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

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.

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。

热休克蛋白（Heat Shock Proteins，HSP）是一类分布广泛且高度保守的蛋白，是生物界经历长期进化保留下来的对细胞内外不良环境因素起保护作用的一类蛋白，在细胞应激（比如受热和氧化应激）过程中大量表达。其中，HSP70（也称HSP72或HSPA1B）是HSP中最保守和重要的一类分子伴侣蛋白。研究表明，HSP70的生物学功能非常多样，作为分子伴侣既参与新生肽链或未折叠蛋白的折叠，也能帮助变性蛋白通过蛋白酶体或自噬途径降解，以维持细胞内环境稳定。作为细胞中非常重要的一种保护性蛋白，HSP70自身蛋白的稳定对于上述功能至关重要。然而，HSP70蛋白稳定性的调控机制并不清楚。近几年有关HSP70的研究还表明它具有保护DNA的作用，但其中具体的分子机制仍不清楚。本研究利用分子靶向药物厄洛替尼或吉非替尼处理非小细胞肺癌临床相关细胞系HCC827细胞，观察到HSP70蛋白发生泛素化降解，并揭示其降解机制与药物处理导致HSP70第41位酪氨酸（Y41）的磷酸化被抑制有关，进一步实验证实HSP70 Y41的磷酸化水平与HSP70蛋白稳定性紧密相关。不仅如此，细胞中HSP70的蛋白水平和HSP70 Y41的磷酸化水平也影响了HCC827细胞中的基因突变率。通过深入分析HSP70与碱基切除修复（BER）途径的关系，我们发现HSP70与BER途径中的多种修复酶相互作用，并通过该蛋白互作促进BER的修复效率和修复准确性。因此，非小细胞肺癌细胞中的HSP70蛋白具有通过促进BER从而抑制基因突变的作用。本项目利用非小细胞肺癌临床相关的细胞系及其临床治疗的药物作为工具，发现EGFR-TKIs药物处理导致肿瘤细胞基因突变率增加，有大量研究表明EGFR基因的继发突变（比如EGFR T790M耐药突变）是造成肿瘤细胞产生耐药反应的主要原因之一，我们的研究成果揭示了产生这类基因突变的一种机制，为临床克服类似基因突变造成的耐药反应提供新的策略并奠定理论基础。