去泛素化酶USP45调节溶酶体功能和细胞自噬过程的机制研究

Deubiquitinating enzymes (DUBs) are proteases that cleave ubiquitin from ubiquitin precursor or target proteins. Like ubiquitination, deubiquitination by DUBs is a highly regulated process that has been implicated in numerous cellular functions, including cell cycle regulation, proteasome-and lysosome-dependent protein degradation, gene expression, DNA repair, and kinase activation. Mutations in several DUBs have been linked to disease ranging from cancer to neurodegeneration. The physiological role of most DUBs is poorly defined despite considerable research efforts. In a forward mosaic screen in Drosophila designed to identify genes required for lysosomal function and productive autophagy, we identified one mutant of deubiquitinase Usp45, as cancer genome analysis has revealed that ~16.7% of diffuse large B-cell lymphoma and ~12% of prostate cancers display deletions, and 37.9% of breast cancer patient xenografts display amplifications in the USP45 gene. Here, we plan to further study the regulation mechanism of Usp45 on lysosomal function and proper autophagy in fly model and culture cells. At the same time, we will also use mouse model，and generate the Usp45-/- mice via the CRISPR/Cas9 system to examine if the regulation function of Usp45 is conserved in mammalians. Furthermore, we will make a preliminary exploration on its relationship with neurodegeneration and cancers. The study of the DUB Usp45 will help us to better understand the regulation of the ubiquitin system on cellular homeostasis, and benefit to the identification of novel diagnostic tools, therapeutic approaches and potential drug targets to the related diseases.

去泛素化酶介导的去泛素化过程受到严密的调控，参与了许多细胞生物学过程，包括细胞周期调控，蛋白酶体和溶酶体依赖的蛋白降解，基因表达，DNA修复和激酶激活。但是，大多数去泛素化酶的生理功能仍不清楚。在一个果蝇正向遗传筛选中，我们发现了一个去泛素化酶Usp45是维持溶酶体功能和细胞自噬过程所必须的。癌症基因组分析显示,有~16.7%的弥漫大B细胞淋巴瘤和~12%的前列腺癌中都存在USP45的缺失。我们将进一步利用果蝇和小鼠模型及体外细胞系分析去泛素化酶USP45在体内对溶酶体功能的调节进而影响细胞自噬过程和胞吞作用的分子机制，系统阐述其生理功能。同时，我们也将对其与肿瘤和神经退行性疾病的关联进行初步的探索。该项研究将完善高等真核细胞中泛素信号系统在维系细胞稳态平衡中所发挥的调节作用,为相关疾病今后诊断工具, 治疗手段和药物靶向的鉴别和开发提供实验和理论依据以及新的思路。

自噬是在进化上高度保守并受到多途径严密调控的细胞生物学过程，对于细胞稳态的维持至关重要，与许多人类疾病的发生发展密切相关。随着细胞自噬调节机制研究的不断深入，越来越多的去泛素化酶被证明在自噬相关的泛素信号调控系统中发挥了重要的作用。本项目发现一个新的编码果蝇去泛素化酶USP45的基因CG4165发生突变会引起果蝇脂肪小体细胞自噬障碍和溶酶体功能障碍，并导致果蝇幼虫视觉神经盘中神经细胞分化的缺陷。USP45在高等哺乳动物中的各组织中是广泛表达的，我们利用USP45基因敲除小鼠模型验证了USP45调节细胞自噬的功能。在体外系统中，细胞过表达USP45会增强细胞自噬流；而且，我们发现USP45能与p62/SQSTM1相互作用，并且在饥饿诱导自噬条件下，两者的相互作用会得到增强，揭示了USP45通过与p62/SQSTM1相互作用促进其自噬活性，来正向调节细胞自噬过程的分子机制。我们通过对癌症基因组学公共数据库进行检索以及对一系列癌症细胞系进行细胞自噬流和USP45表达的检测，发现USP45可能通过调节细胞自噬来影响肿瘤的发生发展以及预后情况。本项目进一步补充了泛素信号系统对细胞自噬所发挥的重要调节作用，为理解细胞自噬相关疾病，如神经退行性疾病和癌症等的发生和发展及其靶向治疗提供了新的思路。