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/小泛素样修饰物（small ubiquitin-like modifier, SUMO）特异性蛋白酶（small ubiquitin-like modifier-specific proteases, SENPs）已被证实与前列腺癌的发生发展密切相关。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底物大多尚未明确。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，并以稳定转染的PC3前列腺癌细胞作为研究模型，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，我们共鉴定出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，我们证实USP39的SUMO化修饰位点为K6、K16、K29、K51及K73。对USP39的这些SUMO修饰位点进行突变后，其对前列腺癌细胞的增殖促进作用进一步增强。本研究获取了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研究。