Proteasomes Modulate Conjugation to the Ubiquitin-like Protein, ISG15

Abstract

ISG15 is a ubiquitin-like protein that is induced by interferon and microbial challenge. Ubiquitin-like proteins are covalently conjugated to cellular proteins and may intersect the ubiquitin-proteasome system via common substrates or reciprocal regulation. To investigate the relationship between ISG15 conjugation and proteasome function, we treated interferon-induced cells with proteasome inhibitors. Surprisingly, inhibition of proteasomal, but not lysosomal, proteases dramatically enhanced the level of ISG15 conjugates. The stimulation of ISG15 conjugates occurred rapidly in the absence of protein synthesis and was most dramatic in the cytoskeletal protein fraction. Inhibition of ISG15 conjugation by ATP depletion abrogated the proteasome inhibitor-dependent increase in ISG15 conjugates, suggesting that the effect was mediated by de novo conjugation, rather than protection from proteasomal degradation or inhibition of ISG15 deconjugating activity. The increase in ISG15 conjugates did not occur through a stabilization of the ISG15 E1 enzyme, UBE1L. Furthermore, simultaneous modification of proteins by both ISG15 and ubiquitin did not account for the proteasome inhibitor-dependent increase in ISG15 conjugates. These findings provide the first evidence for a link between ISG15 conjugation and proteasome function and support a model in which proteins destined for ISG15 conjugation are proteasomeregulated. Ubiquitin (ub)1 is the most highly conserved protein among eucaryotes and functions to post-translationally modify cellular proteins by covalent conjugation. Ub conjugation is carried out by the concerted activities of E1 (ub activation), E2 (ub conjugation), and E3 (ub ligase) enzymes in an ATP-dependent process (1). Sequential transfer of ub-thiol ester intermediates between ubiquitin-conjugating enzymes results in isopeptide bond formation between the ⑀-amino group of a substrate lysine residue and the carboxyl-terminal glycine of ub. The conjugated ub substrate can be targeted for further ubiquitylation in which polyubiquitin chains linked through lysine 48 are formed. Importantly, ubiquitylation is reversible, as ub can be removed from conjugates through the action of deconjugating enzymes (DUBs) (2). DUBs share highly conserved catalytic domains but exhibit great sequence diversity outside of these regions; the heterogeneity of these noncatalytic domains is thought to reflect substrate-specific activity. Thus, ubiquitin conjugation is a dynamic balance between conjugation and deconjugation. In the best characterized outcome of ubiquitylation, proteins conjugated to poly-ub chains of four or greater are targeted for degradation in the 26 S proteasome (3). The proteasome is composed of two 19 S "cap" complexes that bind, de-ubiquitylate, and unfold substrates to facilitate entry into the barrel-shaped 20 S proteolytic core. Identification of the protease activities of the proteasome as chymotrypsin-like, trypsin-like, and caspase-like permitted the development of peptide inhibitors of proteasome function. Stu...

Innate antiviral response targets HIV-1 release by the induction of ubiquitin-like protein ISG15

“…Interestingly, type I IFN stimulates expression not only of ISG15 but also UBEL1, UbcH8, and UBP43 (3,10) and markedly increases ISG15 conjugation. ISG15 targets a large number of cellular proteins (7); however, modification by ISG15 does not typically cause substrate degradation (11).…”

Post-transcriptional control of the interferon system

“…Another post-translational effect of IFN is ubiquitin conjugation, a process that targets proteins for degradation in the 26 S proteasome. ISG15 is an IFN-inducible ubiquitin-like protein that has been experimentally linked to the proteasome [85]. A possible ramification of this pathway has been recently demonstrated in which virus-and IFN-induced ISG15 conjugation leads to inhibition of IRF-3 degradation and therefore increased IRF-3 activity and an enhanced antiviral innate response [86].…”

Down-regulation of UCRP and UBE2L6 in BRCA2 knocked-down human breast cells

“…UCRP conjugation was thought to occur via a similar but distinct pathway compared with Ub conjugation [30]. An activating enzyme for UCRP (the E1 enzyme) was shown to be UBE1L [34,35]. This enzyme is described as specific for UCRP and does not catalyze the activation of ubiquitin [10].…”

“…Most remarkably, UCRP is conjugated to intracellular proteins via an isopeptide bond in a manner similar to ubiquitin (Ub), SUMO, and Nedd8 [33,34]. Conjugation of ubiquitin-like proteins (Ubls) involves a three-step mechanism whereby specific enzymes (or enzyme complexes) activate and covalently link Ubls to their substrates [34,35]. UCRP conjugation was thought to occur via a similar but distinct pathway compared with Ub conjugation [30].…”

“…Several reports demonstrate that UCRP is released by various cell types and can act as a cytokine leading to proliferation of NK cells [6,7,[28][29][30][31]. Most remarkably, UCRP is conjugated to intracellular proteins via an isopeptide bond in a manner similar to ubiquitin (Ub), SUMO, and Nedd8 [33,34]. Conjugation of ubiquitin-like proteins (Ubls) involves a three-step mechanism whereby specific enzymes (or enzyme complexes) activate and covalently link Ubls to their substrates [34,35].…”