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。

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.