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

ERAD途径处理IP3受体的机制及IP3受体-erlin 1/2复合物-RNF170轴的分析

• 批准号: 9383964
• 负责人: RICHARD J H WOJCIKIEWICZ
• 金额: $ 40.5万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9383964
• 关键词: Address; Affect; Autophagosome; Binding; Biochemical; Biology; CRISPR/Cas technology; Cell Line; Cell physiology; Cells; Cellular biology; Complex; Cysteine; Cytosol; Degradation Pathway; Development; Disease; Docking; Electron Transport Complex III; Endoplasmic Reticulum; Endoplasmic Reticulum Degradation Pathway; Ensure; Eukaryotic Cell; Family; Goals; Hereditary Spastic Paraplegia; Human; ITPR1 gene; Impairment; Inositol; Ion Channel; Isoleucine; Lead; Life Cycle Stages; Link; Lipids; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Molecular Biology; Mutagenesis; Mutation; NMR Spectroscopy; Neurodegenerative Disorders; Organelles; Outcome; Pathway Analysis; Pathway interactions; Physiological; Physiological Processes; Play; Point Mutation; Property; Proteins; RNA Interference; Regulation; Resolution; Role; Second Messenger Systems; Signal Pathway; Signal Transduction; Signaling Protein; Spectrometry; Structural Models; Structure; Time; Ubiquitin; Ubiquitination; Work; crosslink; design; human disease; insight; multicatalytic endopeptidase complex; mutant; novel; phosphatidylinositol 3-phosphate; programs; protein function; receptor; tool; trafficking; ubiquitin ligase

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.

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 ER 相关降解（ERAD）途径的处理， 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) 由两种 ER 膜蛋白（称为 erlin1 和 erlin2）组成的复合物，直接与激活的 IP3R 结合 并介导其 ERAD，(ii) erlin1/2 复合物与磷脂酰肌醇 3-磷酸 (PI(3)P) 结合， 脂质对于细胞内细胞器的发育和运输至关重要，并且（iii）人类神经退行性疾病 与疾病相关的 erlin2 点突变会改变复合物的功能。未解决的关键问题 这些发现得出的结果如下：erlin1/2 复合体如何识别激活的 IP3R 并 ERAD 的靶标，PI(3)P 与 erlin1/2 复合体结合的全部意义是什么，以及如何进行 erlin1/2 复合体自然发生的点突变会改变其特性吗？这些问题将会 通过 2 个具体目标来解决。 目标 1) 分析 erlin1/2 复合体和激活的 IP3R 如何相互作用 采取的方法是 在缺乏这些蛋白质的细胞中表达 (i) erlin2 或 (ii) IP3R1 突变体，并评估 突变损害激活的 IP3R1 和 erlin1/2 复合物之间的相互作用，(iii) 识别相互作用 通过交联和质谱分析来确定区域，并且（iv）生成高分辨率的结构模型 erlin1/2 复合体使用冷冻电镜和核磁共振波谱，可以与等效结构对接 激活IP3R1。从这些互补方法获得的结果应确定交互点 激活的 IP3R1 和 erlin1/2 复合体之间的关系，并确定 IP3R1 四聚体激活的情况 触发 erlin1/2 复合物对 ERAD 的识别。这将阐明生命周期中的关键一步 IP3Rs 并将首次详细描述如何识别天然内源信号蛋白 用于通过 ERAD 途径进行处理。 目标 2) 分析 erlin1/2 复合物的 PI(3)P 结合能力及其意义 采取的方法 将确定 (i) PI(3)P 如何通过诱变和 NMR 光谱与 erlin1/2 复合物结合，(ii) IP3R ERAD 是否需要 PI(3)P 与 erlin1/2 复合物结合，以及 (iii) erlin1/2 复合物如何 影响自噬体形成或其他 PI(3)P 依赖性细胞过程。获得的结果将 阐明 erlin1/2 复杂生物学的全新方面，并应确定新的机制细节 关于 IP3R ERAD 和 PI(3)P 依赖性细胞过程。 总之，将首次研究新颖的主题，其结果将推进我们的研究 了解 IP3R-erlin1/2 复合物-RNF170 轴、一般 ERAD 和神经退行性疾病。