Molecular Mechanisms of Endocytosis

内吞作用的分子机制

• 批准号: 7616409
• 负责人: ANDREW J BEAN
• 金额: $ 30.53万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7616409
• 关键词: Actinin; Address; Affect; Affinity; Back; Binding; Biochemical; Biochemical Pathway; Biological Assay; Cell membrane; Cells; Complex; Cues; Cytosol; Dependence; Deubiquitinating Enzyme; Differentiation and Growth; Disease; Endocytosis; Endosomes; Event; Goals; Homologous Gene; Huntington Disease; Ion Channel; Learning; Ligand Binding; Lipids; Lysosomes; Malignant Neoplasms; Measures; Mediating; Membrane; Membrane Protein Traffic; Membrane Proteins; Memory; Molecular; Movement; Multivesicular Body; Mus; Mutation; Nature; Organism; Pathway interactions; Phenotype; Physiological; Piper; Play; Principal Investigator; Process; Proteins; Reaction; Recruitment Activity; Recycling; Regulation; Relative (related person); Research Personnel; Role; Secretory Vesicles; Sorting - Cell Movement; Staging; Surface; TSG101 gene; Testing; Time; Vesicle; Virus; Yeasts; base; bean; extracellular; fly; hepatocyte growth factor-regulated tyrosine kinase substrate; insight; late endosome; membrane activity; neurodevelopment; novel; programs; protein complex; protein degradation; protein transport; receptor; reconstitution; research study; residence; response; trafficking; ubiquitin ligase

DESCRIPTION (provided by applicant): The number of receptors and their residence time on the plasma membrane are critical determinants for the response of a cell to extracellular cues and can control cellular plasticity, growth, and differentiation. Cells require endocytosis to retrieve and sort molecules from the plasma membrane as a result of the fusion of regulated and constitutive secretory vesicles. The endocytic pathway can be separated into numerous stages based on the movement of cargo and the identification of morphologically defined compartments. Early events in the endocytic process include membrane invagination and vesicle budding from the plasma membrane. Later events include transport to the late endosome and lysosome for degradation, and recycling back to various compartments. Following transport from early to late endosomes, proteins to be degraded in the lysosome are internalized into the lumen of the late endosome via membrane invagination and vesicle fission. This fission reaction results in the formation of the multivesicular body (MVB) possessing a limiting membrane and intralumenal vesicles. While much progress has been made in elucidating the molecular processes involved in early endocytic events an equally clear understanding of later events remains elusive. Hrs is an endosomal protein that interacts with proteins previously implicated in membrane trafficking. Deletion or mutation of hrs results in an enlarged endosomal phenotype in mouse, fly, and yeast suggesting that hrs may play a role in cargo sorting/endosomal trafficking. The focus of this proposal is to understand the role of hrs in the molecular mechanisms of cargo sorting at the MVB and to determine which of its many protein interactions are relevant for this function. To decipher the function of hrs and its binding partners, we propose an integrated approach using biochemical and functional experiments. First, we will analyze hrs protein interactions. We will then utilize a novel cell-free assay that measures receptor sorting into the lumen of late endosome/MVBs to elucidate requirements for sorting. Finally, we will probe the role of proteins recruited by hrs to the MVB membrane in receptor sorting and ask whether these proteins act independently or in conjunction with each other, and whether they act sequentially. A better appreciation of the mechanism of endocytic protein sorting will help in understanding learning and memory and diseases in which protein transport is disturbed like cancer, lysosomal storage disorders, and Huntingtons Disease.

描述（由申请人提供）：受体数量及其在质膜上的停留时间是细胞对细胞外提示反应的关键决定因素，并可以控制细胞可塑性，生长和分化。细胞需要内吞作用以从质膜中检索和分类分子，这是由于受调节和组成型分泌囊泡的融合而导致的。根据货物的运动和形态定义的隔室的识别，可以将内吞通路分为多个阶段。内吞过程中的早期事件包括膜的起伏和质膜的囊泡发芽。以后的事件包括运输到晚期内体和溶酶体以降解，并回收回各种隔间。从早期到后期内体运输后，将通过膜的起伏和囊泡裂变内部地将要降解在溶酶体中的蛋白质内部内部化为晚期内体的腔。这种裂变反应导致形成具有限制膜和肉内囊泡的多囊体（MVB）。尽管在阐明早期内吞事件中涉及的分子过程方面取得了很多进展，但对以后事件的同样清楚仍然难以捉摸。 HRS是一种内体蛋白，与以前与膜运输有关的蛋白质相互作用。 HRS的缺失或突变导致小鼠，Fly和酵母中的内体表型增大，这表明HRS可能在货物分类/内体运输中发挥作用。该提案的重点是了解HR在MVB处货物分类的分子机理中的作用，并确定其许多蛋白质相互作用中的哪种与此功能有关。为了破译HRS及其结合伙伴的功能，我们提出了一种使用生化和功能实验的综合方法。首先，我们将分析HRS蛋白相互作用。然后，我们将利用一种新型的无细胞测定法，该测定法测量将受体分类到后期内体/MVB的管腔中，以阐明对分类的需求。最后，我们将探测HRS在受体分类中募集的蛋白质在MVB膜上的作用，并询问这些蛋白质是独立起作用还是相互作用，以及它们是否顺序起作用。对内吞蛋白质分类机制的更好欣赏将有助于理解学习和记忆和疾病，在这些疾病中，蛋白质转运像癌症，溶酶体储存障碍和亨廷顿氏病一样受到干扰。