热休克蛋白70（HSP70）通过促进碱基切除修复抑制基因突变的作用机制研究

项目名称： 热休克蛋白70（HSP70）通过促进碱基切除修复抑制基因突变的作用机制研究

项目编号： No.31500628

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 生物科学

项目作者： 兰蕾

作者单位： 南京师范大学

项目金额： 20万元

中文摘要： HSP70是细胞内重要的分子伴侣蛋白，参与蛋白折叠和降解，以及碱基切除修复（BER），但HSP70自身的蛋白稳定性调控以及HSP70调节BER途径的机制仍不明确。我们发现：HSP70自身稳定性受其磷酸化修饰的影响；抗肿瘤药物厄洛替尼处理抑制了HSP70第41位酪氨酸（Y41）的磷酸化，引起HSP70降解并伴随BER反应效率下降，细胞内源基因突变率增加；过表达HSP70能抑制药物处理导致的基因突变率增加；HSP70分别与BER修复酶Pol β和FEN1结合，其结合量在DNA损伤剂处理后增加。本申请拟探讨药物处理导致HSP70蛋白降解的原因，分析HSP70 Y41磷酸化与HSP70蛋白稳定性的关系，观察HSP70与FEN1和Pol β的相互作用，检测HSP70对FEN1酶活和Pol β聚合酶保真性的促进作用。本研究将揭示HSP70蛋白稳定性与BER修复能力的联系，为上述药物临床应用提供参考。

中文关键词： 蛋白质；磷酸化；蛋白稳定性；碱基切除修复

英文摘要： Heat shock protein 70 (HSP70) is an important molecular chaperone which functions in the protein folding and degradation as well as in the base excision repair (BER), a DNA repair pathway to reduce DNA damage and gene mutation. However, it is largely unknown how HSP70 itself is degraded and the mechanism by which HSP70 modulates BER pathway. . We recently found that the protein stability of HSP70 was affected by its phosphorylation status. By Mass Spectrometry, we found that the treatment of Erlotinib, an antitumor drug clinically used for the treatment of Non-small cell lung cancer (NSCLC), caused the inhibtion of HSP70 phosphorylation at site of tyrosine 41 (Y41), the degradation of HSP70 protein, and the inhibiton of BER capacity, which probably lead to the increase of gene mutation frequency after the Erlotinib treatment. If HSP70 was overexpressed prior to the drug treament, the increase of gene mutation frequency was inhibited. By co-immunoprecipitation, we also found that HSP70 was associated with Pol β and FEN1, respectively. DNA damage regeant MMS induced the increased binding of HSP70 and FEN1. Based on these observations, we speculate that HSP70 can inhibit the gene mutation via promoting BER pathway. . In this study, we plan to investigate how the treatment of Erlotinib causes the degradation of HSP70 protein in NSCLC clinical relevant cell line HCC827 cells, and to determine the relationship between the HSP70 Y41 phosphorylation level and its protein stability. In addition, we also plan to detect the interactions of HSP70 with Pol β or with FEN1, and to analyze the effects of HSP70 on the FEN1 activity and the Pol β fidelity, in order to unveil the mechanism that HSP70 promotes the BER pathway. We believe this project will reveal the relationships between HSP70 protein stability and the BER capacity, in order to provide some clues for the therepeutic use of EGFR tyrosine kinase inhibitors.

英文关键词： protein;phosphorylation;protein stability;base excision repair