Covalent protein modification with ISG15 via a conserved cysteine in the hinge region.
The ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa) is strongly induced by type I interferons and displays antiviral activity. As other ubiquitin-like proteins (Ubls), ISG15 is post-translationally conjugated to substrate proteins by an isopeptide bond between the C-terminal glycine of ISG15 and the side chains of lysine residues in the substrates (ISGylation). ISG15 consists of two ubiquitin-like domains that are separated by a hinge region. In many orthologs, this region contains a single highly reactive cysteine residue. Several hundred potential substrates for ISGylation have been identified but only a few of them have been rigorously verified. In order to investigate the modification of several ISG15 substrates, we have purified ISG15 conjugates from cell extracts by metal-chelate affinity purification and immunoprecipitations. We found that the levels of proteins modified by human ISG15 can be decreased by the addition of reducing agents. With the help of thiol blocking reagents, a mutational analysis and miRNA mediated knock-down of ISG15 expression, we revealed that this modification occurs in living cells via a disulphide bridge between the substrates and Cys78 in the hinge region of ISG15. While the ISG15 activating enzyme UBE1L is conjugated by ISG15 in the classical way, we show that the ubiquitin conjugating enzyme Ubc13 can either be classically conjugated by ISG15 or can form a disulphide bridge with ISG15 at the active site cysteine 87. The latter modification would interfere with its function as ubiquitin conjugating enzyme. However, we found no evidence for an ISG15 modification of the dynamin-like GTPases MxA and hGBP1. These findings indicate that the analysis of potential substrates for ISG15 conjugation must be performed with great care to distinguish between the two types of modification since many assays such as immunoprecipitation or metal-chelate affinity purification are performed with little or no reducing agent present.
Center for Molecular Medicine Cologne-CMMC, Institute for Genetics, University of Cologne, Cologne, Germany.
Veronika N
VN
Bade
Jochen
J
Nickels
Kirstin
K
Keusekotten
Gerrit J K
GJ
Praefcke
D015153:Blotting, Western; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016207:Cytokines / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D034741:RNA, Small Interfering; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014452:Ubiquitins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D016207:Cytokines; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D014452:Ubiquitins; C074034:ISG15 protein, human; D003545:Cysteine