Investigation of UCH-L1 interactions and functions.

泛素C末端水解酶L1（UCH-L1）是神经元中高度稳定、大量丰富的蛋白质，其作为去泛素化酶（DUB）起作用，并且在单泛素稳态中起关键作用。UCH-L1占大脑中总细胞蛋白质的1-5%，尽管其相对较小，但具有迄今为止发现的最复杂的三维结构之一。UCH-L1的确切功能仍然是谜，但打结的骨架已被建议，以防止蛋白酶体解折叠和降解，这表明在蛋白酶体破坏后，在保护和回收从降解的蛋白质释放的泛素中可能发挥作用。UCH-L1缺失或功能障碍的主要表型是轴突营养不良（所谓的“死回”），特别是在脊髓神经元中。与此一致，缺乏功能性UCH-L1的纤细轴突营养不良（gad）小鼠发育正常，但随着年龄的增长和过早死亡，表现出共济失调和进行性瘫痪。这个博士项目的目的是通过使用野生型和gad小鼠的组合，以及在脊髓背根神经节（DRG）神经元培养物中的敲除和分子替代策略（使用我们已经拥有的UCH-L1突变体）来了解UCH-L1在轴突维持和衰老中功能的分子基础，以确定：1.操纵UCH-L1如何影响轴突维持（例如起泡，闭塞和球体形成以及“死亡”的形态学迹象）。这些实验将使用共聚焦成像和微流体室，通过测定标记垂死轴突的球状体/闭塞来分离和直接成像轴突稳定性和维持。我们的目标是将这些表型与转运缺陷联系起来。2. UCH-L1相互作用和/或间接调节的蛋白质使用定量蛋白质组学方法与蛋白质组学设施合作，使用TMT后标记蛋白质组学方法i）比较WT和缺乏UCH-L1的神经元中的蛋白质丰度。这将鉴定受UCH-L1调节的蛋白质，和ii）进行天然UCH-L1的免疫沉淀，并使用外源病毒表达的GFP-UCH-L1的GFP-陷阱来确定与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。蛋白质命中将在单独的下拉测定中进行验证，然后根据其在轴突中的潜在作用进行优先级排序。选择的候选蛋白在轴突维持和/或降解过程中的确切作用将通过生物化学，成像和功能测定来确定。因为我们已经拥有所有必要的UCH-L1工具，并在这些方法中拥有丰富的专业知识，学生将理想地在解开作用机制方面取得重大进展，以及UCH-L1调节泛素稳态的生理和病理生理作用。

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.