Structural and mechanistic basis of AAGAB-controlled AP2 adaptor assembly

AAGAB 控制的 AP2 适配器组件的结构和机械基础

• 批准号: 10215568
• 负责人: Qian Yin
• 金额: $ 34.42万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-13 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10215568
• 关键词: Binding; Binding Proteins; Biochemical; Biochemical Genetics; Biological Assay; Capsid Proteins; Cells; Clathrin; Clathrin Adaptors; Complex; Cryoelectron Microscopy; Data; Defect; Development; Endocytosis; Genetic; Genetic Screening; Glean; Goals; Human; Individual; Knowledge; Light; Mediating; Membrane Proteins; Mutation; Palmoplantar Keratosis; Pathogenesis; Pathway interactions; Physiological; Play; Process; Property; Proteins; Research; Role; Site; Structural Protein; Structure; System; Vesicle; Work; X-Ray Crystallography; base; disease-causing mutation; enhancer-binding protein AP-2; genetic approach; genome-wide; human disease; insight; novel therapeutic intervention; particle; protein complex; protein function; protein transport; reconstitution; recruit; skin disorder; trafficking

PROJECT SUMMARY In clathrin-mediated endocytosis (CME), the coat protein clathrin relies on adaptors to recruit cargo proteins to endocytic sites. The predominant clathrin adaptor in CME is the heterotetrameric AP2 adaptor complex, which is comprised of two large subunits ( and β), one medium subunit (µ), and one small subunit (). It is generally thought that multimeric trafficking adaptors such as AP2 adaptor assemble spontaneously. However, we recently discovered that AP2 adaptor assembly is an ordered process controlled by alpha and gamma adaptin binding protein (AAGAB). Without the assistance of AAGAB, AP2 adaptor fails to form, leading to CME defects. These findings revealed a previously unrecognized pathway in clathrin-mediated trafficking. However, it remains unclear how AAGAB recognizes AP2 subunits and how AAGAB-AP2 interactions drive AP2 adaptor assembly. In our preliminary studies, we expressed and purified free AAGAB and AAGAB:AP2 assembly intermediates, and gained initial insights into their structures and functions. In this research, we will take advantage of these preliminary data to determine the structural and mechanistic basis of AAGAB-controlled AP2 adaptor assembly. We will first characterize the biochemical properties of free AAGAB and AAGAB:AP2 assembly intermediates using reconstituted systems. We will then determine the atomic structures of the proteins and protein complexes using X-ray crystallography and single-particle cryo-electron microscopy. Next, we will validate the physiological relevance of the biochemical and structural findings using cell-based genetic assays. Finally, we will determine how AAGAB functions are impacted by disease-causing mutations. Successful completion of this proposed research will fill a major gap in our knowledge of membrane protein trafficking. This work will also serve as a paradigm for understanding the assembly of trafficking adaptors in general. Ultimately, these findings will facilitate the development of novel therapeutic strategies for human diseases caused by AAGAB mutations.

项目概要 在网格蛋白介导的内吞作用 (CME) 中，外壳蛋白网格蛋白依赖于接头来招募货物蛋白 CME 中主要的网格蛋白接头是异四聚体 AP2 接头复合物。 由两个大亚基（α 和 β）、一个中亚基（μ）和一个小亚基（β）组成。 认为多聚体运输适配器（例如 AP2 适配器）会自发组装。但是，我们最近发现。 发现 AP2 接头组装是由 α 和 γ 接头蛋白结合控制的有序过程 没有 AAGAB 的帮助，AP2 接头就无法形成，从而导致 CME 缺陷。 研究结果揭示了网格蛋白介导的贩运中以前未被识别的途径，但它仍然存在。 目前尚不清楚 AAGAB 如何识别 AP2 亚基以及 AAGAB-AP2 相互作用如何驱动 AP2 适配器组装。 在我们的初步研究中，我们表达并纯化了游离的 AAGAB 和 AAGAB:AP2 组装中间体， 并初步了解了它们的结构和功能，在这项研究中，我们将利用这些。 确定 AAGAB 控制的 AP2 适配器组件的结构和机械基础的初步数据。 我们将首先表征游离 AAGAB 和 AAGAB:AP2 组装中间体的生化特性 然后我们将使用重构的系统确定蛋白质和蛋白质复合物的原子结构。 接下来，我们将使用 X 射线晶体学和单粒子冷冻电子显微镜来验证其生理学。 最后，我们将确定使用基于细胞的遗传测定的生化和结构发现的相关性。 AAGAB 功能如何受到致病突变的影响？成功完成了这一提议。 这项研究将填补我们在膜蛋白运输方面的知识空白。 最终，这些发现将成为理解贩运适配器组装的范式。 促进针对 AAGAB 突变引起的人类疾病的新治疗策略的开发。