Proteomic analysis of ubiquitin modification

1.我们利用MALDI-TOF质谱法建立了一种简便的多肽质量指纹图谱测定泛素链连接的方法：标记到靶蛋白上的多泛素链经过胰蛋白酶消化，并通过MALDI-TOF质谱法测定链类型特异性连接。2.我们利用从酵母中纯化的E1（Uba1）、E2（Ubc4）、E3（Ufd4）和E4（Ufd2）酶重构了泛素融合降解（UFD）途径：UFD底物首先通过Uba1、Ubc4和Ufd4的作用被泛素化，然后由Ufd2催化泛素链的延伸。通过肽质量指纹图谱直接测定多泛素化UFD底物的泛素链连锁类型，发现在UFD通路中，Ufd2通过Lys48连锁催化泛素链的伸长。3.我们发现含有SH3结构域的蛋白Lsb2被多泛素化。通过肽质量指纹图谱直接测定标记在Lsb2上的多泛素链的泛素链连锁类型，发现Lsb2是通过Lys63连锁被多泛素化的。此外，我们发现这种泛素化是由一种HECT型泛素连接酶Rsp5催化的，并且Lys80残基是Lsb2中的泛素化位点。3.我们通过蛋白质组学分析确定了用泛素样蛋白ISG15（一种干扰素刺激的基因产物，分子量为15 kDa）修饰的新靶蛋白。然后，我们研究了ISG15修饰蛋白磷酸酶2Cβ的结果，发现ISG15修饰蛋白磷酸酶2Cβ抑制蛋白磷酸酶2Cβ的活性，抑制TAK1/ tab1诱导的NF-κB活化。4.关于泛素依赖的蛋白水解途径，我们表征了26S蛋白酶体的亚基，26S蛋白酶体是该蛋白水解途径中的一个蛋白水解机器。此外，我们成功地分离了抑制这一蛋白水解途径的新化合物。

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.