前列腺癌细胞SUMO化蛋白的发现和功能研究

发布时间：2018-04-01 12:34

本文选题：前列腺癌　切入点：SUMO　出处：《上海交通大学》2014年博士论文

【摘要】：前列腺癌是影响男性健康的主要疾病之一,SUMO特异性蛋白酶SENP1与前列腺癌的发生和转移等密切相关,但前列腺癌细胞内SUMO修饰底物目前并不清楚。本研究通过计算机虚拟筛选和体外及细胞内酶活性测试,发现小分子化合物SI2是SENP1的抑制剂,同时SI2还能抑制SENP2和SENP3的活性,但对SENP5的活性无明显抑制。SI2作用于前列腺癌PC3细胞,使内源SUMO修饰的底物发生累积,但并不影响SENP1,SENP2和SENP3以及SUMO接合酶UBC9的蛋白水平。SI2不仅能增加内源SUMO修饰底物,也能增加外源转染的Flag-SUMO修饰的底物。我们利用SI2为分子探针结合细胞培养稳定同位素标记氨基酸技术,在PC3细胞内鉴定出900多个潜在的SUMO的底物,其中有231个底物在SI2处理后SUMO化水平升高?1.3倍。我们对此231个蛋白进行信号通路分析,发现它们至少参与到剪切体、蛋白酶体、糖酵解、支链氨基酸的合成、氨基酰-t RNA的生物合成、丙酮酸代谢、支链氨基酸的降解和丙酸代谢等八个通路中。剪切体通路中共富集了20个蛋白,其中HNRNPK,HNRNPM,DDX5是已报道的SUMO底物。同时,我们通过体外SUMO修饰实验验证了此通路中的USP39,HSPA1A和HSPA2是新的SUMO底物。除在体外SUMO反应中能够检测到USP39的SUMO化修饰,在PC3细胞内也能检测到USP39的SUMO化修饰。通过免疫荧光检测,我们发现USP39能够与SUMO1和SUMO2/3共定位。进一步实验发现,USP39的SUMO修饰位点主要集中在RS样结构域,并且第6,16,29,51和73位赖氨酸是其SUMO化修饰的位点。SUMO化对USP39功能具有重要的调节作用,在前列腺癌细胞内过表达野生型USP39(USP39 WT)能够促进细胞的增殖,过表达SUMO修饰位点突变的USP39(USP39 SM),进一步促进了细胞增殖效应。本研究提供了一个先导化合物SI2,为优化和发展SENP特异性的抑制剂提供了结构基础。通过蛋白质组学发现了900多个潜在的SUMO的底物,为认识SENPs在前列腺癌中的作用提供了更多的线索。本研究还发现SUMO对剪切体具有调控作用,并且明确了剪切体因子USP39及其SUMO化对前列腺癌细胞的增殖具有重要作用,为前列腺癌的治疗提供了新的视角和一个潜在的治疗靶点。
[Abstract]:Prostate cancer is one of the major diseases affecting male health. Sumo specific protease SENP1 is closely related to the occurrence and metastasis of prostate cancer, but the SUMO modified substrates in prostate cancer cells are not clear.In this study, we found that small molecular compound SI2 is an inhibitor of SENP1 and SI2 can inhibit the activities of SENP2 and SENP3 by computer virtual screening and enzyme activity test in vitro and in vitro.However, the activity of SENP5 was not inhibited by .SI2 on PC3 cells of prostate cancer, which resulted in the accumulation of endogenous SUMO modified substrates, but did not affect the protein levels of SENP1, SENP2, SENP3 and SUMO conjugate enzyme UBC9. SI2 could not only increase the endogenous SUMO modified substrates.It can also increase the level of Flag-SUMO modified by exogenous transfection.More than 900 potential substrates of SUMO were identified in PC3 cells by using SI2 as a molecular probe and stable isotope labeled amino acid technique in cell culture, of which 231 substrates were identified to increase SUMO level by 1.3-fold after SI2 treatment.We analyzed the signaling pathways of 231 proteins and found that they were at least involved in shearing, proteasome, glycolysis, the synthesis of branched amino acids, the biosynthesis of aminoacyl-t RNA, and pyruvate metabolism.The degradation of branched amino acids and the metabolism of propionic acid were involved in eight pathways.A total of 20 proteins were enriched in the shearing body pathway, among which HNRNPK (HNRNPK) HNRNPMN DDX5 was reported as a SUMO substrate.At the same time, we confirmed that USP39 HSPA1A and HSPA2 in this pathway are new SUMO substrates by in vitro SUMO modification.Not only SUMO modification of USP39 could be detected in SUMO reaction in vitro, but also SUMO modification of USP39 could be detected in PC3 cells.By immunofluorescence detection, we found that USP39 can co-locate with SUMO1 and SUMO2/3.It was further found that the SUMO modification site of USP39 was mainly located in the RS like domain, and the lysine at position 6 ~ (16) ~ (29) and 73 ~ (th) was the site of SUMO modification. Sumo played an important role in regulating the function of USP39.Overexpression of wild-type USP39(USP39 in prostate cancer cells can promote cell proliferation, and overexpression of SUMO modified site mutation USP39(USP39 SMN further promotes cell proliferation.This study provides a leading compound, SI2, which provides a structural basis for the optimization and development of SENP specific inhibitors.More than 900 potential substrates of SUMO have been identified by proteomics, providing further clues to the role of SENPs in prostate cancer.We also found that SUMO can regulate shearing bodies, and that shearing factor USP39 and its SUMO play an important role in the proliferation of prostate cancer cells, which provides a new perspective and a potential therapeutic target for the treatment of prostate cancer.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25

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