Molecular Mechanisms that Regulate Lysosomal Protein Transport

调节溶酶体蛋白质转运的分子机制

基本信息

批准号：
8319784
负责人：
Anjon Audhya
金额：
$ 6.85万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8319784
关键词：
Adaptor Signaling Protein Address Affinity Animals Binding Binding Proteins Biochemical Biological Assay Biological Models Caenorhabditis elegans Cataloging Catalogs Caveolins Cell Surface Receptors Cell membrane Cell surface Cells Clathrin Complex Coupled Cues Cytoplasmic Protein Defect Degradation Pathway Development Disease Down-Regulation Embryo Embryonic Development Endocytosis Endosomes Engineering Eukaryotic Cell Event Exhibits Fertilization Fluorescence Microscopy Future Gene Mutation Genetic Goals Hormone Receptor Hormones Human Development Huntington Disease Hydrolase Immune System Diseases In Vitro Integral Membrane Protein Intervention Investigation Lead Life Location Lysosomes Malignant Neoplasms Mammalian Cell Measurement Mediating Membrane Membrane Protein Traffic Membrane Proteins Microscopy Modeling Modification Molecular Monitor Movement Neurodegenerative Disorders Oocytes Parkinson Disease Pathway interactions Phosphorylation Play Process Protein Binding Proteins RNA Interference Receptor Cell Recruitment Activity Regulation Research Role Signal Transduction Site Sorting - Cell Movement Specificity System Technology Testing Transgenic Organisms Ubiquitin Vesicle base genetic manipulation insight intracellular protein transport lysosomal proteins multicatalytic endopeptidase complex oocyte maturation prevent protein complex protein function protein transport public health relevance reconstitution research study stoichiometry therapeutic target trafficking

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to define molecular mechanisms that regulate the trafficking of integral membrane proteins to the lysosome for degradation. Recent evidence indicates that modification of transmembrane proteins by ubiquitin is sufficient for protein sorting into this pathway. The ESCRT machinery, a set of conserved endosomal protein complexes, is proposed to directly bind to ubiquitinylated membrane proteins and govern their entry into vesicles that bud into the lumen of specialized multivesicular endosomes (MVEs). This process is particularly important for the downregulation of hormone receptors to prevent constitutive signaling, which can lead to developmental abnormalities and disease. How the ESCRT machinery coordinates the efficient capture and transport of ubiquitinylated substrates to MVEs will be addressed in this proposal. The C. elegans germline and early embryo are powerful model systems to study membrane dynamics in an intact, developing animal. Specific proteins can be efficiently depleted from oocytes using RNA interference. Additionally, oocyte maturation and fertilization reproducibly trigger the internalization and degradation of multiple transmembrane proteins, providing an ideal, physiologically relevant system for studying lysosomal protein transport. C. elegans is highly amenable to genetic manipulation and can be engineered to stably express fluorescently tagged proteins, including cell surface receptors that can be monitored by live cell microscopy. Taking advantage of this unique combination of attributes, the specific aims of this proposal are: 1) to determine mechanisms by which the ESCRT machinery recognizes substrates, 2) to define the role of PTH-2, a newly discovered ESCRT-0 binding protein, and 3) to define mechanisms that regulate cargo entry into the ESCRT pathway. Our preliminary genetic and biochemical studies have uncovered new components of the lysosomal transport pathway that associate with the ESCRT machinery. The significance of these interactions will be tested using a combination of fluorescence microscopy-based functional assays, biophysical measurements, and in vitro reconstitution experiments. These studies will provide a framework for future investigation into highly related pathways in mammalian cells. PUBLIC HEALTH RELEVANCE: The directed movement of proteins and membranes between different cellular locations is a fundamental process required for the proper functioning of all eukaryotic cells. Many diseases including cancer, neurodegenerative disorders such as Parkinson's disease and Huntington's disease, and immune dysfunction can be caused by intracellular protein transport defects. The proposed research will determine how membrane trafficking pathways are appropriately regulated, enhancing our fundamental understanding of this process, which should facilitate the future identification of therapeutic targets for disease intervention.

描述（由申请人提供）：该提案的长期目标是定义分子机制，以调节整体膜蛋白的运输到溶酶体降解。最近的证据表明，泛素修饰跨膜蛋白足以使蛋白质分类为该途径。提议ESCRT机械是一组保守的内体蛋白复合物，它直接与泛素化的膜蛋白结合，并控制其进入囊泡中的囊泡，这些囊泡芽到专门的多囊体内体（MVE）的腔内。对于防止构型信号传导的激素受体下调，这一过程尤其重要，这可能导致发育异常和疾病。该提案将如何解决ESCRT机械如何协调泛素化底物向MVE的有效捕获和运输。秀丽隐杆线虫种系和早期胚胎是研究完整的动物中膜动力学的强大模型系统。使用RNA干扰可以有效地从卵母细胞中耗尽特定的蛋白质。另外，卵母细胞成熟和受精可重复触发多种跨膜蛋白的内在化和降解，为研究溶酶体蛋白转运提供了理想的生理相关系统。秀丽隐杆线虫非常适合基因操纵，可以设计以稳定地表达荧光标记的蛋白质，包括可以通过活细胞显微镜监测的细胞表面受体。利用这种属性的独特组合，该提案的具体目的是：1）确定ESCRT机械识别底物的机制，2）定义PTH-2（一种新发现的ESCRT-0结合蛋白的作用），以定义调节Cargo进入ESCRT PATH的机制。我们的初步遗传和生化研究发现了与ESCRT机械相关的溶酶体传输途径的新组成部分。这些相互作用的重要性将通过基于荧光显微镜的功能测定，生物物理测量和体外重构实验的组合进行测试。这些研究将为将来研究哺乳动物细胞中高度相关的途径提供一个框架。公共卫生相关性：不同细胞位置之间蛋白质和膜的定向运动是所有真核细胞正确功能所需的基本过程。许多疾病，包括癌症，神经退行性疾病，例如帕金森氏病和亨廷顿氏病以及免疫功能障碍，可能是由细胞内蛋白质转运缺陷引起的。拟议的研究将确定如何适当调节膜运输途径，从而增强我们对这一过程的基本理解，这应该促进未来对疾病干预治疗靶标的识别。