端粒酶相关的蛋白复合物p19/p45调控端粒酶活性的结构基础

Telomerase has close relationship to aging and cancer. It has also been a potential new target for tumor treatment in recent years. Telomerase is tightly regulated not only by telomerase catalytic subunit TERT, but also by telomerase associated proteins. The three dimensional structure of TERT/RNA/DNA complex which has been resolved recently reaveals the catalytic mechanism of telomerase. However，little is known about the molecular mechanism of the regulation of telomerase activity by telomerase associated proteins. The aim of this project is to solve the three dimensional structure of the telomerase associated protein complex p19/p45 from tetrahymena through X-ray diffraction. Based on the crystal structure, by using biochemical and cellular assays, the molecular mechanism of the interaction between p19 and p45 and the regulation of telomerase activity by the complex will be studied, which could also discover the mechanism how the complex controls telomere length by regulating telomerase activity. The accomplishment of this project will at first time explain how the telomerase associated protein complex p19/p45 regulates telomerase activity. It may lay solid foundation on throughly elucidating the molecular mechanism of the regulation of telomrase activity by telomease associated proteins. Theoretically, it may also provide instruction to some extent on tumor cure by targeting telomerase.

端粒酶与细胞衰老和肿瘤发生密切相关，近年来已经成为了肿瘤治疗的新靶点。端粒酶活性不仅与端粒酶自身的催化亚基有关，它还受端粒酶相关蛋白的调控。虽然端粒酶催化亚基、RNA以及底物DNA的复合物的三维结构最近刚被解析，揭示了端粒酶的催化机理。但是，端粒酶相关蛋白对端粒酶活性的调控机理目前还不清楚。本项目将采用X-射线晶体衍射来解析嗜热四膜虫的一个端粒酶相关蛋白复合物p19/p45的三维结构；利用生物化学方法研究p19和p45的相互作用以及其对端粒酶活性调控的分子机制；利用细胞生物学方法研究该复合物与端粒酶活性及端粒长度的关系。本项目的完成将首次说明端粒酶活性如何受端粒酶相关蛋白复合物p19/p45的调控，为全面阐明端粒酶相关蛋白调控端粒酶的机理奠定坚实的基础，也为以端粒酶为靶点的肿瘤治疗提供理论指导。

端粒酶与细胞衰老和肿瘤发生密切相关，近年来已经成为了肿瘤治疗的新靶点。端粒酶活性不仅与端粒酶自身的催化亚基有关，它还受端粒酶相关蛋白的调控。p19/p45是四膜虫端粒酶全酶的一个亚基复合物，过表达 p19 或 p45 会导致端粒缩短。本项目在前期研究工作基础上，纯化到 p19/p45 的蛋白复合物并结晶，最终解析出 p19/p45 复合物的三维晶体结构。通过结构分析，找到了p19和p45相互作用的关键氨基酸且通过体内体外实验证实，揭示了p19和p45的相互作用机理。野生型和突变体的端粒酶活性实验比较表明 p19 突变影响了端粒酶全酶的组装，从而极大地减弱了端粒酶活性。体内过表达p19、p45或它们的突变体，发现过表达p19突变体不改变端粒C链长度，但是会使G链长度增加，意味着p19调控了端粒C链的合成。总之，本项目通过解析p19/p45 复合物的三维结构，揭示了该复合物调控端粒酶活性以及端粒长度的分子机理。