线性泛素链组装复合物(LUBAC)的调节机制研究

项目名称： 线性泛素链组装复合物(LUBAC)的调节机制研究

项目编号： No.31500597

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 生物科学

项目作者： 刘建平

作者单位： 中国科学院上海有机化学研究所

项目金额： 20万元

中文摘要： 线性泛素化是一类广泛参与炎症和先天免疫的新型泛素化修饰。迄今唯一的线性泛素链E3连接酶LUBAC复合物由调节亚基Sharpin、HOIL1和催化亚基HOIP组成。单独的HOIP处于自抑制状态，但可以被Sharpin或HOIL1激活。然而，HOIP的自抑制和激活、与Sharpin的相互作用机制仍然未知。Sharpin缺失小鼠表现出多器官炎症和TNFα通路异常。为了阐明LUBAC复合物的调控机制，本申请计划解析自抑制和激活状态的HOIP催化核心片段，以及Sharpin/HOIP复合物的结构，揭示HOIP的自抑制和激活机制，并设计点突变解除自抑制或者破坏Sharpin对HOIP的激活，研究相应突变体对细胞死亡或RIP1线性泛素化的影响，以检验我们的假说：Sharpin/HOIP复合物对RIP1的线性泛素化决定了TNFα通路的最终效应——炎症或细胞死亡。现有工作进展为实现上述目标奠定了坚实的基础。

中文关键词： 蛋白质复合物；X射线晶体衍射；泛素化；肿瘤坏死因子TNFα；细胞死亡

英文摘要： Linear ubiquitination, a recently discovered novel type of ubiquitination, is extensively involved in numerous signaling pathways related to inflammation and innate immune. Linear ubiquitin chain assembly complex (LUBAC) is the sole identified E3 ubiquitin ligase that catalyzes the formation of linear poly-ubiquitin chain. It consists of two regulatory subunits Sharpin and HOIL1 as well as one catalytic subunit HOIP. The isolated HOIP is in an auto-inhibited state, but it can be activated by Sharpin or HOIL1 to sufficiently promote the linear ubiquitination of target substrates. However, the molecular mechanisms underlying the the auto-inhibition of HOIP, its activation by Sharpin or HOIL1, and the interaction between HOIP and Sharpin are largely unknown. Previous studies indicated that loss of Sharpin in the mouse caused immunodeficiency and multi-organ inflammation, and severely inhibited the linear ubiquitination of TNFα receptor signaling complex. Based on these findings, we propose a hypothesis: the linear ubiquitination level of RIP1 catalyzed by Sharpin/HOIP complex determines the outcome of TNFα signaling—inflammation or cell death. To further understand the regulatory mechanism of LUBAC complex, we aimed to solve the structures of the catalytic fragment of HOIP either in auto-inhibited or in activated states as well as the Sharpin/HOIP complex, thereby to uncover the auto-inhibition and activation mechanism of HOIP. Based on the solved structures, we will design point mutations to specifically disrupt the auto-inhibition or the trans-activation of HOIP mediated by Sharpin; we will further test our hypothesis by assessing the impact of these mutations on the linear ubiquitination level of RIP1 and the TNFα induced cell death. Our current research progress has laid the foundation for us to pursue the scientific goals in this proposal.

英文关键词： protein complex;X-ray crystallography;ubiquitination;TNFα;cell death