Investigation of UCH-L1 interactions and functions.

泛素C末端水解酶L1(UCH-L1)是神经元中一种高度稳定、含量丰富的蛋白质，作为一种去泛素酶(DUB)，在单泛素稳态中起着关键作用。UCH-L1占大脑细胞总蛋白的1-5%，尽管其体积相对较小，但它拥有迄今发现的最复杂的三维结构之一。UCH-L1的确切功能仍然是个谜，但结节的骨架被认为可以防止蛋白酶体的展开和降解，这表明它可能在蛋白酶体破坏后从降解的蛋白质中释放的泛素的保护和回收中发挥作用。UCH-L1缺失或功能障碍的主要表型是轴索营养不良(所谓的‘死亡型’)，特别是在脊髓神经元。与此一致的是，缺乏功能性UCH-L1的细小轴突营养不良(GAD)小鼠发育正常，但随着年龄的增长表现出共济失调和进行性瘫痪，并过早死亡。这个博士项目的目的是通过使用野生型和Gad小鼠的组合，以及在脊髓背根节(DRG)神经元培养中的击倒和分子替换策略(利用我们已经拥有的UCH-L1突变体)来了解UCH-L1在轴突维持和衰老中的分子基础，以确定：1.操纵UCH-L1如何对轴突维持(例如起泡、闭塞、球体形成以及‘死亡回来’的形态迹象)产生影响。这些实验将使用共聚焦成像和微流体室来分离和直接成像轴突的稳定性和维护，通过分析标记死亡轴突的球体/闭塞。我们的目标将是将这些表型与运输赤字联系起来。2.使用定量蛋白质组学方法与蛋白质组学设施合作使用TMT标记后蛋白质组学方法与UCH-L1相互作用和/或间接调节的蛋白质，以i)比较WT和UCH-L1缺失神经元中的蛋白质丰度。这将识别由UCH-L1调控的蛋白质，ii)对本地UCH-L1进行免疫沉淀，并使用外源病毒表达的GFP-UCH-L1的GFP-Trap来定义与UCH-L1结合的蛋白质，再次使用TMT来鉴定保留的相互作用蛋白质。蛋白质点击将在单独的下拉测试中得到验证，然后根据它们在轴突中的潜在作用进行优先排序。选定的候选蛋白质在涉及轴突维持和/或降解的过程中的确切作用将通过生化、成像和功能分析来确定。由于我们已经拥有所有必要的UCH-L1工具，并在这些方法方面拥有丰富的专业知识，该学生将处于理想的位置，在揭开UCH-L1调节泛素稳态的作用机制以及生理和病理生理学作用方面取得重大进展。

Ubiquitin C-Terminal Hydrolase L1 (UCH-L1) is a highly stable, massively abundant protein in neurons that acts as a deubiquitinating enzyme (DUB) and plays a key role in monoubiquitin homeostasis. UCH-L1 accounts for 1-5% of total cellular protein in the brain and, despite its relatively small size, has one of the most complicated three-dimensional structures yet discovered. The precise functions of UCH-L1 remain enigmatic but the knotted backbone has been suggested to protect against proteasomal unfolding and degradation, suggesting a possible role in protecting and recycling of ubiquitin released from degraded proteins after proteasomal destruction. The major phenotype of UCH-L1 loss or dysfunction is axonal dystrophy (so called 'dying back'), particularly in spinal neurons. Consistent with this, gracile axonal dystrophy (gad) mice, that lack functional UCH-L1 develop normally but show ataxia and progressive paralysis as they age and die prematurely. The aims of this PhD project are to understand the molecular basis of UCH-L1 function in axonal maintenance and ageing by using combinations of wild-type and gad mice, and knockdown and molecular replacement strategies (with UCH-L1 mutants we already have) in spinal cord dorsal root ganglion (DRG) neuronal cultures to identify:1. How manipulating UCH-L1 impacts on axonal maintenance (e.g. blebbing, occlusion and spheroid formation as well as morphological signs of 'dying back'). These experiments will use confocal imaging and microfluidics chambers to isolate and directly image axonal stability and maintenance by assaying for spheroids/occlusions that mark dying axons. Our objective will be to correlate these phenotypes to transport deficits. 2. The proteins that UCH-L1 interacts with and/or indirectly regulates using quantitative proteomics approaches in collaboration with the Proteomic facility using TMT post-labelling proteomics approaches to i) compare protein abundance in WT and UCH-L1 lacking neurons. This will identify proteins which are regulated by UCH-L1 and ii) performing immunoprecipitation of native UCH-L1 and using GFP-Trap of exogenous virally expressed GFP-UCH-L1 to define proteins that bind to UCH-L1, again using TMT to identify the retained interacting proteins. The protein hits will be validated in separate pull-down assays and then prioritised according to their potential roles in axons. The precise roles of the selected candidate proteins in processes involved in axonal maintenance and/or degradation will then be defined using biochemical, imaging and functional assays.Because we already have all of the necessary UCH-L1 tools and have a wealth of expertise in these approaches the student will be ideally placed to make significant progress towards unravelling the mechanisms of action, and physiological and pathophysiological roles of UCH-L1 regulation of ubiquitin homeostasis.