7 research outputs found
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
Multivalent interactions of the SUMO-interaction motifs in RING finger protein 4 determine the specificity for chains of the SUMO
Different types of ISG15 modification of endogenous Ubc13.
<p>HeLa cells were transiently transfected with pCMVb-HA-Ubc13 WT (A) or pCMVb-HA-Ubc13 C87G (B) mutant and other vectors as shown in the figure. 24 h post-transfection the cells were collected and lysed in presence 2-ME. The metal-chelate pull-downs were carried out under denatured conditions with or without 2-ME. Immunoblotting against the S-tag show the levels of ISG15. Equal loading of total protein was verified by anti alpha-tubulin immunoblotting.</p
Amino acid alignment of ISG15 proteins from different vertebrate species with human di-ubiquitin.
<p>The potential functionally important sites are highlighted. The C-terminal site including the double glycine motif for the conjugation to substrates is marked in bold, cysteine residues are indicated in bold and italics and the conserved cysteine residues in hinge region are shaded in grey. Sequence analysis was performed using ClustalW.</p
No evidence for ISG15 modification of MxA, hGBP1 and PML.
<p>(A) HeLa cells were induced with IFN-β for 24 h. The cells were lysed in urea buffer without reducing agent. Cellular lysates were equally aliquoted and 2-ME was added (loading to SDS-PAGE from left to right: 500 mM, 100 mM, 50 mM, 20 mM, 10 mM, 5 mM, 1 mM 2-ME, empty lane, 0 mM 2-ME) and blotted for MxA and hGBP1. (B) HeLa cells were transiently transfected with pCMV2b-Flag-MxA and the components of the ISG15 conjugation machinery as indicated in the figure. 24 h post-transfection, the cells were induced with IFN-β24 post-induction the cells were collected and lysed without 2-ME. Anti-FLAG immunoprecipitations were performed without 2-ME. Eluates were equally split and treated with or without 2-ME before SDS-PAGE (C) HeLa cells were transiently transfected with pCMVb-HA-MxA and components of the ISG15 conjugation machinery as indicated in the figure. 24 h post-transfection, the cells were collected and lysed without 2-ME. Anti-HA immunoprecipitations were performed. Eluates were equally split and treated with or without 2-ME before SDS-PAGE. (D) HeLa cells were transiently transfected with either pCMVb-MRGS-His-ISG15 or pCDNA4/TO/N-MRGS-His-SUMO2. 24 h post-transfection, the cells were induced with IFN-β (1,000 units/ml). Purifications of ISG15 or SUMO2 modified proteins were carried out under denaturating conditions without 2-ME. Eluates were equally split and treated with or without 2-ME before SDS-PAGE.</p
Multivalent interactions of the SUMO-interaction motifs in RING finger protein 4 determine the specificity for chains of the SUMO
RNF4 (RING finger protein 4) is a STUbL [SUMO (small ubiquitin-related modifier)-targeted ubiquitin ligase] controlling PML (promyelocytic leukaemia) nuclear bodies, DNA double strand break repair and other nuclear functions. In the present paper, we describe that the sequence and spacing of the SIMs (SUMO-interaction motifs) in RNF4 regulate the avidity-driven recognition of substrate proteins carrying SUMO chains of variable length