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

Abstract

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 (100 μmol/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.

1

雄激素诱导的棕榈酰化修饰蛋白筛选 A schematic overview of screening of androgen-induced palmitoylated proteins

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前列腺癌细胞中受雄激素诱导的棕榈酰化修饰蛋白功能网络图 Functional network of androgen-induced palmitoylated proteins in LNCaP cells

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在前列腺癌细胞中雄激素促进代谢相关蛋白的棕榈酰化修饰 Androgen promoted the palmitoylation levels of metabolism-related proteins in LNCaP cells

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前列腺癌细胞中雄激素促进UQCRC2棕榈酰化修饰水平 Androgen increased the palmitoylation level of UQCRC2 in LNCaP cellsPalm, palmitoylation; HAM, hydroxylamine; IP, immunoprecipitation; IB, immunoblotting; DMSO, dimethyl sulfoxide.