E3泛素连接酶ARF-BP1新的底物筛选及其调节细胞增殖和肿瘤发生的机制研究

项目名称： E3泛素连接酶ARF-BP1新的底物筛选及其调节细胞增殖和肿瘤发生的机制研究

项目编号： No.31501114

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 生物科学

项目作者： 侯静静

作者单位： 厦门大学

项目金额： 20万元

中文摘要： 泛素化调控的蛋白降解失调会导致包括癌症在内的许多重大疾病。ARF-BP1是一个重要的HECT家族E3泛素连接酶，它通过调控不同底物参与多种信号通路调节，包括基因转录、DNA损伤应答、细胞增殖和细胞凋亡等。然而对ARF-BP1功能的机制研究还很不清楚。前期工作我们筛选出ARF-BP1一个新的结合蛋白TIP60。通过泛素化实验和蛋白半衰期检测，发现TIP60是ARF-BP1一个新的底物。当在U2OS和H1299细胞中用shRNA敲低ARF-BP1时，引起了细胞凋亡和细胞周期阻滞；而同时敲低TIP60，细胞凋亡和周期阻滞则显著减弱了，说明二者存在调控关系；推测缺失ARF-BP1使TIP60水平升高，激活了ATM-CHK-p53细胞监控点及细胞凋亡。本课题将从分子、细胞、小鼠水平结合临床标本，系统研究ARF-BP1调控TIP60稳定性及细胞增殖和凋亡的分子机理，并为肿瘤的防治提供潜在的理论依据。

中文关键词： E3泛素连接酶；细胞凋亡；细胞周期调控；细胞增殖；DNA损伤信号通路

英文摘要： Defects in the ubiquitin-dependent protein degradation system have been shown to be involved in pathogenesis of many human diseases including cancer, and the E3 ubiquitin ligases play critical roles. ARF-BP1 (ARF binding protein 1) is a recently identified HECT domain-containing E3 ubiquitin ligase which is implicated in multiple cellular signaling pathways, including transcriptional regulation, DNA damage response, cell proliferation and cell apoptosis. However, the underlying molecular mechanisms remain obscure. In this project, using Yeast Two-hybrid Screen we identified TIP60 as a novel binding protein of ARF-BP1. In vivo ubiqutination assay revealed that TIP60 is a new substrate of ARF-BP1. Consistent with this, overexpression of ARF-BP1 decreased TIP60 protein level; In contrast, knocking down ARF-BP1 increased cellular TIP60 level. Furthermore, we showed that, in in p53-wild U2oS cells and p53-defect H1299 cells, knocking down ARF-BP1 induces apoptosis and G2/M cell cycle arrest, respectively. This might be due to without ARF-BP1 the activity of TIP60 is enhanced. To verify this hypothesis, we co-depleted ARF-BP1 and TIP60. Downregulation of TIP60 abrogated the apoptosis of U2oS cells and reversed the G2/M arrest and cell growth inhibition of H1299 cells. These results further support that TIP60 lies downstream of ARF-BP1 and is an important substrate of ARF-BP1. In future study, we will further investigate the mechanisms by which ARF-BP1 regulates TIP60 activity at the cellular and molecular levels and using mouse model and clinical samples to validate the role of ARF-BP1 and TIP60 in tumorigenesis. Our study will help to understand the important function of ARF-BP1 and TIP60 in cell proliferation, apoptosis and tumorigenesis, and provide further knowledge for early diagnosis and therapeutic treatment of related cancers using this E3 ubiquitin ligase as a target.

英文关键词： E3 ubiquitin ligase;apoptosis;cell cycle control;cell proliferation;DNA damage signaling pathway