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
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.
| Medienart: |
Artikel |
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| Erscheinungsjahr: |
2004 |
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| Erschienen: |
2004 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:4 |
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| Enthalten in: |
Proteomics - 4(2004), 11 vom: 25. Nov., Seite 3437-45 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Lee, Ah Young [VerfasserIn] |
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| Themen: |
EC 3.4.21.- |
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| Anmerkungen: |
Date Completed 13.04.2005 Date Revised 15.11.2006 published: Print Citation Status MEDLINE |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM15198204X |
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| 100 | 1 | |a Lee, Ah Young |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a 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 |
| 264 | 1 | |c 2004 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ohne Hilfsmittel zu benutzen |b n |2 rdamedia | ||
| 338 | |a Band |b nc |2 rdacarrier | ||
| 500 | |a Date Completed 13.04.2005 | ||
| 500 | |a Date Revised 15.11.2006 | ||
| 500 | |a published: Print | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 7 | |a Serine Endopeptidases |2 NLM | |
| 650 | 7 | |a EC 3.4.21.- |2 NLM | |
| 700 | 1 | |a Goo Park, Sung |e verfasserin |4 aut | |
| 700 | 1 | |a Kho, Chang Won |e verfasserin |4 aut | |
| 700 | 1 | |a Young Park, Sun |e verfasserin |4 aut | |
| 700 | 1 | |a Cho, Sayeon |e verfasserin |4 aut | |
| 700 | 1 | |a Lee, Sang Chul |e verfasserin |4 aut | |
| 700 | 1 | |a Lee, Do Hee |e verfasserin |4 aut | |
| 700 | 1 | |a Myung, Pyung Keun |e verfasserin |4 aut | |
| 700 | 1 | |a Park, Byoung Chul |e verfasserin |4 aut | |
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