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

我们在这项提议中的长期目标是了解内体如何区分 在单个内涵体内平衡相反的活动(循环与降解)， 尤其是关于唯一已知的内体DNA-J结构域的调节 蛋白质RME-8。我们还试图了解RME-8如何有助于 神经退行性疾病。以获得对平衡机制的新见解 在反对内体功能的情况下，我们率先使用了人的体腔细胞 线虫线虫，用于直接显示内体微域，以及 然后应用强大的分子遗传学技术来鉴定和破译后生动物- 控制内体功能和功能微域的特定机制 分离。然后，我们将这项工作扩展到哺乳动物系统。在之前的研究中，我们 鉴定出J-结构域蛋白RME-8是后生动物特有的内吞体调控子。我们 进一步表明RME-8与逆转录组份SNx-1/Snx1一起工作，并且 伴侣Hsc70，以促进线虫和线虫体内逆转录物质的循环利用 哺乳动物。最近，我们发现RME-8允许逆转录病毒负性调节 ESCRT在内体上的组装，这是防止回收和回收混合所需的过程 退化的微域。在这里，我们建议定义SNX-1如何调控RME-8， 测试RME-8激活的去抑制模型。然后我们将重点放在机械学上 关于RME-8如何通过抑制ESCRT微区扩展的问题 内体扁平网状蛋白晶格和新发现的RME-8相互作用蛋白最后， 我们使用原代小鼠神经元和体内线虫分析来破译神经元 分析一个家族性帕金森病相关等位基因揭示RME-8的功能 RME-8。我们建议定义疾病等位基因如何影响RME-8功能 内体循环和微域维持，并确定RME-8如何 调节内小体的长距离轴突运输，这是一个额外的运输步骤 内吞货物分拣所需的神经元。更好地从机制上理解 这些内小体的调节机制将在识别 与内溶酶体功能障碍相关疾病作斗争的治疗目标。

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.