Mechanism of IP3 receptor processing by the ERAD pathway and analysis of the IP3 receptor-erlin 1/2 complex-RNF170 axis

我的长期目标是了解肌醇1，4，5-三磷酸的分子生物学和调控 受体(IP3R)，特别是它们通过内质网相关降解(ERAD)途径的处理，a 泛素-蛋白酶体途径的一个方面。近年来，我们发现(I)一个非常大的(~2MDa) 由两个内质网膜蛋白组成的复合体，称为erlin1和erlin2，直接与激活的IP3Rs结合 并介导其ERAD，(Ii)erlin1/2复合体与磷脂酰肌醇3-磷酸(PI(3)P)结合，a 对细胞内细胞器发育和运输至关重要的脂类，以及(Iii)人类神经退行性变 与疾病相关的erlin2点突变会改变复合体的功能。尚未解决的关键问题 这些发现的来源如下：erlin1/2复合体如何识别激活的IP3R和 靶向ERAD，PI(3)P与erlin1/2复合体结合的全部意义是什么，以及如何 Erlin1/2复合体的自然发生的点突变会改变其性质吗？这些问题将是 通过两个具体目标来解决。 目标1)分析erlin1/2复合体与激活的IP3R如何相互作用，将采取的方法是 在缺乏这些蛋白的细胞中表达(I)erlin2或(Ii)IP3R1突变体，并评估 突变损害了激活的IP3R1和erlin1/2复合体之间的相互作用，(Iii)识别相互作用 (Iv)生成一个高分辨率的结构模型 利用低温EM和核磁共振波谱技术对Erlin1/2络合物进行了研究，该络合物可以与 激活了IP3R1。从这些互补方法获得的结果应该确定相互作用点 在激活的IP3R1和erlin1/2复合体之间，并确定IP3R1四聚体激活是什么 触发ERAD的erlin1/2复合体识别。这将照亮生命周期中的关键一步 IP3Rs，并将首次详细描述天然内源信号蛋白是如何被识别的 通过ERAD途径进行处理。 目的2)分析Erlin1/2复合体与PI(3)P的结合能力及其意义 将确定(I)PI(3)P如何通过突变和核磁共振光谱与Erlin1/2复合体结合，(Ii) IP3R ERAD是否需要PI(3)P与erlin1/2复合体结合，以及(Iii)erlin1/2复合体如何 影响自噬小体的形成，或其他依赖PI(3)P的细胞过程。所取得的成果将 阐明erlin1/2复杂生物学的一个全新的方面，并应确定新的机制细节 关于IP3R、ERAD和PI(3)依赖P的细胞过程。 总而言之，将首次研究新的主题，结果将推动我们的 了解IP3R-erlin1/2复合体-RNF170轴、一般ERAD和神经退行性疾病。

My long-term goal is to understand the molecular biology and regulation of inositol 1,4,5-trisphosphate receptors (IP3Rs) and, in particular, their processing by the ER-associated degradation (ERAD) pathway, a facet of the ubiquitin-proteasome pathway. In recent years we have discovered (i) that a very large (~2MDa) complex composed of two ER membrane proteins, termed erlin1 and erlin2, binds directly to activated IP3Rs and mediates their ERAD, (ii) that the erlin1/2 complex binds to phosphatidylinositol 3-phosphate (PI(3)P), a lipid critical to intracellular organelle development and trafficking, and (iii) that human neurodegenerative disease-linked point mutations to erlin2 alter the functionality of the complex. The key unresolved questions that flow from these findings are as follows: how does the erlin1/2 complex recognize activated IP3Rs and target them for ERAD, what is the full significance of PI(3)P binding to the erlin1/2 complex, and how do naturally-occurring point mutations to the erlin1/2 complex alter its properties? These questions will be addressed through 2 Specific Aims. Aim 1) Analysis of how the erlin1/2 complex and activated IP3Rs interact The approach taken will be to express (i) erlin2, or (ii) IP3R1 mutants in cells lacking these proteins and assess the extent to which the mutations impair the interaction between activated IP3R1 and the erlin1/2 complex, (iii) identify the interacting regions by cross-linking followed by mass spectrometry, and (iv) generate a high resolution structural model of the erlin1/2 complex using cryo-EM and NMR spectroscopy that can be docked with an equivalent structure of activated IP3R1. Results obtained from these complementary approaches should identify the interaction points between activated IP3R1 and the erlin1/2 complex and establish what it is about IP3R1 tetramer activation that triggers its recognition by the erlin1/2 complex for ERAD. This will illuminate a key step in the life cycle of IP3Rs and will provide the first detailed description of how a native endogenous signaling protein is recognized for processing by the ERAD pathway. Aim 2) Analysis of the PI(3)P-binding capacity of the erlin1/2 complex and its significance The approach taken will be to determine (i) how PI(3)P binds to the erlin1/2 complex from mutagenesis and NMR spectroscopy, (ii) whether PI(3)P binding to the erlin1/2 complex is required for IP3R ERAD, and (iii) how the erlin1/2 complex affects autophagosome formation, or other PI(3)P-dependent cellular processes. Results obtained will illuminate a completely novel aspect of erlin1/2 complex biology and should identify new mechanistic details about IP3R ERAD and PI(3)P-dependent cellular processes. In summary, novel topics will be investigated for the first time, with outcomes that will advance our understanding of the IP3R-erlin1/2 complex-RNF170 axis, ERAD in general and neurodegenerative disorders.