去泛素化酶ataxin-3参与DNA修复通路的分子机制研究

Ataxin-3 is a member of deubiquitinating enzymes (DUBs) of the cysteine protease family. Currently, the actual physiological substrate(s) of ataxin-3 remains largely unexplored, which limits our understanding of its normal cellular function. The abnormal expansion of polyglutamine (polyQ) at the C-terminus of ataxin-3 is the pathological cause of an autosomal dominantly inherited neurodegenerative disease---Spinocerebellar ataxia type 3 (SCA3). Our preliminary results show that, for the first time, ataxin-3 functions in the DNA repair process after DNA damage. Furthermore, we have identified p53, an important executor in the early stage of DNA repair, is associated with ataxin-3 by Mass Spectrometry analysis, pull-down and immunoprecipitation experiments. DNA damage treatment results in a significantly increased interaction between these two proteins. Importantly, ataxin-3 is found to regulate the ubiquitination levels of p53 as well as p53-dependent cell death. These preliminary data have suggested that ataxin-3 plays a crucial role in the DNA damage checkpoint pathway. Therefore, in the present project, we will focus on the molecular mechanism study of how ataxin-3 functions in the DNA damage checkpoint pathway, and whether the polyQ expansion in the pathological ataxin-3 affects its normal function and thus leading to neuronal cell death. This project is going to identify a novel regulation mechanism of p53, which will certainly help us for better understanding of the pathogenic mechanism of SCA3, and for offering new potential therapeutic targets for the disease cure.

Ataxin-3是属于半胱氨酸蛋白酶家族的一种去泛素化酶。Ataxin-3蛋白C端的多聚谷氨酰胺异常延长可以导致脊髓小脑性共济失调症三型 (SCA3)。目前ataxin-3在细胞内的生理底物尚不明了，因此大大限制了我们对其正常细胞生物学功能的了解。我们的前期研究首次发现ataxin-3参与DNA损伤后的修复过程，并与DNA检查点通路中的重要蛋白p53存在相互作用。更为重要的是，ataxin-3在生理和DNA损伤情况下均可调控p53的泛素化水平和依赖于p53的细胞凋亡，提示ataxin-3在DNA损伤检查点通路中可能发挥着重要作用。因此，本项目将致力于研究ataxin-3参与DNA修复的确切分子机制，以及ataxin-3的致病突变如何影响其DNA修复功能并诱导神经细胞的丢失。预期结果将阐明一个新的调控p53的分子机制，并有助于进一步了解SCA3的发病机制，最终为SCA3的治疗提供新的靶点。

三型脊髓小脑性共济失调症（SCA3）是一种致命的常染色体遗传神经退行性疾病，ataxin-3（ATX-3）蛋白C端多聚谷氨酰胺（polyQ）异常延长是SCA3的遗传致病因素。ATX-3的生理底物尚不清楚，这限制了人们对SCA3发病机制的了解和治疗手段的研发。本项目聚焦在研究ATX-3参与DNA损伤检查点通路的确切分子机制，以及ATX-3的致病突变如何影响这一功能并诱导神经元的丢失。通过本项目的实施，我们发现并鉴定了p53是ataxin-3的一个新底物。ATX-3在生理状态下和DNA损伤情况下均可通过调控p53的泛素化水平，进而密切调节p53的稳定性、活性和功能。更为重要的是，polyQ 异常延长增强了ATX-3与p53的结合，通过上调p53的功能调节，诱导更多的晚期细胞凋亡/细胞坏死，进而导致p53依赖性的神经细胞死亡和神经退行。活体动物水平的结果也完全支持我们的结论。此项研究不仅发现了p53稳定性和活性调控的新机制，还阐明了p53在SCA3的病理发生中所起的重要作用，为了解SCA3的发病机制提供了新视角，也为SCA3的治疗提供了新靶点。此外，我们还发现了ATX-3调控其他DNA损伤应答因子（如Chk1）的相关机制。目前已经发表与该项目相关的SCI文章12篇，包括PloS Biol (2016)、Nucleic Acids Res (2017a,b，2018)、Nature Commun (2017)、J Cell Sci (2016)等。