Important Role of SUMOylation of Spliceosome Factors in Prostate Cancer Cells

Sentrin/SUMO (small ubiquitin-like modifier)-specific proteases (SENPs) have been implicated in the development of prostate cancer. However, due to the low abundance of SUMO-modified proteins and high activity of SENPs, the SUMO substrates affected by SENPs in prostate cancer cells are largely unknown. Here, we identified SI2, a novel cell-permeable SENP-specific inhibitor, by high-throughput screening. Using SI2 as a way of inhibiting the activity of SENPs and the SUMO stably transfected PC3 cells as a prostate cancer model, in combination with the stable isotope labeling with amino acids (SILAC) quantitative proteomic technique, we identified more than 900 putative target proteins of SUMO, in which 231 proteins were further subjected to bioinformatic analysis. In the highly enriched spliceosome pathway, we validated that USP39, HSPA1A, and HSPA2 were novel target proteins of SUMO. Furthermore, we demonstrated that K6, K16, K29, K51, and K73 were the SUMOylation sites of USP39. Mutation of these SUMO modification sites of USP39 further promoted the proliferation-enhancing effect of USP39 on prostate cancer cells. This study provides the SUMOproteome of PC3 cells and reveals that SUMOylation of spliceosome factors may be implicated in the pathogenesis of prostate cancer. Optimization of SI2 for isotype-specific SENP inhibitors warrants further investigation.Sentrin/SUMO（类泛素小修饰蛋白，small ubiquitin-like modifier）特异性蛋白酶（SENPs）已被证实参与前列腺癌的发生发展。然而，由于SUMO修饰蛋白丰度较低且SENPs活性较高，前列腺癌细胞中受SENPs调控的SUMO底物目前仍尚未明确。本研究通过高通量筛选鉴定出一种新型细胞通透性SENPs特异性抑制剂SI2。以SI2抑制SENPs活性，并以稳定过表达SUMO的前列腺癌细胞系PC3作为前列腺癌模型，结合稳定同位素标记氨基酸（stable isotope labeling with amino acids, SILAC）定量蛋白质组学技术，本研究共鉴定出900余种潜在SUMO靶蛋白，其中231种蛋白进一步开展了生物信息学分析。在高度富集的剪接体通路中，我们验证了USP39、HSPA1A及HSPA2为新型SUMO靶蛋白。此外，本研究证实USP39的SUMO化修饰位点为K6、K16、K29、K51及K73。对USP39的上述SUMO修饰位点进行突变后，可进一步增强USP39对前列腺癌细胞的增殖促进作用。本研究解析了PC3细胞的SUMO修饰蛋白质组，并揭示剪接体因子的SUMO化修饰可能参与前列腺癌的发病机制。针对亚型特异性SENPs抑制剂的SI2优化工作仍有待进一步探索。