E3泛素连接酶PARK2通过单泛素化修饰RAD50促进同源重组修复的分子机制及功能研究

Homologous recombination repair is a major way to resolving DNA double strand break. Inhibiting Homologous recombination repair signal pathway can significantly enhance tumor cell sensitivity towards PARP inhibitor. So it is meaningful to improve therapy effect of PARP inhibitor by identifing the mechanism of homologous recombination repair. In the previous study, we found that:1) E3 ligase PARK2 can interact with the DNA damage sensor protein RAD50 and promote its mono-ubiquitination, which enhancing the recruitment of RAD50 to DNA damage sites and facilities the homologous recombination repair; 2) Knockdown of E3 ligase PARK2 in breast cancer cells significantly increased the sensitivity of PARP inhibitors; 3) TCGA data analysis revealed that high-amplitude PARK2 deletion was observed in 18.74% of breast cancer patients. Accordingly, we hypothesized that PARK2 deficiency inhibited the monou-biquitination of RAD50, which repressed the recruiment of RAD50 to DNA damage sites and impaired homologous recombination repair, thus caused hyper sensitive to PARP inhibitors, suggesting that the breast cancer patients with PARK2 deletion may benefit a great in PARP inhibitor treatment. In this project, we will investigate the effects of PARK2 in PARP inhibitor sensitivity and uncover the mechanism of PARK2 facilitating homologous recombination repair, and providing novel theoretical basis for PARP inhibitor targeted therapy in breast cancer.

同源重组修复是DNA双链断裂的主要修复方式之一。抑制肿瘤细胞的同源重组信号通路可增强其对PARP抑制剂的敏感性，因此研究同源重组修复的分子机制对于提高PARP抑制剂疗效具有重要意义。我们预实验发现：1）DNA双链断裂时，E3泛素连接酶PARK2促进损伤感应蛋白RAD50的单泛素化修饰，增强RAD50在DNA损伤处的募集，提高同源重组修复效率；2）敲低PARK2显著增强乳腺癌细胞对PARP抑制剂敏感性；3）TCGA数据显示18.74%乳腺癌患者的PARK2基因拷贝数高度缺失。据此，我们提出假设：敲低PARK2抑制RAD50的单泛素化修饰，减弱后者募集至DNA损伤处能力而使同源重组修复受阻，进而对PARP抑制剂敏感，这提示PARK2基因缺失的乳腺癌患者是PARP抑制剂潜在获益人群。本项目将深入研究PARK2参与同源重组修复机制及其与PARP抑制剂敏感性的关系，为PARP抑制剂合理使用提供依据。

DNA修复对于维持基因组稳定性具有十分重要的作用，本研究不仅发现PARK2具有调控DNA损伤修复及化疗敏感性的能力，我们还对损伤感应蛋白RAD50参与同源重组修复的分子机制有了新的认识。我们研究发现PARK2能与BCL-2相互作用，通过促进BCL-2的泛素化降解方式调控肿瘤线粒体凋亡的能力，而乳腺癌化疗相关药物一方面可通过抑制STAT3信号通路而促进PARK2的转录激活，另一方面激活了PARK2对抗凋亡分子Bcl-2识别，从而增强PARK2调控肿瘤细胞线粒体凋亡的能力。在同源重组修复过程中，RAD50是识别、传递、放大DNA损伤信号的关键分子，其在DNA损伤处的正确募集对于顺利完成同源重组修复必不可少。我们在研究过程中发现染色质重塑复合物PBAF亚基BRD7是DNA损伤感应蛋白RAD50的相互作用蛋白，进一步的深入研究发现，DNA双链断裂会引起BRD7第263位丝氨酸和515位苏氨酸被磷酸化修饰，这磷酸化修饰信号可招募RAD50及PRC2、NuRD复合物到DSBs参与DNA损伤应答和转录抑制，维持基因组稳定性，BRD7的表达量与乳腺癌PARPi敏感性密切相关。综上所述，我们不仅发现PARK2是预测乳腺癌化疗药物敏感性的新型分子标志物，还阐明了PBAF复合物亚基BRD7通过增强DNA损伤效应蛋白的募集，从而维持基因组完整性的新功能。

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