新型Nedd8连接酶Smurf1的底物鉴定及其作为Nedd8连接酶的体内功能研究

Neddylation, the covalent attachment of the ubiquitin-like protein Nedd8, is essential for the viability of most organisms and is involved in the development of cancer. The currently reported Nedd8 ligases is less than ten, which are all RING-type E3 ligases, and no more than twenty neddylated proteins were found out.Recently, we found Smurf1 and its yeast homologue Rsp5 were thioester bond-type Nedd8 ligases to catalyze their own neddylation, and the autoneddylation of Smurf1 needs an active site C426 in the HECT N-lobe. However, the neddylated substrates of Smurf1 remains unknown, and its physiological function as a nedd8 ligase is also not clear. In this project, we plan to intensivly identify the neddylated substrates of Smurf1 using NEDP1-/- konck out mice. Neddylation modification is thought to be more stable and easier detected after koncking out NEDP1, which is a cysteine protease specific for Nedd8. In addtion, we intend to intensivly investigate the physiological function of Smurf1 as a nedd8 ligase taking advantage of Smurf1 C426A mutant mice. Our project could shed new light on the neddylation function and mechanism.

类泛素蛋白Nedd8介导的Neddylation为个体发育、存活所必需，其异常与肿瘤关系密切。已知Nedd8连接酶数量不足10个且均为RING类型，修饰底物数量也仅有20个左右，均亟待挖掘与研究。我们新近发现HECT类泛素连接酶Smurf1及其酵母同源物Rsp5是一类新型的可形成硫脂键中间体的Nedd8连接酶，C426为Smurf1的Nedd8连接酶活性中心。但其修饰底物还不清楚，Smurf1作为泛素与类泛素双连接酶，其Nedd8连接酶的体内功能也未揭示。本项目将利用去Nedd8酶NEDP1 knockout小鼠可上调Neddylation修饰本底水平的特殊优势，鉴定Smurf1的修饰底物；利用Smurf1-C426A knockin小鼠模型，精确研究Smurf1这一酶活性的体内功能及与泛素连接酶活性的crosstalk。本研究对于拓宽Nedd8修饰的功能与机制认识具有重要的科学意义。

类泛素蛋白Nedd8介导的Neddylation为个体发育、存活所必需，其异常与肿瘤关系密切。前期工作发现HECT类泛素连接酶Smurf1是一类新型的可形成硫脂键中间体的Nedd8连接酶，本项目进一步揭示了Smurf1的同源家族蛋白Itch和Smurf2也是HECT类Neddylation连接酶，其中Itch自我催化mono-neddylation修饰，并发现其下游底物JunB被Itch催化neddylation修饰，Neddylation修饰将抑制JunB的转录活性；Smurf2自我催化poly-neddylation修饰并促进了它的泛素化降解。本项目利用Smurf1-C426A knockin小鼠模型鉴定到Smurf1的neddylation修饰底物Smad5，ING2，RhoA等，同时发现Smurf1-C426A knock in小鼠呈现骨密度降低，骨量减少的表型。这对于拓宽Nedd8修饰的功能与机制认识具有重要的科学意义。