细胞蛋白质组学和代谢组学整合策略表征散斑型BTB/POZ蛋白质突变调控的关键代谢通路

散斑型BTB/POZ蛋白(SPOP)是前列腺癌中突变率最高的蛋白质之一。该研究通过整合细胞蛋白质组学和代谢组学的方法,揭示SPOP突变引起的代谢紊乱及其调控的代谢通路。首先,系统地研究了LNCaP SPOP野生型及突变型高表达细胞中的代谢变化。代谢组学结果显示,SPOP野生型和突变型(SPOP_Y87N和SPOP_F133L)导入的LNCaP细胞在偏最小二乘法判别分析(PLS-DA)得分图上得到了很好的区分。进一步通过单因素方差分析发现,SPOP突变引起富马酸、苹果酸、柠檬酸、天冬氨酸和天冬酰胺等代谢物含量的增加。蛋白质组学共发现909种蛋白质在两种LNCaP SPOP突变体细胞中发生变化。分别对差异代谢物和差异蛋白质进行通路富集分析,发现三羧酸循环、氨酰基-转运核糖核酸生物合成在代谢组学和蛋白质组学分析中都发生了明显改变。最后,在SPOP敲除的Du145细胞中验证了上述研究结果。该研究证明SPOP突变可促进三羧酸循环。

Identification of speckle type BTB/POZ protein mutation regulated key metabolic pathways by cell based proteomics and metabolomics

Speckle type BTB/POZ protein (SPOP) is one of the most frequently mutated protein in prostate cancer. In this study, proteomics and metabolomics were integrated to study the effects of SPOP mutation on metabolism. First, LNCaP control (CON), SPOP wild-type (SPOP_WT), and SPOP mutation (SPOP_Y87N and SPOP_F133L) cells were subjected to a metabolomics study. The metabolomics data of LNCaP CON, SPOP_WT, SPOP_Y87N, and SPOP_F133L cells were evaluated by partial least squares-discriminant analysis (PLS-DA). Four groups could be clearly differentiated with an explanation ability of R²X=0.512, R²Y=0.616 and predictive ability of Q²=0.475. Totally, 36 differential metabolites were defined with variable importance for the projection (VIP) value > 1. Then, the 36 metabolites were subjected to one-way ANOVA analysis. Fumaric acid, malic acid, citric acid, aspartic acid, and asparagine were increased in LNCaP SPOP mutation cells compared to that in LNCaP SPOP_WT cells. Using a proteomics study, 909 differential proteins were found in LNCaP SPOP_Y87N and SPOP_F133L cells. MetaboAnalyst 3.0 was used to enrich metabolic pathways by using differential metabolites. KOBAS 3.0 was used to enrich metabolic pathways by using differential proteins. Both metabolomics and proteomics analysis showed that the tricarboxylic acid (TCA) cycle and aminoacyl-tRNA biosynthesis were significantly changed. To validate these findings, gas chromatography-mass spectrometry (GC-MS)-based metabolomics was performed in Du145 SPOP knock-out cells. The results indicated that the TCA cycle was activated in Du145 SPOP knock-out cells. Collectively, this study found that SPOP mutation significantly promoted TCA cycle in prostate cancer cells.