| Project/Area Number |
24659099
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| Research Category |
Grant-in-Aid for Challenging Exploratory Research
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| Allocation Type | Single-year Grants |
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| Research Field |
General physiology
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| Research Institution | Kyoto University |
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Principal Investigator |
HATTORI Akira 京都大学, 薬学研究科(研究院), 准教授 (50300893)
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| Co-Investigator(Kenkyū-buntansha) |
OISHI Shinya 京都大学, 薬学研究科, 講師 (80381739)
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| Co-Investigator(Renkei-kenkyūsha) |
NISHIMURA Shinichi 京都大学, 薬学研究科, 助教 (30415260)
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| Project Period (FY) |
2012-04-01 – 2014-03-31
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| Project Status |
Completed (Fiscal Year 2013)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000)
Fiscal Year 2013: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2012: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
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| Keywords | ユビキチン / 脱ユビキチン化酵素 / activity-based probe / プローブ / システインプロテアーゼ |
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| Research Abstract |
Recently, it has been reported that unanchored Lys63-linked polyubiquitin chains can act as a scaffold protein linking TAK1 and TAB2 in the NF-kappaB signaling pathway. In this study, we synthesized a novel fluorogenic substrate, x3Ub(G76V)-Ub-AMC and an activity-based probe, x3Ub(G76V)-Ub-VS, which enable detection of the unanchored polyubiquitin chain-liberating activity by a native chemical ligation strategy. We showed that the activity profile for the x3Ub(G76V)-Ub-AMC hydrolysis in MonoQ fractions of HeLa cell extract was different from those for Ub-AMC and Ub-GrB hydrolysis, suggesting a possibility of the substrate for endo-cleavage type deubiquitinating enzymes. We also revealed that the x3Ub(G76V)-Ub-AMC hydrolyzing activity in HT1080 cells was attenuated by tumor necrosis factor stimulation, suggesting an involvement of endo-cleavage type deubiquitinating enzymes in inflammatory responses.
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