Regulatory mechanisms of clathrin dependant traffic at the TGN and endosomes

TGN 和内体上网格蛋白依赖性交通的调节机制

• 批准号: 8107878
• 负责人: Mara C Duncan
• 金额: $ 27.92万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8107878
• 关键词: Adaptor Signaling Protein; Address; Affinity; Alleles; Animals; Behavior; Binding; Biochemical; Biochemical Genetics; Biochemistry; Biological Assay; Biological Models; Capsid Proteins; Cell Adhesion Molecules; Cell Fractionation; Cell Survival; Cell physiology; Cells; Cellular Assay; Clathrin; Clathrin Adaptors; Cyclic AMP-Dependent Protein Kinases; Data; Development; Disease; Endosomes; Ensure; Environment; Enzymes; Eukaryota; Event; Exclusion; Feedback; Fluorescence Microscopy; Functional disorder; Genetic Transcription; Goals; Health; Homeostasis; Human; Human Development; In Vitro; Inherited; Injury; Lead; Life; Link; Malignant Neoplasms; Mediating; Membrane; Membrane Protein Traffic; Mental Retardation; Microscopy; Mitosis; Modeling; Modification; Molecular; Molecular Biology; Mutation; Nutrient; Oranges; Organelles; Peptide Hydrolases; Phosphorylation; Play; Process; Property; Protein Isoforms; Proteins; Regulation; Risk Factors; Role; Saccharomyces cerevisiae; Schizophrenia; Signal Pathway; Signal Transduction; Staging; Structure; System; Testing; Therapeutic Intervention; Time; Work; Yeasts; adhesion receptor; cell behavior; cell motility; genome wide association study; human disease; in vitro Assay; in vivo; insight; intercellular communication; pathogen; programs; protein transport; research study; response; trafficking; trans-Golgi Network; yeast genetics

DESCRIPTION (provided by applicant): Differences in cellular behavior are central to normal human development and how injury, pathogens and mutations cause dysfunction. Many aspects of differential cellular behavior can be attributed to differences in protein localization such as reduced adhesion molecules or increased secretion of enzymes. Clathrin dependent traffic between the trans-Golgi network (TGN) and endosomes plays an important role in localization of proteins important for cell migration, cell signaling and survival. The experiments in this proposal fit into a long term goal to understand how developmental programs, mutations and cell signaling act on the traffic machinery to cause differential cellular behavior. This is the first step will be understand the molecular mechanisms leading to efficient and accurate traffic at the TGN and endosomes. It is still unclear how clathrin and its many adaptor and accessory proteins combine to provide all activities required for the complicated steps involved in traffic. These steps include selecting and concentrating cargo, generating and targeting a transport carrier in vivo. Previous identification of a network of interacting clathrin adaptors that act at TGN and endosomes has opened new avenues for understanding the molecular mechanism of clathrin dependent traffic. In this proposal, molecular mechanisms leading to fidelity in membrane traffic and a signaling pathway regulating membrane traffic in yeast will be examined. In aim1, specific hypotheses will be tested about how clathrin and adaptors ensure coats assemble at the correct membrane, that events occur in the correct order and the correct proteins are transported. Hypotheses to be tested are that 1) cooperative binding and 2) competition for space in the tightly packed clathrin coat determine when an adaptor functions. A combined approach to test these hypotheses will use in vitro biochemistry, cell fractionation, assays of membrane traffic and live-cell microscopy of specific mutations in adaptor proteins. In Aim2, a newly identified regulation of clathrin adaptors in low nutrient conditions will be investigated. Hypotheses to be tested are that 1) low nutrients inhibits membrane traffic, 2) inhibition acts at the level of adaptor modification and 3) conserved signaling pathways coordinate traffic with other cellular responses to low nutrients. PUBLIC HEALTH RELEVANCE: Mutations of clathrin adaptors are observed in cancer and inherited mental retardation, and genome wide association studies links an adaptor to schizophrenia. Understanding how mutations in adaptors lead to dysfunction at the cellular level may suggest therapeutic interventions, and determine whether adaptor alleles are risk factors for cancer, schizophrenia and other diseases.

描述（由申请人提供）：细胞行为的差异对正常的人类发育以及损伤、病原体和突变如何导致功能障碍至关重要。差异细胞行为的许多方面可归因于蛋白质定位的差异，例如粘附分子减少或酶分泌增加。跨高尔基体网络（trans-Golgi network，TGN）和内体之间的网格蛋白依赖性运输在对细胞迁移、细胞信号传导和存活重要的蛋白质的定位中起重要作用。该提案中的实验符合一个长期目标，即了解发育程序、突变和细胞信号传导如何作用于交通机制，从而导致不同的细胞行为。这是第一步，将了解导致TGN和内体高效、准确运输的分子机制。目前还不清楚网格蛋白和它的许多衔接蛋白和辅助蛋白联合收割机如何结合，以提供交通中涉及的复杂步骤所需的所有活动。这些步骤包括选择和浓缩货物，在体内产生和靶向运输载体。先前对作用于TGN和内体的相互作用网格蛋白衔接子网络的鉴定为理解网格蛋白依赖性交通的分子机制开辟了新途径。在这个提议中，将研究导致膜交通保真度的分子机制和调节酵母膜交通的信号通路。在aim 1中，将测试关于网格蛋白和衔接子如何确保外套在正确的膜上组装，事件以正确的顺序发生以及正确的蛋白质被运输的特定假设。待测试的假设是1）协同结合和2）在紧密堆积的网格蛋白外壳中对空间的竞争决定衔接子何时起作用。一个综合的方法来测试这些假设将使用体外生物化学，细胞分级分离，膜交通和活细胞显微镜的衔接蛋白的特定突变的测定。在目标2中，将研究低营养条件下网格蛋白衔接子的新鉴定的调节。待检验的假设是：1）低营养素抑制膜运输，2）抑制作用在衔接子修饰水平，3）保守的信号传导途径与其他细胞对低营养素的反应协调运输。 公共卫生关系：在癌症和遗传性精神发育迟滞中观察到网格蛋白衔接子的突变，并且全基因组关联研究将衔接子与精神分裂症联系起来。了解适配器突变如何导致细胞水平的功能障碍可能会建议治疗干预，并确定适配器等位基因是否是癌症，精神分裂症和其他疾病的风险因素。