Discovery of a pyruvylated peptide-metabolizing enzyme using a fluorescent substrate-based protein discovery technique.
Discovery of a pyruvylated peptide-metabolizing enzyme using a fluorescent substrate-based protein discovery technique.
复制标题
使用基于荧光底物的蛋白质发现技术发现丙酮酰化肽代谢酶。
作者：
K. Yoshioka;Toru Komatsu;K. Hanaoka;Tasuku Ueno;Takuya Terai;T. Nagano;Y. Urano
我们采用基于荧光底物的靶标发现方法来筛选酶组对乙酰氨基化肽的代谢活性，并鉴定了酰基氨基酸释放酶（APEH）。过表达APEH的细胞对探针N-乙酰-亮氨酰-7-氨基-4-甲基香豆素（Pyr-Leu-AMC）表现出更高的代谢活性，而选择性APEH抑制剂AA 74 -1阻断了该反应。AA 74 -1几乎完全阻断了肝脏裂解物中各种乙酰化肽的代谢。丙酮酸肽的合成是为了应对氧化应激，但其生物学作用知之甚少;确定其代谢的关键因素应刺激对从氧化应激到蛋白质修饰和生物输出的途径的研究。
We employed a fluorescent substrate-based target discovery approach to screen the enzymome for metabolic activity towards pyruvyl-amidated peptides, and identified an acylamino acid-releasing enzyme (APEH). Cells overexpressing APEH exhibited higher metabolic activity towards the probe, N-pyruvyl-leucyl-7-amido-4-methylcoumarin (Pyr-Leu-AMC), while the selective APEH inhibitor AA74-1 blocked the reaction. Metabolism of various pyruvylated peptides in liver lysate was almost completely blocked by AA74-1. Pyruvyl peptides are synthesized in response to oxidative stress, but their biological role is poorly understood; identification of a key contributor to their metabolism should stimulate research on pathways leading from oxidative stress to protein modification and biological output.