去泛素化酶USP4调节SMAD4蛋白单泛素化并调控TGF-β/Activin信号的研究

TGF-β signaling plays critical roles in embryonic development and the maintenance of tissue homeostasis in all metazoans. Deregulation of TGF-β activity results in cancer development. In normal and premalignant cells, TGF-β enforces homeostasis and suppresses tumor progression directly through cell autonomous regulation of apoptosis and growth arrest or indirectly through blockage of paracrine factor production in the tumor stroma. In advanced cancers, the TGF-β pathway acts as an oncogenic factor and is considered to be a therapeutic target. Deubiquitination enzymes are druggable. In previous study, we screened out USP4 as a TGF-β/Activin pathway activator. USP4 interacted with SMAD4 and could remove the mono-ubiquitin-modification from SMAD4. The mono-ubiquitination modification on SMAD4 was reported to potently inhibit transcriptional activity of SMAD complex. We hypothesized that USP4 activates both TGF-β and Activin signaling via removing the mono-Ubiquitin modification on SMAD4. We aim to identify the mechanism of which USP4 activates TGF-β/Activin pathway and figure out whether USP4 promotes TGF-β/Activin-mediated EMT (epithelial to mesenchymal transition), migration and metastasis. We also aim to build up a drug screen model targeting deubiquitination enzymes (USP4, in this study）in tumor therapy. Upon successful conclusion of this work, we will have proposed a new TGF-β/Activin regulation model and provided reference for clinical therapy in the future.

TGF-β超家族信号通路在多细胞生物胚胎发育和维持组织内稳态方面发挥着重要的作用，该通路的失调会导致癌症的发生。TGF-β抑制早期肿瘤的发生但在肿瘤发生后期却促进肿瘤的转移。因而在恶性肿瘤中该通路被视为抗肿瘤药物的作用靶点。为此，在肿瘤发展晚期特异地抑制TGF-β信号的研究显得尤为重要。去泛素化酶（DUB）是一类优良的小分子药物靶点。申请人前期通过对DUB家族的筛选发现以USP4为代表的几个去泛素化酶蛋白可激活TGF-β和Activin信号。USP4与SMAD4相互作用,很可能去除抑制SMAD4转录活性的单泛素化修饰从而同时上调TGF-β和Activin信号并促进肿瘤细胞的迁移，侵润和转移。本项目旨在阐明USP4调控肿瘤转移的分子机理和它增强TGF-β和Activin信号的原因。以此为基础搭建以DUB活性为读出的小分子药物筛选模型，为指导临床实践和以DUB活性为目的的药物筛选提供理论基础。

SMAD4是TGF-β家族细胞因子的常见细胞内效应物，但其活性受动态调节的机制尚不清楚。本项目的研究证实去泛素化酶USP4能通过去除SMAD4 K519位点抑制性的单泛素化修饰从而强烈诱导Activin/BMP信号传导。在配体诱导的调节(R)-SMAD-SMAD4复合物形成后，E3连接酶SMURF2被募集到SMAD4来触发该单泛素化修饰。尽管负调节因子c-SKI的相互作用抑制SMAD4单泛素化，但配体刺激SMURF2向c-SKI-SMAD2复合物的募集引发c-SKI泛素化和降解。因此，SMURF2在终止和启动TGF-β家族信号传导中均起作用。在USP4敲低的小鼠胚胎干细胞（mESC）中单泛素化SMAD4的增加降低了BMP-和激活素诱导的胚胎干细胞命运的变化。USP4在早期斑马鱼胚胎中激活Actin和BMP介导的形态发生事件，维持SMAD4活性。此外，敲低USP4的斑马鱼表现出有缺陷的细胞迁移和较慢的协调细胞运动，这两部分功能的改变都可以被SMAD4所回补。因此，USP4是SMAD4活性的关键决定因素。本项目不仅阐明了USP4调控肿瘤转移的分子机理和它增强TGF-β和Activin信号的原因，还以此为基础搭建以DUB活性为读出的小分子药物筛选模型，为指导临床实践和以DUB活性为目的的药物筛选提供理论基础。