Gan-Erdene Tudeviin, Nagamalleswari Kolli, Yin Luming, Wu Kenneth, Pan Zhen-Qiang, Wilkinson Keith D
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
J Biol Chem. 2003 Aug 1;278(31):28892-900. doi: 10.1074/jbc.M302890200. Epub 2003 May 19.
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 kcat of7s-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.
为了鉴定去泛素化酶（即对Nedd8衍生物水解具有特异性的蛋白酶），开发了一种简便的方法来合成Nedd8氨甲基香豆素（一种底物）和Nedd8乙烯砜（一种抑制剂）。去泛素化酶活性对于逆转Nedd8与cullin的缀合是必需的，这种修饰至少调节一些泛素连接酶。Nedd8乙烯砜与L-M(TK-)小鼠成纤维细胞裂解物的反应鉴定出两种去泛素化酶。去泛素化酶UCH-L3被泛素乙烯砜和Nedd8乙烯砜标记。相比之下，第二种更具选择性的酶仅被Nedd8乙烯砜标记。这种蛋白质DEN1是一种221个氨基酸的硫醇蛋白酶，由先前注释为SENP8的开放阅读框编码。重组人DEN1对Nedd8具有显著的特异性，催化Nedd8氨甲基香豆素的水解，Km为51 nM，kcat为7 s-1。DEN1作用于泛素氨甲基香豆素的催化效率是Nedd8氨甲基香豆素的6×10-4，其对SUMO-1氨甲基香豆素的活性无法检测到。这种选择性出乎意料，因为DEN1与催化去SUMO化的酶关系最为密切。这一观察结果使能够处理Nedd8 C末端的DUB家族成员数量增加到四个。
