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Heat-shock cognate 70 is required for the activation of heat-shock factor 1 in mammalian cells.

ABSTRACT: HSF1 (heat-shock factor 1) plays an essential role in mediating the appropriate cellular response to diverse forms of physiological stresses. However, it is not clear how HSF1 is regulated by interacting proteins under normal and stressful conditions. In the present study, Hsc70 (heat-shock cognate 70) was identified as a HSF1-interacting protein using the TAP (tandem affinity purification) system and MS. HSF1 can interact with Hsc70 in vivo and directly in vitro. Interestingly, Hsc70 is required for the regulation of HSF1 during heat stress and subsequent target gene expression in mammalian cells. Moreover, cells transfected with siRNAs (small interfering RNAs) targeted to Hsc70 showed greatly decreased HSF1 activation with expression of HSF1 target genes being dramatically reduced. Finally, loss of Hsc70 expression in cells resulted in an increase in stress-induced apoptosis. These results indicate that Hsc70 is a necessary and critical regulator of HSF1 activities.

SUBMITTER: Ahn SG

PROVIDER: S-EPMC1317673 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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Heat-shock cognate 70 is required for the activation of heat-shock factor 1 in mammalian cells.

Ahn Sang-Gun SG Kim Soo-A SA Yoon Jung-Hoon JH Vacratsis Panayiotis P

The Biochemical journal 20051101 Pt 1

HSF1 (heat-shock factor 1) plays an essential role in mediating the appropriate cellular response to diverse forms of physiological stresses. However, it is not clear how HSF1 is regulated by interacting proteins under normal and stressful conditions. In the present study, Hsc70 (heat-shock cognate 70) was identified as a HSF1-interacting protein using the TAP (tandem affinity purification) system and MS. HSF1 can interact with Hsc70 in vivo and directly in vitro. Interestingly, Hsc70 is require ...[more]

PMID: 16050811

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Project description:BackgroundHepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Because of the limited effectiveness of existing vaccines and drugs, development of novel antiviral strategies is urgently needed. Heat stress cognate 70 (Hsc70) is an ATP-binding protein of the heat stress protein 70 family. Hsc70 has been found to be required for HBV DNA replication. Here we report, for the first time, that combined siRNAs targeting viral gene and siHsc70 are highly effective in suppressing ongoing HBV expression and replication.MethodsWe constructed two plasmids (S1 and S2) expressing short hairpin RNAs (shRNAs) targeting surface open reading frame of HBV(HBVS) and one plasmid expressing shRNA targeting Hsc70 (siHsc70), and we used the EGFP-specific siRNA plasmid (siEGFP) as we had previously described. First, we evaluated the gene-silencing efficacy of both shRNAs using an enhanced green fluorescent protein (EGFP) reporter system and flow cytometry in HEK293 and T98G cells. Then, the antiviral potencies of HBV-specific siRNA (siHBV) in combination with siHsc70 in HepG2.2.15 cells were investigated. Moreover, type I IFN and TNF-α induction were measured by quantitative real-time PCR and ELISA.ResultsCotransfection of either S1 or S2 with an EGFP plasmid produced an 80%-90% reduction in EGFP signal relative to the control. This combinational RNAi effectively and specifically inhibited HBV protein, mRNA and HBV DNA, resulting in up to a 3.36 log10 reduction in HBV load in the HepG2.2.15 cell culture supernatants. The combined siRNAs were more potent than siHBV or siHsc70 used separately, and this approach can enhance potency in suppressing ongoing viral gene expression and replication in HepG2.2.15 cells while forestalling escape by mutant HBV. The antiviral synergy of siHBV used in combination with siHsc70 produced no cytotoxicity and induced no production of IFN-α, IFN-β and TNF-α in transfected cells.ConclusionsOur combinational RNAi was sequence-specific, effective against wild-type and mutant drug-resistant HBV strains, without triggering interferon response or producing any side effects. These findings indicate that combinational RNAi has tremendous promise for developing innovative therapy against viral infection.

Dataset Information

Heat-shock cognate 70 is required for the activation of heat-shock factor 1 in mammalian cells.

Publications

Heat-shock cognate 70 is required for the activation of heat-shock factor 1 in mammalian cells.

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