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Heat shock cognate 70 gene in Haliotis diversicolor: responses to pathogen infection and environmental stresses and its transcriptional regulation analysis.

ABSTRACT: Heat shock cognate 70 (HSC70) is a class of highly conserved proteins which functions as a molecular chaperon, participates in tolerance processes, and is involved in protein folding, degradation, targeting, translocation, and protein complex remodeling. In this study, the mRNA expression level of the Haliotis diversicolor HSC70 (HdHSC70) gene was detected by quantitative real-time PCR in different tissues and under different stresses. The results showed that the HdHSC70 gene was ubiquitously expressed in seven selected tissues. The highest expression level was detected in gills (P < 0.05). The expression level of the HdHSC70 gene was significantly upregulated by thermal stress, hypoxia stress, Vibrio parahaemolyticus infection, and combined thermal and hypoxia stress. The upregulation occurred at the early stage of stress. These results indicated that the HdHSC70 is an important component in the immune system of H. diversicolor and is involved in the early stress response. Meanwhile, 5'-flanking region sequence (2013 bp) of the HdHSC70 gene was cloned; it contains a putative core promoter region, heat shock element, CpG, and transcription elements including NF-1, Sp1, Oct-1, interferon consensus sequence binding protein (ICSBP), etc. In HEK 293T cells, the 5'-flanking region sequence is able to drive expression of the enhanced green fluorescent protein (EGFP), proving its promoter function. The promoter activity increased after high-temperature treatment, which may be the immediate reason why the expression of the HdHSC70 gene was significantly upregulated by thermal stress. After the ICSBP-binding site was mutated, we found the luciferase activity significantly reduced, which suggested that the ICSBP-binding site has a certain enhancement effect on the activity of the HdHSC70 promoter.

SUBMITTER: Li Y

PROVIDER: S-EPMC5904075 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Heat shock cognate 70 gene in Haliotis diversicolor: responses to pathogen infection and environmental stresses and its transcriptional regulation analysis.

Li Yuting Y Zhang Tao T Zhang Xin X Wang Guodong G Wang Yilei Y Zhang Ziping Z

Cell stress & chaperones 20170923 3

Heat shock cognate 70 (HSC70) is a class of highly conserved proteins which functions as a molecular chaperon, participates in tolerance processes, and is involved in protein folding, degradation, targeting, translocation, and protein complex remodeling. In this study, the mRNA expression level of the Haliotis diversicolor HSC70 (HdHSC70) gene was detected by quantitative real-time PCR in different tissues and under different stresses. The results showed that the HdHSC70 gene was ubiquitously ex ...[more]

PMID: 28939948

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Project description:BackgroundThe effects of diverse stresses ultimately alter the structures and functions of proteins. As molecular chaperones, heat shock proteins (HSPs) are a group of highly conserved proteins that help in the refolding of misfolded proteins and the elimination of irreversibly damaged proteins. They are mediated by a family of transcription factors called heat shock factors (HSFs). The small abalone Haliotis diversicolor is a species naturally distributed along the southern coast of China. In this study, the expression of HdHSF1 was inhibited by RNAi in hemocytes in order to further elucidate the regulatory roles of HdHSF1 on heat shock responsive genes in abalone. Meanwhile, to understand the transcriptional regulation of the HdHSF1 gene, the 5'-upstream regulatory region of HdHSF1 was characterized, and the relative promoter activity was examined by dual-luciferase reporter gene assay system in HEK293T cell lines.ResultsAfter the inhibition of the H. diversicolor HSF1 gene (HdHSF1) by dsRNA (double-stranded RNA), the expression of most heat shock related-genes was down-regulated (p < 0.05). It indicated the importance of HdHSF1 in the heat shock response of H. diversicolor. Meanwhile, 5'-flanking region sequence (2633 bp) of the HdHSF1 gene was cloned; it contained a putative core promoter region, TATA box, CAAT box, CpG island, and many transcription elements. In HEK293T cells, the 5'-flanking region sequence can drive expression of the enhanced green fluorescent protein (EGFP), proving its promoter function. Exposure of cells to the high-temperature (39 °C and 42 °C) resulted in the activation of HdHSF1 promoter activity, which may explain why the expression of the HdHSF1 gene participates in heat shock response. Luciferase activity of different recombinant plasmids, which contained different truncated promoter fragments of the HdHSF1 gene in HEK293T cells, revealed the possible active regions of the promoter. To further identify the binding site of the critical transcription factor in the region, an expression vector with the site-directed mutation was constructed. After being mutated on the GATA-1 binding site, we found that the luciferase activity was significantly increased, which suggested that the GATA-1 binding site has a certain weakening effect on the activity of the HdHSF1 promoter.ConclusionsThese findings suggest that GATA-1 may be one of the transcription factors of HdHSF1, and a possible signaling pathway mediated by HdHSF1 may exist in H. diversicolor to counteract the adverse effects of heat shock stress.

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 gene in Haliotis diversicolor: responses to pathogen infection and environmental stresses and its transcriptional regulation analysis.

Publications

Heat shock cognate 70 gene in Haliotis diversicolor: responses to pathogen infection and environmental stresses and its transcriptional regulation analysis.

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