Gamma radiation induced changes in expression of heat shock proteins (Hsc70 and Hsp83) in the dengue vector Aedes aegypti (L.)

We aimed to assess the effect of gamma radiation on the expression of heat shock proteins Hsc70 and Hsp83 in Aedes aegypti. Adult males were irradiated with 50Gy of gamma radiation, and changes in the expression of proteins in SDS-PAGE gel bands corresponding to molecular weights ~60–75kDa and ~80–95kDa were analyzed at two different time points 6 and 12-hour post-irradiation, using a temporal mass spectrometry based semi-quantitative analysis. A 2-3-fold increase was observed in both proteins Hsc70 and Hsp83, at both time points. In addition, the experiment also revealed the overexpression of several other molecules such as Arginine Kinase - known to be upregulated in certain insects during stress, Esterase B1- implicated in insecticide resistance, and also down-regulation of the 26S proteasome non-ATPase regulatory subunit 1 and ubiquitin-activating enzyme E1 - both known to be involved in ubiquitin-mediated protein degradation. The results taken together with existing data on Hsp83 and Hsc70, indicate that these proteins may enhance the survival of Ae. aegypti following gamma radiation and could serve as molecular markers for the detection of radiation-induced stress.

Clinical significance of expression of tumor immunogenic cell death related molecules / 国际肿瘤学杂志

Tumor immunogenic cell death is a type of regulatory cell death, which is driven by stress including chemotherapy drugs, radiotherapy, oncolytic virus, nano carrier drugs and photodynamic force. It can induce specific immune response to tumor death cell antigen. The further study can provide theoretical basis and new ideas for anti-tumor immunity and clinical immunotherapy of tumor.

Effects of HDAC6 regulation of HSC70 acetylation level on bone loss in osteoporosis after oophorectomy / 中华内分泌外科杂志

Objective:To investigate the effects of histone deacetylase 6 (histone deacetylase 6, HDAC6) on oopherectomy (OOX) induced osteoporosis (OP) bone loss by binding to the promoter region of heat-shock protein 70 (HSC70) and regulating it’s acetylation.Methods:OP mouse model was established by using OOX methods. Then the mice were divided into sham operation group, OOX group, OOX+shHDAC6 group, OOX+shNC group and OOX+shHDAC6+shHSC70 group. The micro-CT system and Western blot experiment were used to detect the bone microscopic parameters of the mouse right femur and the protein expression levels of osteoblast-specific transcription factors. In vitro experiments, Westwen blot, alkaline phosphatase (ALP) staining and Alizarin Red S (ARS) staining were used to determine the effects of HDAC6 and HSC70 on the osteogenic differentiation of MC3T3-E1 cells. QRT-PCR was used to detect the expression levels of HDAC6 and HSC70 in tissue or cells. The relationship between HDAC6 and HSC70 was analyzed by ChIP experiment.Results:Compared with sham group, the expression of bone mineral density (BMD) , trabecular bone number (Tb. N) , trabecular thickness (Tb.th) and bone volume fraction (BV/TV) in the right femur of OOX group mice were decreased, the expression of TB. Sp was increased, protein expression of OSX and RUNX2 was increased. At the same time, compared with sham group (1±0.11) , the expression of HDAC6 was increased in OOX group (2.33±0.19) ( t=10.56, P<0.001) . Compared with pcDNA3.1-NC group, the protein level of Osterix (OSX) and runt-related transcription factor 2 (RUNX2) , ALP activity and mineralized area in pcDNA3.1-HDAC6 group were decreased (all P<0.05) . ChIP analysis showed that compared with the pcDNA3.1-NC group (5.26±0.47) , the acetylation level of the HSC70 promoter region in the pcDNA3.1-HDAC6 group (2.37±0.21) was significantly reduced ( t=9.72, P<0.001) . Compared with pcDNA3.1-HDAC6 group, the expression of OSX and RUNX2, ALP activity and mineralization were increased in pcDNA3.1-HDAC6+ pcDNA3.1-HSC70 group (all P<0.05) . Compared with OOX+shHDAC6 group, the expression of OSX and RUNX2 protein, BMD, Tb.N, Tb.th and BV/TV were decreased but the expression of Tb. Sp was increased in OOX+ shHDAC6+ shHSC70 group. Conclusions:HDAC6 regulates the acetylation level of HSC70 and then affects OOX-induced OP bone loss. Inhibition of HDAC6 can significantly improve OP bone loss.

Metformin activates chaperone-mediated autophagy and improves disease pathologies in an Alzheimer disease mouse model

Chaperone-mediated autophagy (CMA) is a lysosome-dependent selective degradation pathway implicated in the pathogenesis of cancer and neurodegenerative diseases. However, the mechanisms that regulate CMA are not fully understood. Here, using unbiased drug screening approaches, we discover Metformin, a drug that is commonly the first medication prescribed for type 2 diabetes, can induce CMA. We delineate the mechanism of CMA induction by Metformin to be via activation of TAK1-IKKα/β signaling that leads to phosphorylation of Ser85 of the key mediator of CMA, Hsc70, and its activation. Notably, we find that amyloid-beta precursor protein (APP) is a CMA substrate and that it binds to Hsc70 in an IKKα/β-dependent manner. The inhibition of CMA-mediated degradation of APP enhances its cytotoxicity. Importantly, we find that in the APP/PS1 mouse model of Alzheimer's disease (AD), activation of CMA by Hsc70 overexpression or Metformin potently reduces the accumulated brain Aβ plaque levels and reverses the molecular and behavioral AD phenotypes. Our study elucidates a novel mechanism of CMA regulation via Metformin-TAK1-IKKα/β-Hsc70 signaling and suggests Metformin as a new activator of CMA for diseases, such as AD, where such therapeutic intervention could be beneficial.

The sequential switch from macroautophagy to chaperone-mediated autophagy in Raji cells induced by starvation / 中国药理学通报

Aim To research the cross-talk and conversion between macroautophagy and chaperone-media-ted autophagy ( CMA) in cultured Burkitt lymphoma Raji cells induced by starvation. Methods The autophagic vacuoles were observed by fluorescence microscopy and confocal laser-scanning microscopy with monodansylcadaverine staining. The expression of autophagy associated-proteins were determined by West-em blot. Results Both macroautophagy and CMA were activated sequentially instead of simultaneously in starvation-induced Raji cells, and macroautophagy was quickly activated and peaked during the first hours of near baseline. With starvation persisted, CM A progressively increased along with the decline of macroautoph- A gy. Conclusions Macroautophagy and CMA are maximally activated during different stages of starvation. Activation of these two pathways is often sequential. The sequential switch from macroautophagy to CMA might be conducive to the adaption of cancer cells to miscellaneous intracellular or extracellular changes, maintaining their own growth and proliferation.

Decreased rotavirus infection of MA104 cells via probiotic extract binding to Hsc70 and ß3 integrin receptors / Disminución de la infección por rotavirus en células MA104 por la unión de proteínas de extractos probióticos a receptores celulares integrina ß3 y Hsc70 / Diminuição da infecção por rotavírus em células MA104 pela união de proteínas de extratos probióticos a receptores integrina ß3 e Hsc70

Abstract Probiotic bacteria are microorganisms beneficial to human health, useful to improving biological conditions. Thanks to probiotic bacteria the symptoms of viral infections can be alleviated. Different mechanisms whereby probiotic bacteria exert they antiviral effect have been proposed. The aim of this study was to determine whether probiotic bacteria extracts bind to receptors of host cells susceptible of rotavirus (RV) infection. To accomplish this objective, four probiotic bacterial strains of Lactobacillus spp. and Bifidobacterium spp. were tested. Probiotic extracts were obtained after bacterial growth, cell lysis and centrifugation. Obtained probiotic extracts were used in assays to interfere with adhesion and penetration of a RV strain in the mammal cell line MA104. Furthermore, the interaction between probiotic extracts and MA104 cell receptors was evaluated by co-immunoprecipitation assays using anti-β3-integrins and anti-Hsc70 antibodies. All four probiotic, protein-rich, extracts reduced RV infections in MA104 cells, suggesting a successful antiviral activity mediated by these probiotic extracts. All probiotic extracts significantly exerted their antiviral activity by interfering with RV adhesion on MA104 cell receptors, with proteins in probiotic extracts competitively interacting with cell surface receptors necessary to RV infection. Co-immunoprecipitation assay results showed that proteins in probiotic extracts were able to bind to β3-integrinsand Hsc70, which are two cellular receptors required to viral infection. The most significant contribution of this study is an insight into the mechanisms of probiotic antiviral activity, thus expanding current probiotics fundamental knowledge.

Resumen Las bacterias probióticas son microorganismos con efectos positivos en la salud humana, gracias a las bacterias probióticas los síntomas de infecciones virales pueden mitigarse. Al respecto, varios mecanismos antivirales de las bacterias probióticas han sido propuestos. El propósito de este estudio fue determinar, de manera experimental, si extractos de bacterias probióticas reducen la infección rotavírica al interferir con la unión entre el rotavirus y sus receptores celulares blanco. Extractos de cuatro cepas probióticas de Lactobacillus spp. y Bifidobacterium spp. fueron obtenidos a partir de cultivos bacterianos lisados y centrifugados. Cada uno de los extractos fue usado en experimentos para determinar si estos interfieren con la adhesión y penetración del rotavirus en células de mamífero MA104. Además, la interacción entre extractos probióticos y receptores de las células MA104 fue evaluada con ensayos de co-inmunoprecipitación, usando anticuerpos anti-integrina β3 y anti-Hsc70. Se observó que los cuatro extractos probióticos, ricos en proteínas, redujeron significativamente la infección de rotavirus en las células MA104. También se estableció que la que la actividad antiviral de los extractos probióticos es mediada por la interacción competitiva de sus proteínas con los receptores integrina β3 y Hsc70 de las células MA104, necesarios para iniciar la infección por rotavirus. Estos hallazgos constituyen un aporte al conocimiento de los mecanismos básicos de acción antiviral de las bacterias probióticas.

Resumo Bactérias probióticas são microrganismos com efeitos positivos na saúde humana, úteis na melhora de certas condições biológicas. Gracas a bactérias probióticas os sintomas de uma infecção viral podem ser aliviados. Diferentes mecanismos pelos quais as bactérias probióticas exercem seus efeitos antivirales têm sido propostos. O objetivo de este estudo foi determinar se extratos de bactérias probióticas reduzem a infecção de rotavírus (RV) ao interferir com a união entre o RV e seus receptores celulares alvo. Quatro cepas probióticas de Lactobacillus spp. e Bifidobacterium spp. foram testadas. Os extratos probióticos foram obtidos após o crescimento bacteriano, lise celular e centrifugação. Os extratos probióticos obtidos foram utilizados em ensaios para determinar se interferem com a adesão e penetração de uma cepa de RV em células de mamífero MA104. Adicionalmente, a interação entre os extratos probióticos e os receptores das células MA104 foi avaliada por ensaios de co-imunoprecipitação usando anticorpos anti-integrina β3 e anti- Hsc70. Os quatro extratos probióticos, ricos em proteínas, reduziram as infecções por RV em células MA104, sugerindo uma atividade antiviral mediada por estes extratos. Todos os extratos interferiram na adesão do RV aos receptores de células MA104, sendo que as proteínas presentes nos extratos mostraram uma interação competitiva com os receptores integrina β3 e Hsc70 das células MA104, necessários para iniciar a infecção por RV. Estes resultados contribuem para o conhecimento dos mecanismos básicos de ação antiviral de bactérias probióticas.

Protein disulfide isomerase and heat shock cognate protein 70 interactions with rotavirus structural proteins using their purified recombinant versions / Interacciones de las proteínas disulfuro isomerasa y de choque térmico Hsc70 con proteínas estructurales recombinantes purificadas de rotavirus

Introduction. Rotavirus entry into cells seems to be mediated by sequential interactions between viral structural proteins and some cell surface molecules. However, the mechanisms by which rotavirus infects target cell are still not well understood. There is some evidence showing that rotavirus structural proteins VP5* and VP8* interact with some cell surface molecules. The availability of recombinant rotavirus structural proteins in sufficient quantity has become very important for the identification of the specific virus-cell receptor interactions during the early events of the infectious process. Objective. The aim of the present work is to perform an analysis of the interactions between recombinant rotavirus structural proteins VP5*, VP8* and VP6, and cellular proteins Hsc70 and PDI using their purified recombinant versions. Materials and methods. Rotavirus recombinant VP5* and VP8*, and cellular recombinant proteins Hsc70 and PDI were expressed in E. coli BL21(DE3) while VP6 was expressed in recombinant vaccinia virus-transfected MA104 cells. The interaction between rotavirus and cellular proteins was studied using ELISA, co-immunoprecipitation and SDS-PAGE/Western blotting analysis. Results. The optimal conditions for expression of recombinant proteins were determined and antibodies were raised against them. The findings suggested that viral proteins rVP5* and rVP6 interact with Hsc70 and PDI in vitro. These viral recombinant proteins were also found to interact with raft-associated Hsc70 in a cell culture system. The treatment of cells with either rVP6 or DLPs produced significantly inhibition of rotavirus infection. Conclusion. The results allow us to conclude that rVP5* and rVP6 interact with Hsc70 and PDI during the rotavirus infection process.

Introducción. La entrada de rotavirus a las células parece estar mediado por interacciones secuenciales entre las proteínas estructurales virales y algunas moléculas de la superficie celular. Sin embargo, los mecanismos por los cuales el rotavirus infecta la célula diana aún no se comprenden bien. Existe alguna evidencia que muestra que las proteínas estructurales de rotavirus VP5* y VP8* interactúan con algunas moléculas de la superficie celular. La disponibilidad de las proteínas estructurales de rotavirus recombinantes en cantidad suficiente se ha convertido en un aspecto importante para la identificación de las interacciones específicas de los receptores virus-célula durante los eventos tempranos del proceso infeccioso. Objetivo. El propósito del presente trabajo es realizar un análisis de las interacciones entre las proteínas estructurales de rotavirus recombinante VP5*, VP8* y VP6, y las proteínas celulares Hsc70 y PDI utilizando sus versiones recombinantes purificadas. Materiales y métodos. Las proteínas recombinantes de rotavirus VP5* y VP8* y las proteínas recombinantes celulares Hsc70 y PDI se expresaron en E. coli BL21 (DE3), mientras que VP6 se expresó en células MA104 con virus vaccinia recombinante transfectada. La interacción entre el rotavirus y las proteínas celulares se estudió mediante ELISA, co-inmunoprecipitación y SDS-PAGE/ Western. Resultados. Las condiciones óptimas para la expresión de proteínas recombinantes se determinaron y se generaron anticuerpos contra ellas. Los resultados sugirieron que las proteínas virales rVP5* y rVP6 interactúan con Hsc70 y PDI in vitro. También se encontró que éstas proteínas virales recombinantes interactúan con Hsc70 en las balsas lipídicas ("Rafts") en un cultivo celular. El tratamiento de las células, ya sea con DLP o rVP6 produjo significativamente la inhibición de la infección por rotavirus. Conclusión. Los resultados permiten concluir que rVP5 * y rVP6 interactúan con Hsc70 y PDI durante el proceso de la infección por rotavirus.

Expression and significance of heat shock protein 70 in maternal serum, umbilical cord blood and placenta of patients with hypertensive disorders complicating pregnancy / 中华妇产科杂志

Objective To investigate the expression of heat shock protein 70 (HSP70) in maternal serum,umbilical cord blood and placenta of patients with hypertensive disorders complicating pregnancy (HDCP) and to discuss its role in the pathogenesis of HDCP.Methods Totally 90 patients with HDCP were recruited as HDCP group,and were devided into three subgroups,including gestational hypertension group (30 cases),mild preeclampsia group(30 cases) and severe preeclampsia group(30 cases).A totally of 30 cases of healthy pregnant women were defined as the control group.All of them were admitted to Xuzhou Maternity and Child Health Care Hospital from August 2011 to December 2012.ELISA was used to detect the expression of HSP70 in maternal serum and umbilical cord blood.Immunohistochemistry streptavidin peroxidase(SP) was used to detect the protein in placenta,and semi-quantitative reverse transcription (RT)-PCR was used to detect the expression of HSP70 mRNA.Results (1) The levels of HSP70 in maternal serum and cord blood of mild preeclampsia group were (2.61±0.98) and (0.78±0.27)μg/L,respectively; and were (3.10± 1.18) and (0.96±0.28)μg/L in severe preeclampsia group.The levels of HSP70 in mild and severe preeclampsia groups were significantly higher than those in the control group [(1.88±0.79) and (0.61±0.15) μg/L,respectively] and gestational hypertension group [(2.13 ± 0.71) and (0.64 ± 0.18) μg/L,respectively;P＜0.05].The level of HSP70 in severe preeclampsia group was significantly higher than that in mild preeclampsia group (P＜0.05).And the level of HSP70 in gestational hypertension group was higher than that in the control group,but there was no statistical difference (P＞0.05).(2)The positive rate of placental HSP70 in gestational hypertension group,mild and severe preeclampsia group [83％(25/30),90％ (27/30) and 100％(30/30)],respectively were significantly higher than those in the control group(43％,13/30;P＜0.05).The positive rate of placental HSP70 in severe preeclampsia group was significantly higher than that in gestational hypertension group and mild preeclampsia group(P＜0.05).(3)The expression of placental HSP70 mRNA in gestational hypertension group,mild and severe preeclampsia group (0.82±0.27,0.92± 0.26 and 1.36±0.29,respectively) were significantly higher than that in the control group (0.45±0.18),with statistically significant differences (P＜0.05).The expression of placental HSP70 mRNA in severe preeclampsia group was significantly higher than that in gestational hypertension group and mild preeclampsia group(P＜0.05).Conclusion The expression of HSP70 increased significantly in maternal serum,umbilical cord blood and placenta of patients with HDCP,and it had positive correlation with the severity of the disease,indicating that HSP70 may play a role in the pathogenesis of HDCP.

Interaction of Hsc73 with CXCR4 in nuclei of renal cell carcinoma A498 cells / 第二军医大学学报

Objective: To investigate the role of protein Hsc73 (heat shock cognate protein 73 000) in renal cell cancer metastasis promoted by SDF-1/CXCR4 axis, so as to determine whether Hsc73 participates in CXCR4 nuclear localization. Methods: Western blotting analysis was used to observe the expression of Hsc73 in A498 cells over-expressing CXCR4. The location of Hsc73 and the interaction of CXCR4 with Hsc73 were investigated in SDF-1-stimulated A498 cells by immunohistochemical staining, Co-IP (Co-Immunoprecipitation) experiment, etc. Results: Hsc73 was up-regulated in A498 cells over-expressing CXCR4. Hsc73 was mainly found in the cytoplasm of A498 cells; after stimulation with SDF-1, some Hsc73 appeared in the nuclei. Hsc73 protein was found in the nuclei of A498 cells after Co-IP with anti-CXCR4 antibody. Conclusion: Hsc73 as a common molecular chaperone participates in the intra-cellular translocation of CXCR4; Hsc73 also plays a key role in the activation of SDF-1/CXCR4 signal pathway and may be involved in the nuclear translocalization of CXCR4.

Identification of a Major Cytoplasmic Laminin Binding Protein as the Heat Shock Cognate Protein 70 in Highly Metastatic Human Colon Carcinoma Cells / 대한면역학회지

"It is not clear yet how the laminin is made in cytoplasm and is secreted by cells, though the significance of the interaction between laminin and cells during the development, cell differentiation, tumor growth, and metastasis is well known. Furthermore, it has been reported that aberrant expression. and secretion of laminin in malignant tumor cells, In this study I found a major cytoplasmic laminin binding protein (LBP), which was revealed as the heat shock cognate protein 70 (hsc70), in human colon carcinoma cells by cell surface labeling with '""I, laminin and gelatin affinity chromatography, peptide microsequencing, and immunoblot experiments. This suggests another function of hsc70 as the cytoplasmic LBP, which may play an important role in biosynthesis, assembly, and secretion of laminin in tumor cells."