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基本信息

批准号：
10182761
负责人：
Michael Ailion
金额：
$ 20万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-02-01 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10182761
关键词：
Attention deficit hyperactivity disorder Beta Cell Binding Biochemical Biogenesis Biogenic Amines Biological Biological Assay Biological Process Blood Glucose Caenorhabditis elegans Calcium Cell Line Cells Chemicals Complex Defect Dense Core Vesicle Development Diabetes Mellitus Disease Eating Disorders Endocrine Equipment Genetic Genetic Screening Goals Golgi Apparatus Growth Factor Hormone secretion Hormones Human In Vitro Lead Mediating Membrane Mental Depression Mental disorders Metabolic Diseases Molecular Monomeric GTP-Binding Proteins Mood Disorders Nematoda Nerve Neuroendocrine Cell Neurons Neuropeptides Obesity Organelles Process Proteins Role Schizophrenia Sorting - Cell Movement Synaptic Vesicles Synaptic plasticity Testing Work blood glucose regulation golgin in vivo insight mutant neuron development neuronal survival novel therapeutics pain sensation peptide hormone rab GTP-Binding Proteins response trafficking trans-Golgi Network vesicular release

项目摘要

Project summary The dense-core vesicle is an organelle that releases peptide hormones, growth factors, and biogenic amines from neurons and neuroendocrine cells in response to increases in calcium. Dense-core vesicle cargos regulate a variety of biological processes including neuronal survival and development, pain sensation, blood glucose homeostasis, and synaptic plasticity. As a consequence, numerous diseases such as mood disorders, obesity, and diabetes are caused by defects in neuropeptide and hormone secretion. Thus, it is important to determine the molecular machinery and mechanisms that orchestrate the biogenesis and release of these vesicular carriers. However, in comparison to other vesicular compartments such as synaptic vesicles, little is known about the molecular mechanisms of dense-core vesicle biogenesis, trafficking, and release. Dense-core vesicles are generated at the trans-Golgi and gain their compartmental identity in a poorly defined maturation process that occurs post-Golgi, but few molecules have been identified that function in these processes. Cargo sorting to dense-core vesicles remains a puzzle. The long-term goal of this project is to understand the molecular mechanisms by which dense-core vesicles are formed, sort cargos, and gain their compartmental identity. A first step towards this goal is to identify molecules required for dense-core vesicle biogenesis. We performed a genetic screen in the nematode C. elegans for mutants defective in dense-core vesicle function and identified a number of new proteins that act in dense-core vesicle biogenesis, including the small GTPase RAB-2 and CCCP-1, a RAB-2 effector, as well as the EARP endosomal trafficking complex. In this project, we will test how RAB-2 and its effectors interact with EARP to mediate cargo sorting to dense-core vesicles. In Aims 1 and 2, we will use genetic, biochemical and cell biological approaches in C. elegans and in the mammalian insulin-secreting cell line INS-1 832/13 to determine how and where the RAB-2 and EARP complexes are localized, whether they physically and functionally interact, and determine their precise roles in the sorting, processing, and secretion of dense-core vesicle cargos. In Aim 3, we will use a combination of in vitro biochemical approaches and in vivo functional assays to determine how the golgin-like coiled-coil protein CCCP-1 binds membranes and functions to mediate dense-core vesicle biogenesis. These studies will advance our understanding of how dense-core vesicles are generated and sort cargos, and provide general insights into mechanisms of membrane trafficking and cargo sorting controlled by multisubunit complexes that mediate trafficking between endosomal compartments and the trans-Golgi network.

项目概要致密核心囊泡是一种释放肽激素、生长因子和生物胺的细胞器来自神经元和神经内分泌细胞对钙增加的反应。致密核心囊泡货物调节多种生物过程，包括神经元存活和发育、痛觉、血液葡萄糖稳态和突触可塑性。因此，许多疾病如情绪障碍、肥胖和糖尿病是由神经肽和激素分泌缺陷引起的。因此，重要的是确定协调这些物质的生物发生和释放的分子机制和机制囊泡载体。然而，与其他囊泡区室（例如突触小泡）相比，几乎没有了解致密核心囊泡生物发生、运输和释放的分子机制。致密核心囊泡在反高尔基体处产生，并在不明确的成熟过程中获得其区室特性发生在高尔基体后的过程，但很少有分子被发现在这些过程中发挥作用。货物对致密核心囊泡的分类仍然是一个谜。该项目的长期目标是了解形成致密核心囊泡、对货物进行分类并获得其隔室的分子机制身份。实现这一目标的第一步是确定致密核心囊泡生物发生所需的分子。我们对线虫秀丽隐杆线虫进行了遗传筛选，寻找致密核心囊泡功能缺陷的突变体并鉴定了许多在致密核心囊泡生物发生中起作用的新蛋白质，包括小 GTPase RAB-2 和 CCCP-1（RAB-2 效应子）以及 EARP 内体运输复合物。在这个项目中，我们将测试 RAB-2 及其效应器如何与 EARP 相互作用以介导货物分选至致密核心囊泡。在目标 1 和 2，我们将在秀丽隐杆线虫和哺乳动物胰岛素分泌细胞系 INS-1 832/13 以确定 RAB-2 和 EARP 的方式和位置复合物是局部的，无论它们在物理上和功能上相互作用，并决定它们在致密核心囊泡货物的分类、加工和分泌。在目标 3 中，我们将结合使用体外生化方法和体内功能测定，以确定高尔金样卷曲螺旋蛋白如何 CCCP-1 结合膜并发挥介导致密核心囊泡生物发生的作用。这些研究将增进我们对致密核心囊泡如何生成和货物分类的理解，并提供一般性信息深入了解多亚基复合物控制的膜运输和货物分类机制介导内体区室和跨高尔基体网络之间的运输。