Ataxin-3对PCNA泛素化的调控机制及抗肿瘤耐药性的作用研究

Chemo-resistance is a burning question in cancer therapy. A certain type of cancer cells with hyperactive translesion synthesis(TLS), which is regulated by the mono-ubiquitination of PCNA, show chemo-resistance to bifunctional alkylating and platinum based drugs. Inhibition of the mono-ubiquitination of PCNA could induce hypersensitivity to these chemotherapeutic drugs. Therefore, proteins regulating the ubiquitination of PCNA could be promising targets for cancer therapy. Recently, we first identified a deubiquitinase ATX3, which enhanced the damage induced oligo/poly ubiquitination of PCNA, thereby inhibited the mono-ubiquitination of PCNA. Accordingly, we found that knockdown of ATX3 promoted the mono-ubiquitination of PCNA and lead to an increased mutation rate for cells. Everything indicated that ATX3 may be involved in DNA damage response by regulating the ubiquitination of PCNA. Next step, we project to further study how ATX3 regulates PCNA ubiquitination and the consequences of the regulation on PCNA. For ATX3 does not bind to PCNA, we need to find a molecule which mediates the interaction between ATX3 and PCNA, and make it clear how ATX3 regulates PCNA via the unknown protein. Our study will finally figure out whether ATX3 decrease the chemo-resistance by triggering template switch pathway and inhibiting translesion DNA synthesis pathway, which were both regulated by PCNA ubiquitination. Given that deubiquitinases are cell-type and substrates specific, ATX3 could be an attractive target in the design of anti-chemoresistance drugs. Our research would contribute to better understanding the mechanism of PCNA ubiquitination and may provide a novel therapeutic approach for overcoming chemo-resistance of cancer cells to a broad spectrum of drugs.

增殖细胞核抗原（PCNA）介导的跨损伤合成（TLS）是癌细胞产生获得性耐药性的机制之一，抑制PCNA单泛素化可以提高癌细胞对交联类药物的敏感性。我们前期研究发现去泛素化酶ATX3参与多种DNA损伤后修复通路，并发现ATX3增强PCNA多泛素化并抑制其单泛素化。ATX3缺失不仅升高PCNA单泛素化水平并造成细胞突变率增加。上述结果提示我们ATX3可能通过调控PCNA泛素化参与调控PCNA介导的TLS通路。本项目将在前期研究基础上进一步探索ATX3对PCNA泛素化调控的机制及作用。由于ATX3不直接与PCNA结合，我们拟筛选中间分子，探究ATX3如何通过中间分子调控PCNA泛素化。我们将最终揭示ATX3能否通过抑制PCNA单泛素化抑制TLS，从而降低肿瘤抗药性。本研究将有助于深入了解PCNA介导的损伤耐受通路，同时为开发抗癌药物提供新靶点。

的机制之一，抑制PCNA单泛素化可以提高癌细胞对交联类药物的敏感性。我们发现当采用甲基磺酸甲酯处理Hela细胞时，ataxin-3（ATX3）大大增强PCNA的多泛素化。通过免疫共沉淀和质谱分析发现了ATX3的多个结合蛋白，ATX3与PCNA并无强相互作用。其中ATX3与调控PCNA泛素化通路中的两个重要泛素连接酶（E3）Rad18和SHPRH 存在较强的相互作用，且DNA损伤后对两者的泛素化状态具有调控作用。进一步通过生化分子和细胞学实验，我们发现ATX3能与Rad18和SHPRH形成复合物，在MMS损伤后增强Rad18与SHPRH之间的相互作用，同时抑制Rad18的长泛素链形成，并促进SHPRH的多泛素化。据此，我们推测ATX3可能作为中间分子，介导了由Rad18向SHPRH的泛素链的转移，此为一种PCNA多泛素化的新调控机制。ATX3通过调控PCNA的泛素化，抑制了PCNA和Polκ到损伤位点的招募，并降低基因的突变率。为能够深入研究耐药性肿瘤细胞的治疗作用，我们设计并合成了一系列靶向肿瘤的纳米递药体系和递送DNA体系，以期通过表达polyQ-ATX3联合其他抗肿瘤方法，抑制耐药性肿瘤的生长。此项研究不仅发现了PCNA 活性和稳定性调控的新机制，还阐明了ATX3在PCNA介导的肿瘤耐受性中所起的重要作用，为开发新型耐药性肿瘤治疗的提供了新思路。