Hwang In-Wook, Makishima Yu, Kato Tatsuya, Park Sungjo, Terzic Andre, Park Enoch Y
Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan.
Appl Microbiol Biotechnol. 2014 Oct;98(19):8201-9. doi: 10.1007/s00253-014-5715-6. Epub 2014 Apr 17.
Biotin-dependent human acetyl-CoA carboxylases (ACCs) are integral in homeostatic lipid metabolism. By securing posttranslational biotinylation, ACCs perform coordinated catalytic functions allosterically regulated by phosphorylation/dephosphorylation and citrate. The production of authentic recombinant ACCs is heeded to provide a reliable tool for molecular studies and drug discovery. Here, we examined whether the human ACC2 (hACC2), an isoform of ACC produced using the silkworm BmNPV bacmid system, is equipped with proper posttranslational modifications to carry out catalytic functions as the silkworm harbors an inherent posttranslational modification machinery. Purified hACC2 possessed genuine biotinylation capacity probed by biotin-specific streptavidin and biotin antibodies. In addition, phosphorylated hACC2 displayed limited catalytic activity whereas dephosphorylated hACC2 revealed an enhanced enzymatic activity. Moreover, hACC2 polymerization, analyzed by native page gel analysis and atomic force microscopy imaging, was allosterically regulated by citrate and the phosphorylation/dephosphorylation modulated citrate-induced hACC2 polymerization process. Thus, the silkworm BmNPV bacmid system provides a reliable eukaryotic protein production platform for structural and functional analysis and therapeutic drug discovery applications implementing suitable posttranslational biotinylation and phosphorylation.
生物素依赖性人乙酰辅酶A羧化酶（ACC）在脂质稳态代谢中不可或缺。通过确保翻译后生物素化，ACC发挥协同催化功能，受磷酸化/去磷酸化和柠檬酸变构调节。生产出真实的重组ACC对于为分子研究和药物发现提供可靠工具至关重要。在此，我们研究了利用家蚕BmNPV杆粒系统生产的ACC同工型人ACC2（hACC2）是否具备适当的翻译后修饰，以发挥催化功能，因为家蚕拥有内在的翻译后修饰机制。用生物素特异性抗生蛋白链菌素和生物素抗体检测纯化的hACC2具有真正的生物素化能力。此外，磷酸化的hACC2催化活性有限，而去磷酸化的hACC2酶活性增强。而且，通过非变性聚丙烯酰胺凝胶分析和原子力显微镜成像分析，hACC2的聚合受柠檬酸变构调节，磷酸化/去磷酸化调节柠檬酸诱导的hACC2聚合过程。因此，家蚕BmNPV杆粒系统为实施适当的翻译后生物素化和磷酸化的结构和功能分析及治疗药物发现应用提供了可靠的真核蛋白生产平台。
