Cellular localization domains of a rabbit and a human carboxylesterase: influence on irinotecan (CPT-11) metabolism by the rabbit enzyme.
Cellular localization domains of a rabbit and a human carboxylesterase: influence on irinotecan (CPT-11) metabolism by the rabbit enzyme.
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发表时间：
1998-08
期刊：
影响因子：
11.2
通讯作者：
P. Potter;J. S. Wolverton;C. Morton;M. Wierdl;M. Danks
中科院分区：
文献类型：
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作者：
P. Potter;J. S. Wolverton;C. Morton;M. Wierdl;M. Danks
前药的酶激活以提高特定抗癌药物的治疗指数是目前化疗方案的一个有吸引力的替代方案。本研究探讨了重组羧酸酯酶(CES)激活伊立替康(CPT-11)的可能性。CES是一类普遍存在的酶，被认为参与了外源生物的解毒。它们的初级氨基酸序列表明这些蛋白质应该定位于内质网。通过对兔肝和人肺泡巨噬细胞CE基因在COS7细胞中的表达和免疫组织化学定位，我们确定了18个氨基酸的NH2末端疏水信号肽负责这些蛋白在内质网的定位。通过类似的方法，我们已经证明了COOH末端的氨基酸HIIL阻止蛋白质从细胞中分泌。去除NH2末端结构域后，酶活性丧失；然而，在表达COOH末端截短蛋白的细胞的培养液中可以检测到活性酶。灯盏花素A可以阻止缺乏6个COOH末端氨基酸的CEs的分泌，证实这些被截短的酶是通过内质网介导的胞吐作用被加工和释放出来的。NH2-和COOH-末端缺失的双截断突变酶的免疫染色模式与NH2末端截短的蛋白质相似，也缺乏CE活性。在所有情况下，经典酯酶底物邻硝基苯乙酸酯的新陈代谢预测了表达兔CE的细胞对抗癌剂CPT-11的敏感性。此外，分泌的酶使COS7细胞对该药物敏感，表明底物代谢不需要与脂质双层结合的蛋白质。
Enzyme activation of prodrugs to improve the therapeutic index of specific anticancer agents is an attractive alternative to current chemotherapy regimens. This study addresses the potential for activating irinotecan (CPT-11) with recombinant carboxylesterases (CEs). CEs are a ubiquitous class of enzymes thought to be involved in the detoxification of xenobiotics. Their primary amino acid sequence indicates that these proteins should be localized to the endoplasmic reticulum. By PCR-mediated mutagenesis of a rabbit liver and a human alveolar macrophage CE cDNA, expression in Cos7 cells, and subsequent immunohistochemical localization, we have determined that an 18-amino acid NH2-terminal hydrophobic signal peptide is responsible for the localization of these proteins to the endoplasmic reticulum. By similar approaches, we have demonstrated that the COOH-terminal amino acids HIEL prevent secretion of the proteins from the cell. Enzymatic activity was lost by removing the NH2-terminal domain; however, active enzyme could be detected in the culture media of cells expressing the COOH-terminally truncated proteins. Secretion of CEs lacking the six COOH-terminal amino acids could be prevented with brefeldin A, confirming that these truncated enzymes were processed and released from cells by endoplasmic reticulum-mediated exocytosis. Double-truncation mutant enzymes lacking both NH2- and COOH-terminal sequences demonstrated immunostaining patterns similar to those of the NH2-terminally truncated proteins and also lacked CE activity. In all cases, metabolism of the classic esterase substrate o-nitrophenyl acetate predicted the sensitivity of cells expressing the rabbit CE to the anticancer agent CPT-11. In addition, the secreted enzyme sensitized Cos7 cells to this drug, indicating that protein association with a lipid bilayer is not required for substrate metabolism.