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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Title
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
Author(s)
Ah Young Lee; Sung Goo Park; Chang Won Kho; Sun Young Park; S Y Cho; Sang Chul Lee; Do Hee Lee; P K Myung; Byoung Chul Park
Bibliographic Citation
Proteomics, vol. 4, no. 11, pp. 3437-3445
Publication Year
2004
Abstract
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 CIpC and EF-Tu as putative Isp-1 substrates and studied their in vitro degradation. CIpC and EF-Tu contain putative cleavage sites for Isp-1. N-terminal sequencing of in vitro proteolytic fragments of CIpC and EF-Tu revealed that these sites are indeed recognized and cleaved by Isp-1. Moreover, the cellular levels of CIpC 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 CIpC 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.
Keyword
DegradomicsIsp-1Serine proteaseTwo-dimensional gel
ISSN
1615-9853
Publisher
Wiley
DOI
http://dx.doi.org/10.1002/pmic.200400997
Type
Article
Appears in Collections:
Division of Biomedical Research > Disease Target Structure Research Center > 1. Journal Articles
Division of Biomedical Research > Metabolic Regulation Research Center > 1. Journal Articles
Critical Diseases Diagnostics Convergence Research Center > 1. Journal Articles
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