Lys63多聚泛素化是一种非蛋白降解的翻译后修饰和活性调节方式，而Ubc13是迄今已知唯一能介导Lys63多聚泛素化的泛素结合酶。我们发现，斑马鱼中存在Ubc13a、Ubc13b、Uev1A和Mms2，它们协同作用对靶蛋白进行Lys63多聚泛素化。此外，Ubc13敲除小鼠及其敲降的斑马鱼胚胎均死亡，但尚不清楚Lys63泛素化调控脊椎动物发育的分子机制。本项目主要内容为: 1）研制Ubc13a、Ubc13b、Uev1A和Mms2的突变体，研究它们在胚胎细胞凋亡和脊索形成中的作用；2）利用突变体胚胎和ZF4细胞，研究它们对E3酶如Mib的Lys63多聚泛素化作用；3）利用突变体胚胎和ZF4细胞，研究它们对Notch通路因子的修饰和活性调控。该项目的获准与实施，将阐明这些因子在胚胎细胞凋亡和Notch通路活性调控中的功能差异和分子机制，为揭示Lys63泛素化在斑马鱼组织器官发育中的功能奠定基础。

Lys63-polyubiquitination mediates the post-translational modification of target proteins in a non-degradation manner and thus regulates their activities. So far, Ubc13 is the only known ubiquitin-conjugating enzyme (E2) that can work together with its cofactors (Uev1 or Mms2) to mediate Lys63-polyubiquitination. Our previous studies have shown that zebrafish contains Ubc13a, Ubc13b, Uev1A and Mms2, their synergic action can cause Lys63-polyubiquitination of target proteins. In addition, developing embryos of Ubc13-knockout mice and Ubc13-knockdown zebrafish are lethal, but it remains largely unclear about molecular mechanisms underlying the regulation of embryonic development by Lys63-polyubiquitination in vertebrates. The main contents of this project include: 1) the generation of Ubc13a-, Ubc13b-, Uev1A- or/and Mms2-knockout zebrafish mutants for investigation of their roles in embryonic cell apoptosis and notochord formation; 2) the Lys63-polyubiquitination of known E3 enzymes such as Mib in mutant embryos and ZF4 cells; and 3) the roles of Lys63-polyubiquitination in modification of Notch signaling factors and activity of Notch pathway in mutant embryos and ZF4 cells. Allowed the implementation of the project, we will clarify the differential roles of Ubc13a, Ubc13b, Uev1A and Mms2 in the control of embryonic cell apoptosis and Notch signaling pathway activity, reveal the molecular mechanisms for Lys63-polyubiquitination in zebrafish, and thus lay the foundation for a full uncover the function and mechanism of Lys63-polyubiquitination during the development of tissues and organs.