多聚泛素化在胚胎干细胞的多能性维持与神经分化中的作用机理研究

泛素化通过共价结合不同的泛素链到底物蛋白上来影响它们的功能。泛素化参与调节几乎所有的真核细胞功能，其中包括胚胎干细胞多能性的维持与分化。申请人的前期工作在蛋白质组水平上系统地报道了泛素信号通路的相互作用蛋白，鉴定了一系列可以选择性结合特异泛素化链的蛋白，并发现了第一个K27多聚泛素化链的选择性结合蛋白UCHL3。申请人课题组近期的进一步研究发现了单泛素化H2A的去泛素化酶复合体（PR-DUB）可以特异地结合K29和K63泛素化链。本项目拟在此基础上，利用选择性结合不同多聚泛素化链的蛋白结构域，结合定量蛋白质谱分析，系统地研究泛素信号通路在胚胎干细胞与神经前体细胞中泛素化蛋白的水平与功能。同时研究PR-DUB与UCHL3调控胚胎干细胞DNA损伤反应的分子机制。

Ubiquitination entails covalent attachment of different ubiquitin chains to protein substrates. It plays pivotal roles in almost every process in eukaryotic cells via altering the functions of the protein substrates, including pluripotency maintenance and differentiation of embryonic stem cells. Our previous studies provided an interaction landscape of ubiquitin signalling in a proteome-wide level. We identified a series of linkage-selective ubiquitin chains interactors, and verified UCHL3 as the first K27 linkage-selective interactor. Furthermore, our recent studies have identified that the polycomb repressor-deubiquitinase (PR-DUB) complex, which deubiquitinates monoubiquitinated-H2A, selectively interacts with K29 and K63 ubiquitin linkages. The purposes of this proposal are to explore the dynamics of ubiquitylome and the functions of ubiquitin signalling in embryonic stem cells and neural progenitor cells. To achieve that, we will make use of linkage-selective ubiquitin binding domains to enrich ubiquitinated proteins followed by mass spectrometry measurement. In the meantime, we will investigate the molecular mechanisms of how PR-DUB and UCHL3 regulate the DNA damage response in embryonic stem cells.