Li W Q, Ren S M, Long X B, Tian Y Q
The Key Laboratory of Geriatrics, Beijing Institute of Geriatrics, Beijing Hospital, National Center of Gerontology, National Health Commission; Institute of Geriatric Medicine, Chinese Academy of Medical Science, Beijing 100730, China.
National Center for Clinical Laboratory, Beijing Hospital, National Center of Gerontology; Institute of Geriatric Medicine, Chinese Academy of Medical Science, Beijing 100730, China.
Beijing Da Xue Xue Bao Yi Xue Ban. 2020 Apr 18;52(2):227-233. doi: 10.19723/j.issn.1671-167X.2020.02.006.
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.
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.
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.
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.
通过在LNCaP细胞中筛选雄激素在棕榈酰化修饰水平诱导的蛋白质，探索前列腺癌除雄激素剥夺治疗之外的潜在治疗靶点。
用雄激素（甲基三烯醇酮，R1881，5 nmol/L）或二甲基亚砜（DMSO）处理LNCaP细胞24小时，然后用炔基棕榈酸Alk-C16（100 μmol/L）进行标记。之后，收集细胞，裂解，提取总蛋白，加入用叠氮化物标记的琼脂糖珠（1 mmol/L），并在室温下进行点击化学反应1小时。利用叠氮化物与炔基的点击化学反应形成的共价键，在琼脂糖珠上富集棕榈酰化蛋白质。采用无标记定量（LFQ）比较R1881处理组和未处理组细胞的蛋白质棕榈酰化水平，以筛选雄激素在棕榈酰化修饰水平诱导的蛋白质。
本实验共鉴定出907个潜在的棕榈酰化蛋白质（Mascot得分>2，P<0.05），其中430个蛋白质的LFQ值至少有两次不为零。在这430个蛋白质中，92个候选蛋白的棕榈酰化水平经雄激素处理后升高，且在≥2个雄激素处理的LNCaP细胞样本与未处理细胞样本中，它们的LFQ值显著上调（>1.5倍，P<0.05）。我们还使用Cytoscape软件对这92个蛋白质进行分类，发现其已知功能的蛋白质可分为三类：代谢相关、蛋白质折叠相关和翻译起始相关。其中，代谢相关蛋白质包括脂质代谢（6个）、葡萄糖代谢（7个）和呼吸电子传递链（8个），以及少量氨基酸代谢（2个）和其他代谢相关蛋白质（2个）。值得注意的是，细胞色素b-c1复合物亚基2（UQCRC2）的LFQ比值在雄激素处理的细胞中比未处理的细胞显著更高（>3倍，P<0.05），表明UQCRC2的棕榈酰化水平受雄激素增强的程度最显著。其次是与脂质代谢相关的长链酰基辅酶A脱氢酶（ACADVL）和与葡萄糖代谢相关的6-磷酸葡萄糖脱氢酶（PGD），但其LFQ比值小于3倍。
对代谢的棕榈酰化机制的研究，尤其是与呼吸电子传递链相关的蛋白质，将为前列腺癌的诊断和治疗以及靶向药物的开发提供新的指导。
