Protein Droplets as Catalysts of Coated Vesicle Assembly

蛋白质液滴作为包被囊泡组装的催化剂

• 批准号: 9760358
• 负责人: Kasey Jill Day
• 金额: $ 6.12万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-30 至 2022-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9760358
• 关键词: Actins; Adaptor Signaling Protein; Behavior; Binding; Biochemical; Biophysical Process; Biophysics; Capsid Proteins; Catalysis; Cell Surface Receptors; Cell membrane; Cells; Clathrin; Clathrin Adaptors; Clathrin-Coated Vesicles; Coated vesicle; Complex; Cues; Data; Diabetes Mellitus; Discipline; Disease; Endocytic Vesicle; Endocytosis; Event; Goals; Health; Homeostasis; Human; In Vitro; Kinetics; Knowledge; Laboratories; Light; Liquid substance; Malignant Neoplasms; Measures; Mediating; Membrane; Membrane Protein Traffic; Microfilaments; Microscopy; Mission; Molecular; Nutrient; Outcome; Outcomes Research; Pathway interactions; Phase; Phase Transition; Play; Property; Proteins; Public Health; Receptor Signaling; Research; Role; Signal Transduction; Site; Surface; System; T-Cell Receptor; Temperature; Tertiary Protein Structure; Testing; Thermodynamics; Thinking; Time; Training; Transcription Factor AP-2 Alpha; Transmembrane Transport; Transport Process; United States National Institutes of Health; Ursidae Family; Vesicle; WNT Signaling Pathway; Work; biophysical properties; catalyst; coated pit; developmental disease; experimental study; in vivo; intersectin 1; live cell microscopy; member; mutant; optogenetics; pre-doctoral; prevent; protein complex; recruit; response; skills; solute; spatiotemporal; tool; trafficking

PROJECT SUMMARY By internalizing cell surface receptors, clathrin-mediated endocytosis enables cellular responses to external cues, regulates nutrient availability, and controls cell signaling. Clathrin-coated pits (CCPs) form at the plasma membrane through the coordinated assembly of dozens of adaptor and coat proteins into a mesh-like network that surrounds a budding vesicle. Within seconds of initiation, nascent CCPs will either mature productively into vesicles or abortively disassemble. What differentiates productive CCPs from their abortive counterparts? It has been suggested that productive CCPs pass through a critical checkpoint, but the checkpoint criteria remain poorly understood. Toward explaining the mechanism that drives CCP progression, preliminary work shows that key CCP initiator proteins, Fcho1 and Eps15, assemble together at membrane surfaces into liquid droplets. The formation of protein droplets via phase separation has recently been shown to provide spatiotemporal control over the catalysis of several pathways including membrane receptor signaling and actin filament nucleation. In a similar way, assembly of a protein droplet at the CCP could function to locally catalyze endocytosis. Fcho1 and Eps15, which are among the earliest factors to arrive at CCPs, recruit other clathrin adaptors and are collectively essential for timely CCP initiation and maturation. Importantly, these initiator proteins bear two hallmarks of phase separating proteins: multivalent interaction motifs and intrinsically disordered regions. Recently I have discovered that Fcho1 and Eps15 assemble into protein liquid droplets at membrane surfaces. This exciting result has the potential to explain both the stochastic assembly of nascent CCPs at discrete endocytic sites and the robust recruitment of a protein network to these sites. Specifically, phase separation of initiator proteins could provide a plausible mechanistic explanation for CCP initiation and maturation. Therefore, the goal of the proposed work is to understand the mechanism by which assembly of the Fcho1/Eps15 initiator proteins contributes to robust endocytosis. Work in Aim 1 will evaluate the biochemical properties of the Fcho1/Eps15 network, testing the working hypothesis that Fcho1 and Eps15 enhance recruitment of each other to the membrane through specific multivalent interactions, and thereby enhance recruitment of downstream binding partners. Work in Aim 2 will evaluate the biophysical properties of the Fcho1/Eps15 network, testing the working hypothesis that the thermodynamic and kinetic properties of Fcho1/Eps15 droplets are consistent with a phase separated system. Work in Aim 3 will evaluate the properties of the Fcho1/Eps15 network in live cells, testing the working hypothesis that the liquid-like behavior of Fcho1/Eps15 is essential for effective catalysis of CCP maturation in cells. The outcome of this research will be a characterization of the presently unknown thermodynamics and kinetics of the CCP initiator complex. More broadly, this work has the potential to introduce a new biophysical paradigm for understanding the roles of protein networks during vesicle formation events throughout the cell.

项目摘要通过内化细胞表面受体，网状蛋白介导的内吞作用使细胞 对外界信号的反应，调节营养的供应，并控制细胞信号。笼状蛋白包被坑(CCP) 通过几十个接头和外壳蛋白的协调组装在质膜上形成 围绕着萌发中的小泡的网状网络。在启动的几秒钟内，新生的CCP将要么成熟 高效地变成囊泡或流产地分解。生产的CCP与其流产的CCP的区别是什么 对应方？有人建议，生产性CCP通过关键检查点，但检查点 人们对标准仍然知之甚少。关于解释推动CCP进步的机制，初步 研究表明，关键的CCP启动子蛋白Fcho1和Eps15在膜表面组装成 液滴。最近研究表明，通过相分离形成蛋白质液滴可以提供 包括膜受体、信号转导和肌动蛋白在内的几条通路催化的时空调控 细丝成核。以类似的方式，在CCP组装蛋白质液滴可以起到局部催化的作用 内吞作用。Fcho1和Eps15是最早到达CCP的因素之一，它们招募了其他网状蛋白 适配接头是及时启动和成熟CCP的共同要素。重要是，这些引发剂 蛋白质具有相分离蛋白质的两个特征：多价相互作用基序和内在 无序的区域。最近我发现Fcho1和Eps15在10点组装成蛋白质液滴 膜表面。这一令人兴奋的结果有可能解释新生生物的随机组装 离散内吞部位的CCP以及蛋白质网络向这些部位的强劲募集。具体来说， 启动蛋白的相分离可以为CCP的启动提供一个合理的机制解释 成熟。因此，拟议工作的目标是了解 Fcho1/Eps15启动子蛋白有助于强健的内吞作用。目标1的工作将评估生化 Fcho1/Eps15网络的特性，测试Fcho1和Eps15增强的工作假设 通过特定的多价相互作用将彼此募集到膜上，从而增强 招募下游有约束力的合作伙伴。目标2的工作将评估生物物理特性 Fcho1/Eps15网络，测试工作假设，热力学和动力学性质 Fcho1/Eps15液滴与相分离体系一致。AIM 3中的工作将评估这些属性 Fcho1/Eps15网络在活细胞中的作用，检验了Fcho1/Eps15的类液体行为 Fcho1/Eps15是有效催化细胞内CCP成熟所必需的。这项研究的结果将是 目前未知的CCP引发剂络合物的热力学和动力学的表征。更多 总的来说，这项工作有可能引入一种新的生物物理范式来理解蛋白质的作用。 在整个细胞的囊泡形成过程中形成网络。