心脏高表达基因hhole通过ERK结合位点抑制心肌肥厚信号分子ANF的转录活性

Heart high-expressing gene hhole regulated the transcriptional activity of cardiac hypertrophic signaling molecule ANF by its ERK binding site

目的探讨心脏高表达基因hhole调控心肌肥厚信号分子ANF的转录活性的分子机制。方法利用生物信息学在线软件分析hhole蛋白的分子结构。利用酶切法和环状诱变技术突变得到hhole基因的突变体重组质粒。将突变体和野生型的重组质粒分别转染HEK293细胞系,测定荧光素酶报告基因ANF的转录活性。结果生物信息学分析发现hhole蛋白含有ERK结合位点和多聚脯氨酸序列。利用酶切法构建了4个截短突变体重组质粒,环状质粒定点诱变的方法构建了3个定点突变的突变体。荧光报告系统分析发现pCMV-tag2B-TD与pCMV-tag2B-hhole全长均能强烈地抑制ANF的转录活性。结论hhole蛋白主要通过其ERK结合位点抑制心肌肥厚信号分子ANF的转录活性。

Aim To study the molecular mechanism of heart high-expressing gene hhole on regulating the transcriptional activity of cardiac hypertrophic signaling molecule ANF.Methods Bioinformatics online software was used to analyse the molecular structure of hhole protein.Several mutants of the recombinant plasmid pCMV-tag2 B-hhole were constructed by restriction digestion and site-directed mutagenesis.The mutant and wild-type recombinant plasmids were transfected into HEK293 cell line respectively to determine the transcriptional activity of luciferase reporter gene ANF.Results The bioinformatics analysis reveals that hhole protein contains a binding site of ERK(D-domain)and proline-rich SH3 binding motifs.Four mutants of the recombinant plasmid pCMV-tag2 B-hhole have been constructed by restriction digestion and three mutants have been constructed by site-directed mutagenesis.The report assay showed that recombinant plasmids pCMV-tag2 B-TD and pCMV-tag2 B-hhole could suppress strongly the transcriptional activities of ANF.Conclusion The binding site of ERK-D-domain of hhole protein played a crucial role in inhibiting the transcriptional activities of hypertrophic signal molecule ANF.