title: Precursor processing of pro-ISG15/UCRP, an interferon-beta-induced ubiquitin-like protein.
abstract: Induction of the 17-kDa ubiquitin-like protein ISG15/UCRP and its subsequent conjugation to cellular targets is the earliest response to type I interferons. The polypeptide is synthesized as a precursor containing a carboxyl-terminal extension whose correct processing is required for subsequent ligation of the exposed mature carboxyl terminus. Recombinant pro-ISG15 is processed in extracts of human lung fibroblasts by a constitutive 100-kDa enzyme whose activity is unaffected by type I interferon stimulation. The processing enzyme has been purified to apparent homogeneity by a combination of ion exchange and hydrophobic chromatography and found to be stimulated 12-fold by micromolar concentrations of ubiquitin. Analysis of the products of pro-ISG15 processing enzyme demonstrates specific cleavage exclusively at the Gly(157)-Gly(158) peptide bond to generate a mature ISG15 carboxyl terminus. Irreversible inhibition of pro-ISG15 processing activity by thiol-specific alkylating agents and a pH rate dependence conforming to titration of a single group of pK(a) 8.1 indicate the 100-kDa enzyme is a thiol protease. Partial sequencing of a trypsin-derived peptide indicates the enzyme is either the human ortholog of yeast Ubp1 or a Ubp1-related protein. As yeast do not contain ISG15, these results suggest that a ubiquitin-specific enzyme was recruited for pro-ISG15/UCRP processing by adaptive divergence.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Potter
authors.forename: J
authors.initials: J
authors.lastname: Narasimhan
authors.forename: L
authors.initials: L
authors.lastname: Mende-Mueller
authors.forename: A L
authors.initials: AL
authors.lastname: Haas
mesh_terms: D003546:Cysteine Endopeptidases / Q000737:chemistry*; D016207:Cytokines; D010450:Endopeptidases / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016899:Interferon-beta / Q000494:pharmacology*; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D016207:Cytokines; D004791:Enzyme Inhibitors; D011498:Protein Precursors; D011994:Recombinant Proteins; D014452:Ubiquitins; C074034:ISG15 protein, human; D016899:Interferon-beta; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C059237:ubiquitin-Nalpha-protein hydrolase