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.

摘要 内吞再循环对于许多生理事件都是至关重要的，包括营养摄取、细胞运动 和极性，细胞内信号和胞质分裂。我们一直在研究一种被毛复合体 转运囊泡在再循环途径中，其涉及ACAP 1（Arfgap with Coil-coil，Ankyrin repeat和PH 结构域类型1）作为内部组分，网格蛋白作为外部包衣。最近，我们做了一个 这是一个了不起的发现，发现蛋白激酶Akt是这种外壳复合物的另一种成分。Akt 也具有直接弯曲膜的能力，这是前所未有的发现，因为已知没有激酶 拥有这种能力。我们最近的发现也使我们重新构建了由这种外套形成的囊泡 复杂.总而言之，这些发现使我们提出三个主要目标。第一，最终理解 蛋白质如何弯曲膜是通过高分辨率冷冻电子显微镜（EM）实现的。 解决蛋白质在膜上组装的结构的方法。因为这是函数形式， 外壳蛋白，我们将与一个小组合作，在EM为基础的方法， 从分子上详细阐明外壳因子如何在膜上组装成蛋白质晶格结构， 膜弯曲第二，尽管基于蛋白质的囊泡转运机制正在被强烈地 尽管已经研究过，但基于脂质的机制还远未被探索。为了解决这一根本性缺陷， 我们最近研究了囊泡重构系统，以鉴定囊泡重构所需的脂质酶。 由ACAP 1包衣复合物形成。因此，我们将阐明囊泡形成的具体阶段， 需要一种特殊的酶此外，为了更全面地了解脂质产物是如何 一个特定的酶的行为，我们将探讨是否产生脂质的几何形状影响ACAP 1囊泡 形成，以及特定的脂质几何形状是否促进ACAP 1包被因子弯曲的能力 膜的第三，我们最近对重组的ACAP 1囊泡进行了质谱分析， 涉及ACAP 1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 进一步审查，因为确认他们使用ACAP 1途径将提供特别令人信服的 支持我们的方法已经识别出ACAP 1途径的真正货物。具体地说，我们将改变 ACAP 1涂层复合体识别这些意外货物中的序列，然后确认分拣 进入ACAP 1通路被抑制。我们还将寻求一个统一的解释意外货物 使用ACAP 1途径，通过确定这种转运是否导致它们被递送到侵袭伪足， 是局部的细胞表面结构，集中了基质降解的关键因素，这是一个需要的过程， 细胞侵入组织。我们预期这些研究完成后， 对内吞再循环的基本理解，但也对生理作用提供了新的见解 通过ACAP 1途径。