Honda Shotaro, Murakami Chiaki, Yamada Haruka, Murakami Yuki, Ishizaki Ayuka, Sakane Fumio
Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba, 263-8522, Japan.
Lipids. 2019 Nov;54(11-12):763-771. doi: 10.1002/lipd.12201. Epub 2019 Nov 17.
Specific inhibitors of diacylglycerol kinase (DGK) ζ can be promising anticancer medications via the activation of cancer immunity. Although the detection of cellular activities of target enzymes is essential for drug screening in addition to in vitro assays, 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.
二酰基甘油激酶（DGK）ζ的特异性抑制剂可通过激活癌症免疫成为有前景的抗癌药物。尽管除体外试验外，检测靶酶的细胞活性对于药物筛选至关重要，但在细胞中检测DGKζ的活性却很困难。在本研究中，我们构建了在5'端带有共有N-肉豆蔻酰化序列的AcGFP-DGKζ cDNA（Myr-AcGFP-DGKζ），以使DGKζ靶向细胞膜。使用液相色谱（LC）-串联质谱（MS/MS）（LC-MS/MS），我们发现Myr-AcGFP-DGKζ，而非没有肉豆蔻酰化序列的AcGFP-DGKζ，能显著提高几种磷脂酸（PtdOH）的水平。与Myr-AcGFP-DGKζ相反，其无活性突变体并未表现出PtdOH产量增加，这表明PtdOH产量的增加依赖于DGK活性。该方法将有助于筛选针对DGKζ的药物开发的化合物，并且可应用于各种可溶性（非膜结合）脂质代谢酶，包括其他DGK同工酶。
