Abstract
Gene products that modify the apoptotic susceptibility of cancer cells may offer novel drug response markers or therapeutic targets. In this study, we probed the contribution of 53 different isopeptidases to apoptosis triggered by bortezomib and etoposide. USP18, a type I IFN-induced protein that deconjugates the ubiquitin-like modifier ISG15 from target proteins, was found to limit apoptotic susceptibility to IFN-alpha or bortezomib. Ablating USP18 in cells treated with IFN-alpha increased tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) production; upregulated expression of transcription factors IFN-regulatory factor (IRF)-1, IRF-7, and IRF-9; and promoted the extrinsic pathway of apoptosis. The proapoptotic effects of ablating USP18 were abrogated by FLIP overexpression or TRAIL silencing. However, in bortezomib-treated cells, weak spontaneous signaling from type I IFNs was implicated in the proapoptotic effect of USP18 ablation. Ectopic USP18 repressed apoptotic signaling by IFN-alpha, TRAIL, or bortezomib. Similar effects were produced by a catalytically inactive USP18 mutant, indicating that the antiapoptotic function of USP18 is independent of its catalytic activity. These findings suggest that USP18 may significantly limit operation of the extrinsic apoptotic pathway triggered by type I IFN and drugs.
Highlights
Post-translational modification of proteins by covalently linked ubiquitin (Ub) or Ub-like modifiers (Ubl) is a regulatory mechanism involved in many cellular processes [1]
Mice lacking USP18 (Ubp43) accumulate poly-ISGylated proteins and show a severe phenotype characterized by premature death, brain cell injury, and hydrocephalus [28]
This severe phenotype cannot be rescued by the simultaneous absence of ISG15 [29], arguing for catalysis-independent or ISG15-independent functions of USP18
Summary
Post-translational modification of proteins by covalently linked ubiquitin (Ub) or Ub-like modifiers (Ubl) is a regulatory mechanism involved in many cellular processes [1]. Ub and Ubl molecules are attached to substrates by isopeptide linkage generally through the sequential activity of the three conjugating enzymes E1, E2, and E3 [2]. This linkage is reversible and the Ub or the Ubl moieties can be released from the substrates by deconjugating enzymes belonging to the isopeptidase family [3]. Isopeptidase inhibition, in addition to blocking proteasomemediated protein turnover, can alter many Ub-dependent functions unrelated to protein degradation. These alterations generate cellular stresses that cause cell death. Nonselective isopeptidase inhibitors can trigger apoptosis in Authors' Affiliation: Dipartimento di Scienze e Tecnologie Biomediche, Universita' di Udine, Udine, Italy
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