Hook V Y, Schiller M R, Azaryan A V
Department of Medicine, University of California, San Diego 92103, USA.
Arch Biochem Biophys. 1996 Apr 1;328(1):107-14. doi: 10.1006/abbi.1996.0149.
Proteases of cysteine, aspartic, and subtilisin classes have been indicated as candidate prohormone processing enzymes. The chromaffin granule proenkephalin processing proteases have been characterized as the novel cysteine protease prohormone thiol protease (PTP), a 70-kDa aspartic proteinase, and the subtilisin-like PC1/3 and PC2 enzymes. The goal of this study was to assess whether these processing proteases possess preference(s) for prohormone substrates. The recombinant prohormones proenkephalin, proneuropeptide Y (pro-NPY), and proopiomelanocortin (POMC) were expressed in Escherichia coli using the T7 expression system and purified for in vitro processing studies. Results indicated that the chromaffin granule processing proteases possess selectivity for particular prohormones. PTP preferred proenkephalin, with good cleavage of pro-NPY and slow processing of POMC. In contrast, the 70-kDa aspartic proteinase cleaved POMC most readily, with cleavage of proenkephalin and some processing of pro-NPY. PC1/3 and PC2 preferred POMC among the prohormones tested. Importantly, these results indicate that prohormone selectivity of processing proteases may be an important factor in predicting the primary and rate-limiting protease(s) required for processing a particular prohormone.
半胱氨酸类、天冬氨酸类和枯草杆菌蛋白酶类蛋白酶已被指出是候选激素原加工酶。嗜铬颗粒前脑啡肽加工蛋白酶已被鉴定为新型半胱氨酸蛋白酶激素原硫醇蛋白酶（PTP）、一种70 kDa的天冬氨酸蛋白酶以及枯草杆菌蛋白酶样PC1/3和PC2酶。本研究的目的是评估这些加工蛋白酶是否对激素原底物具有偏好性。使用T7表达系统在大肠杆菌中表达重组激素原前脑啡肽、前神经肽Y（pro-NPY）和阿黑皮素原（POMC），并进行纯化以用于体外加工研究。结果表明，嗜铬颗粒加工蛋白酶对特定激素原具有选择性。PTP偏好前脑啡肽，对pro-NPY有良好的切割作用，对POMC的加工作用缓慢。相比之下，70 kDa的天冬氨酸蛋白酶最容易切割POMC，对前脑啡肽有切割作用，对pro-NPY有一些加工作用。在测试的激素原中，PC1/3和PC2偏好POMC。重要的是，这些结果表明，加工蛋白酶的激素原选择性可能是预测加工特定激素原所需的主要和限速蛋白酶的一个重要因素。
