NEDD8激活酶UBA3在干细胞多能性获得与维持中的作用机制

The reprogramming process serves as an ideal platform for understanding cell fate decision. Ubiquitin and Ubiquitin like modifications play important roles in various biological processes via affecting the activity and stability of the targets. However, the systematical understanding of the role of ubiquitin and ubiquitin like modifications in pluripotency acquisition and maintenance is extremely lacking. We have established pluripotency research platform based on iPS mice, and noticed the importance of ubiquitin modification in pluripotency regulation. In recent studies, we performed functional screening of E1s of ubiquitin and ubiquitin-like modifications, found that UBA3 which serves as the E1 of NEDD8 negatively regulated the iPSC derivation and influence the protein level of pluripotency associated genes. Then we performed the proteome and ubiquitinated proteome analysises of Uba3 knock-down ESCs. Based on these studies, we want to further examine the function of the enzymes involved in the NEDD8 modification in reprogramming, pluripotency maintenance and differentiation, find the substrate of NEDD8 modification in ESCs using pull down, explore the downstream proteins and pathways by analyzing the substrates together with the proteome data, and then finally construct the regulation network associated with NEDD8 modification in pluripotency acquisition and maintenance. Thus, provide new clues for understanding the molecular mechanism of reprogramming and NEDD8 modification.

体细胞重编程是探索细胞命运决定机制的理想平台。泛素化和类泛素化通过影响蛋白活性和稳定性在各生理病理过程具有重要功能，而在干细胞多能性获得与维持中缺乏系统认识。申请人已成功建立基于iPS小鼠的多能性研究平台，并初步发现泛素化修饰在多能性调控中发挥作用。近期研究对泛素化和类泛素化的E1酶进行功能筛选，发现NEDD8激活酶UBA3负调控iPSC形成、影响ESC多能性相关蛋白水平，进而初步分析了UBA3敲低的ESC蛋白质组和泛素化蛋白质组。基于此，拟在重编程及ESC多能性和分化中研究NEDD8修饰相关酶的功能，利用Pull-down寻找NEDD8的修饰底物，结合蛋白质组数据分析下游蛋白和通路，解析它们在重编程及ESC多能性和分化中的功能，并建立敲除小鼠模型对关键蛋白进行体内功能研究，以此构建NEDD8修饰在干细胞多能性获得与维持中的调控网络，为理解多能性调控及NEDD8修饰的作用方式提供科学依据。

泛素化和类泛素化通过影响蛋白活性和稳定性在各生理病理过程具有重要功能，而在干细胞干性获得与维持中缺乏系统认识。本项目通过对类泛素化E1酶的表达和功能筛选，发现NEDD8修饰E1酶编码基因Uba3的敲低能有效提高iPS形成效率，从Nedd8最主要的修饰底物Cullin蛋白家族中筛选到Cul5能够影响重编程效率。利用SILAC对比分析Uba3敲低前后的蛋白质组变化，我们找到了细胞应激通路，进而找到该通路的Chek2和Mtr是Uba3影响重编程的重要下游因子，并通过一系列联合作用功能实验，基本完成了UBA3-CUL5-CHEK2/MTR的调控链，揭示了类泛素化NEDD8修饰在重编程中的作用和机制，为理解重编程机制及NEDD8在干性调控中的作用方式提供科学依据。此外，卵母细胞的母源因子和早期胚胎发育过程对于理解细胞命运决定和体细胞重编程具有重要指导意义，我们发现并阐释了母源因子Surf4通过激活细胞内质网应激反应在重编程中起到的积极作用，揭示了小鼠合子胚胎中雌雄原核的组蛋白不对称分布和修饰机制，利用不同研究系统为理解干性建立和维持机制提供助力。