Defining the Function of RME-8 in Endosomal Regulation During Health and Disease

定义 RME-8 在健康和疾病期间内体调节中的功能

基本信息

批准号：
10565681
负责人：
Qian Cai
金额：
$ 33.79万
依托单位：
RUTGERS, THE STATE UNIV OF N.J.
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-01 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10565681
关键词：
Acute Affect Alleles Animals Architecture Area Auxins Axon Axonal Transport Binding Biological Assay Biological Process C-terminal Caenorhabditis elegans Cell physiology Cells Clathrin Clathrin Heavy Chains Complex DNA Data Diameter Disease Dynein ATPase Endosomes Equilibrium Essential Tremor Fluorescence Functional disorder Genetic Genetic Techniques Gilles de la Tourette syndrome Goals Golgi Apparatus Health Human Impairment In Vitro Individual Learning Life Link Lysosomes Maintenance Mammals Membrane Membrane Microdomains Methods Modeling Molecular Molecular Chaperones Molecular Genetics Motor Movement Mus Nematoda Neurodegenerative Disorders Neurons Organelles Organism Parkinson Disease Pathway interactions Patients Peripheral Point Mutation Process Proteins Recycling Regulation Research Role Sorting System Techniques Tertiary Protein Structure Testing Time Transmembrane Transport Variant Visible Radiation Visualization Work cell motility combat derepression dynactin experimental study in vivo in vivo Model insight light microscopy mutant nervous system disorder neuronal cell body novel prevent retrograde transport scaffold therapeutic target ultra high resolution virtual

项目摘要

Our long-term goal in this proposal is to understand how endosomes differentially balance opposing activities (recycling versus degradation) within a single endosome, especially with respect to regulation by the only known endosomal DNA-J domain protein RME-8. We also seek to understand how RME-8 contributes to neurodegenerative disease. To gain new insights into the mechanisms that balance opposing endosomal functions, we have pioneered the use of the coelomocyte cells of the nematode C. elegans for the direct visualization of endosomal microdomains, and then applied powerful molecular genetic techniques to identify and decipher metazoan- specific mechanisms that control endosome function and functional microdomain separation. We then extend this work into mammalian systems. In previous studies, we identified the J-domain protein RME-8 as a metazoan specific endosome regulator. We further showed that RME-8 functions with Retromer component SNX-1/Snx1, and the chaperone Hsc70, to promote the recycling of retromer cargo in C. elegans and mammals. Most recently, we showed that RME-8 allows Retromer to negatively regulate ESCRT assembly on endosomes, a process required to prevent mixing of recycling and degradative microdomains. Here we propose to define how SNX-1 regulates RME-8, testing a de-repression model for RME-8 activation. We then focus on mechanistic questions of how RME-8 inhibits ESCRT microdomain expansion through the endosomal flat clathrin lattice and newly identified RME-8 interacting proteins. Finally, we use primary mouse neurons and in vivo C. elegans analysis to decipher neuronal functions of RME-8 as revealed by analysis of a familial Parkinson’s associated allele of RME-8. We propose to define how the disease allele affects RME-8 function in endosomal recycling and microdomain maintenance, and determine how RME-8 regulates long-distance axonal transport of endosomes, an additional transport step in neurons required for endocytic cargo sorting. A better mechanistic understanding of these regulatory mechanisms of endosomes will be profoundly important in identifying therapeutic targets to combat diseases associated with endolysosomal dysfunction.

我们在该提案中的长期目标是了解内体如何差异化平衡单个内体内的相反活动（回收与降解），特别是关于唯一已知的内体 DNA-J 结构域的调节我们还试图了解 RME-8 蛋白如何发挥作用。获得对平衡机制的新见解。与内体功能相反，我们率先使用了体腔细胞用于直接可视化内体微域的线虫 C. elegans，以及然后应用强大的分子遗传学技术来识别和破译后生动物控制内体功能和功能微区的特定机制然后我们将这项工作扩展到哺乳动物系统中。鉴定出 J 结构域蛋白 RME-8 是后生动物特异性内体调节剂。进一步表明 RME-8 与 Retromer 成分 SNX-1/Snx1 一起发挥作用，并且分子伴侣 Hsc70，促进线虫中逆转录酶货物的回收利用最近，我们发现 RME-8 可以对Retromer 进行负调节。内体上的 ESCRT 组装，这是一个防止回收和混合的过程在这里，我们建议定义 SNX-1 如何调节 RME-8，测试 RME-8 激活的去抑制模型然后我们关注机制。 RME-8 如何通过内体扁平网格蛋白晶格和新鉴定的 RME-8 蛋白相互作用。我们使用原代小鼠神经元和体内秀丽隐杆线虫分析来破译神经元通过对家族性帕金森病相关等位基因的分析揭示了 RME-8 的功能我们建议定义疾病等位基因如何影响 RME-8 的功能。内体回收和微区维护，并确定 RME-8 如何调节内体的长距离轴突运输，这是内体的一个额外运输步骤更好地理解内吞货物分类所需的神经元。内体的这些调节机制对于识别目标是对抗与治疗性内溶酶体功能障碍相关的疾病。