E3泛素连接酶Smurf1调节经典Wnt信号通路的机制研究

Ubiquitination plays essential and diverse roles in modulating protein functions, and its abnormality is linked to many human diseases. As a C2-WW-HECT type ubiquitin ligase, Smad ubiquitination regulatory factor 1 (Smurf1) commonly serves to regulate ubiquitin-dependent protein degradation in a number of signaling pathways. Our previous work has identified Smurf1 as a novel E3 ligase of Axin by ubiquitinating Axin via K29-linked poly-ubiquitin (Ub) chains. This ubiquitination does not lead to Axin degradation but interfering with its function in Wnt signaling and then inhibiting Wnt activity. The special ubiquitination type and the non-degradation destination distinguish Axin from Smurf1’s other substrates; however, why Smurf1 specifically attaches K29 ubiquitin chains to Axin is poorly understood. In this project, we aim to unveil the molecular mechanism underlying Smurf1-mediated K29-linked poly-ubiquitination of Axin through the following two aspects: 1) clarification of the non-canonical interaction pattern between Smurf1 and Axin; and 2) identification of the related E2 ubiquitin ligase responsible for Smurf1-mediated Axin ubiquitination. Our preliminary data suggest that, unlike other substrates of Smurf1, the PY motifs of Axin are not required for Smurf1 binding, implying a non-canonical interaction pattern between the two proteins. Therefore, firstly, we will construct several fragments of Smurf1 and Axin to analyze their involvement in Smurf1-Axin interaction. In this process, in vivo immunoprecipitation assays, in combination with in vitro GST pull down assays, will be applied to clarify their interaction pattern. Considering that the non-canonical interaction pattern might endow the protein complex with unique conformation which prefers a K29 Ub-linkage, we will try to purify the Smurf1-Axin complex by using in vitro system and then to analyze its three-dimensional structure. Alternatively, since the different linkages of poly-ubiquitination could be achieved by associating with different E2 ligases, we will also initiate an effort to identify which E2, among more than 30 known human E2 proteins, is involved by knocking down each one of them. In this process, Smad7, a well-studied substrate of Smurf1, will be included as a control. Taken together, this project will not only help us to gain new insights into the specific characteristics of Smurf1-mediated Axin ubiquitination, but also further our understanding of the molecular mechanisms for Wnt signaling pathway, and of the wide range functions of Smurf1.

细胞内蛋白质的泛素化修饰在调节蛋白质功能方面具有重要的作用，其调控异常和很多疾病的发生相关。Smurf1是一个重要的E3泛素化连接酶，能够通过泛素化修饰作用促进底物蛋白质的降解，参与调控多条信号通路。我们实验室早前发现Smurf1能够对Wnt信号通路中关键分子Axin进行泛素分子Lys29连接的多聚泛素化修饰，并通过影响Axin的功能而不是其稳定性进而抑制Wnt信号的传递。Smurf1对Axin的这种非降解修饰关系使得Axin成为Smurf1众多底物中较为特殊的一个。在本研究中，我们将针对Smurf1和Axin间这种特殊的修饰关系，从蛋白质相互作用模式、蛋白质复合物的结构以及参与修饰反应的E2泛素化结合酶等方面揭示产生这种特殊泛素化修饰的内在机理。此项工作的完成将进一步完善对细胞内泛素化修饰系统和对Wnt信号转导机制的认识，并促进对Smurf1分子功能研究的拓展。