Analytical Method for Diacylglycerol Kinase ζ Activity in Cells Using Protein Myristoylation and Liquid Chromatography?Tandem Mass Spectrometry
Analytical Method for Diacylglycerol Kinase ζ Activity in Cells Using Protein Myristoylation and Liquid Chromatography?Tandem Mass Spectrometry
复制标题
使用蛋白质肉豆蔻酰化和液相色谱串联质谱分析细胞中二酰基甘油激酶 z 活性的分析方法
作者：
Honda Shotaro;Murakami Chiaki;Yamada Haruka;Murakami Yuki;Ishizaki Ayuka;Sakane Fumio
甘油二酯激酶（DGK）的特异性抑制剂可通过激活肿瘤免疫而成为很有前途的抗癌药物.虽然除了体外试验外，检测靶酶的细胞活性对于药物筛选是必不可少的，但是在细胞中检测DGK酶的活性是困难的。在本研究中，我们产生了在5′端具有共有N-豆蔻酰化序列的AcGFP-DGKcDNA（Myr-AcGFP-DGKcDNA），以靶向DGKcDNA至膜。使用液相色谱（LC）-串联质谱（MS/MS）（LC-MS/MS），我们发现Myr-AcGFP-DGK酶，而不是没有豆蔻酰化序列的AcGFP-DGK酶，显著增加了几种磷脂酸（PtdOH）物质的水平。与Myr-AcGFP-DGK β相反，其失活突变体没有表现出PtdOH产生的增加，表明PtdOH产生的增加是DGK活性依赖性的。该方法将用于化合物选择，以开发靶向DGK β的药物，并可适用于各种可溶性（非膜结合）脂质代谢酶，包括其他DGK同工酶。
Specific inhibitors of diacylglycerol kinase (DGK) ζ can be promising anticancer medicationsviathe activation of cancer immunity. Although the detection of cellular activities of target enzymes is essential for drug screening in addition toin vitroassays, it is difficult to detect the activity of DGKζ in cells. In the present study, we generated AcGFP‐DGKζ cDNA with a consensus N‐myristoylation sequence at the 5′ end (Myr‐AcGFP‐DGKζ) to target DGKζ to membranes. Using liquid chromatography (LC)‐tandem mass spectrometry (MS/MS) (LC–MS/MS), we showed that Myr‐AcGFP‐DGKζ, but not AcGFP‐DGKζ without the myristoylation sequence, substantially augmented the levels of several phosphatidic acid (PtdOH) species. In contrast to Myr‐AcGFP‐DGKζ, its inactive mutant did not exhibit an increase in PtdOH production, indicating that the increase in PtdOH production was DGK activity‐dependent. This method will be useful in chemical compound selection for the development of drugs targeting DGKζ and can be applicable to various soluble (nonmembrane bound) lipid‐metabolizing enzymes, including other DGK isozymes.