Positional-scanning fluorigenic substrate libraries reveal unexpected specificity determinants of DUBs (deubiquitinating enzymes).
Positional-scanning fluorigenic substrate libraries reveal unexpected specificity determinants of DUBs (deubiquitinating enzymes).
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DOI：
10.1042/bj20080779
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发表时间：
2008-11-01
期刊：
影响因子：
--
通讯作者：
Salvesen GS
中科院分区：
文献类型：
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作者：
Drag M;Mikolajczyk J;Bekes M;Reyes-Turcu FE;Ellman JA;Wilkinson KD;Salvesen GS
去泛素化酶（DUB）是负责特异性去除附着于靶蛋白的泛素并因此控制该分子的游离细胞库的蛋白酶家族。DUB活性通常使用全长泛素测定，并且这些酶通常对构成泛素的P4-P1 LRGG C-末端基序的小底物显示低活性。为了深入了解DUBs对泛素C-末端识别区域的影响，我们合成了荧光四肽的位置扫描文库，并在三个人DUBs（OTU-1，Iso-T和UCH-L3）和一种病毒泛素特异性蛋白酶-来自西尼罗病毒的Plpro上对其进行了测试。在大多数情况下，结果显示P4位置的灵活性，P3位置的Arg和P2 - in雅阁的非常高的特异性与天然底物泛素的序列一致。令人惊讶的是，P2位置的筛选显示，与所有其它测试的DUB相反，UCH-L3在P2处表现出对Ala和瓦尔的显著耐受性，并且使用全长泛素的适当突变的平行分析证实了这一点。我们还使用了一个最佳的四肽底物，Ac-LRGG-AFC研究泛素和高盐浓度激活DUBs的机制。总之，我们的结果揭示了1）底物特异性和2）遍在蛋白结合机制在决定这组蛋白酶去遍在蛋白化中的双重特征的重要性。
Deubiquitinating enzymes (DUBs) are a family of proteases responsible for the specific removal of ubiquitin attached to target proteins and thus control the free cellular pools of this molecule. DUB activity is usually assayed using full-length ubiquitin, and these enzymes generally show low activity on small substrates that constitute the P4-P1 LRGG C-terminal motif of ubiquitin. To gain insight into the C-terminal recognition region of ubiquitin by DUBs we synthesized positional scanning libraries of fluorogenic tetrapeptides and tested them on three examples of human DUBs (OTU-1, Iso-T and UCH-L3) and one viral ubiquitin specific protease – Plpro from West Nile virus. In most cases the results show flexibility in the P4 position, very high specificity for Arg in P3 position and Gly at P2 – in accord with the sequence of the natural substrate ubiquitin. Surprisingly, screening of the P2 position revealed that UCH-L3 in contrast to all the other tested DUBs, demonstrates substantial tolerance of Ala and Val at P2, and a parallel analysis using the appropriate mutation of the full-length ubiquitin confirms this. We have also used an optimal tetrapeptide substrate, Ac-LRGG-AFC to investigate the activation mechanism of DUBs by ubiquitin and elevated salts concentration. Together, our results reveal the importance of the dual features of 1) substrate specificity and 2) the mechanism of ubiquitin binding in determining deubiquitination by this group of proteases.