Molecular mechanisms of coat assembly and regulation in membrane trafficking pathways

膜运输途径中外壳组装和调节的分子机制

• 批准号: 9488020
• 负责人: Lauren Parker Jackson
• 金额: $ 39.23万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9488020
• 关键词: Adaptor Signaling Protein; Affinity; Alzheimer' s Disease; Animal Model; Binding; Biochemical; Capsid Proteins; Cell Line; Cell physiology; Cell surface; Cellular Membrane; Cellular biology; Clathrin; Coat Protein Complex I; Complex; Data; Defect; Destinations; Disease; Electron Microscopy; Ensure; Goals; Health; Hereditary Spastic Paraplegia; Human; Integral Membrane Protein; Link; Lipids; Location; Malignant Neoplasms; Maps; Membrane; Methods; Molecular; Molecular Structure; Movement; Parkinson Disease; Pathway interactions; Phenotype; Physiological Processes; Proteins; Proteomics; Regulation; Spastic Paraplegia; Structural Models; Structure; Time; Tubular formation; Vesicle; Work; X-Ray Crystallography; biophysical techniques; design; experimental study; genetic regulatory protein; human disease; nervous system disorder; protein complex; protein protein interaction; protein transport; tool; trafficking; virtual

Project Summary The timely delivery of membrane-bound vesicles and tubules bearing transmembrane protein and lipid cargo to discrete cellular locations via specific trafficking pathways is fundamental to cell biology and human health. Many proteins associated with trafficking pathways are linked to serious and crippling human diseases, including neurological disorders like Alzheimer's and the hereditary spastic paraplegias. Although certain trafficking proteins and pathways are well characterized, we lack direct evidence or have only partial evidence for other pathways that we infer must exist between membranes. This constitutes an enormous gap in our current understanding of fundamental cell biology. Our goal is to elucidate the molecular structures and functions of important coat protein complexes that initiate trafficking pathways by forming coats around vesicles or tubules at specific membranes. Coat proteins recognize and package relevant cargoes, and they promote efficient assembly of additional required protein components, like SNAREs. While clathrin coats have been extensively studied, functions of certain non-clathrin coats remain virtually unknown. Increasing evidence indicates non-clathrin coats assemble using distinct mechanisms, suggesting clathrin cannot be considered a paradigm for coat assembly. We investigate non- clathrin coat complexes, including adaptor protein 4 (AP4), coat protein complex I (COPI), and retromer, by using a variety of tools to ascertain molecular mechanisms of coat assembly and regulation. Biochemical and proteomic approaches allow us to identify new components of coated structures, especially cargo molecules, accessory, and regulatory proteins. Structural methods like X-ray crystallography, NMR, and electron microscopy reveal at the molecular level how coats interact with key protein partners and allow us to map specific binding interfaces. Biophysical techniques enable us to quantify binding affinities and to probe interfaces identified in structural models. With collaborators, we use molecular data to design experiments in cultured cell lines and in model organisms to explore how a variety of protein-protein interactions drive phenotypes at the cellular and organismal levels. Ultimately, we hope to gain a molecular understanding of how non-clathrin coats assemble at distinct membranes to drive different trafficking pathways. We anticipate this work will reveal new mechanisms of cargo recognition, coat assembly, and regulation. We further aim to uncover the molecular basis of specific diseases associated with these coat proteins.

项目摘要 及时递送的膜结合的囊泡和带有跨膜蛋白和脂质货物的小管 通过特定的运输途径离散的细胞位置是细胞生物学和人类健康的基础。 许多与贩运途径相关的蛋白质都与严重和严重的人类疾病有关， 包括阿尔茨海默氏症和遗传性痉挛性瘫痪等神经系统疾病。虽然确定 贩运蛋白和途径的特征很好，我们缺乏直接证据或只有部分证据 对于我们推断的其他途径，必须存在膜之间。这构成了我们的巨大差距 当前对基本细胞生物学的理解。 我们的目标是阐明启动重要外套蛋白复合物的分子结构和功能 通过在特定膜上形成囊泡或小管周围的大衣，运输途径。外套蛋白质 识别和包装相关的货物，它们促进有效组装额外的必需蛋白 组成部分，像贪食一样。虽然已经对网状蛋白涂层进行了广泛的研究，但某些非加拉链蛋白的功能 大衣几乎是未知的。越来越多的证据表明非加拉蛋白涂层使用不同的 表明网格蛋白的机制不能被视为外套组件的范式。我们调查非 - 网格蛋白涂层配合物，包括衔接蛋白4（AP4），外套蛋白质复合物I（COPI）和替代物，通过 使用多种工具来确定外套组装和调节的分子机制。生化和 蛋白质组学方法使我们能够鉴定涂层结构的新组成部分，尤其是货物分子，，即 附件和调节蛋白。 X射线晶体学，NMR和电子等结构方法 显微镜在分子水平上揭示了涂层如何与关键蛋白质伴侣相互作用并允许我们映射 特定的绑定界面。生物物理技术使我们能够量化结合亲和力并进行探测 在结构模型中确定的接口。与合作者一起，我们使用分子数据来设计实验 培养的细胞系和模型生物中，以探索各种蛋白质 - 蛋白质相互作用如何驱动 细胞和生物水平的表型。最终，我们希望对 非加拉蛋白外套如何在不同的膜上组装以驱动不同的运输途径。我们期待 这项工作将揭示货物识别，外套组件和调节的新机制。我们进一步的目标 发现与这些外套蛋白相关的特定疾病的分子基础。