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Molecular regulation of the AP2 clathrin adaptor complex

AP2 网格蛋白接头复合物的分子调控

基本信息

批准号：
9900825
负责人：
Gunther Hollopeter
金额：
$ 36.53万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9900825
关键词：
Address Alzheimer&apos s Disease Animals Ankyrin Repeat Binding Biochemical Biological Assay Caenorhabditis elegans Cardiovascular Diseases Cardiovascular system Cell membrane Cell physiology Cells Cellular biology Cholesterol Clathrin Clathrin Adaptors Collaborations Complex Coupled Cryoelectron Microscopy Data Disease Ear Endocytosis Ensure Eukaryotic Cell Event Face Functional disorder Genes Genetic Genetic Screening Goals Growth Factor Health Heart Hepatitis Image Influenza Knock-out Ligands Liposomes Malignant Neoplasms Mediating Medical Membrane Missense Mutation Modeling Molecular Molecular Conformation Molecular Machines Molting Mutagenesis Names Nephronophthisis Neurodegenerative Disorders Neurologic Neuromodulator Outcome Paper Pathologic Pattern Peptide Hydrolases Phenocopy Phospholipids Phosphorylation Phosphotransferases Physiological Physiology Process Protein Family Proteins Proteomics Receptor Signaling Recycling Regulation Research Role Route Signal Transduction Structure System Testing Therapeutic Time Tissues Vesicle Viral Virus Virus Diseases base design enhancer-binding protein AP-2 in vitro Assay in vivo innovation insight macromolecule molecular rearrangement mutant neoplastic novel particle polymerization receptor binding spatiotemporal stem structural biology tool trafficking

项目摘要

Abstract Clathrin-mediated endocytosis is the main port of entry into our cells for medically relevant substances including cholesterol-laden particles and viruses such as influenza and hepatitis. By engulfing signaling receptors, this fundamental cellular process also tunes our sensitivity to the potentially pathological actions of growth factors and neuromodulators. As such, understanding how the underlying endocytic machinery is regulated promises to reveal novel mechanisms that could be harnessed to control neoplastic, neurodegenerative, cardiovascular, and viral diseases. At the heart of the endocytic process lies the AP2 clathrin adaptor complex which appears to undergo a conformational change during vesicle formation to actively couple membrane and cargo to the clathrin coat. Despite the central role of AP2, we lack critical details about how this molecular machine is regulated in vivo and how this regulation influences multicellular systems. To address this need, we have developed innovative tools in C. elegans that allow us to quantify AP2 activity at multiple levels and have employed deep genetic screens to identify three conserved protein families that appear to govern AP2 conformation and activity. Our goal is to illuminate how these allosteric regulators of the endocytic machinery function mechanistically. In Aim 1 we will validate our hypothesis that adaptiN-Ear-Binding Coat-Associated Proteins (NECAP)s counteract the active (open) conformation of AP2 to ensure proper recycling of adaptor complexes. We have discovered that AP2 accumulates in an active state in NECAP mutants, and that NECAPs specifically bind open, phosphorylated forms of AP2. Using cryo- EM we have determined that the phosphorylated AP2 core bound to NECAP is conformationally inactive. We will validate this structure in vivo and whether it reflects the end product of NECAP activity. Previously it was thought that membrane phospholipids, cytosolic cargo domains, and phosphorylation by the AP2-associated kinase (AAK1) activate AP2. Our preliminary data indicate that a conserved region of the membrane- associated Fer/Cip4 Homology Domain-only (FCHo) proteins is required to promote endocytosis by converting AP2 to an active complex. We have named this functionally important domain the AP2 Activator, or APA. In Aim 2 we will determine where the APA binds AP2 using cryo-EM and test whether the APA is sufficient to induce a structural rearrangement of AP2, as well as defining the roles of membrane, cargo, and phosphorylation in that process. We will evaluate the physiological significance of AP2 phosphorylation by characterizing kinase mutants. In our new Aim 3 we will examine how membrane trafficking influences tissue physiology using our suite of assays to study a novel mutant in a tissue patterning inversin/nephronophthisis- 2 protein called MLT-4 that phenocopies loss of AP2 activity. The long-term impact of the proposed research will be to clarify how fundamental cellular machinery is controlled with spatiotemporal precision in metazoans, where misregulation leads to important diseases.

摘要网格蛋白介导的内吞作用是进入我们的细胞进行医学相关的主要端口物质，包括富含胆固醇的颗粒和病毒，例如流感和肝炎。通过吞没信号受体，这个基本的细胞过程也调整我们对潜在病理的敏感性生长因子和神经调节剂的作用。因此，了解潜在的内吞作用是如何进行的机械受到监管，有望揭示可用于控制肿瘤的新颖机制，神经退行性疾病、心血管疾病和病毒性疾病。 AP2 是内吞过程的核心网格蛋白接头复合物似乎在囊泡形成过程中经历构象变化主动将膜和货物耦合到网格蛋白涂层上。尽管 AP2 发挥着核心作用，但我们缺乏关键的关于这种分子机器如何在体内调节以及这种调节如何影响多细胞的详细信息系统。为了满足这一需求，我们开发了线虫创新工具，使我们能够量化 AP2 活性在多个水平上，并采用深度遗传筛选来鉴定三种保守蛋白质似乎控制 AP2 构象和活性的家族。我们的目标是阐明这些变构如何内吞机制的调节器以机械方式发挥作用。在目标 1 中，我们将验证我们的假设适应耳结合涂层相关蛋白 (NECAP) 抵消 AP2 的活性（开放）构象确保适配器复合物的正确回收。我们发现 AP2 在活性物质中积累 NECAP 突变体中的状态，并且 NECAP 特异性结合 AP2 的开放磷酸化形式。使用冷冻- EM 我们已确定与 NECAP 结合的磷酸化 AP2 核心在构象上无活性。我们将在体内验证该结构以及它是否反映了 NECAP 活动的最终产品。以前是认为膜磷脂、胞质货物结构域和 AP2 相关的磷酸化激酶 (AAK1) 激活 AP2。我们的初步数据表明，膜的一个保守区域- 需要关联的 Fer/Cip4 同源域 (FCHo) 蛋白来促进内吞作用将 AP2 转化为活性复合物。我们将这个功能上重要的域命名为 AP2 激活器，或 APA。在目标 2 中，我们将使用冷冻电镜确定 APA 与 AP2 结合的位置，并测试 APA 是否足以诱导 AP2 的结构重排，以及定义膜、货物和物质的作用该过程中的磷酸化。我们将通过以下方式评估 AP2 磷酸化的生理意义表征激酶突变体。在我们的新目标 3 中，我们将研究膜运输如何影响组织生理学使用我们的检测套件来研究组织模式反转/肾结核中的新型突变体- 2 种称为 MLT-4 的蛋白质，可表现 AP2 活性的丧失。拟议研究的长期影响将阐明后生动物中基本的细胞机器是如何通过时空精度进行控制的，监管不当会导致重大疾病。