癌基因DDA1促进p21蛋白的泛素-蛋白酶体降解的分子机制研究

The ubiquitin-proteasome degradation pathway plays an important role in maintaining the intracellular protein homeostasis and the pathogenesis of major diseases such as malignant tumors. The DDA1 gene, which is highly conserved among different species, is a novel gene that we have screened with cDNA libraries. We found that DDA1 is highly expressed in several cancers including lung cancer, colon cancer. It is a subunit of CRL4-E3 ubiquitin ligase complexes and regulates ubiquitinated protein degradation. These targets are involved in the regulation of cell cycle-related proteins, proliferation and it can promote tumor growth with the characteristics of oncogene. In our previous study, we found that knocking out DDA1 in lung cancer cells leads to accumulation of p21 protein, cell cycle arrest, senescence, and polyploidy, because DDA1 promotes the poly-ubiquitylation and degradation of p21 protein, but how DDA1 promotes ubiquitination and degradation of p21 protein is unclear. In this project, Quantitative proteomics of whole ubiquitinated protein, functional domain of protein and amino acid point-mutation, multiplexed immunohistochemistry and CoIP will be used to clarify the direct target of DDA1 and its regulatory mechanism of ubiquitination of p21 protein. The mechanism of degradation and how to regulate the cell cycle and growth of tumors will lay a foundation for further understanding of the function of DDA1 gene.

泛素蛋白酶体降解途径在保持胞内蛋白稳态以及恶性肿瘤等重大疾病的发病过程中起重要作用。DDA1是我们筛选cDNA文库得到的新基因，它在不同物种中序列高度保守，我们发现它在肺癌、结肠癌等肿瘤中高表达，参与细胞增殖、周期的调控并促进肿瘤生长，具有癌基因特征，有研究发现DDA1参与形成CRL4-E3泛素连接酶复合物并调控蛋白降解。我们进一步利用全蛋白泛素化修饰定量蛋白质组学技术等，发现在肺癌细胞敲除DDA1会导致重要的细胞周期蛋白依赖性激酶抑制剂p21蛋白积累、细胞周期阻滞并产生多倍体，原因可能是DDA1促进p21泛素化修饰并被蛋白酶体降解，但DDA1如何调控p21蛋白泛素化降解尚不清楚，也未见文献报导。本课题将开展蛋白片段缺失和氨基酸定点突变、CoIP、泛素化蛋白富集等研究，希望能阐明DDA1的直接作用靶点和如何调控p21蛋白泛素化降解以及肺癌细胞周期和生长，为深入理解DDA1基因功能奠定基础。

经过课题组成员3年的研究，我们已基本完成了该课题的研究目标，阐明了DDA1基因调控p21泛素化修饰并降解的分子机制，相关原创性研究结果正在投稿中，该论文将标注的第一且唯一的基金号是该课题编号（81902822）。在该课题的资助下，我们首次在肺癌细胞中阐明了敲除DDA1后会导致p21蛋白积累、细胞周期阻滞及多倍体的形成，DDA1表达水平与p21的表达水平呈负相关，原因是DDA1可促进p21 蛋白的多聚泛素化修饰并被泛素蛋白酶体降解。经过对p21的点突变研究发现，DDA1影响p21多个氨基酸残基的泛素化修饰。被修饰氨基酸突变后，p21的泛素化水平降低，降解受到抑制，蛋白丰度增加。本课题研究将为未来肺癌以及其他肿瘤的治疗提供了新的思路，为DDA1基因的功能的相关研究提供参考。