本文選題：前列腺癌　切入點(diǎn)：SUMO　出處：《上海交通大學(xué)》2014年博士論文

【摘要】：前列腺癌是影響男性健康的主要疾病之一,SUMO特異性蛋白酶SENP1與前列腺癌的發(fā)生和轉(zhuǎn)移等密切相關(guān),但前列腺癌細(xì)胞內(nèi)SUMO修飾底物目前并不清楚。本研究通過計(jì)算機(jī)虛擬篩選和體外及細(xì)胞內(nèi)酶活性測(cè)試,發(fā)現(xiàn)小分子化合物SI2是SENP1的抑制劑,同時(shí)SI2還能抑制SENP2和SENP3的活性,但對(duì)SENP5的活性無明顯抑制。SI2作用于前列腺癌PC3細(xì)胞,使內(nèi)源SUMO修飾的底物發(fā)生累積,但并不影響SENP1,SENP2和SENP3以及SUMO接合酶UBC9的蛋白水平。SI2不僅能增加內(nèi)源SUMO修飾底物,也能增加外源轉(zhuǎn)染的Flag-SUMO修飾的底物。我們利用SI2為分子探針結(jié)合細(xì)胞培養(yǎng)穩(wěn)定同位素標(biāo)記氨基酸技術(shù),在PC3細(xì)胞內(nèi)鑒定出900多個(gè)潛在的SUMO的底物,其中有231個(gè)底物在SI2處理后SUMO化水平升高?1.3倍。我們對(duì)此231個(gè)蛋白進(jìn)行信號(hào)通路分析,發(fā)現(xiàn)它們至少參與到剪切體、蛋白酶體、糖酵解、支鏈氨基酸的合成、氨基酰-t RNA的生物合成、丙酮酸代謝、支鏈氨基酸的降解和丙酸代謝等八個(gè)通路中。剪切體通路中共富集了20個(gè)蛋白,其中HNRNPK,HNRNPM,DDX5是已報(bào)道的SUMO底物。同時(shí),我們通過體外SUMO修飾實(shí)驗(yàn)驗(yàn)證了此通路中的USP39,HSPA1A和HSPA2是新的SUMO底物。除在體外SUMO反應(yīng)中能夠檢測(cè)到USP39的SUMO化修飾,在PC3細(xì)胞內(nèi)也能檢測(cè)到USP39的SUMO化修飾。通過免疫熒光檢測(cè),我們發(fā)現(xiàn)USP39能夠與SUMO1和SUMO2/3共定位。進(jìn)一步實(shí)驗(yàn)發(fā)現(xiàn),USP39的SUMO修飾位點(diǎn)主要集中在RS樣結(jié)構(gòu)域,并且第6,16,29,51和73位賴氨酸是其SUMO化修飾的位點(diǎn)。SUMO化對(duì)USP39功能具有重要的調(diào)節(jié)作用,在前列腺癌細(xì)胞內(nèi)過表達(dá)野生型USP39(USP39 WT)能夠促進(jìn)細(xì)胞的增殖,過表達(dá)SUMO修飾位點(diǎn)突變的USP39(USP39 SM),進(jìn)一步促進(jìn)了細(xì)胞增殖效應(yīng)。本研究提供了一個(gè)先導(dǎo)化合物SI2,為優(yōu)化和發(fā)展SENP特異性的抑制劑提供了結(jié)構(gòu)基礎(chǔ)。通過蛋白質(zhì)組學(xué)發(fā)現(xiàn)了900多個(gè)潛在的SUMO的底物,為認(rèn)識(shí)SENPs在前列腺癌中的作用提供了更多的線索。本研究還發(fā)現(xiàn)SUMO對(duì)剪切體具有調(diào)控作用,并且明確了剪切體因子USP39及其SUMO化對(duì)前列腺癌細(xì)胞的增殖具有重要作用,為前列腺癌的治療提供了新的視角和一個(gè)潛在的治療靶點(diǎn)。
[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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.25

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本文編號(hào)：1695632