteins, which include many oncogenic protein kinases. This provides the rationale for HSP90 inhibitors as cancer therapeutics. However, the mechanism by which HSP90 ATPase inhibition triggers ubiquitylation is not understood, and the E3 ubiquitin ligases involved are largely unknown. Using a siRNA screen, we have identified components of two independent degradation pathways for the HSP90 client kinase CRAF. The first requires CUL5, Elongin B, and Elongin C, while the second requires the E3 ligase HECTD3, which is also involved in the degradation of MASTL and LKB1. HECTD3 associates with HSP90 and CRAF in cells via its N-terminal DOC domain, which is mutationally disrupted in tumor cells with activated MAP kinase signaling. Our data implicate HECTD3 as a tumor suppressor modulating the activity of this important oncogenic signaling pathway.

Hsp90 depletion goes wild

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Siegal, Mark L; Masel, Joanna

2012-01-01

Abstract Hsp90 reveals phenotypic variation in the laboratory, but is Hsp90 depletion important in the wild? Recent work from Chen and Wagner in BMC Evolutionary Biology has discovered a naturally occurring Drosophila allele that downregulates Hsp90, creating sensitivity to cryptic genetic variation. Laboratory studies suggest that the exact magnitude of Hsp90 downregulation is important. Extreme Hsp90 depletion might reactivate transposable elements and/or induce aneuploidy, in addition to r...

Distinct radioprotective activities of major heat shock proteins in irradiated mammalian cells

International Nuclear Information System (INIS)

Kabakov, Alexander; Malyutina, Yana; Kudryavtsev, Vladimir

2008-01-01

Full text: Several years ago we have suggested that heat shock proteins (Hsps) can be involved in cellular and tissue mechanisms of protection from ionizing radiation. At present, the accumulated experimental data do allow us to characterize three major mammalian Hsps, Hsp70, Hsp27 and Hsp90, as specific endogenous radioprotectors which are able to prevent or minimize cell death resulting from the radiation exposure. It follows from the many findings that the radioprotective effect of these Hsps is particularly manifested in their ability to attenuate apoptosis in various normal and tumor cells irradiated in vivo or in vitro. The obtained data already enable to suggest three main mechanisms of the radioprotection conferred by the excess Hsps: 1) Modulation of the intracellular signaling so that the apoptotic signal transduction is blocked, whereas the 'cell survival' signal transduction is stimulated; 2) Suppression of the radiation-associated free radical generation and apoptosis induced by reactive oxygen species (ROS); 3) Attenuation of the genotoxic impact of ionizing radiation. The latter suggested mechanism seems particularly intriguing and implies that the excess Hsps can somehow contribute to protection/repair of genomic DNA from radiation-induced damage. According to our recent results, Hsp90 is indeed involved in the post-irradiation repair of nuclear DNA, while excess Hsp70 can beneficially affect the p53-mediated DNA damage response in irradiated cells to ensure their long-term survival and recovery. As for Hsp27, we found that its accumulation in target cells increases their radioresistance by enhancing the irradiation-responsive activation of anti apoptotic pathways. While the Hsp70 and Hsp27 seem to perform different functions in irradiated cells, the synergistic enhancement of radioprotection was clearly observed in the cells enriched by the both the Hsps. In vivo, such radioprotective activities of the major mammalian Hsps may play a role in

A novel biomarker for marine environmental pollution of HSP90 from Mytilus coruscus

International Nuclear Information System (INIS)

Liu, Huihui; Wu, Jiong; Xu, Mengshan; He, Jianyu

2016-01-01

Heat shock protein 90 (HSP90) is a conserved molecular chaperone contributing to cell cycle control, organism development and the proper regulation of cytosolic proteins. The full-length HSP90 cDNA of Mytilus coruscus (McHSP90, KT946644) was 2420 bp, including an ORF of 2169 bp encoding a polypeptide of 722 amino acids with predicted pI/MW 4.89/83.22 kDa. BLASTp analysis and phylogenetic relationship strongly suggested McHSP90 was a member of HSP90 family, and it was highly conserved with other known HSP90, especially in the HSP90 family signatures, ATP/GTP-Binding sites and ‘EEVD’ motif. The mRNA of McHSP90 in haemolymph was upregulated in all treatments including Vibrio alginolyticus and Vibrio harveyi challenge, metals stresses (copper and cadmium) and 180 CST fuel exposure. All the results implied the expression of McHSP90 could be affected by Vibrio challenge and environmental stress, which might help us gain more insight into the molecular mechanism of HSP against adverse stresses in mollusca. - Highlights: • A novel HSP90 (McHSP90) was identified from Mytilus coruscus. • McHSP90 significantly affected by Vibrio challenge for immune defense. • McHSP90 mRNA was obviously up-regulated under stress of heavy metals and 180CST fuel. • McHSP90 might be an ideal marine pollution indicator.

Hsp70 cochaperones HspBP1 and BAG-1M differentially regulate steroid hormone receptor function.

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Regina T Knapp

Full Text Available Hsp70 binding protein 1 (HspBP1 and Bcl2-associated athanogene 1 (BAG-1, the functional orthologous nucleotide exchange factors of the heat shock protein 70 kilodalton (Hsc70/Hsp70 chaperones, catalyze the release of ADP from Hsp70 while inducing different conformational changes of the ATPase domain of Hsp70. An appropriate exchange rate of ADP/ATP is crucial for chaperone-dependent protein folding processes. Among Hsp70 client proteins are steroid receptors such as the glucocorticoid receptor (GR, the mineralocorticoid receptor (MR, and the androgen receptor (AR. BAG-1 diversely affects steroid receptor activity, while to date the influence of HspBP1 on steroid receptor function is mostly unknown. Here, we compared the influence of HspBP1 and BAG-1M on Hsp70-mediated steroid receptor folding complexes and steroid receptor activity. Coimmunoprecipitation studies indicated preferential binding of Hsp40 and the steroid receptors to BAG-1M as compared to HspBP1. Furthermore, Hsp70 binding to the ligand-binding domain of GR was reduced in the presence of HspBP1 but not in the presence of BAG-1M as shown by pull-down assays. Reporter gene experiments revealed an inhibitory effect on GR, MR, and AR at a wide range of HspBP1 protein levels and at hormone concentrations at or approaching saturation. BAG-1M exhibited a transition from stimulatory effects at low BAG-1M levels to inhibitory effects at higher BAG-1M levels. Overall, BAG-1M and HspBP1 had differential impacts on the dynamic composition of steroid receptor folding complexes and on receptor function with important implications for steroid receptor physiology.

Geranylgeranylacetone ameliorates lung ischemia/reperfusion injury by HSP70 and thioredoxin redox system: NF-kB pathway involved.

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Cao, Weijun; Li, Manhui; Li, Jianxiong; Li, Chengwei; Xu, Xin; Gu, Weiqing

2015-06-01

Geranylgeranylacetone (GGA) has been clinically used as an anti-ulcer drug. In the present study, we explored the protective effects of GGA on lung ischemia/reperfusion injury (IRI) and the underlying mechanism. The results demonstrated that GGA ameliorated the lung biochemical and histological alterations induced by IRI, which was reversed by HSP70 inhibition. To further explore the mechanism of GGA action, we focused on NF-kB and thioredoxin (Trx) redox system. It was shown that GGA induced the HSP70 and Trx-1 expression, NF-kB nuclear translocation and activated thioredoxin reductase (TrxR). The Trx-1 expression and TrxR activity was suppressed by HSP70 and NF-kB inhibition, while the nuclear NF-kB p65 expression was suppressed by HSP70 inhibitor. These results indicated that GGA may protect rat lung against IRI by HSP70 and Trx redox system, in which NF-kB pathway may be involved. Copyright © 2015 Elsevier Ltd. All rights reserved.

Hsp10, Hsp70, and Hsp90 immunohistochemical levels change in ulcerative colitis after therapy

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G. Tomasello

2011-10-01

Full Text Available Ulcerative colitis (UC is a form of inflammatory bowel disease (IBD characterized by damage of large bowel mucosa and frequent extra-intestinal autoimmune comorbidities. The role played in IBD pathogenesis by molecular chaperones known to interact with components of the immune system involved in inflammation is unclear. We previously demonstrated that mucosal Hsp60 decreases in UC patients treated with conventional therapies (mesalazine, probiotics, suggesting that this chaperonin could be a reliable biomarker useful for monitoring response to treatment, and that it might play a role in pathogenesis. In the present work we investigated three other heat shock protein/molecular chaperones: Hsp10, Hsp70, and Hsp90. We found that the levels of these proteins are increased in UC patients at the time of diagnosis and decrease after therapy, supporting the notion that these proteins deserve attention in the study of the mechanisms that promote the development and maintenance of IBD, and as biomarkers of this disease (e.g., to monitor response to treatment at the histological level.

Geldanamycin induces heat shock protein 70 and protects against MPTP-induced dopaminergic neurotoxicity in mice.

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Shen, Hai-Ying; He, Jin-Cai; Wang, Yumei; Huang, Qing-Yuan; Chen, Jiang-Fan

2005-12-02

As key molecular chaperone proteins, heat shock proteins (HSPs) represent an important cellular protective mechanism against neuronal cell death in various models of neurological disorders. In this study, we investigated the effect as well as the molecular mechanism of geldanamycin (GA), an inhibitor of Hsp90, on 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity, a mouse model of Parkinson disease. Neurochemical analysis showed that pretreatment with GA (via intracerebral ventricular injection 24 h prior to MPTP treatment) increased residual dopamine content and tyrosine hydroxylase immunoreactivity in the striatum 24 h after MPTP treatment. To dissect out the molecular mechanism underlying this neuroprotection, we showed that the GA-mediated protection against MPTP was associated with a reduction of cytosolic Hsp90 and an increase in Hsp70, with no significant changes in Hsp40 and Hsp25 levels. Furthermore, in parallel with the induction of Hsp70, striatal nuclear HSF1 levels and HSF1 binding to heat shock element sites in the Hsp70 promoter were significantly enhanced by the GA pretreatment. Together these results suggested that the molecular cascade leading to the induction of Hsp70 is critical to the neuroprotection afforded by GA against MPTP-induced neurotoxicity in the brain and that pharmacological inhibition of Hsp90 may represent a potential therapeutic strategy for Parkinson disease.

Effects of temperature-humidity index and chromium supplementation on antioxidant capacity, heat shock protein 72, and cytokine responses of lactating cows.

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Zhang, F J; Weng, X G; Wang, J F; Zhou, D; Zhang, W; Zhai, C C; Hou, Y X; Zhu, Y H

2014-07-01

Heat stress adversely affects the productivity and immune status of dairy cows. The temperature-humidity index (THI) is commonly used to indicate the degree of heat stress on dairy cattle. We investigated the effects of different THI and Cr supplementation on the antioxidant capacity, the levels of heat shock protein 72 (Hsp72), and cytokine responses of lactating cows. The study used a total of 24 clinically healthy uniparous midlactation Holstein cows, which were randomly divided into 2 groups (n = 12 per group), and was conducted in 3 designated THI periods: low THI period (LTHI; THI = 56.4 ± 2.5), moderate THI period (MTHI; THI = 73.9 ± 1.7), and high THI period (HTHI; THI = 80.3 ± 1.0). The 2 groups of cows were fed corn and corn silage based basal diet supplemented chromium picolinate to provide 3.5 mg of Cr/cow daily (Cr+) or basal diet with no Cr (Cr-). The experiment was a 3 × 2 factorial design. The numbers of leukocytes (P Cows supplemented with Cr had lower (P = 0.009) serum concentrations of cholesterol but greater (P cows supplemented with Cr had greater (P = 0.038) expression of the inhibitor of nuclear factor kappa B α (IκBα) in peripheral blood mononuclear cells (PBMC) compared with those without Cr supplementation in the HTHI, whereas the expression of Hsp72 in PBMC was unaltered. Data indicate that there is a decrease in glucose and increases in BUN and creatinine in the serum of midlactation cows under hot conditions during the summer and that these cows have a lowered oxidative capacity but an elevated antioxidant capacity. In addition, Cr may play an anti-inflammatory role in lactating cows by promoting the release of Hsp72, increasing the production of IL-10, and inhibiting the degradation of IκBα under hot conditions during the summer.

Characterization of CaHsp70-1, a Pepper Heat-Shock Protein Gene in Response to Heat Stress and Some Regulation Exogenous Substances in Capsicum annuum L.

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Guo, Meng; Zhai, Yu-Fei; Lu, Jin-Ping; Chai, Lin; Chai, Wei-Guo; Gong, Zhen-Hui; Lu, Ming-Hui

2014-01-01

Pepper (Capsicum annuum L.) is sensitive to heat stress (HS). Heat shock proteins 70 (Hsp70s) play a crucial role in protecting plant cells against HS and control varies characters in different plants. However, CaHsp70-1 gene was not well characterized in pepper. In this study, CaHsp70-1 was cloned from the pepper thermotolerant line R9, which encoded a protein of 652 amino acids, with a molecular weight of 71.54 kDa and an isoelectric point of 5.20. CaHsp70-1 belongs to the cytosolic Hsp70 subgroup, and best matched with tomato SlHsp70. CaHsp70-1 was highly induced in root, stem, leaf and flower in R9 with HS treatment (40 °C for 2 h). In both thermosensitive line B6 and thermotolerant line R9, CaHsp70-1 significantly increased after 0.5 h of HS (40 °C), and maintained in a higher level after 4 h HS. The expression of CaHsp70-1 induced by CaCl2, H2O2 and putrescine (Put) under HS were difference between B6 and R9 lines. The different expression patterns may be related to the differences in promoters of CaHsp70-1 from the two lines. These results suggest that CaHsp70-1 as a member of cytosolic Hsp70 subgroup, may be involved in HS defense response via a signal transduction pathway contained Ca2+, H2O2 and Put. PMID:25356507

The Exported Chaperone PfHsp70x Is Dispensable for the Plasmodium falciparum Intraerythrocytic Life Cycle.

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Cobb, David W; Florentin, Anat; Fierro, Manuel A; Krakowiak, Michelle; Moore, Julie M; Muralidharan, Vasant

2017-01-01

Export of parasite proteins into the host erythrocyte is essential for survival of Plasmodium falciparum during its asexual life cycle. While several studies described key factors within the parasite that are involved in protein export, the mechanisms employed to traffic exported proteins within the host cell are currently unknown. Members of the Hsp70 family of chaperones, together with their Hsp40 cochaperones, facilitate protein trafficking in other organisms, and are thus likely used by P. falciparum in the trafficking of its exported proteins. A large group of Hsp40 proteins is encoded by the parasite and exported to the host cell, but only one Hsp70, P. falciparum Hsp70x (PfHsp70x), is exported with them. PfHsp70x is absent in most Plasmodium species and is found only in P. falciparum and closely related species that infect apes. Herein, we have utilized clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing in P. falciparum to investigate the essentiality of PfHsp70x. We show that parasitic growth was unaffected by knockdown of PfHsp70x using both the dihydrofolate reductase (DHFR)-based destabilization domain and the glmS ribozyme system. Similarly, a complete gene knockout of PfHsp70x did not affect the ability of P. falciparum to proceed through its intraerythrocytic life cycle. The effect of PfHsp70x knockdown/knockout on the export of proteins to the host red blood cell (RBC), including the critical virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), was tested, and we found that this process was unaffected. These data show that although PfHsp70x is the sole exported Hsp70, it is not essential for the asexual development of P. falciparum . IMPORTANCE Half of the world's population lives at risk for malaria. The intraerythrocytic life cycle of Plasmodium spp. is responsible for clinical manifestations of malaria; therefore, knowledge of the parasite's ability to survive within the erythrocyte is

Activation of Hsp90 Enzymatic Activity and Conformational Dynamics through Rationally Designed Allosteric Ligands.

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Sattin, Sara; Tao, Jiahui; Vettoretti, Gerolamo; Moroni, Elisabetta; Pennati, Marzia; Lopergolo, Alessia; Morelli, Laura; Bugatti, Antonella; Zuehlke, Abbey; Moses, Mike; Prince, Thomas; Kijima, Toshiki; Beebe, Kristin; Rusnati, Marco; Neckers, Len; Zaffaroni, Nadia; Agard, David A; Bernardi, Anna; Colombo, Giorgio

2015-09-21

Hsp90 is a molecular chaperone of pivotal importance for multiple cell pathways. ATP-regulated internal dynamics are critical for its function and current pharmacological approaches block the chaperone with ATP-competitive inhibitors. Herein, a general approach to perturb Hsp90 through design of new allosteric ligands aimed at modulating its functional dynamics is proposed. Based on the characterization of a first set of 2-phenylbenzofurans showing stimulatory effects on Hsp90 ATPase and conformational dynamics, new ligands were developed that activate Hsp90 by targeting an allosteric site, located 65 Å from the active site. Specifically, analysis of protein responses to first-generation activators was exploited to guide the design of novel derivatives with improved ability to stimulate ATP hydrolysis. The molecules' effects on Hsp90 enzymatic, conformational, co-chaperone and client-binding properties were characterized through biochemical, biophysical and cellular approaches. These designed probes act as allosteric activators of the chaperone and affect the viability of cancer cell lines for which proper functioning of Hsp90 is necessary. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Administration of M. leprae Hsp65 interferes with the murine lupus progression.

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Eliana B Marengo

Full Text Available The heat shock protein [Hsp] family guides several steps during protein synthesis, are abundant in prokaryotic and eukaryotic cells, and are highly conserved during evolution. The Hsp60 family is involved in assembly and transport of proteins, and is expressed at very high levels during autoimmunity or autoinflammatory phenomena. Here, the pathophysiological role of the wild type [WT] and the point mutated K(409A recombinant Hsp65 of M. leprae in an animal model of Systemic Lupus Erythematosus [SLE] was evaluated in vivo using the genetically homogeneous [NZBxNZW]F(1 mice. Anti-DNA and anti-Hsp65 antibodies responsiveness was individually measured during the animal's life span, and the mean survival time [MST] was determined. The treatment with WT abbreviates the MST in 46%, when compared to non-treated mice [p<0.001]. An increase in the IgG2a/IgG1 anti-DNA antibodies ratio was also observed in animals injected with the WT Hsp65. Incubation of BALB/c macrophages with F(1 serum from WT treated mice resulted in acute cell necrosis; treatment of these cells with serum from K(409A treated mice did not cause any toxic effect. Moreover, the involvement of WT correlates with age and is dose-dependent. Our data suggest that Hsp65 may be a central molecule intervening in the progression of the SLE, and that the point mutated K(409A recombinant immunogenic molecule, that counteracts the deleterious effect of WT, may act mitigating and delaying the development of SLE in treated mice. This study gives new insights into the general biological role of Hsp and the significant impact of environmental factors during the pathogenesis of this autoimmune process.

Increased levels of IgG antibodies against human HSP60 in patients with spondyloarthritis

DEFF Research Database (Denmark)

Hjelholt, Astrid; Carlsen, Thomas Gelsing; Deleuran, Bent Winding

2013-01-01

Introduction: Spondyloarthritis (SpA) comprises a heterogeneous group of inflammatory diseases, with strong association to human leukocyte antigen (HLA)-B27. SpA is suggested triggered by bacterial infection, and bacterial heat shock protein (HSP) seems to be a strong T cell antigen. Since...... against human HSP60, but not antibodies against bacterial HSP60, were elevated in the SpA group compared with the control group. Association between IgG3 antibodies against human HSP60 and BASMI was shown in HLA-B27+ patients. Only weak correlation between antibodies against bacterial and human HSP60...... was seen, and there was no indication of cross-reaction. Conclusion: These results suggest that antibodies against human HSP60 is associated with SpA, however, the theory that antibodies against human HSP60 is a specific part of the aetiology, through cross-reaction to bacterial HSP60, cannot be supported...

Heat shock protein HSP60 and the perspective for future using as vaccine antigens

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Joanna Bajzert

2015-10-01

Full Text Available Heat Shock Proteins (HSPs are widely spread in nature, highly conserved proteins, found in all prokaryotic and eukaryotic cells. HSPs have been classified in 10 families, one of them is the HSP60 family. HSP60 function in the cytoplasm as ATP-dependent molecular chaperones by assisting the folding of newly synthesised polypeptides and the assembly of multiprotein complexes. There is a large amount of evidence which demonstrate that HSP60 is expressed on the cell surface. Especially in bacteria the expression on the surface occurs constitutively and increases remarkably during host infection. HSP60 also play an important role in biofilm formation. In the extracellular environment, HSP60 alone or with self or microbial proteins can acts not only as a link between immune cells, but also as a coordinator of the immune system activity. This protein could influence the immune system in a different way because they act as an antigen, a carrier of other functional molecules or as a ligand for receptor. They are able to stimulate both cells of the acquired (naïve, effector, regulatory T lymphocyte, B lymphocyte and the innate (macrophages, monocytes, dendritic cells immune system. HSPs have been reported to be potent activators of the immune system and they are one of the immunodominant bacterial antigens they could be a good candidate for a subunit vaccine or as an adjuvant.

Hsp90 depletion goes wild

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Siegal Mark L

2012-02-01

Full Text Available Abstract Hsp90 reveals phenotypic variation in the laboratory, but is Hsp90 depletion important in the wild? Recent work from Chen and Wagner in BMC Evolutionary Biology has discovered a naturally occurring Drosophila allele that downregulates Hsp90, creating sensitivity to cryptic genetic variation. Laboratory studies suggest that the exact magnitude of Hsp90 downregulation is important. Extreme Hsp90 depletion might reactivate transposable elements and/or induce aneuploidy, in addition to revealing cryptic genetic variation. See research article http://wwww.biomedcentral.com/1471-2148/12/25

Cytosine deletion at AP2-box region of HSP70 promoter and its ...

Indian Academy of Sciences (India)

of thermotolerance in cells (Leung et al. 1996). ... region of HSP70 significantly affected cellular thermotol- erance ... The double PCR-RFLP using ScrFI confirmed the occur- ... suade of HSP70 on defense of proteins related to respiration.

An RNA aptamer specific to Hsp70-ATP conformation inhibits its ATPase activity independent of Hsp40.

Science.gov (United States)

Thirunavukarasu, Deepak; Shi, Hua

2015-04-01

The highly conserved and ubiquitous molecular chaperone heat shock protein 70 (Hsp70) plays a critical role in protein homeostasis (proteostasis). Controlled by its ATPase activity, Hsp70 cycles between two conformations, Hsp70-ATP and Hsp70-ADP, to bind and release its substrate. Chemical tools with distinct modes of action, especially those capable of modulating the ATPase activity of Hsp70, are being actively sought after in the mechanistic dissection of this system. Here, we report a conformation-specific RNA aptamer that binds only to Hsp70-ATP but not to Hsp70-ADP. We have refined this aptamer and demonstrated its inhibitory effect on Hsp70's ATPase activity. We have also shown that this inhibitory effect on Hsp70 is independent of its interaction with the Hsp40 co-chaperone. As Hsp70 is increasingly being recognized as a drug target in a number of age related diseases such as neurodegenerative, protein misfolding diseases and cancer, this aptamer is potentially useful in therapeutic applications. Moreover, this work also demonstrates the feasibility of using aptamers to target ATPase activity as a general therapeutic strategy.

Overexpression of a heat shock protein (ThHSP18.3) from Tamarix hispida confers stress tolerance to yeast.

Science.gov (United States)

Gao, Caiqiu; Jiang, Bo; Wang, Yucheng; Liu, Guifeng; Yang, Chuanping

2012-04-01

It is well known that plant heat shock proteins (HSPs) play important roles both in response to adverse environmental conditions and in various developmental processes. However, among plant HSPs, the functions of tree plant HSPs are poorly characterized. To improve our understanding of tree HSPs, we cloned and characterized an HSP gene (ThHSP18.3) from Tamarix hispida. Sequence alignment reveals that ThHSP18.3 belongs to the class I small heat shock protein family. A transient expression assay showed that ThHSP18.3 protein was targeted to the cell nucleus. Treatment of Tamarix hispida with cold and heat shock highly induced ThHSP18.3 expression in all studied leaves, roots and stems, whereas, treatment of T. hispida with NaCl, NaHCO(3), and PEG induced ThHSP18.3 expression in leaves and decreased its expression in roots and stems. Further, to study the role of ThHSP18.3 in stress tolerance under different stress conditions, we cloned ThHSP18.3 into the pYES2 vector, transformed and expressed the vector in yeast Saccharomyces cerevisiae. Yeast cells transformed with an empty pYES2 vector were employed as a control. Compared to the control, yeast cells expressing ThHSP18.3 showed greater tolerance to salt, drought, heavy metals, and both low and high temperatures, indicating that ThHSP18.3 confers tolerance to these stress conditions. These results suggested that ThHSP18.3 is involved in tolerance to a variety of stress conditions in T. hispida.

Resistência à Síndrome Ascítica, Competência Homeotérmica e Níveis de Hsp70 no Coração e Pulmão de Frangos de Corte Resistance to Ascites Syndrome, Homoeothermic Competence and Levels of Hsp70 in the Heart and Lung of Broilers

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Renata Hernandes

2002-06-01

Full Text Available Como em outros seres vivos, também nas células das aves ocorre a síntese das proteínas de baixo peso molecular (Hsp, cujo aumento é induzido sob condições de estresse. As Hsps têm um papel importante na manutenção da integridade celular, questiona-se o seu envolvimento no mecanismo de proteção celular de órgãos alvos na ocorrência da síndrome ascítica (SA. Este trabalho objetivou avaliar a temperatura corporal e os níveis da Hsp70 no coração e pulmão de frangos de corte Hubbard (sensível à SA e caipira de pescoço-pelado (resistente, criados em termoneutralidade (25°C e frio (16°C entre 10 e 45 dias de idade. Foram utilizados 192 pintos machos, 96 de cada linhagem. Não houve mortalidade por SA nas aves caipiras. Nas aves Hubbard, a mortalidade devida à SA foi de 4% e 41% em ambiente termoneutro e frio, respectivamente. Em ambiente frio, a temperatura corporal das aves Hubbard foi menor que a das caipiras. A temperatura corporal e o nível de Hsp70 do coração das aves Hubbard diminuíram com o aumento da idade, mas não nas aves caipiras, os quais se mantiveram constantes, inclusive a Hsp70 do pulmão. Independente da idade ou da temperatura, o nível de Hsp70 no pulmão das aves caipiras era superior ao das aves Hubbard. Em relação às aves Hubbard, as caipiras são homeotérmicas mais competentes e apresentam uma maior indução de Hsp70 nos órgãos primariamente afetados na SA, mas este não parece ser o sistema de proteção contra SA, a qual as aves de pescoço pelado são resistentes.Similar to other living animals, the cells of the birds also synthesize small proteins (heat shock protein, Hsp, which increasing levels can be induce by stress. The Hsp have a relevant function in maintaining the integrity of the cell, and we question if they are involved in the mechanism of the cellular protection of target organs affected by ascites syndrome (AS. The objective of this study was to evaluate the body temperature

Denaturation of membrane proteins and hyperthermic cell killing

NARCIS (Netherlands)

Burgman, Paulus Wilhelmus Johannes Jozef

1993-01-01

Summarizing: heat induced denaturation of membrane proteins is probably related to hyperthermic cell killing. Induced resistance of heat sensitive proteins seems to be involved in the development of thermotolerance. Although many questions remain still to be answered, it appears that HSP72, when

Conformational Activation of Argonaute by Distinct yet Coordinated Actions of the Hsp70 and Hsp90 Chaperone Systems.

Science.gov (United States)

Tsuboyama, Kotaro; Tadakuma, Hisashi; Tomari, Yukihide

2018-05-17

Loading of small RNAs into Argonaute, the core protein in RNA silencing, requires the Hsp70/Hsp90 chaperone machinery. This machinery also activates many other clients, including steroid hormone receptors and kinases, but how their structures change during chaperone-dependent activation remains unclear. Here, we utilized single-molecule Förster resonance energy transfer (smFRET) to probe the conformational changes of Drosophila Ago2 mediated by the chaperone machinery. We found that empty Ago2 exists in various closed conformations. The Hsp70 system (Hsp40 and Hsp70) and the Hsp90 system (Hop, Hsp90, and p23) together render Ago2 into an open, active form. The Hsp70 system, but not the Hsp90 system alone, is sufficient for Ago2 to partially populate the open form. Instead, the Hsp90 system is required to extend the dwell time of Ago2 in the open state, which must be transiently primed by the Hsp70 system. Our data uncover distinct and coordinated actions of the chaperone machinery, where the Hsp70 system expands the structural ensembles of Ago2 and the Hsp90 system captures and stabilizes the active form. Copyright © 2018 Elsevier Inc. All rights reserved.

Hsf1p and Msn2/4p cooperate in the expression of Saccharomyces cerevisiae genes HSP26 and HSP104 in a gene- and stress type-dependent manner.

Science.gov (United States)

Amorós, M; Estruch, F

2001-03-01

Saccharomyces cerevisiae possesses several transcription factors involved in the transcriptional activation of stress-induced genes. Among them, the heat shock factor (Hsf1p) and the zinc finger proteins of the general stress response (Msn2p and Msn4p) have been shown to play a major role in stress protection. Some heat shock protein (HSP) genes contain both heat shock elements (HSEs) and stress response elements (STREs), suggesting the involvement of both transcription factors in their regulation. Analysis of the stress-induced expression of two of these genes, HSP26 and HSP104, reveals that the contribution of Hsf1p and Msn2/4p is different depending on the gene and the stress condition.

Targeting Hsp90-Cdc37: A Promising Therapeutic Strategy by Inhibiting Hsp90 Chaperone Function.

Science.gov (United States)

Wang, Lei; Li, Li; Gu, Kai; Xu, Xiao-Li; Sun, Yuan; You, Qi-Dong

2017-01-01

The Hsp90 chaperone protein regulates the folding, maturation and stability of a wide variety of oncoproteins. In recent years, many Hsp90 inhibitors have entered into the clinical trials while all of them target ATPase showing similar binding capacity and kinds of side-effects so that none have reached to the market. During the regulation progress, numerous protein- protein interactions (PPI) such as Hsp90 and client proteins or cochaperones are involved. With the Hsp90-cochaperones PPI networks being more and more clear, many cancerous proteins have been reported to be tightly correlated to Hsp90-cochaperones PPI. Among them, Hsp90-Cdc37 PPI has been widely reported to associate with numerous protein kinases, making it a novel target for the treatment of cancers. In this paper, we briefly review the strategies and modulators targeting Hsp90-Cdc37 complex including direct and indirect regulation mechanism. Through these discussions we expect to present inspirations for new insights into an alternative way to inhibit Hsp90 chaperone function. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Heat shock protein 70 modulates neural progenitor cells dynamics in human neuroblastoma SH-SY5Y cells exposed to high glucose content.

Science.gov (United States)

Salimi, Leila; Rahbarghazi, Reza; Jafarian, Vahab; Biray Avci, Çıgır; Goker Bagca, Bakiye; Pinar Ozates, Neslihan; Khaksar, Majid; Nourazarian, Alireza

2018-01-18

In the current experiment, detrimental effects of high glucose condition were investigated on human neuroblastoma cells. Human neuroblastoma cell line SH-SY5Y were exposed to 5, 40, and 70 mM glucose over a period of 72 h. Survival rate and the proliferation of cells were analyzed by MTT and BrdU incorporation assays. Apoptosis was studied by the assays of flow cytometry and PCR array. In order to investigate the trans-differentiation capacity of the cell into mature neurons, we used immunofluorescence imaging to follow NeuN protein level. The transcription level of HSP70 was shown by real-time PCR analysis. MMP-2 and -9 activities were shown by gelatin Zymography. According to data from MTT and BrdU incorporation assay, 70 mM glucose reduced cell viability and proliferation rate as compared to control (5 mM glucose) and cells treated with 40 mM glucose (P Cell exposure to 70 mM glucose had potential to induced apoptosis after 72 h (P SH-SY5Y cells to detrimental effects of high glucose condition during trans-differentiation into mature neuron-like cells. Real-time PCR analysis confirmed the expression of HSP70 in cells under high content glucose levels, demonstrating the possible cell compensatory response to an insulting condition (p control vs 70 mM group  cells being exposed to 70 mM glucose. High glucose condition could abrogate the dynamics of neural progenitor cells. The intracellular level of HSP70 was proportional to cell damage in high glucose condition. © 2018 Wiley Periodicals, Inc.

Synergistic role of HSP90α and HSP90β to promote myofibroblast persistence in lung fibrosis.

Science.gov (United States)

Bellaye, Pierre-Simon; Shimbori, Chiko; Yanagihara, Toyoshi; Carlson, David A; Hughes, Philip; Upagupta, Chandak; Sato, Seidai; Wheildon, Nolan; Haystead, Timothy; Ask, Kjetil; Kolb, Martin

2018-02-01

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the lung parenchyma, causing significant morbidity through worsening dyspnoea and overall functional decline. IPF is characterised by apoptosis-resistant myofibroblasts, which are a major source for the excessive production of extracellular matrix (ECM) overtaking normal lung tissue. We sought to study the role of heat shock protein (HSP) isoforms HSP90α and HSP90β, whose distinct roles in lung fibrogenesis remain elusive.We determined the level of circulating HSP90α in IPF patients (n=31) and age-matched healthy controls (n=9) by ELISA. The release of HSP90α and HSP90β was evaluated in vitro in primary IPF and control lung fibroblasts and ex vivo after mechanical stretch on fibrotic lung slices from rats receiving adenovector-mediated transforming growth factor-β1.We demonstrate that circulating HSP90α is upregulated in IPF patients in correlation with disease severity. The release of HSP90α is enhanced by the increase in mechanical stress of the fibrotic ECM. This increase in extracellular HSP90α signals through low-density lipoprotein receptor-related protein 1 (LRP1) to promote myofibroblast differentiation and persistence. In parallel, we demonstrate that the intracellular form of HSP90β stabilises LRP1, thus amplifying HSP90α extracellular action.We believe that the specific inhibition of extracellular HSP90α is a promising therapeutic strategy to reduce pro-fibrotic signalling in IPF. Copyright ©ERS 2018.

Targeting GRP75 improves HSP90 inhibitor efficacy by enhancing p53-mediated apoptosis in hepatocellular carcinoma.

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Weiwei Guo

Full Text Available Heat shock protein 90 (HSP90 inhibitors are potential drugs for cancer therapy. The inhibition of HSP90 on cancer cell growth largely through degrading client proteins, like Akt and p53, therefore, triggering cancer cell apoptosis. Here, we show that the HSP90 inhibitor 17-AAG can induce the expression of GRP75, a member of heat shock protein 70 (HSP70 family, which, in turn, attenuates the anti-growth effect of HSP90 inhibition on cancer cells. Additionally, 17-AAG enhanced binding of GRP75 and p53, resulting in the retention of p53 in the cytoplasm. Blocking GRP75 with its inhibitor MKT-077 potentiated the anti-tumor effects of 17-AAG by disrupting the formation of GRP75-p53 complexes, thereby facilitating translocation of p53 into the nuclei and leading to the induction of apoptosis-related genes. Finally, dual inhibition of HSP90 and GRP75 was found to significantly inhibit tumor growth in a liver cancer xenograft model. In conclusion, the GRP75 inhibitor MKT-077 enhances 17-AAG-induced apoptosis in HCCs and increases p53-mediated inhibition of tumor growth in vivo. Dual targeting of GRP75 and HSP90 may be a useful strategy for the treatment of HCCs.

Hsp90 molecular chaperone: structure, functions and participation in cardio-vascular pathologies

Directory of Open Access Journals (Sweden)

Kroupskaya I. V.

2009-10-01

Full Text Available The review is devoted to the analysis of structural and functional properties of molecular chaperon Hsp90. Hsp90 is a representative of highly widespread family of heat shock proteins. The protein is found in eubacteria and all branches of eukarya, but it is apparently absent in archaea. It is one of key regulators of numerous signalling pathways, cell growth and development, apoptosis, induction of autoimmunity, and progression of heart failure. The full functional activity of Hsp90 shows up in a complex with other molecular chaperones and co-chaperones. Molecular interactions between chaperones, different signalling proteins and protein-partners are highly crucial for the normal functioning of signalling pathways and their destruction causes an alteration in the cell physiology up to its death.

Expression profile of HSP genes during different seasons in goats (Capra hircus).

Science.gov (United States)

Dangi, Satyaveer Singh; Gupta, Mahesh; Maurya, Divakar; Yadav, Vijay Prakash; Panda, Rudra Prasanna; Singh, Gyanendra; Mohan, Nitai Haridas; Bhure, Sanjeev Kumar; Das, Bikash Chandra; Bag, Sadhan; Mahapatra, Ramkrishna; Taru Sharma, Guttalu; Sarkar, Mihir

2012-12-01

The present study has demonstrated the expression of HSP60, HSP70, HSP90, and UBQ in peripheral blood mononuclear cells (PBMCs) during different seasons in three different age groups (Groups I, II, and III with age of 0-2, 2-5, and >5 years, respectively) of goats of tropical and temperate regions. Real-time polymerase chain reaction was applied to investigate mRNA expression of examined factors. Specificity of the desired products was documented using analysis of the melting temperature and high-resolution gel electrophoresis to verify that the transcripts are of the exact molecular size predicted. The mRNA expression of HSP60, HSP90, and UBQ was significantly higher (P tropical and temperate region goats. HSP70 mRNA expression was significantly higher (P tropical region goats. However, in the temperate region, in goats from all the three age groups studied, a non-significant difference of HSP70 expression between summer and winter seasons was noticed. In conclusion, results demonstrate that (1) HSP genes are expressed in caprine PBMCs and (2) higher expression of HSPs during thermal stress suggest possible involvement of them to ameliorate deleterious effect of thermal stress so as to maintain cellular integrity and homeostasis in goats.

Increased HSF activation in muscles with a high constitutive Hsp70 expression

OpenAIRE

Locke, Marius; Tanguay, Robert M.

1996-01-01

Stress-induced transcriptional regulation of the Hsps is mediated by trimerization and binding of a pre-existing heat shock transcription factor (HSF1) to a specific DNA sequence located in the 5′ region of hsp genes, known as the heat shock element. Hsp70 has been implicated in regulating the activation of the HSF and, according to cell culture models, high steady-state levels of Hsp70 are inversely correlated with HSF activation. To determine if this applies in an intact animal, muscles of ...

Inhibition of Hsp90 acts synergistically with topoisomerase II poisons to increase the apoptotic killing of cells due to an increase in topoisomerase II mediated DNA damage.

Science.gov (United States)

Barker, Catherine R; McNamara, Anne V; Rackstraw, Stephen A; Nelson, David E; White, Mike R; Watson, Alastair J M; Jenkins, John R

2006-01-01

Topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and meiosis and is a highly attractive target for chemotherapeutic agents. We have identified previously topoisomerase II and heat shock protein 90 (Hsp90) as part of a complex. In this paper we demonstrate that drug combinations targeting these two enzymes cause a synergistic increase in apoptosis. The objective of our study was to identify the mode of cell killing and the mechanism behind the increase in topoisomerase II mediated DNA damage. Importantly we demonstrate that Hsp90 inhibition results in an increased topoiosmerase II activity but not degradation of topoisomerase II and it is this, in the presence of a topoisomerase II poison that causes the increase in cell death. Our results suggest a novel mechanism of action where the inhibition of Hsp90 disrupts the Hsp90-topoisomerase II interaction leading to an increase in and activation of unbound topoisomerase II, which, in the presence of a topoisomerase II poison leads to the formation of an increased number of cleavable complexes ultimately resulting in rise in DNA damage and a subsequent increase cell death.

Reduced heat shock response in human mononuclear cells during aging and its association with polymorphisms in HSP70 genes

DEFF Research Database (Denmark)

Singh, Ripudaman; Kølvraa, Steen; Bross, Peter

2006-01-01

Age-dependent changes in heat shock response (HSR) were studied in mononuclear cells (monocytes and lymphocytes) collected from young (mean age = 22.6 +/- 1.7 years) and middle-aged (mean age = 56.3 +/- 4.7 years) subjects after 1 hour of heat shock at 42 degrees C. Genotype-specific HSR...... was measured by genotyping the subjects for 3 single nucleotide polymorphisms, HSPA1A(A-110C), HSPA1B(A1267G), and HSPA1L(T2437C), 1 each in the 3 HSP70 genes. A significant age-related decrease in the induction of Hsp70 occurred after heat shock in both monocytes and lymphocytes. The noninducible...

Characterization of HSP27 phosphorylation sites in human atherosclerotic plaque secretome

DEFF Research Database (Denmark)

Durán, Mari-Carmen; Boeri-Erba, Elisabetta; Mohammed, Shabaz

2007-01-01

spectrometry (MS). Among the identified proteins, two isoforms of heat shock protein 27 (HSP27), a protein recently described as a potential biomarker of atherosclerosis, were detected. However, the putative mechanisms in which HSP27 isoforms could be involved in the atherosclerotic process are unknown. Thus......, the role that phosphorylated HSP27 could play in the atherosclerotic process is actually under study. The present work shows the strategies employed to characterize the phosphorylation in the HSP27 secreted by atheroma plaque samples. The application of liquid chromatography tandem mass spectrometry (MS......-lymphocytes). These interactions can be mediated by proteins secreted from these cells, which therefore exert an important role in the atherosclerotic process. We recently described a novel strategy for the characterization of the human atherosclerotic plaque secretome, combining two-dimensional gel electrophoresis and mass...

Preliminary X-Ray Crystallographic Studies of the N-Terminal Domains of Hsp104 from Yeast Candida albicans and Saccharomyces cerevisiae

Science.gov (United States)

Wang, P.; Li, J.; Sha, B.

2017-12-01

Yeast Hsp104 is an ATP-dependent molecular chaperone, which can solublize and rescue denatured proteins from aggregates into active form by cooperating with Hsp70 and Hsp40 chaperones. Moreover, overexpression of Hsp104 of Saccharomyces cerevisiae (ScHsp104) cures the yeast [ PSI +] prion due to the completely dissolution of the prion seeds, demonstrating ScHsp104's potential to clear amyloid-like protein aggregates, thus making ScHsp104 a promising medication approach for human amyloidogenic neurodegenerative diseases. Because the working mechanisms for ScHsp104's activities have not been clearly elucidated yet, crystallographic determination of ScHsp104 stands for great significance. Here, the expression, purification and crystallization of the N-terminal domains of Hsp104 from yeast Candida albicans (CaHsp104N) and S. cerevisiae (ScHsp104N) are described. The CaHsp104N crystals diffracted to 1.54 Å and belonged to the sp. gr. P3221 or P3121, with unit cell parameters of a = 55.213 Å, c = 109.451 Å. The data of the ScHsp104N crystals were collected to the resolution of 2.53 Å in the sp. gr. C2, with unit cell parameters a = 148.587 Å, b = 66.255 Å, c = 74.577 Å, β = 107.369°. The phase of ScHsp104N is determined by the molecular replacement method using CaHsp104N as the search model.

Localization of Heat Shock Protein 27 (Hsp27) in the Rat Gingiva and its Changes with Tooth Eruption

International Nuclear Information System (INIS)

Sasaki, Au; Yamada, Tohru; Inoue, Katsuyuki; Momoi, Tomoko; Tokunaga, Hiroshi; Sakiyama, Koji; Kanegae, Haruhide; Suda, Naoto; Amano, Osamu

2011-01-01

Heat shock protein 27 kDa (Hsp27) functions as a molecular chaperon to prevent apoptosis as well as to contribute to the regulation of cell proliferation and differentiation during development. In the present study, the localization of Hsp27 in the oral epithelium of rats and its expression change during formation of the gingiva with the tooth eruption were examined immunohistochemically to elucidate the roles of Hsp27 in the oral mucosa. In adult rats, Hsp27-immunoreactivity was localized in the prickle and granular layers but absent in the basal and horny layers of the oral epithelium. On the other hand, in the outer and sulcular epithelia of the free gingival, Hsp27-immunoreactivity was detected in the whole layers, while it was not found in the proliferation zone of the junctional epithelium immunoreactive for Ki67. In immature rats on 10th postnatal day, Hsp27-immunoreactivity was intense in the prickle and granular layers of the oral epithelium, but was not detected in its basal layer. In rats at the eruptive phase on 15th postnatal day, Hsp27-immunoreactivity was detected in sites of the basal layer adjacent to where the dental cusps penetrated through the oral epithelium. Although the immunoreactivity for Ki67 was found in the basal layer of the oral epithelium, it was not localized in the Hsp27-immunopositive sites of tooth-penetration in the basal layer. Just after the tooth-eruption on 20th postnatal day, Hsp27-immunoreactivity was not found in the stratified squamous epithelium at the dentogingival junction, whereas it was intense in a single layer of cuboidal epithelial cells attached to the tooth neck. Ki67-positive cells were scattered in the stratified squamous epithelium at the dentogingival junction, whereas no positive cells were found in the portion of a single layer of cuboidal epithelial cells. These findings suggest that the outer and sulcular epithelia of the free gingiva have a relatively slower rate of proliferation than other gingival and

Design and Construction of a Cloning Vector Containing the hspX Gene of Mycobacterium tuberculosis.

Science.gov (United States)

Yaghoubi, Atieh; Aryan, Ehsan; Zare, Hosna; Alami, Shadi; Teimourpour, Roghayeh; Meshkat, Zahra

2016-10-01

Tuberculosis (TB) is a major cause of death worldwide. Finding an effective vaccine against TB is the best way to control it. Several vaccines against this disease have been developed but none are completely protective. The aim of this study was to design and construct a cloning vector containing the Mycobacterium tuberculosis (M. tuberculosis) heat shock protein X (hspX) . First, an hspX fragment was amplified by PCR and cloned into plasmid pcDNA3.1(+) and recombinant vector was confirmed. A 435 bp hspX fragment was isolated. The fragment was 100% homologous with hspX of M. tuberculosis strain H37Rv in GenBank. In this study, the cloning vector pcDNA3.1(+), containing a 435-bp hspX fragment of M. tuberculosis , was constructed. This could be used as a DNA vaccine to induce immune responses in animal models in future studies.

Adenoviral transfer of HSP-70 into pulmonary epithelium ameliorates experimental acute respiratory distress syndrome.

Science.gov (United States)

Weiss, Yoram G; Maloyan, Alina; Tazelaar, John; Raj, Nichelle; Deutschman, Clifford S

2002-09-01

The acute respiratory distress syndrome (ARDS) provokes three pathologic processes: unchecked inflammation, interstitial/alveolar protein accumulation, and destruction of pulmonary epithelial cells. The highly conserved heat shock protein HSP-70 can limit all three responses but is not appropriately expressed in the lungs after cecal ligation and double puncture (2CLP), a clinically relevant model of ARDS. We hypothesize that restoring expression of HSP-70 using adenovirus-mediated gene therapy will limit pulmonary pathology following 2CLP. We administered a vector containing the porcine HSP-70 cDNA driven by a CMV promoter (AdHSP) into the lungs of rats subjected to 2CLP or sham operation. Administration of AdHSP after either sham operation or 2CLP increased HSP-70 protein expression in lung tissue, as determined by immunohistochemistry and Western blot hybridization. Administration of AdHSP significantly attenuated interstitial and alveolar edema and protein exudation and dramatically decreased neutrophil accumulation, relative to a control adenovirus. CLP-associated mortality at 48 hours was reduced by half. Modulation of HSP-70 production reduces pathologic changes and may improve outcome in experimental ARDS.

Ammonia stress under high environmental ammonia induces Hsp70 and Hsp90 in the mud eel, Monopterus cuchia.

Science.gov (United States)

Hangzo, Hnunlalliani; Banerjee, Bodhisattwa; Saha, Shrabani; Saha, Nirmalendu

2017-02-01

The obligatory air-breathing mud eel (Monopterus cuchia) is frequently being challenged with high environmental ammonia (HEA) exposure in its natural habitats. The present study investigated the possible induction of heat shock protein 70 and 90 (hsp70, hsc70, hsp90α and hsp90β) genes and more expression of Hsp70 and Hsp90 proteins under ammonia stress in different tissues of the mud eel after exposure to HEA (50 mM NH 4 Cl) for 14 days. HEA resulted in significant accumulation of toxic ammonia in different body tissues and plasma, which was accompanied with the stimulation of oxidative stress in the mud eel as evidenced by more accumulation of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) during exposure to HEA. Further, hyper-ammonia stress led to significant increase in the levels of mRNA transcripts for inducible hsp70 and hsp90α genes and also their translated proteins in different tissues probably as a consequence of induction of hsp70 and hsp90α genes in the mud eel. However, hyper-ammonia stress was neither associated with any significant alterations in the levels of mRNA transcripts for constitutive hsc70 and hsp90β genes nor their translated proteins in any of the tissues studied. More abundance of Hsp70 and Hsp90α proteins might be one of the strategies adopted by the mud eel to defend itself from the ammonia-induced cellular damages under ammonia stress. Further, this is the first report of ammonia-induced induction of hsp70 and hsp90α genes under hyper-ammonia stress in any freshwater air-breathing teleost.

Altered Cross-Linking of HSP27 by Zerumbone as a Novel Strategy for Overcoming HSP27-Mediated Radioresistance

International Nuclear Information System (INIS)

Choi, Seo-Hyun; Lee, Yoon-Jin; Seo, Woo Duck; Lee, Hae-June; Nam, Joo-Won; Lee, Yoo Jin; Kim, Joon; Seo, Eun-Kyoung; Lee, Yun-Sil

2011-01-01

Purpose: HSP27 or HSP25 negatively regulates apoptosis pathways after radiation or chemotherapeutic agents. Abrogation of HSP27 function may be a candidate target for overcoming radio- and chemoresistance. Methods and Materials: Zerumbone (ZER), a cytotoxic component isolated from Zingiber zerumbet smith. Clonogenic survival assay and flow cytometry after Annexin V staining were performed to determine in vitro sensitization effects of ZER with ionizing radiation. A nude mouse xenografting system was also applied to detect in vivo radiosensitizing effects of ZER. Results: ZER produced cross-linking of HSP27, which was dependent on inhibition of the monomeric form of HSP27. ZER was directly inserted between the disulfide bond in the HSP27 dimer and modified normal HSP27 dimerization. Pretreatment with ZER before radiation inhibited the binding affinity between HSP27 and apoptotic molecules, such as cytochrome c and PKCδ, and induced sensitization in vitro and in an in vivo xenografted nude mouse system. Structural analogs lacking only the carbonyl group in ZER, such as α-humulene (HUM) and 8-hydroxy-humulen (8-OH-HUM), did not affect normal cross-linking of HSP27 and did not induce radiosensitization. Conclusions: We suggest that altered cross-linking of HSP27 by ZER is a good strategy for abolishing HSP27-mediated resistance.

Hsp104 suppresses polyglutamine-induced degeneration post onset in a drosophila MJD/SCA3 model.

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Mimi Cushman-Nick

Full Text Available There are no effective therapeutics that antagonize or reverse the protein-misfolding events underpinning polyglutamine (PolyQ disorders, including Spinocerebellar Ataxia Type-3 (SCA3. Here, we augment the proteostasis network of Drosophila SCA3 models with Hsp104, a powerful protein disaggregase from yeast, which is bafflingly absent from metazoa. Hsp104 suppressed eye degeneration caused by a C-terminal ataxin-3 (MJD fragment containing the pathogenic expanded PolyQ tract, but unexpectedly enhanced aggregation and toxicity of full-length pathogenic MJD. Hsp104 suppressed toxicity of MJD variants lacking a portion of the N-terminal deubiquitylase domain and full-length MJD variants unable to engage polyubiquitin, indicating that MJD-ubiquitin interactions hinder protective Hsp104 modalities. Importantly, in staging experiments, Hsp104 suppressed toxicity of a C-terminal MJD fragment when expressed after the onset of PolyQ-induced degeneration, whereas Hsp70 was ineffective. Thus, we establish the first disaggregase or chaperone treatment administered after the onset of pathogenic protein-induced degeneration that mitigates disease progression.

Perbedaan Kadar HSP90 pada Preeklamsi Berat dengan Kehamilan Normal

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Soetrisno

2015-06-01

Full Text Available Severe pre-eclampsia is the second highest cause of maternal mortality. Free radicals that stimulate heat shock protein 90 (HSP 90 are believed to determine severe pre-eclampsia. HSP90 is an important protein that helps the establishment and maintenance of other proteins. It also increases the life time of cells after various pathological conditions (chaperone function. The chaperone function is the adaptation key factor to endogenous stress in tissues. By recognizing HSP90 level in early detection of severe pre-eclampsia, prevention and management can be started early. This study aimed to prove that the HSP90 level in pregnancy with severe pre-eclampsia is higher than normal pregnancy. This was a quantitative study using cross sectional approach by testing the HSP90 level. The study was conducted during the period of September to November 2013, at the Obstetrics and Gynecological Unit, Moewardi Hospital Surakarta and Prodia Laboratory Jakarta. The number of subjects was 30 patients, consisting of 15 normal pregnant mothers and 15 pregnant mothers with pre-eclampsia . The calculation of serum HSP90 level was conducted using enzyme-linked immunosorbent assay (ELISA. Data were analyzed using t-test using SPSS for Windows version 17 for Windows. The mean of HSP90 in the severe pre-eclampsia group was 131.91±26.66 while the mean in the normal pregnancy group was 80.28±13.39 with p=0.00 (p<0.05. Level of HSP90 serum in severe pre-eclampsia is higher than in normal pregnancy, due to the occurrence of oxidative stress in severe pre-eclampsia

Genome-wide identification and analysis of biotic and abiotic stress regulation of small heat shock protein (HSP20) family genes in bread wheat.

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Muthusamy, Senthilkumar K; Dalal, Monika; Chinnusamy, Viswanathan; Bansal, Kailash C

2017-04-01

Small Heat Shock Proteins (sHSPs)/HSP20 are molecular chaperones that protect plants by preventing protein aggregation during abiotic stress conditions, especially heat stress. Due to global climate change, high temperature is emerging as a major threat to wheat productivity. Thus, the identification of HSP20 and analysis of HSP transcriptional regulation under different abiotic stresses in wheat would help in understanding the role of these proteins in abiotic stress tolerance. We used sequences of known rice and Arabidopsis HSP20 HMM profiles as queries against publicly available wheat genome and wheat full length cDNA databases (TriFLDB) to identify the respective orthologues from wheat. 163 TaHSP20 (including 109 sHSP and 54 ACD) genes were identified and classified according to the sub-cellular localization and phylogenetic relationship with sequenced grass genomes (Oryza sativa, Sorghum bicolor, Zea mays, Brachypodium distachyon and Setaria italica). Spatio-temporal, biotic and abiotic stress-specific expression patterns in normalized RNA seq and wheat array datasets revealed constitutive as well as inductive responses of HSP20 in different tissues and developmental stages of wheat. Promoter analysis of TaHSP20 genes showed the presence of tissue-specific, biotic, abiotic, light-responsive, circadian and cell cycle-responsive cis-regulatory elements. 14 TaHSP20 family genes were under the regulation of 8 TamiRNA genes. The expression levels of twelve HSP20 genes were studied under abiotic stress conditions in the drought- and heat-tolerant wheat genotype C306. Of the 13 TaHSP20 genes, TaHSP16.9H-CI showed high constitutive expression with upregulation only under salt stress. Both heat and salt stresses upregulated the expression of TaHSP17.4-CI, TaHSP17.7A-CI, TaHSP19.1-CIII, TaACD20.0B-CII and TaACD20.6C-CIV, while TaHSP23.7-MTI was specifically induced only under heat stress. Our results showed that the identified TaHSP20 genes play an important role under

Hsp27 promotes ABCA1 expression and cholesterol efflux through the PI3K/PKCζ/Sp1 pathway in THP-1 macrophages.

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Kuang, Hai-Jun; Zhao, Guo-Jun; Chen, Wu-Jun; Zhang, Min; Zeng, Gao-Feng; Zheng, Xi-Long; Tang, Chao-Ke

2017-09-05

Heat shock protein 27 (Hsp27) is a putative biomarker and therapeutic target in atherosclerosis. This study was to explore the potential mechanisms underlying Hsp27 effects on ATP-binding cassette transporter A1 (ABCA1) expression and cellular cholesterol efflux. THP-1 macrophage-derived foam cells were infected with adenovirus to express wild-type Hsp27, hyper-phosphorylated Hsp27 mimic (3D Hsp27), antisense Hsp27 or hypo-phosphorylated Hsp27 mimic (3A Hsp27). Wild-type and 3D Hsp27 were found to up-regulate ABCA1 mRNA and protein expression and increase cholesterol efflux from cells. Expression of antisense or 3A Hsp27 suppressed the expression of ABCA1 and cholesterol efflux. Furthermore, over-expression of wild-type and 3D Hsp27 significantly increased the levels of phosphorylated specificity protein 1 (Sp1), protein kinase C ζ (PKCζ) and phosphatidylinositol 3-kinase (PI3K). In addition, the up-regulation of ABCA1 expression and cholesterol efflux induced by 3D Hsp27 was suppressed by inhibition of Sp1, PKCζ and PI3K with specific kinase inhibitors. Taken together, our results revealed that Hsp27 may up-regulate the expression of ABCA1 and promotes cholesterol efflux through activation of the PI3K/PKCζ/Sp1 signal pathway in THP-1 macrophage-derived foam cells. Our findings may partly explain the mechanisms underlying the anti-atherogenic effect of Hsp27. Copyright © 2017 Elsevier B.V. All rights reserved.

The expression and function of hsp30-like small heat shock protein genes in amphibians, birds, fish, and reptiles.

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Heikkila, John J

2017-01-01

Small heat shock proteins (sHSPs) are a superfamily of molecular chaperones with important roles in protein homeostasis and other cellular functions. Amphibians, reptiles, fish and birds have a shsp gene called hsp30, which was also referred to as hspb11 or hsp25 in some fish and bird species. Hsp30 genes, which are not found in mammals, are transcribed in response to heat shock or other stresses by means of the heat shock factor that is activated in response to an accumulation of unfolded protein. Amino acid sequence analysis revealed that representative HSP30s from different classes of non-mammalian vertebrates were distinct from other sHSPs including HSPB1/HSP27. Studies with amphibian and fish recombinant HSP30 determined that they were molecular chaperones since they inhibited heat- or chemically-induced aggregation of unfolded protein. During non-mammalian vertebrate development, hsp30 genes were differentially expressed in selected tissues. Also, heat shock-induced stage-specific expression of hsp30 genes in frog embryos was regulated at the level of chromatin structure. In adults and/or tissue culture cells, hsp30 gene expression was induced by heat shock, arsenite, cadmium or proteasomal inhibitors, all of which enhanced the production of unfolded/damaged protein. Finally, immunocytochemical analysis of frog and chicken tissue culture cells revealed that proteotoxic stress-induced HSP30 accumulation co-localized with aggresome-like inclusion bodies. The congregation of damaged protein in aggresomes minimizes the toxic effect of aggregated protein dispersed throughout the cell. The current availability of probes to detect the presence of hsp30 mRNA or encoded protein has resulted in the increased use of hsp30 gene expression as a marker of proteotoxic stress in non-mammalian vertebrates. Copyright © 2016 Elsevier Inc. All rights reserved.

Silica Nanoparticles Induce Oxidative Stress and Autophagy but Not Apoptosis in the MRC-5 Cell Line

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Sorina Nicoleta Petrache Voicu

2015-12-01

Full Text Available This study evaluated the in vitro effects of 62.5 µg/mL silica nanoparticles (SiO2 NPs on MRC-5 human lung fibroblast cells for 24, 48 and 72 h. The nanoparticles’ morphology, composition, and structure were investigated using high resolution transmission electron microscopy, selected area electron diffraction and X-ray diffraction. Our study showed a decreased cell viability and the induction of cellular oxidative stress as evidenced by an increased level of reactive oxygen species (ROS, carbonyl groups, and advanced oxidation protein products after 24, 48, and 72 h, as well as a decreased concentration of glutathione (GSH and protein sulfhydryl groups. The protein expression of Hsp27, Hsp60, and Hsp90 decreased at all time intervals, while the level of protein Hsp70 remained unchanged during the exposure. Similarly, the expression of p53, MDM2 and Bcl-2 was significantly decreased for all time intervals, while the expression of Bax, a marker for apoptosis, was insignificantly downregulated. These results correlated with the increase of pro-caspase 3 expression. The role of autophagy in cellular response to SiO2 NPs was demonstrated by a fluorescence-labeled method and by an increased level of LC3-II/LC3-I ratio. Taken together, our data suggested that SiO2 NPs induced ROS-mediated autophagy in MRC-5 cells as a possible mechanism of cell survival.

Hsp60-induced tolerance in the rotifer Brachionus plicatilis exposed to multiple environmental contaminants.

Science.gov (United States)

Wheelock, C E; Wolfe, M F; Olsen, H; Tjeerdema, R S; Sowby, M L

1999-04-01

Hsp60 induction was selected as a sublethal endpoint of toxicity for Brachionus plicatilis exposed to a water accommodated fraction (WAF) of Prudhoe Bay crude oil (PBCO), a PBCO/dispersant (Corexit 9527(R)) fraction and Corexit 9527(R) alone. To examine the effect of multiple stressors, exposures modeled San Francisco Bay, where copper levels are approximately 5 microgram/L, salinity is 22 per thousand, significant oil transport and refining occurs, and petroleum releases have occurred historically. Rotifers were exposed to copper at 5 microgram/L for 24 h, followed by one of the oil/dispersant preparations for 24 h. Batch-cultured rotifers were used in this study to model wild populations instead of cysts. SDS-PAGE with Western Blotting using hsp60-specific antibodies and chemiluminescent detection were used to isolate, identify, and measure induced hsp60 as a percentage of control values. Both PBCO/dispersant and dispersant alone preparations induced significant levels of hsp60. However, hsp60 expression was reduced to that of controls at high WAF concentrations, suggesting interference with protein synthesis. Rotifers that had been preexposed to copper maintained elevated levels of hsp60 upon treatment with WAF at all concentrations. Results suggest that induction of hsp60 by chronic low-level exposure may serve as a protective mechanism against subsequent or multiple stressors and that hsp60 levels are not additive for the toxicants tested in this study, giving no dose-response relationship. The methods employed in this study could be useful for quantifying hsp60 levels in wild rotifer populations.

Design and Construction of a Cloning Vector Containing the hspX Gene of Mycobacterium tuberculosis