DESI2的去SUMO化功能在DNA损伤应答中的作用及调控机制

DESI2 (also known as PNAS-4) has recently been identified as an important pro-apoptotic gene. We reported for the first time that DESI2 overexpression causes DNA damage, and induces S-phase arrest as well as apoptosis by activating checkpoint kinases. Furthermore, knockdown of DESI2 partially abolished cisplatin-induced DNA damage effect, implying that except for its ability for inducing DNA damage, DESI2 could also regulate DNA damage response. However, the regulating mechanism remains unclear. We further found that DESI2 has desumoylating ability, and that DNA damage induces translocation of DESI2 from the cytoplasm to the nucleus. More importantly, wild-type DESI2 enhances cisplatin-induced DNA damage effect and inhibits DNA damage repair, whereas DESI2 mutation (loss of desumoylation function) has little effect on cisplatin-induced DNA damage effect. Based on these observations, we speculate that DESI2 first cause DNA damage, which in turn promotes DESI2 to translocate from the cytoplasm to the nucleus, then DESI2 in the nucleus regulates DNA damage repair through desumoylation. However, the regualting mechanism remains to be elucidated. In this project, we will reveal the action mechanism for regulating DNA damage response by DESI2 through desumoylation. This project will provide the theoretical basis for the potentially clinical application of DESI2 in cancer gene therapy.

DESI2基因(又名PNAS-4)是参与肿瘤细胞凋亡的重要基因。我们首次报道，DESI2基因过表达引发肿瘤细胞DNA损伤、激活周期检查点激酶而导致S-期阻滞，并诱导凋亡；干扰DESI2基因则减少顺铂的DNA损伤效应。这些结果表明，DESI2除引起DNA损伤外，还对DNA损伤应答起调控作用，但机制不清楚。我们进一步研究发现，DESI2具有去SUMO化功能；DNA损伤使DESI2从细胞质转运到细胞核；而且，野生型DESI2增强顺铂的DNA损伤效应、抑制DNA修复，而其突变体(去SUMO化活性缺失)则影响不大。由此，我们推测DESI2引起DNA损伤，使其转运到细胞核，并通过去SUMO化修饰调控DNA损伤应答，但具体的调控机制有待进一步研究。本项目拟使用多种实验技术揭示DESI2的去SUMO化功能在DNA损伤应答中的作用及调控机制，为进一步探讨DESI2基因的临床应用前景提供理论依据。

DESI2基因是参与细胞凋亡的重要基因。我们首次报道，DESI2基因过表达引发肿瘤细胞DNA损伤、激活周期检查点激酶而导致S-期阻滞，并诱导凋亡；干扰DESI2基因则减少顺铂的DNA损伤效应。这些结果表明，DESI2除引起DNA损伤外，还对DNA损伤应答起调控作用，但机制不清楚。本项目探讨了DESI2的去SUMO化功能在DNA损伤应答中的作用，取得一些新的发现, (一). DESI2为一个新的去SUMO化异肽酶：(1).DESI2无SUMO1、SUMO2 及SUMO3 前体加工活性； (2).DESI2 有SUMO2/3 链的deconjugation活性; (3). DESI2具有半胱氨酸催化位点；(4). DESI2蛋白的去SUMO化异肽酶活性中心的半胱氨酸Cys108为其关键氨基酸，DESI2-C108S突变体蛋白无去SUMO化异肽酶的相关活性。(二).DNA损伤诱导下DESI2蛋白从细胞质穿梭到细胞核的现象是其发挥去SUMO化功能所必需的，并鉴定了质核穿梭的关键结构域：(1). DNA损伤诱导下野生型DESI2（ DESI2-WT）蛋白从细胞质穿梭到细胞核，而其去SUMO化功能突变体（DESI2-C108S）则无质核穿梭现象；(2). DESI2蛋白含活性中心的结构域部分【DESI2-（7-148）】是其质核穿梭的关键结构域；(三).机制上，DESI2蛋白通过对DNA损伤诱导下发生SUMO化修饰的相互作用蛋白XRCC5/XRCC6进行去SUMO化修饰，负调控DNA-PKcs参与的非同源末端连接（NHEJ）介导的DNA损伤修复，从而在DNA损伤应答中发挥重要作用；(四). DESI2蛋白的去SUMO化功能在DNA损伤试剂杀伤肿瘤细胞中发挥重要作用：(1). DESI2的去SUMO化活性增加DNA损伤试剂的体外肿瘤细胞杀伤效应，而DESI2-C108S的增敏作用则不明显; (2).野生型DESI2蛋白除了本身造成S期阻滞外，还进一步增加顺铂的S期阻滞效应；DESI2-C108S突变体的作用不如DESI2-WT；(3). 体内DESI2的去SUMO化活性显著抑制肺腺癌成瘤并增加顺铂的敏感性，而其突变体DESI2-C108S的抑瘤效应及顺铂增敏性均明显不如野生型DESI2蛋白。我们的研究初步阐明了DESI2的去SUMO化功能在DNA损伤应答中的作用及调控机制。