The function of UBEIL2, a novel ubiquitin specific activating enzyme (E1)

泛素需要在其C末端被激活，才能与目标蛋白共价连接。自1981年发现泛素特异性激活酶UBE1以来，实验证据表明，只存在一种泛素的EL激活酶(命名为UBE1)。在寻找泛素样蛋白FAT10的EL激活酶时，我们鉴定了一种迄今鲜有研究的EL酶，命名为MOP4或UBE1L2。这种酶在体外和体内激活泛素的能力与U-BE1一样强，并能够将激活的泛素转移到E2酶UBCHSb上，进而转移到E3酶和底物p53上。这些发现提出了一个问题，为什么泛素的第二种EL酶已经进化，以及它是否具有UBE1无法实现的功能。在本项目中，我们将研究UBE1L2在mRNA和蛋白质水平的表达水平，我们将建立UBE1L2的敲除方法，并确定其在细胞中对蛋白质降解、多泛素化、增殖、存活、抗辐射和抗逆性的影响。此外，我们将尝试识别UBE1缺陷细胞中的泛素化底物，并调查这些底物是否依赖UBE1L2变得泛素化。此外，UBE1L2缺陷细胞中积累的蛋白质将通过2D-DGE技术进行研究。将UBE1L2和UBE1L2特异的E2酶USEL进行酵母双杂交筛选，以寻找与UBE1L2结合的E2酶和与USEL作用的E3酶。最后，我们将建立器官特异性和可诱导的基因敲除小鼠，以阐明UBE1L2在体内的功能。

Ubiquitin needs to be activated at its C-terminus in order to be covalently linked to target proteins. Since the discovery of the ubiquitin specific activation enzyme UBE1 in 1981 experimental evidence has suggested that only a single El activating enzyme for ubiquitin (named UBE1) exists. While sear-ching for the El activating enzyme of the ubiquitin-like protein FAT 10 we characterized a so far barely investigated El enzyme named MOP4 or UBE1L2. This enzyme activated ubiquitin as potently as U-BE1 in vitro and in vivo and was able to transfer activated ubiquitin onto the E2 enzyme UBCHSb and further onto E3 enzymes and the substrate p53. These findings pose the question why a second El enzyme for ubiquitin has evolved and whether it serves a function that UBE1 can not fulfil. In this pro-ject we will investigate the expression levels of UBE1L2 on mRNA and protein level, we will establish a knock down of UBE1L2 and determine its effects on protein degradation, polyubiquitylation, proliferation, survival, radiation resistance, and stress resistance in cells. In addition, we will try to identify ubiquitylation substrates in UBE1 deficient cells and investigate if these rely on UBE1L2 to become ubiquitylated. Moreover, proteins that accumulate in UBE1L2 deficient cells will be investiga-ted by 2D-DIGE technology. A yeast two hybrid screen will be performed with UBE1L2 and the UBE1L2 specific E2 enzyme Usel in order to find E2 enzymes that bind to UBE1L2 and E3 enzymes that interact with Usel. Finally we will generate organ specific and inducible knock out mice in order to elucidate the in vivo functions of UBE1L2.