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 内吞再循环对于广泛的生理事件至关重要，包括营养吸收、细胞运动 以及极性、细胞内信号传导和胞质分裂。我们一直在研究形成的外套复合物 循环途径中的运输囊泡，涉及 ACAP1（Arfgap 与 Coil-coil、Ankyrin 重复序列和 PH） 结构域类型 1) 作为内部成分，网格蛋白作为外部涂层。最近，我们做了一个 一项了不起的发现，发现蛋白激酶 Akt 是该外壳复合物的另一个组成部分。阿克特 还具有直接弯曲膜的能力，这是前所未有的发现，因为目前还没有激酶可以弯曲膜。 具备这个能力。我们最近的发现还使我们能够通过这层外壳重建囊泡的形成 复杂的。总而言之，这些发现使我们提出了三个主要目标。一、最终的认识 通过高分辨率冷冻电子显微镜 (EM) 实现蛋白质如何弯曲膜 解决膜上组装的蛋白质结构的方法。因为这是函数形式 外壳蛋白，我们将与一个在基于 EM 的方法方面具有专业知识的团队合作 从分子细节阐明外壳因子如何组装成膜上的蛋白质晶格结构以实现 膜弯曲。其次，虽然基于蛋白质的囊泡运输机制正在被广泛研究。 研究表明，基于脂质的机制的探索要少得多。为了解决这个根本性的缺陷， 我们最近研究了囊泡重建系统来鉴定囊泡所需的脂质酶 由 ACAP1 外壳复合物形成。因此，我们将阐明囊泡形成的特定阶段 需要一种特殊的酶。此外，为了更全面地了解脂质产物如何 特定酶的作用，我们将探讨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 ACAP1 外壳因子弯曲的能力 膜。第三，我们最近对重组的 ACAP1 囊泡进行了质谱分析，以 涉及 ACAP1 途径的许多货物。为了验证这一发现，我们将重点关注意外货物 进行进一步审查，因为确认他们使用 ACAP1 途径将提供特别令人信服的证据 支持我们的方法已经确定了 ACAP1 途径的真正货物。具体来说，我们将改变 这些意外货物中的序列被 ACAP1 外壳复合物识别，然后确认排序 进入 ACAP1 途径被抑制。对于意外的货物我们也会寻求统一的解释 使用 ACAP1 途径确定这种运输是否导致它们递送至侵袭伪足，这 是局部细胞表面结构，集中了基质降解的关键因素，这是基质降解所需的过程 细胞侵入组织。我们预计这些研究的完成不仅将进一步推进 对内吞再循环的基本理解，同时也对所服务的生理作用提供了新的见解 通过 ACAP1 途径。