鉴定调控ULK1泛素化的新分子及其在调节自噬中的功能

ULK1 is essential for autophagy initiation. It plays a key role in mitophagy and starvation-induced autophagy. Post-translational modifications of ULK1 are essential for its biological function. However, major signaling pathways modulating the steady state of ULK1 is largely unknown. We previously reported that ULK1 underwent K48-linked polyubiquitination. We found p32 is critical for maintaining the steady state of ULK1 by inhibiting its K48-linked polyubiquitination and the subsequent proteasome-mediated degradation. The deubiquitinating enzyme (DUB) that can modulate the polyubiquitination of ULK1 is unknown. We have constructed 85 DUB expression constructs and identified DUB-X as a binding partner of ULK1. We further demonstrated that DUB-X may influence the K48-linked polyubiquitination of ULK1 and its protein stability. Moreover, our preliminary data indicated that DUB-X depletion resulted in autophagy defect. In this application, we will elucidate the detailed molecular mechanisms underlying the regulation of ULK1 by DUB-X and investigate how DUB-X-ULK1 axis controls autophagy and mitophagy. We will further determine the importance of DUB-X-dependent autophagy in cell survival and tumorigenesis. The correlation between DUB-X and ULK1 in clinical tumor samples will be examined. These studies will lead to the discovery of a novel signaling pathway centered by ULK1 regulating autophagy and mitophagy under stress conditions.

ULK1是调控自噬起始的核心元件，对饥饿诱导的自噬和线粒体自噬至关重要。ULK1的蛋白翻译后修饰对其功能活性至关重要，然而调控其蛋白稳定性的关键通路只有零星报道。我们前期率先报道ULK1蛋白K48位泛素化修饰，发现p32通过抑制K48位泛素化修饰正向调控ULK1稳定性，揭示p32-ULK1是调控自噬的关键通路。调控ULK1的去泛素化酶（DUB）至今尚无报道。我们前期构建85个已知的DUB表达载体，初筛发现DUB-X能够结合ULK1，并证明该分子调控ULK1的泛素化水平和蛋白稳定性，同时还发现DUB-X对自噬有重要影响。本申请书将重点研究DUB-X调控ULK1泛素化的分子机理，明确DUB-X-ULK1主轴调控自噬的机制，揭示该通路的下调在调控细胞存活以及肿瘤发生中的功能；还将探讨 DUB-X-ULK1主轴与临床肿瘤的相关性。以上工作将首次揭示DUB-X-ULK1主轴在调控自噬中的核心地位。

原课题拟研究“鉴定调控ULK1泛素化的新分子及其在调节自噬中的功能”，项目实施过程中，该工作被其他研究小组先发表 (Kim et al. EMBO Reports, 2018)，于是调整课题。.N6-甲基腺嘌呤(m6A)是真核生物RNA最常见的一种RNA转录后修饰，RNA甲基化修饰约占RNA修饰的60%以上。METTL3是m6A甲基转移酶复合物中负责催化甲基化反应的甲基转移酶。目前已有大量研究证明METTL3通过调节基因表达，蛋白翻译等过程调控细胞内多种生命活动，m6A介导的表观转录修饰与生理病理过程息息相关。然而，关于METTL3自身的翻译后修饰及其表达调控的机制研究目前仍是空白领域。我们课题组聚焦于METTL3的乙酰化修饰，包括鉴定METTL3的乙酰基转移酶AT-1，通过构建一系列突变表达载体，发现AT-1对METTL3 KX和KY位点进行乙酰化修饰；发现药物compound-3能够在体内和体外诱导METTL3的乙酰化修饰；利用昆虫杆状病毒包装系统在体外重构METTL3的功能复合体，发现乙酰化修饰并不影响METTL3甲基转移酶活性, 但是改变其细胞内定位并降低总体m6A水平。同时我们发现METTL3一旦发生乙酰化修饰，能够显著抑制乳腺癌细胞的体外迁移侵袭能力。这些原创性发现揭示乙酰化修饰在METTL3细胞内定位以及作为甲基转移酶调控m6A水平中的重要地位，我们进一步发现肿瘤微环境能够抑制METTL3的乙酰化修饰，从而促进肿瘤的转移能力。我们将利用乳腺癌肺转移的体内转移模型，论证METTL3乙酰化对肿瘤转移的调控能力以及Compound-3通过促进METTL3乙酰化抑制肿瘤转移的表型和机制。以上数据目前在整理中，力争2021年完成后续的肿瘤微环境调控METTL3乙酰化的机制研究和动物实验，即可撰文投稿。