Precursor Processing of Pro-ISG15/UCRP, an Interferon-β-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 thiolspecific 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.Type I interferons (interferons-␣ and -␤) induce a specific subset of proteins responsible for mediating the antiviral and broad pleiotropic effects of these cytokines, reviewed in Ref. 1. One of the earliest responses to type I interferons is the increased transcription of the ISG15 1 gene, which is controlled by a 5Ј interferon-stimulated response element (1, 2). The sequence of the resulting 17.1-kDa ISG15 protein, also known as the ubiquitin cross-reactive protein (UCRP), is composed of two ubiquitin-like domains that retain the canonical LRLRGG sequence required for conjugation of ubiquitin to intracellular targets as the committed step for 26 S proteasome-dependent degradation (3-5). The biological effects of ISG15 are similarly mediated by its covalent conjugation to a small subset of cellular proteins through an enzyme pathway that is distinct from that of ubiquitin ligation (5, 6). Both free and conjugated ISG15 pools are significantly induced following exposure to interferon-␣/␤; however, in contrast to the homeostatic mechanism that maintains a constant ratio of free to conjugated ubiquitin (7), the two pools of ISG15 exhibit a characteristic biphasic induction during which free polypeptide increases early during the interferon response and then undergoes increased conjugation from 12 to 72 h (5). The ISG15 polypeptide acts in trans to noncovalently bind associated target proteins and model chimeric constructs to intermediate filaments 2 ; in contrast, free ISG15 shows only marginal affinity for binding to these cytoskeletal components (8). Neither free nor conjugated ubiquitin levels are affected by interferon t...

A New SUMO-1-specific Protease, SUSP1, That Is Highly Expressed in Reproductive Organs

“…In addition, SUSP1 showed no evident amino acid sequence similarity to any known deubiquitinating enzymes. On the other hand, it has recently been reported that the partial peptide sequence of an enzyme processing UCRP, a member of Ulps, from human lung carcinoma cells shows a significant similarity to yeast Ub-specific protease, Ubp1 (18). Thus, it is likely that SUSPs described in the present study form a novel family distinct from USPs and UCRP-processing enzymes.…”

Identification and characterization of two homologues of interferon-stimulated gene ISG15 in crucian carp

“…Like ubiquitin, ISG15 can be conjugated to target proteins via its C-terminal end [7]. Due to existence of the additional C-terminal amino acids, ISG15 proteins from human and mouse are synthesized as a 17-kDa precursor and subsequently processed to 15-kDa proteins by a specific protease that has not been identified so far [34]. This process results in exposure of diglycine residues at the carboxyl terminal of ISG15, which is critical for subsequent conjugation to target proteins [34] and its antiviral activity [17].…”

Serpin 2a Is Induced in Activated Macrophages and Conjugates to a Ubiquitin Homolog

“…It contains two ubiquitin-like domains with 43 and 62% homology to ubiquitin (10,14). The mechanism of conjugation of ISG15 to cellular substrates has been proposed to be analogous to that for ubiquitin involving homologous but not identical enzymes (22,23,43,44). Ubiquitin can form polyubiquitin chains from one lysine residue in a target protein, and it is possible that the spi2a-ISG15 complex with two ISG15 molecules is the result of a di-ISG15 chain.…”

“…It has been shown to conjugate to cellular proteins in a process analogous to that for ubiquitin, using homologous, but distinct, enzymes (22)(23)(24). Thus, the 65-kDa form of spi2a likely represents spi2a (50 kDa) covalently bound to one molecule of ISG15, whereas the 80-kDa form represents spi2a bound to two molecules of ISG15.…”