泛素化肿瘤抑制因子p53的化学合成及特异性去泛素化研究

Ubiquitination of the tumor suppressor p53 has recently been found to play a key regulatory role in a variety of intracellular biological processes. However, the difficulty for obtaining ubiquitinated p53 protein greatly limits its development in biochemistry and biophysics studies. This project plans to use the transpeptidase Sort A assisted protein semisynthesis method for the chemical synthesis of different types of ubiquitinated p53 with uniform structure and properties. The synthetic protein acts as a tool to accurately reconstruct in vitro biochemical research system, and then to explore biochemical processes and biophysical mechanisms of specific deubiquitination for the deubiquitinase CYLD. The specific plan is: a. Optimize Sort A mediated protein hydrazide synthesis method to efficiently obtain a C-terminal hydrazide-containing p53 fragment; b. Ubiquitin chain enzymatic synthesis system and auxiliary-mediated ligation strategy were used to obtain the C-terminal p53 fragments with different site, chain length. The full length ubiquitinated p53 can be obtained through hydrazide based native chamical ligation between the C-terminal p53 fragments with p53 hydrazide fragment; c. Using synthetic protein as a tool to establish a precise in vitro biochemistry and biophysical research system and explore the selective hydrolysis mechanism of deubiquitinase CYLD for the hydrolysis of different ubiquitinated p53 with different site, chain length. Through the implementation of this project, we try to expand the enzymatic assisted protein semi-synthesis method and elucidate the selective hydrolysis mechanism of deubiquitinase for ubiquitinated p53, and will also exhibit the important value of protein chemical synthesis in the study of post-translational modified proteins.

肿瘤抑制因子p53的泛素修饰被发现在多种细胞内生物过程中发挥关键调控作用。然而泛素化p53的获取困难限制了其生化机制研究的开展。本项目拟在前期工作基础上运用转肽酶Sort A催化的酶促合成方法实现性质均一的不同类型泛素化p53的化学合成，并以合成蛋白为工具重构体外生化研究体系，探索去泛素化酶特异性去泛素化的生物化学机制。具体内容为：a. 优化Sort A催化的蛋白酰肼酶促合成方法，高效获得C端酰肼的p53片段；b. 运用泛素链酶促合成及辅基介导的连接方法获得不同链长及连接类型泛素修饰的p53片段，并与p53酰肼片段进行连接获得性质均一的泛素修饰p53；c. 建立精准的体外生物化学及生物物理研究体系，探索去泛素化酶对泛素修饰p53的特异性水解机制。通过本项目的实施力求拓展蛋白质酶促合成方法体系，阐明泛素修饰p53的去泛素化机制，也将充分展示蛋白质化学合成在翻译后修饰蛋白研究中的重要价值。

以肿瘤抑制因子p53为代表的泛素化底物蛋白被体内的泛素修饰酶（E1,E2, E3）和去泛素化酶调控其泛素化水平。这些酶的功能异常或缺失将导致底物蛋白在细胞内的含量异常，进而引发诸如癌症等多种疾病。通过发展泛素化底物蛋白合成新方法，获取均一的泛素化蛋白样品，进一步构建精准的体外生化研究系统，成为了理解泛素酶系统调控底物蛋白功能的一个可行且高效的方法。在本项目的支持下，主要开展了两个方向的研究内容：a)发展泛素化底物蛋白合成新方法，主要包括基于新型辅基的自然化学链接策略、E1催化的化学酶法策略、微波辅助合成策略等；b) 将体外重构精准的泛素化蛋白用于泛素酶学系统的生化研究，主要包括去泛素化酶（DUB）活性的分析、结构机制的解析等。目前由本课题组发展泛素化底物蛋白的合成方法和构建的泛素酶学系统的生化研究体系已得到国际学术界的广泛应用。