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

泛素需要在其C-末端被激活，以便共价连接到靶蛋白。自1981年发现泛素特异性激活酶UBE 1以来，实验证据表明，只存在一种泛素的E1激活酶（命名为UBE 1）。在寻找泛素样蛋白FAT 10的El激活酶时，我们表征了迄今为止几乎没有研究的El酶，命名为MOP 4或UBE 1 L2。这种酶在体外和体内与U-BE 1一样有效地活化泛素，并且能够将活化的泛素转移到E2酶UBCHSb上，并进一步转移到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UBE 1无法实现的功能。本课题将从mRNA和蛋白水平研究UBE 1 L2的表达水平，建立UBE 1 L2基因敲除模型，并研究其对细胞蛋白降解、多聚泛素化、增殖、存活、抗辐射和抗应激的影响。此外，我们将尝试在UBE 1缺陷细胞中鉴定泛素化底物，并研究这些底物是否依赖于UBE 1 L2而被泛素化。此外，将通过2D-DIGE技术研究在UBE 1 L2缺陷细胞中积累的蛋白质。将用UBE 1 L2和UBE 1 L2特异性E2酶Usel进行酵母双杂交筛选，以发现与UBE 1 L2结合的E2酶和与Usel相互作用的E3酶。最后，我们将产生器官特异性和可诱导的敲除小鼠，以阐明UBE 1 L2的体内功能。

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.