Mechanisms of endocytic recycling

内吞再循环机制

• 批准号: 10584055
• 负责人: VICTOR W HSU
• 金额: $ 41.71万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10584055
• 关键词: Address; Affect; Ankyrin Repeat; Capsid Proteins; Cell Polarity; Cell surface; Cells; Clathrin; Collaborations; Complex; Cryoelectron Microscopy; Cytokinesis; Diglycerides; Disease; Electron Microscopy; Enzymes; Event; Geometry; Goals; Invaded; Investigation; Lipids; Mass Spectrum Analysis; Mediating; Membrane; Molecular; Monomeric GTP-Binding Proteins; Mutate; Nutrient; PH Domain; Pathway interactions; Phosphatidic Acid; Phosphotransferases; Physiological; Play; Process; Protein Kinase; Proteins; Recycling; Resolution; Role; Signal Transduction; Sorting; Structure; Surface; System; Techniques; Tissues; Validation; Vesicle; cell motility; endosome membrane; in vivo; insight; lipid phosphate phosphatase; mutant; phospholipase D2; reconstitution; uptake; vesicle transport

ABSTRACT Endocytic recycling is critical for a broad range of physiologic events, including nutrient uptake, cell motility and polarity, intracellular signaling, and cytokinesis. We have been studying a coat complex that forms transport vesicles in a recycling pathway, which involves ACAP1 (Arfgap with Coil-coil, Ankyrin repeat and PH domain type 1) acting as the inner component and clathrin as the outer coating. Recently, we have made a remarkable discovery, finding that the protein kinase Akt acts as another component of this coat complex. Akt also possesses a direct ability to bend membrane, a finding that is unprecedented, as no kinase is known to possess this capability. Our recent discovery has also led us to reconstitute vesicle formation by this coat complex. Altogether, these findings lead us to propose three major goals. First, an ultimate understanding of how a protein bends membrane is being achieved through a high-resolution cryo-electron microscopy (EM) approach that solves the structure of the protein assembled on membrane. As this is the functional form of coat proteins, we will collaborate with a group having demonstrated expertise in the EM-based approach to elucidate in molecular detail how coat factors assemble into a protein lattice structure on membrane to achieve membrane bending. Second, whereas protein-based mechanisms of vesicular transport are being intensely investigated, lipid-based mechanisms have been far less explored. Addressing this fundamental shortcoming, we have recently pursued the vesicle reconstitution system to identify lipid enzymes needed for vesicle formation by the ACAP1 coat complex. Thus, we will elucidate the specific stage of vesicle formation that requires a particular enzyme. Moreover, to achieve a more complete understanding of how the lipid product of a particular enzyme acts, we will explore whether the geometry of the produced lipid affects ACAP1 vesicle formation, and also whether a particular lipid geometry promotes the ability of the ACAP1 coat factors to bend membrane. Third, we have recently performed mass spectrometry on the reconstituted ACAP1 vesicles to implicate many cargoes of the ACAP1 pathway. To validate this finding, we will focus on unexpected cargoes for further scrutiny, as confirmation that they use the ACAP1 pathway will provide particularly compelling support that our approach has identified true cargoes of the ACAP1 pathway. Specifically, we will mutate the sequence in these unexpected cargoes recognized by the ACAP1 coat complex and then confirm that sorting into the ACAP1 pathway is inhibited. We will also pursue an unifying explanation for the unexpected cargoes using the ACAP1 pathway by determining whether this transport results in their delivery to invadopodia, which are localized cell-surface structures that concentrate key factors for matrix degradation, a process needed for cell invasion into tissue. We anticipate that the completion of these studies will not only advance a further fundamental understanding of endocytic recycling, but also shed new insights into the physiologic roles served by the ACAP1 pathway.

抽象的 内吞回收对广泛的生理事件至关重要，包括营养吸收，细胞运动 和极性，细胞内信号传导和细胞因子。我们一直在研究形成的外套配合物 在回收途径中的传输囊泡，该途径涉及ACAP1（带线圈，arnkyrin重复和pH的ARFGAP 域类型1）充当内部成分，而网格蛋白作为外涂层。最近，我们做了一个 显着的发现，发现蛋白激酶AKT充当了这种涂层配合物的另一个组成部分。 akt 还具有弯曲膜的直接能力，这是前所未有的发现，因为没有任何激酶 拥有这种能力。我们最近的发现也使我们通过这件大衣重建囊泡形成 复杂的。总之，这些发现使我们提出了三个主要目标。首先，对 蛋白质如何通过高分辨率的低分子电子显微镜（EM）实现膜弯曲 解决蛋白质结构的方法。因为这是功能形式 外套蛋白质，我们将与一个在基于EM的方法中展示专业知识的小组合作 阐明分子细节如何在膜上组装成蛋白质晶格结构以实现 膜弯曲。其次，而囊泡转运的基于蛋白质的机制是强烈的 研究的基于脂质的机制的探索程度要少得多。解决这个基本的缺点， 我们最近追求了囊泡重建系统，以鉴定囊泡所需的脂质酶 ACAP1外套复合物的形成。因此，我们将阐明囊泡形成的特定阶段 需要特定的酶。此外，要更全面地了解如何 一种特定的酶作用，我们将探索产生的脂质的几何形状是否影响ACAP1囊泡 形成，以及特定的脂质几何形状是否促进了ACAP1外壳因子弯曲的能力 膜。第三，我们最近在重构的ACAP1囊泡上进行了质谱。 暗示了ACAP1途径的许多货物。为了验证这一发现，我们将专注于意外的货物 为了进一步审查，确认他们使用ACAP1途径将提供特别引人注目的 支持我们的方法已经确定了ACAP1途径的真正货物。具体来说，我们将变异 ACAP1外套复合物认可的这些意外货物中的顺序，然后确认排序 抑制进入ACAP1途径。我们还将为意外货物提供统一的解释 通过确定这种运输是否导致其输送到Invadopia，使用ACAP1途径 是集中矩阵降解的关键因素的局部细胞表面结构，这是一个需要的过程 细胞入侵组织。我们预计这些研究的完成不仅将进一步发展 对内吞回收的基本理解，但也对所服务的生理角色有了新的见解 通过ACAP1途径。