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.

项目摘要 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衔接子来募集货物蛋白， 内吞部位CME中的主要网格蛋白衔接子是异源四聚体AP 2衔接子复合物， 由两个大亚基（β和β）、一个中亚基（μ）和一个小亚基（μ）组成。一般 认为多聚体运输衔接子如AP 2衔接子自发组装。然而，我们最近 发现AP 2适配器组装是由α和γ适配蛋白结合控制的有序过程 蛋白（AAGAB）。没有AAGAB的帮助，AP 2衔接子无法形成，导致CME缺陷。这些 研究结果揭示了网格蛋白介导的运输中以前未被认识的途径。但委员会仍 目前尚不清楚AAGAB如何识别AP 2亚基以及AAGAB-AP 2相互作用如何驱动AP 2适配器组装。 在我们的初步研究中，我们表达并纯化了游离AAGAB和AAGAB：AP 2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AP 2适配器组装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：AP 2组装中间体的生化性质 使用重组系统。然后我们将确定蛋白质和蛋白质复合物的原子结构 使用X射线晶体学和单粒子低温电子显微镜。接下来，我们将验证 使用基于细胞的遗传分析的生化和结构发现的相关性。最后，我们将确定 AAGAB功能如何受到致病突变的影响。圆满完成本次拟 这项研究将填补我们对膜蛋白运输知识的一个主要空白。这项工作也将作为一个 这是理解一般贩运适配器组装的范例。最终，这些发现将 有助于开发用于由AAGAB突变引起的人类疾病的新治疗策略。