| Budget Amount *help |
¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000)
Fiscal Year 2014: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2013: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2012: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
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| Outline of Final Research Achievements |
The Skp1-Cul1-F-box protein (SCF) complex, one of the best-characterized ubiquitin ligases, is composed of three invariable components and a variable component F-ox protein that serves as the substrate recognition subunit. Among the over 70 F-box proteins found in humans, less than half have been characterized. To identify substrates for uncharacterized F-box proteins, I developed a comprehensive method for identification of ubiquitinated substrates using the trypsin-resistant tandem ubiquitin-binding entity (TR-TUBE). The coexpression of TR-TUBE with a F-box protein stabilizes the ubiquitinated substrates by masking the ubiquitin chains. Using a combination of two strategies for enriching ubiquitinated substrates, TR-TUBE and ubiquitin Lys-e-Gly-Gly (diGly) remnant antibody, I successfully identified specific ubiquitin ligase-substrate pairs. Using this method, I identified target substrates of FBXO21, an uncharacterized and ubiquitiously expressed F-box protein.
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