Identification and characterization of DEN1, a deneddylase of the ULP family
Identification and characterization of DEN1, a deneddylase of the ULP family
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DOI：
10.1074/jbc.m302890200
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发表时间：
2003-08-01
影响因子：
4.8
通讯作者：
Wilkinson, KD
中科院分区：
文献类型：
--
作者：
Gan-Erdene, T;Nagamalleswari, K;Wilkinson, KD
为了鉴定对Nedd8衍生物具有特异性水解的去eddylases，我们开发了一种简便的方法来合成Nedd8酰胺甲基香豆素（底物）和Nedd8乙烯基砜（抑制剂）。Deneddylase的活性对于逆转Nedd8与cullin的结合是必要的，这种修饰至少可以调节一些泛素连接酶。Nedd8乙烯基砜与L-M（TK-）小鼠成纤维细胞裂解物反应鉴定出两种去醛酶。去泛素化酶UCH-L3由泛素乙烯砜和Nedd8乙烯砜标记。相比之下，第二种选择性更强的酶只被Nedd8乙烯砜标记。该蛋白DEN1是一种221个氨基酸的硫醇蛋白酶，由一个开放阅读框编码，先前注释为SENP8。重组人DEN1对Nedd8具有显著的特异性，并能催化Nedd8酰胺甲基香豆素的水解，Km为51 nM， k（cat）为7 s（-1）。DEN1对泛素酰胺甲基香豆素的催化效率是Nedd8酰胺甲基香豆素的6 × 10(-4)，而其对SUMO-1酰胺甲基香豆素的活性未检测到。这种选择性是出乎意料的，因为DEN1与催化去氧基化的酶最密切相关。这一观察结果扩展到四个DUB家族，其成员可以处理Nedd8的C端。
To identify deneddylases, proteases with specificity for hydrolysis of Nedd8 derivatives, a facile method was developed for the synthesis of Nedd8 amidomethylcoumarin ( a substrate) and Nedd8 vinyl sulfone ( an inhibitor). Deneddylase activity is necessary to reverse the conjugation of Nedd8 to cullin, a modification that regulates at least some ubiquitin ligases. The reaction of Nedd8 vinyl sulfone with L-M( TK-) mouse fibroblast lysates identified two deneddylases. The deubiquitinating enzyme UCH-L3 is labeled by both ubiquitin vinyl sulfone and Nedd8 vinyl sulfone. In contrast, a second and more selective enzyme is labeled only by Nedd8 vinyl sulfone. This protein, DEN1, is a 221-amino acid thiol protease that is encoded by an open reading frame previously annotated as SENP8. Recombinant human DEN1 shows significant specificity for Nedd8 and catalyzes the hydrolysis of Nedd8 amidomethylcoumarin with a Km of 51 nM and a k(cat) of 7 s(-1). The catalytic efficiency of DEN1 acting upon ubiquitin amidomethylcoumarin is 6 x 10(-4) that of Nedd8 amidomethylcoumarin and its activity on SUMO-1 amidomethylcoumarin is undetectable. This selectivity was unexpected as DEN1 is most closely related to enzymes that catalyze desumoylation. This observation expands to four the number of DUB families with members that can process the C terminus of Nedd8.