Covalent Protein Modification with ISG15 via a Conserved Cysteine in the Hinge Region

Abstract

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.

90K Glycoprotein Promotes Degradation of Mutant β-Catenin Lacking the ISGylation or Phosphorylation Sites in the N-terminus

“…Therefore, to identify the residue(s) in the N-terminus of β-catenin that is(are) responsible for ISG15 conjugation, we compared the N-terminal aa sequence of β-catenin with the sequences published in previous studies reporting the consensus motif required for conjugation of ISG15. ISG15 conjugation occurs on the ε-amine group of lysine residues [17] or on cysteine residues [18] . As shown in Figure 3 A , we identified two lysine residues (Lys-19 and Lys-49); however, the 86 aa N-terminus of β-catenin contains no cysteine residues.…”

“…ISG15 conjugation occurs primarily between the C-terminal glycine of ISG15 and the ε-amino group of a lysine side chain within the substrate protein [23] . However, conjugation of ISG15 can occur via a disulfide bridge between the Cys87 residue of Ubc13 and the Cys78 at the hinge region of ISG15 [18] . It is likely that canonical ubiquitin conjugation occurs between the C-terminal Gly76 of ubiquitin and the ε-amino group of a lysine residue within the target substrate [19] .…”

Decoding the Ubiquitin-Mediated Pathway of Arthropod Disease Vectors

“…Ubiquitin-like proteins were first discovered in 1979 and named after the interferon-stimulated gene 15 (ISG15) [29]. Since its discovery, several other ubiquitin-like proteins have been identified.…”

Isg15 controls p53 stability and functions

“…Isg15 preferentially modifies lysine residues and, to a lesser extent, cysteines. 31 To verify whether cysteines are modified by Isg15, we incubated purified Isg15-modified p53 in the presence of a reducing agent, mercaptoethanol, and found that this was not sufficient to remove the modification (data not shown). Thus, we turned to the analysis of the lysine residues on p53.…”