Zou Weiguo, Wang Ji, Zhang Dong-Er
Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Biochem Biophys Res Commun. 2007 Mar 2;354(1):321-7. doi: 10.1016/j.bbrc.2006.12.210. Epub 2007 Jan 8.
The function of ubiquitin-like protein ISG15 and protein modification by ISG15 (ISGylation) has been an enigma for many years. Recently, the research of ISGylation has been accelerated by the identification of the enzymes involved in the ISG15 conjugation process. Our previous study identified the interferon inducible protein EFP as an ISG15 isopeptide ligase (E3) for 14-3-3sigma. In this study, we show that ISG15 E3 ligase EFP can be modified by ISG15. Two ubiquitin E2 conjugating enzymes, UbcH6 and UbcH8, can support ISGylation of EFP. The Ring-finger domain of EFP is important for its ISGylation. Full-length EFP can enhance the ISGylation of Ring domain deleted EFP, indicating EFP can function as an ISG15 E3 ligase for itself. We also determined the ISGylation site of EFP and created its ISGylation resistant mutant EFP-K117R. Compared to the wild-type EFP, this mutant further increases the ISGylation of 14-3-3sigma. Thus we propose that autoISGylation of EFP negatively regulates its ISG15 E3 ligase activity for 14-3-3sigma.
泛素样蛋白ISG15的功能以及ISG15介导的蛋白质修饰（ISGylation）多年来一直是个谜。最近，参与ISG15缀合过程的酶的鉴定加速了对ISGylation的研究。我们之前的研究确定干扰素诱导蛋白EFP是14-3-3σ的ISG15异肽连接酶（E3）。在本研究中，我们发现ISG15 E3连接酶EFP可被ISG15修饰。两种泛素E2缀合酶UbcH6和UbcH8可支持EFP的ISGylation。EFP的环指结构域对其ISGylation很重要。全长EFP可增强缺失环结构域的EFP的ISGylation，表明EFP可作为自身的ISG15 E3连接酶发挥作用。我们还确定了EFP的ISGylation位点，并创建了其ISGylation抗性突变体EFP-K117R。与野生型EFP相比，该突变体进一步增加了14-3-3σ的ISGylation。因此，我们提出EFP的自身ISGylation负向调节其对14-3-3σ的ISG15 E3连接酶活性。
