| Budget Amount *help |
¥13,500,000 (Direct Cost: ¥13,500,000)
Fiscal Year 2005: ¥4,800,000 (Direct Cost: ¥4,800,000)
Fiscal Year 2004: ¥8,700,000 (Direct Cost: ¥8,700,000)
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| Research Abstract |
1.We established a simple method for determination of the ubiquitin chain linkage by peptide mass fingerprinting using MALDI-TOF mass spectrometry : Polyubiquitin chains tagged to target proteins are subjected to trypsin digestion and the chain type-specific linkages are determined by MALDI-TOF mass spectrometry. 2.We reconstituted the ubiquitin fusion degradation (UFD) pathway using E1(Uba1), E2(Ubc4), E3(Ufd4), and E4(Ufd2) enzymes purified from yeast : UFD substrates are first ubiquitinated by actions of Uba1, Ubc4, and Ufd4 and elongation of the ubiquitin chains is then catalyzed by Ufd2. Direct determination of the ubiquitin chain linkage type in polyubiquitinated UFD substrates by peptide mass fingerprinting revealed that Ufd2 catalyzes elongation of the ubiquitin chain through Lys48 linkage in the UFD pathway. 3.We found that Lsb2, an SH3 domain-containing protein, is polyubiquitinated. Direct determination of the ubiquitin chain linkage type in polyubiquitin chains tagged to Lsb2 by peptide mass fingerprinting revealed that Lsb2 is polyubiquitinated through Lys63 linkage. In addition, we found that this polyubiquitination is catalyzed by Rsp5, a HECT type ubiquitin ligase and that the Lys80 residue is the ubiquitination site in Lsb2. 3.We determined novel target proteins for modification with a ubiquitin-like protein ISG15 (an interferon-stimulated gene product with a molecular mass of 15 kDa) by a proteomnic analysis. We then studied the consequence of ISG15 modification of protein phosphatase 2Cβ, one of the target proteins identified, and found that ISG15 modification of protein phosphatase 2Cβ suppresses the activity of protein phosphatase 2Cβ against TAK1/TAB1-induced NF-κB activation. 4.With respect to the ubiquitin-dependent proteolytic pathway, we characterized subunits of the 26S proteasome, a proteolytic machine in this proteolytic pathway. In addition, we succeeded in isolating novel compounds inhibiting this proteolytic pathway.
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