DNA-PKcs蛋白翻译后修饰的相互调节及在细胞放射损伤反应中的功能机制研究

Upon ionizing radiation, the pathways of DNA damage signaling and repairs are majorintrinsic mechanisms of determining the fate of injured tissues cells and the performance of radiation health effects, and maintaining the genomic stability. The available research knowledge and our previous research results demonstrated that the DNA-PKcs is an important player in multiple points throughthe whole pathways of DNA DSB damage signaling, repair and cellular responses. This project will focus on the DNA-PKcs and the interactive regulation effects of the post-translation modifications inDNA-PKcs molecules. The following key points will be studied upon to the stress of DNA damage induced by ionizing radiation: the effect and patterns of post-translation modifications occurred in DNA-PKcs, and interactive effect among different modifications; The effect of PARylation (PARsltion) on the phosphorylation at different sites of DNA-PKcs, and also on the kinase activity. The sumoylation of TIP60 and its effect on the autophosphorylation and function of DNA-PKcs via acetylation modification; the role and mechanism of post-translation modificationson the function of DNA-PKcs involved on the choice of HR and NHEJ pathways of DNA double-strand break repair; the signal pathways of DNA-PKcs on regulating G2/M checkpoint and mitotic progression in response to irradiation. The frontier theory and knowledge achieved in this project will provide valuable guidance for the development of Innovative measures or drugs to protect and cure the acute radiation tissue damage, and prevent the harmful health effects of ionizing radiation such as carcinogenesis.

DNA损伤信号响应与修复是决定组织细胞放射损伤后命运、基因组稳定性和整体放射生物效应的主要功能机制。已有信息和我们前期研究表明，DNA-PKcs是贯穿细胞DNA损伤应答全过程多环节的关键调节分子，其自身多个位点也发生多种翻译后修饰。本项目以DNA-PKcs为核心，以蛋白翻译后修饰相互调节为重点，深入如下研究：在放射致DNA损伤下，DNA-PKcs发生PAR化、磷酸化、乙酰化等修饰的效应规律，不同类型翻译后修饰之间的相互调节作用；PAR化修饰机制及对DNA-PKcs不同位点磷酸化、乙酰化修饰和激酶活性的影响；TIP60 SUMO化修饰及通过乙酰化介导对DNA-PKcs自磷酸化和功能的调节作用；翻译后修饰对DNA-PKcs在DNA双链断裂HR和NHEJ修复通路的选择、细胞G2/M阻滞和有丝分裂进程的协同调节和作用机制。本项目将为急性放射组织损伤防治、有害健康效应防护的创新措施研究提供前沿理论。

DNA损伤信号响应与修复是机体决定组织细胞放射损伤后命运、基因组稳定性和整体放射生物效应的主要功能机制。DNA-PKcs是贯穿细胞DNA损伤 应答全过程多环节的关键调节分子，其自身多个位点也发生多种翻译后修饰。本项目以DNA-PKcs为核心，以蛋白翻译后修饰相互调节为重点，研究内容主要有：在放射致DNA损伤下，DNA-PKcs发生PAR化、磷酸化、乙酰化等修饰的效应规律，不同类型翻译后修饰之间的 相互调节作用；PAR化修饰机制及对DNA-PKcs不同位点磷酸化、乙酰化修饰和激酶活性的影响；TIP60 SUMO化修饰及通过乙酰化介导对DNA-PKcs自磷酸化和功能的调节作用；翻译后修饰对DNA-PKcs在DNA双链断裂HR和NHEJ修复通路的选择、细胞G2/M阻滞和有丝分裂进 程的协同调节和作用机制。通过本研究，确定了在细胞电离辐射损伤反应过程中，DNA-PKcs蛋白发生PAR、乙酰化和磷酸化等翻译后修饰的主要特征规律；揭示了DNA-PKcs及其翻译后修饰调控DNA双链断裂修复忠实性的新机制；拓展研究了靶向激活DNA-PKcs的急性放射损伤防治新技术措施。取得的重要结果：① 揭示了TIP60与DNA-PKcs相互作用，促进后者S2056位点磷酸化激活，而在S期细胞中TIP60蛋白K430位点发生SUMO化修饰，其与DNA-PKcs相互作用被解除，DNA-PKcs失活，从而促进了DNA双链断裂的同源重组修复；② DNA-PKcs通过BRX1/Cullin1 SCF E3连接酶通路，介导G1期细胞核酸酶EXO1的泛素化降解，从而抑制了DNA双链断裂末端单链DNA(ssDNA)生成，有效阻止G1细胞选择同源重组修复通路。项目组还发现DNA-PKcs乙酰化修饰，在维持纺锤体结构稳定性和细胞有丝分裂中的作用。在此基础上，拓展研发了DNA-PKcs作为放射损伤防护的生物学靶标，获得了促进DNA-PKcs S2056位点磷酸化、激活DNA-PKcs、增强DNA修复能力的香兰素衍生物VND3207,据此筛选获得了对肠道放射损伤有显著保护和促进修复作用的新型放射防护剂。