棕榈酰化蛋白质组学分析揭示前列腺癌细胞中雄激素促进代谢相关蛋白棕榈酰化修饰

Palmitoylome profiling indicates that androgens promote the palmitoylation of metabolism-related proteins in prostate cancer-derived LNCaP cells

目的:筛选棕榈酰化修饰水平受雄激素诱导的蛋白,探索前列腺癌去雄激素治疗外其他潜在的治疗靶点。方法:以LNCaP细胞为研究对象,雄激素(Methyltrienolone,R1881,5 nmol/L)或DMSO(dimethyl sulfoxide)处理LNCaP细胞24 h,同时利用人工合成的炔基棕榈酸Alk-C16(100μmol/L)对细胞进行代谢标记,收集细胞,裂解,提取总蛋白,加入标记有叠氮化物的琼脂糖珠(1 mmol/L),室温反应1 h,利用叠氮化物与Alk-C16末端炔基发生点击化学反应形成的共价键将棕榈酰化修饰蛋白富集在琼脂糖珠上,进行蛋白质谱非标记定量分析(label-free quantitation,LFQ),比较R1881处理和非处理细胞蛋白棕榈酰化修饰变化情况,筛选棕榈酰化修饰水平受雄激素诱导的蛋白。结果:实验共鉴定出907个潜在的棕榈酰化修饰蛋白(mascot score>2,P<0.05),其中有430个蛋白LFQ值至少有2次不为0。在这430个蛋白中有92个蛋白R1881处理与非处理样品LFQ比值大于1.5(P<0.05),说明雄激素能够显著促进该蛋白棕榈酰化修饰。利用Cytoscape软件对92个蛋白进行功能富集分类,发现已知功能蛋白可分为代谢相关、蛋白折叠相关和翻译起始相关3类,其中,代谢相关蛋白包括脂代谢(6个)、糖代谢(7个)和呼吸电子传递链(8个)3部分,另外还有少量氨基酸代谢(2个)和其他代谢相关蛋白(2个)。参与呼吸电子传递链的细胞色素b-c1复合体亚基2(cytochrome b-cl complex subunit2,UQCRC2)雄激素R1881处理和未处理样品LFQ比值最高(>3,P<0.05),说明该蛋白棕榈酰化修饰受雄激素诱导最为明显。LFQ比值最高为UQCRC2,其次为脂代谢相关的长链特异性酰基辅酶A脱氢酶(very long-chain specific acyl-CoA dehydrogenase,ACADVL)和糖代谢相关的6-磷酸葡萄糖酸脱氢酶(6-phosphogluconate dehydrogenase,PGD),但其LFQ比值均未超过3。结论:代谢尤其是呼吸电子传递链相关蛋白的棕榈酰化调控机制的研究可能将为前列腺癌的诊疗和靶向药的研发提供新的指导思路。

Objective:To explore potential therapeutic targets other than androgen-deprivation treatment for prostate cancer by screening the proteins induced by androgen at palmitoylation modification level in LNCaP cells.Methods:The LNCaP cells were treated with androgen(Methyltrienolone,R1881,5 nmol/L)or dimethyl sulfoxide(DMSO)for 24 h,and then labeled with alkynyl palmitic acid Alk-C16(100μmol/L).After that,the cells were collected,lysed,the total protein was extracted,agarose beads labeled with azide(1 mmol/L)were added,and the click-chemistry reaction was carried out at room temperature for 1 h.The covalent bond formed by click-chemistry reaction of azide and alkynyl group was used to enrich the palmitoylated proteins on agarose beads.Label-free quantitation(LFQ)was used to compare the protein palmitoylation level of R1881 treated and untreated cells to screen the proteins induced by androgen at palmitoylation modification level.Results:In this experiment,907 potential palmitoylated proteins(mascot score>2,P<0.05)were identified,among which 430 proteins had LFQ values not zero at least twice.Among the 430 proteins,the palmitoylation levels of 92 candidates were increased by androgen treatment,and their LFQ values were significantly upregulated(>1.5-fold,P<0.05)in≥2 samples of androgen-treated vs.untreated LNCaP cells.We also used the software of cytoscape to classify the 92 proteins,and found that the known functional proteins of them could be divided into three categories:metabolism related,protein folding related and translation initiation related.Among them,metabolism related proteins included lipid metabolism(6),glucose metabolism(7)and respiratory electron transport chain(8),and a small amount of amino acid metabolism(2)and other metabolism related proteins(2).Notably,the ratio of LFQ of cytochrome b-c1 complex subunit 2(UQCRC2)was significantly(>3-fold,P<0.05)higher in androgen-treated cells compared with untreated cells,indicating that the palmitoylation level of UQCRC2 was enhanced by androgen most significantly than that of others.The second was long-chain acyl CoA dehydrogenase(ACADVL)related to lipid metabolism and glucose 6-phosphate dehydrogenase(PGD)related to glucose metabolism,but the LFQ ratio of them was less than 3-fold.Conclusion:The research on palmitoylation mechanism of metabolism,especially the proteins related to respiratory electron transport chain,will provide a new guidance for the diagnosis and treatment of prostate cancer and the development of targeted drugs.