Identification of the degradome of Isp-1, a major intracellular serine protease of Bacillus subtilis, by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight analysis
Identification of the degradome of Isp-1, a major intracellular serine protease of Bacillus subtilis, by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight analysis
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DOI：
10.1002/pmic.200400997
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发表时间：
2004-11-01
期刊：
影响因子：
3.4
通讯作者：
Park, BC
中科院分区：
文献类型：
--
作者：
Lee, AY;Park, SG;Park, BC
胞内丝氨酸蛋白酶-1（Isp-1）是枯草芽孢杆菌（Bacillus subtilis）胞内主要的丝氨酸蛋白酶，其功能尚不清楚。此外，生理底物尚未确定。为了确定Isp-1底物，我们消化提取物从Isp-1缺陷的芽孢杆菌突变体纯化的Isp-1和检查消除或减少斑点的二维凝胶和基质辅助激光解吸/电离飞行时间分析。被Isp-1降解的蛋白质，称为Isp-1降解组，参与多种细胞功能，如DNA包装，遗传能力和蛋白质分泌。从degradome中，我们选择ClpC和EF-Tu作为推定的Isp-1底物，并研究了它们的体外降解。ClpC和EF-Tu含有Isp-1的推定切割位点。ClpC和EF-Tu的体外蛋白水解片段的N-末端测序显示，这些位点确实被Isp-1识别和切割。此外，ClpC和EF-Tu的细胞水平在稳定期后期显著降低，其中Isp-1的表达水平大大增加。这些结果表明，由Isp-1调节的ClpC蛋白水解在稳定期适应性反应中起着重要作用。这种降解组的方法可以提供一个强大的工具，发现许多蛋白水解酶的功能仍有待确定的生理底物。
Intracellular serine protease-1 (Isp-1) is a major intracellular serine protease of Bacillus subtilis, whose functions still remain largely unknown. Furthermore, physiological substrates are yet to be determined. To identify Isp-1 substrates, we digested extract obtained from an Isp-1 deficient Bacillus mutant with purified Isp-1 and examined eliminated or decreased spots by two-dimensional gel and matrix-assisted laser desorption/ionization-time of flight analyses. Proteins degraded by Isp-1, termed the Isp-1 degradome, are involved in a variety of cellular functions such as DNA packing, genetic competence, and protein secretion. From the degradome we selected ClpC and EF-Tu as putative Isp-1 substrates and studied their in vitro degradation. ClpC and EF-Tu contain putative cleavage sites for Isp-1. N-terminal sequencing of in vitro proteolytic fragments of ClpC and EF-Tu revealed that these sites are indeed recognized and cleaved by Isp-1. Moreover, the cellular levels of ClpC and EF-Tu were dramatically reduced at the late stationary phase, where the expression level of Isp-1 was greatly increased. These results suggest that the regulated proteolysis of ClpC by Isp-1 plays an important role in the stationary phase adaptive response. This degradomic approach could provide a powerful tool for finding physiological substrates of many proteolytic enzymes whose functions remain to be determined.