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Hiroshi Takaku - One of the best experts on this subject based on the ideXlab platform.
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induction of heat shock Protein 70 by prostaglandin a1 inhibits hiv 1 vif mediated degradation of apobec3g
Antiviral Research, 2013Co-Authors: Ryuichi Sugiyama, Makoto Abe, Hironori Nishitsuji, Yuko Murakami, Hiroaki Takeuchi, Hiroshi TakakuAbstract:Abstract Previous studies have demonstrated that cyclopentenone prostaglandins (cyPGs) inhibit human immunodeficiency virus type 1 (HIV-1) replication in various cell types. This antiviral activity has been associated with the induction of Heat-Shock Protein 70 (HSP70) in infected cells. We investigated a new role of prostaglandin A 1 (PGA 1 ) in the replication of HIV-1 in non-permissive cells. Because overexpression of HSP70 blocks the viral infectivity factor (Vif)-mediated degradation of APOBEC3G (A3G) via the ubiquitin–proteasome pathway, we examined the effects of PGA 1 on A3G and HIV-1 replication. The induction of HSP70 synthesis by PGA 1 blocked Vif-mediated A3G degradation and enhanced the incorporation of A3G into both wild-type and Vif-deficient viruses. Furthermore, we determined the viral titer of HIV-1 particles produced from PGA 1 -treated 293T cells. The induction of HSP70 synthesis by PGA 1 significantly reduced the viral titer in the presence of A3G. Additionally, the p24 Gag antigen levels were dramatically reduced in non-permissive cells treated once or repeatedly with PGA 1 . Thus, we showed that PGA 1 inhibits HIV-1 replication, at least in part, by blocking Vif-mediated A3G degradation.
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heat shock Protein 70 inhibits hiv 1 vif mediated ubiquitination and degradation of apobec3g
Journal of Biological Chemistry, 2011Co-Authors: Ryuichi Sugiyama, Hironori Nishitsuji, Hiroaki Takeuchi, Ayako Furukawa, Masato Katahira, Yuichiro Habu, Akihide Ryo, Hiroshi TakakuAbstract:The cytidine deaminase APOBEC3G, which is incorporated into nascent virus particles, possesses potent antiviral activity and restricts Vif-deficient HIV-1 replication at the reverse transcription step through deamination-dependent and -independent effects. HIV-1 Vif counteracts the antiviral activity of APOBEC3G by inducing APOBEC3G polyubiquitination and its subsequent proteasomal degradation. In this study, we show that overexpression of heat shock Protein 70 (HSP70) blocked the degradation of APOBEC3G in the ubiquitin-proteasome pathway by HIV-1 Vif, rendering the viral particles non-infectious. In addition, siRNA targeted knock-down of HSP70 expression enhanced the Vif-mediated degradation of APOBEC3G. A co-immunoprecipitation study revealed that overexpression of HSP70 inhibited APOBEC3G binding to HIV-1 Vif. Thus, we provide evidence for a host Protein-mediated suppression of HIV-1 replication in an APOBEC3G-dependent manner.
Ryuichi Sugiyama - One of the best experts on this subject based on the ideXlab platform.
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induction of heat shock Protein 70 by prostaglandin a1 inhibits hiv 1 vif mediated degradation of apobec3g
Antiviral Research, 2013Co-Authors: Ryuichi Sugiyama, Makoto Abe, Hironori Nishitsuji, Yuko Murakami, Hiroaki Takeuchi, Hiroshi TakakuAbstract:Abstract Previous studies have demonstrated that cyclopentenone prostaglandins (cyPGs) inhibit human immunodeficiency virus type 1 (HIV-1) replication in various cell types. This antiviral activity has been associated with the induction of Heat-Shock Protein 70 (HSP70) in infected cells. We investigated a new role of prostaglandin A 1 (PGA 1 ) in the replication of HIV-1 in non-permissive cells. Because overexpression of HSP70 blocks the viral infectivity factor (Vif)-mediated degradation of APOBEC3G (A3G) via the ubiquitin–proteasome pathway, we examined the effects of PGA 1 on A3G and HIV-1 replication. The induction of HSP70 synthesis by PGA 1 blocked Vif-mediated A3G degradation and enhanced the incorporation of A3G into both wild-type and Vif-deficient viruses. Furthermore, we determined the viral titer of HIV-1 particles produced from PGA 1 -treated 293T cells. The induction of HSP70 synthesis by PGA 1 significantly reduced the viral titer in the presence of A3G. Additionally, the p24 Gag antigen levels were dramatically reduced in non-permissive cells treated once or repeatedly with PGA 1 . Thus, we showed that PGA 1 inhibits HIV-1 replication, at least in part, by blocking Vif-mediated A3G degradation.
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heat shock Protein 70 inhibits hiv 1 vif mediated ubiquitination and degradation of apobec3g
Journal of Biological Chemistry, 2011Co-Authors: Ryuichi Sugiyama, Hironori Nishitsuji, Hiroaki Takeuchi, Ayako Furukawa, Masato Katahira, Yuichiro Habu, Akihide Ryo, Hiroshi TakakuAbstract:The cytidine deaminase APOBEC3G, which is incorporated into nascent virus particles, possesses potent antiviral activity and restricts Vif-deficient HIV-1 replication at the reverse transcription step through deamination-dependent and -independent effects. HIV-1 Vif counteracts the antiviral activity of APOBEC3G by inducing APOBEC3G polyubiquitination and its subsequent proteasomal degradation. In this study, we show that overexpression of heat shock Protein 70 (HSP70) blocked the degradation of APOBEC3G in the ubiquitin-proteasome pathway by HIV-1 Vif, rendering the viral particles non-infectious. In addition, siRNA targeted knock-down of HSP70 expression enhanced the Vif-mediated degradation of APOBEC3G. A co-immunoprecipitation study revealed that overexpression of HSP70 inhibited APOBEC3G binding to HIV-1 Vif. Thus, we provide evidence for a host Protein-mediated suppression of HIV-1 replication in an APOBEC3G-dependent manner.
Marja Jäättelä - One of the best experts on this subject based on the ideXlab platform.
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Human heat shock Protein 70 (Hsp70) as a peripheral membrane Protein
Biochimica et Biophysica Acta, 2014Co-Authors: Ajay K. Mahalka, Marja Jäättelä, Thomas Kirkegaard, Laura T.i. Jukola, Paavo K.j. KinnunenAbstract:Abstract While a significant fraction of heat shock Protein 70 (Hsp70) is membrane associated in lysosomes, mitochondria, and the outer surface of cancer cells, the mechanisms of interaction have remained elusive, with no conclusive demonstration of a Protein receptor. Hsp70 contains two Trps, W90 and W580, in its N-terminal nucleotide binding domain (NBD), and the C-terminal substrate binding domain (SBD), respectively. Our fluorescence spectroscopy study using Hsp70 and its W90F and W580F mutants, and Hsp70-∆SBD and Hsp70-∆NBD constructs, revealed that binding to liposomes depends on their lipid composition and involves both NBD and SBD. Association of Hsp70 with phosphatidylcholine (PC) liposomes is weak, with insertion of its Trps into the bilayer hydrocarbon region. In the presence of cardiolipin (CL), bis-monoacylglycero phosphate (BMP), or phosphatidylserine (PS) Hsp70 attaches to membranes peripherally, without penetration. Our data suggest that the organelle distribution of Hsp70 is determined by their specific lipid compositions, with Hsp70 associating with the above lipids in mitochondria, lysosomes, and the surface of cancer cells, respectively. NBD and SBD attach to lipids by extended phospholipid anchorage, with specific acidic phospholipids associating with Hsp70 in the extended conformation with acyl chains inserting into hydrophobic crevices within Hsp70, and other chains remaining in the bilayer. This anchorage is expected to cause a stringent orientation of Hsp70 on the surface. Our data further suggest that acidic phospholipids induce a transition of SBD into the molten globule state, which may be essential to allow SBD–substrate interaction also within the hydrophobic bilayer interior acyl chain region.
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the heat shock Protein 70 family highly homologous Proteins with overlapping and distinct functions
FEBS Letters, 2007Co-Authors: Mads Daugaard, Mikkel Rohde, Marja JäätteläAbstract:The human heat shock Protein 70 (Hsp70) family contains at least eight homologous chaperone Proteins. Endoplasmatic reticulum and mitochondria have their specific Hsp70 Proteins, whereas the remaining six family members reside mainly in the cytosol and nucleus. The requirement for multiple highly homologous although different Hsp70 Proteins is still far from clear, but their individual and tissue-specific expression suggests that they are assigned distinct biological tasks. This concept is supported by the fact that mice knockout for different Hsp70 genes display remarkably discrete phenotypes. Moreover, emerging data suggest that individual Hsp70 Proteins can bring about non-overlapping and chaperone-independent functions essential for growth and survival of cancer cells. This review summarizes our present knowledge of the individual members of human Hsp70 family and elaborate on the functional differences between the cytosolic/nuclear representatives.
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members of the heat shock Protein 70 family promote cancer cell growth by distinct mechanisms
Genes & Development, 2005Co-Authors: Mikkel Rohde, Mads Daugaard, Jesper Nylandsted, Mette Hartvig Jensen, Kristian Helin, Marja JäätteläAbstract:Whereas the stress-inducible Heat-Shock Protein 70 (Hsp70) has gained plenty of attention as a putative target for tumor therapy, little is known about the role of other Hsp70 Proteins in cancer. Here we present the first thorough analysis of the expression and function of the cytosolic Hsp70 Proteins in human cancer cells and identify Hsp70-2, a Protein essential for spermatogenesis, as an important regulator of cancer cell growth. Targeted knock-down of the individual family members by RNA interference revealed that both Hsp70 and Hsp70-2 were required for cancer cell growth, whereas the survival of tumorigenic as well as nontumorigenic cells depended on Hsc70. Cancer cells depleted for Hsp70 and Hsp70-2 displayed strikingly different morphologies (detached and round vs. flat senescent-like), cell cycle distributions (G2/M vs. G1 arrest) and gene expression profiles. Only Hsp70-2 depletion induced the expression of macrophage inhibitory cytokine-1 that was identified as a target of P53 tumor-suppressor Protein and a mediator of the G1 arrest and the senescent phenotype. Importantly, concomitant depletion of Hsp70 and Hsp70-2 had a synergistic antiproliferative effect on cancer cells. Thus, highly homologous Hsp70 Proteins bring about nonoverlapping functions essential for cell growth and survival.
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heat shock Protein 70 promotes cell survival by inhibiting lysosomal membrane permeabilization
Journal of Experimental Medicine, 2004Co-Authors: Jesper Nylandsted, Gabriele Multhoff, Mikkel Rohde, Mads Gyrdhansen, Agnieszka Danielewicz, Nicole Fehrenbacher, Ulrik Lademann, Maria Hoyerhansen, Ekkehard Weber, Marja JäätteläAbstract:Heat shock Protein 70 (Hsp70) is a potent survival Protein whose depletion triggers massive caspase-independent tumor cell death. Here, we show that Hsp70 exerts its prosurvival function by inhibiting lysosomal membrane permeabilization. The cell death induced by Hsp70 depletion was preceded by the release of lysosomal enzymes into the cytosol and inhibited by pharmacological inhibitors of lysosomal cysteine proteases. Accordingly, the Hsp70-mediated protection against various death stimuli in Hsp70-expressing human tumor cells as well as in immortalized Hsp70 transgenic murine fibroblasts occurred at the level of the lysosomal permeabilization. On the contrary, Hsp70 failed to inhibit the cytochrome c-induced, apoptosome-dependent caspase activation in vitro and Fas ligand-induced, caspase-dependent apoptosis in immortalized fibroblasts. Immunoelectron microscopy revealed that endosomal and lysosomal membranes of tumor cells contained Hsp70. Permeabilization of purified endo/lysosomes by digitonin failed to release Hsp70, suggesting that it is physically associated with the membranes. Finally, Hsp70 positive lysosomes displayed increased size and resistance against chemical and physical membrane destabilization. These data identify Hsp70 as the first survival Protein that functions by inhibiting the death-associated permeabilization of lysosomes.
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Heat-Shock Protein 70 antagonizes apoptosis-inducing factor
Nature Cell Biology, 2001Co-Authors: Luigi Ravagnan, Santos A Susin, Sandeep Gurbuxani, Marja Jäättelä, Carine Maisse-paradisi, Eric Daugas, Naoufal Zamzami, Tak Mak, Josef M. Penninger, Carmen GarridoAbstract:Heat-Shock Protein 70 (Hsp70) has been reported to block apoptosis by binding apoptosis protease activating factor-1 (Apaf-1), thereby preventing constitution of the apoptosome, the Apaf-1/cytochrome c/caspase-9 activation complex [1,2]. Here we show that overexpression of Hsp70 protects Apaf-1-/- cells against death induced by serum withdrawal, indicating that Apaf-1 is not the only target of the anti-apoptotic action of Hsp70. We investigated the effect of Hsp70 on apoptosis mediated by the caspase-independent death effector apoptosis inducing factor (AIF), which is a mitochondrial intermembrane flavoProtein [3,4]. In a cell-free system, Hsp70 prevented the AIF-induced chromatin condensation of purified nuclei. Hsp70 specifically interacted with AIF, as shown by ligand blots and co-immunoprecipitation. Cells overexpressing Hsp70 were protected against the apoptogenic effects of AIF targeted to the extramitochondrial compartment. In contrast, an anti-sense Hsp70 complementary DNA, which reduced the expression of endogenous Hsp70, increased sensitivity to the lethal effect of AIF. The ATP-binding domain of Hsp70 seemed to be dispensable for inhibiting cell death induced by serum withdrawal, AIF binding and AIF inhibition, although it was required for Apaf-1 binding. Together, our data indicate that Hsp70 can inhibit apoptosis by interfering with target Proteins other than Apaf-1, one of which is AIF.
Jun Ho Chung - One of the best experts on this subject based on the ideXlab platform.
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a teratoproteomics analysis heat shock Protein 70 is upregulated in mouse forelimb bud by methoxyacetic acid treatment
Birth Defects Research Part A-clinical and Molecular Teratology, 2005Co-Authors: Aceng Ruyani, Sri Sudarwati, Lien A. Sutasurya, Sony H. Sumarsono, Dong Jo Kim, Jun Ho ChungAbstract:Methoxyacetic acid (MAA) causes fetal limb abnormalities when the substance is administrated on gestation day (GD) 11 in mice. Limb abnormalities are caused mainly by extensive cell death in the mesoderm of the limb plate. This investigation focused on identifying a Protein that is linked with mouse limb teratogenicity.A single dose of MAA at 10 mmol/kg body weight was administered by gavage on GD 11; controls were administered vehicle only. Dams were killed by cervical dislocation 4 hr after treatment and forelimb buds were isolated from both the control and treated embryos. Proteins in forelimb buds GD 11 + 4 hr were precipitated out using 40-60% ammonium sulfate and were then analyzed by 2D SDS-PAGE. Excised Protein spots were identified by mass spectrometry and amino acid internal sequence analysis. Identified Protein was further confirmed by Western blotting.Two-dimensional gel analysis indicated that 1 Protein spot of 81.7 kDa/pI 7.3 was overexpressed, and the Protein matched heat shock Protein 70 (HSP70; accession no. P08109, SwissProt).The results suggest that MAA, when administered to pregnant mice, upregulates HSP70 in the forelimb buds.
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A teratoproteomics analysis: heat shock Protein 70 is upregulated in mouse forelimb bud by methoxyacetic acid treatment
'Wiley', 2005Co-Authors: Ruyani Aceng, Sudarwati Sri, Sutasurya, Lien A., Sumarsono, Sony H., Kim Dong-jo, Jun Ho ChungAbstract:BACKGROUND: Methoxyacetic acid (MAA) causes fetal limb abnormalities when the substance is administrated on gestation day (GD) 11 in mice. Limb abnormalities are caused mainly by extensive cell death in the mesoderm of the limb plate. This investigation focused on identifying a Protein that is linked with mouse limb teratogenicity. METHODS: A single dose of MAA at 10 mmol/kg body weight was administered by gavage on GD 11; controls were administered vehicle only. Dams were killed by cervical dislocation 4 hr after treatment and forelimb buds were isolated from both the control and treated embryos. Proteins in forelimb buds GD 11 + 4 hr were precipitated out using 40-60% ammonium sulfate and were then analyzed by 2D SDS-PAGE. Excised Protein spots were identified by mass spectrometry and amino acid internal sequence analysis. Identified Protein was further confirmed by Western blotting. RESULTS: Two-dimensional gel analysis indicated that 1 Protein spot of 81.7 kDa/pI 7.3 was overexpressed, and the Protein matched heat shock Protein 70 (HSP70; accession no. P08109, SwissProt). CONCLUSIONS: The results suggest that MAA, when administered to pregnant mice, upregulates HSP70 in the forelimb buds
Cristina C Clement - One of the best experts on this subject based on the ideXlab platform.
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heat shock Protein 70 inhibitors 1 2 5 thiodipyrimidine and 5 phenylthio pyrimidine acrylamides as irreversible binders to an allosteric site on heat shock Protein 70
Journal of Medicinal Chemistry, 2014Co-Authors: Yanlong Kang, Tony Taldone, Hardik J Patel, Pallav D Patel, Anna Rodina, Alexander Gozman, Ronnie Maharaj, Cristina C Clement, Maulik R Patel, Jeffrey L BrodskyAbstract:Heat shock Protein 70 (Hsp70) is an important emerging cancer target whose inhibition may affect multiple cancer-associated signaling pathways and, moreover, result in significant cancer cell apoptosis. Despite considerable interest from both academia and pharmaceutical companies in the discovery and development of druglike Hsp70 inhibitors, little success has been reported so far. Here we describe structure-activity relationship studies in the first rationally designed Hsp70 inhibitor class that binds to a novel allosteric pocket located in the N-terminal domain of the Protein. These 2,5'-thiodipyrimidine and 5-(phenylthio)pyrimidine acrylamides take advantage of an active cysteine embedded in the allosteric pocket to act as covalent Protein modifiers upon binding. The study identifies derivatives 17a and 20a, which selectively bind to Hsp70 in cancer cells. Addition of high nanomolar to low micromolar concentrations of these inhibitors to cancer cells leads to a reduction in the steady-state levels of Hsp70-sheltered oncoProteins, an effect associated with inhibition of cancer cell growth and apoptosis. In summary, the described scaffolds represent a viable starting point for the development of druglike Hsp70 inhibitors as novel anticancer therapeutics.
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heat shock Protein 70 inhibitors 2 2 5 thiodipyrimidines 5 phenylthio pyrimidines 2 pyridin 3 ylthio pyrimidines and 3 phenylthio pyridines as reversible binders to an allosteric site on heat shock Protein 70
Journal of Medicinal Chemistry, 2014Co-Authors: Tony Taldone, Yanlong Kang, Hardik J Patel, Pallav D Patel, Anna Rodina, Alexander Gozman, Ronnie Maharaj, Maulik R Patel, Y C Patel, Cristina C ClementAbstract:The discovery and development of heat shock Protein 70 (Hsp70) inhibitors is currently a hot topic in cancer. In the preceding paper in this issue (10.1021/jm401551n), we have described structure–activity relationship studies in the first Hsp70 inhibitor class rationally designed to bind to a novel allosteric pocket located in the N-terminal domain of the Protein. These ligands contained an acrylamide to take advantage of an active cysteine embedded in the allosteric pocket and acted as covalent Protein modifiers upon binding. Here, we perform chemical modifications around the irreversible inhibitor scaffold to demonstrate that covalent modification is not a requirement for activity within this class of compounds. The study identifies derivative 27c, which mimics the biological effects of the irreversible inhibitors at comparable concentrations. Collectively, the back-to-back manuscripts describe the first pharmacophores that favorably and selectively interact with a never explored pocket in Hsp70 and pro...
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design of a flexible cell based assay for the evaluation of heat shock Protein 70 expression modulators
Assay and Drug Development Technologies, 2011Co-Authors: James H Ahn, Yanlong Kang, Anna Rodina, Ronnie Maharaj, Cristina C Clement, Wenjie Luo, Joungnam Kim, Julia Aguirre, Weilin Sun, Kamalika MoulickAbstract:Heat shock Protein 70 (Hsp70) is a chaperone Protein that helps protect against cellular stress, a function that may be co-opted to fight human diseases. In particular, the upregulation of Hsp70 can suppress the neurotoxicity of misfolded Proteins, suggesting possible therapeutic strategies in neurodegenerative diseases. Alternatively, in cancer cells where high levels of Hsp70 inhibit both intrinsic and extrinsic apoptotic pathways, a reduction in Hsp70 levels may induce apoptosis. To evaluate and identify, in a single assay format, small molecules that induce or inhibit endogenous Hsp70, we have designed and optimized a microtiter assay that relies on whole-cell immunodetection of Hsp70. The assay utilizes a minimal number of neuronal or cancer cells, yet is sufficiently sensitive and reproducible to permit quantitative determinations. We further validated the assay using a panel of Hsp70 modulators. In conclusion, we have developed an assay that is fast, robust, and cost efficient. As such, it can be implemented in most research laboratories. The assay should greatly improve the speed at which novel Hsp70 inducers and inhibitors of expression can be identified and evaluated.