线性泛素链组装复合物(LUBAC)的调节机制研究

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.

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。

线性泛素化是一种在先天免疫和炎症信号通路中具有重要功能的蛋白质翻译后修饰，线性泛素化功能的缺陷与众多的人类疾病密切相关，比如炎症、自身免疫疾病、神经退行性疾病和癌症等。人们目前只发现一种E3连接酶能够催化线性泛素链的合成，称为线性泛素链组装复合体(LUBAC)，它由核心蛋白质亚基HOIP与两个调节性蛋白质亚基SHARPIN和HOIL1组成。单独的HOIP蛋白质处于自抑制状态。LUBAC正确行使功能需要SHARPIN和HOIL1与HOIP的直接结合，激活HOIP E3活性，并把LUBAC定位到底物蛋白质区域。. 本项目的主要通过蛋白质结构解析、生物化学和细胞生物学实验，研究SHARPIN结合并激活HOIP的分子机制。我们发现HOIP的UBA结构域通过不同的区域同时结合SHARPIN和HOIL1的UBL结构域，并且发生了很大的构象变化，从而解除了它对催化核心的遮盖，促进了E2~Ub的结合。利用细胞生物学实验，我们进一步证明在细胞内HOIP对NF-kB通路的激活需要SHARPIN或HOIL1，而且二者具有协同增强的效果。. 我们的研究细致的阐释了LUBAC复合物的组装和激活机制，为深入理解该线性泛素链的合成以及在NF-kB通路中的作用提供了可靠的基础。