Identification And Functional Characterization Of SUMO Target Proteins In Prostate Cancer Cells

Prostate cancer is one of the most serious disease to man. SUMO(Small Ubiquitin-like Modifier)-specific protease 1(SENP1) has relationship with development and metastasis of prostate cancer. But the SUMO targets regulated by SENPs in prostate cancer are largely unknown. Here, we discovered a small molecule SI2 as the inhibitor of SENP1 by computer screening and activity tests of SENP1 in vitro de SUMO assay. But SI2 also suppressed the activity of SENP2 and SENP3 but not SENP5. SI2 could inhibit the activities of endogenous SENPs and increase the SUMOylated targets of endogenous SUMO1-3 or exogenous Flag-SUMO1-3, but did not affect the protein level of the unique conjugating enzyme UBC9, SENP1, SENP2 and SENP3. Utilizing SI2 as a molecular probe, and combined with the stable isotope labeling with amino acids in cell culture(SILAC) quantitative proteomic technique, we identified more than 900 putative SUMO targets, in which the SUMOylation level of 231 proteins were changed more than 1.3 fold. These 231 proteins were selected for further bioinformatic analysis and the Spliceosome pathway, Proteasome pathway, Glycolysis / Gluconeogenesis pathway, Valine, leucine and isoleucine biosynthesis pathway, Aminoacyl-t RNA biosynthesis pathway, Pyruvate metabolism pathway, Valine, leucine and isoleucine degradation pathway and Propanoate metabolism pathway were involved. The mainly enriched spliceosome pathway contained 20 proteins, involving three known SUMOylated proteins(HNRNPK, HNRNPM, DDX5) Among them, three spliceosome factors USP39, HSPA1 A and HSPA2 were confirmed as new SUMO substrates. Further experiments demonstrated that USP39 could be SUMOylated in PC3 cells and could colocalized with SUMO1 and SUMO2/3. The SUMO acceptor sites of USP39 located in RS-like domain. Further tests indicated that the K6, K16, K29, K51 and K73 were SUMO sites. SUMO could regulate the function of USP39 in prostate cancer cells. Overexpression of the wild type USP39(USP39 WT) in prostate cancer cells PC3, the cells growed more faster than vector stably transfected PC3 cells. When the SUMO sites mutated USP39(USP39 SM) was overexpressed in PC3 cells, the growth effect were enhanced compared with the USP39 WT.In this study, we provided a leading compound for further developing more specific inhibitors of SENPs. And we also provided more than 900 putative SUMO targets for studying the mechanism of SENPs affecting prostate cancer. In addition, we discovered SUMO could regulate spliceosome pathway. Moreover, the spliceosome factor USP39 and its SUMOylation could affect the growth of prostate cancer cells. These would provide a view to treat prostate cancer patients or a new target for therapy.