Mechanism of ubiquitin chain synthesis employed by a HECT domain ubiquitin ligase
Mechanism of ubiquitin chain synthesis employed by a HECT domain ubiquitin ligase
复制标题
DOI：
10.1074/jbc.m117.789479
复制
发表时间：
2017-06-23
影响因子：
4.8
通讯作者：
Hunter, Tony
中科院分区：
文献类型：
--
作者：
French, Michael E.;Klosowiak, Julian L.;Hunter, Tony
同源性至E6 AP C-末端（HECT）泛素（Ub）连接酶（E3）是结合至其底物并通过形成Ub硫酯中间体催化泛素化的一大类酶。这些E3在其底物上组装聚泛素链的机制仍然不清楚。我们在这里报告Nedd 4家族HECT E3，WWP 1，组装含有Lys-63，Lys-48和Lys-11键的底物连接的Ub链（Lys-63 > Lys-48 > Lys-11）。我们的研究结果表明，WWP 1催化通过顺序添加机制，其中Ub单体被转移到一个连续的方式基板的Ub链的形成，并通过WWP 1的泛素化需要一个低亲和力，非共价的Ub结合位点内的HECT域的存在。出乎意料的是，我们发现WWP 1形成Ub链发生在两个不同的阶段。在第一阶段中，链以单向方式合成，并且仅通过Ub的Lys-63连接。在第二阶段，链以多方向的方式伸长，其特征在于形成混合的Ub连接和分支结构。我们的研究结果为Nedd 4家族HECT E3所采用的Ub链形成机制提供了新的见解，并为理解E3家族如何产生在蛋白酶体非依赖性和蛋白酶体依赖性途径中发挥作用的Ub信号提供了一个框架。
Homologous to E6AP C-terminal (HECT) ubiquitin (Ub) ligases (E3s) are a large class of enzymes that bind to their substrates and catalyze ubiquitination through the formation of a Ub thioester intermediate. The mechanisms by which these E3s assemble polyubiquitin chains on their substrates remain poorly defined. We report here that the Nedd4 family HECT E3, WWP1, assembles substrate-linked Ub chains containing Lys-63, Lys-48, and Lys-11 linkages (Lys-63 > Lys-48 > Lys-11). Our results demonstrate that WWP1 catalyzes the formation of Ub chains through a sequential addition mechanism, in which Ub monomers are transferred in a successive fashion to the substrate, and that ubiquitination by WWP1 requires the presence of a low-affinity, noncovalent Ub-binding site within the HECT domain. Unexpectedly, we find that the formation of Ub chains by WWP1 occurs in two distinct phases. In the first phase, chains are synthesized in a unidirectional manner and are linked exclusively through Lys-63 of Ub. In the second phase, chains are elongated in a multidirectional fashion characterized by the formation of mixed Ub linkages and branched structures. Our results provide new insight into the mechanism of Ub chain formation employed by Nedd4 family HECT E3s and suggest a framework for understanding how this family of E3s generates Ub signals that function in proteasome-independent and proteasome-dependent pathways.