USP16 is an ISG15 cross-reactive deubiquitinase that targets pro-ISG15 and ISGylated proteins involved in metabolism

Abstract

Interferon-induced ubiquitin (Ub)-like modifier ISG15 covalently modifies host and viral proteins to restrict viral infections. Its function is counteracted by the canonical deISGylase USP18 or Ub-specific protease 18. Notwithstanding indications for the existence of other ISG15 cross-reactive proteases, these remain to be identified. Here, we identify deubiquitinase USP16 as an ISG15 cross-reactive protease by means of ISG15 activity-based profiling. Recombinant USP16 cleaved pro-ISG15 and ISG15 isopeptide-linked model substrates in vitro, as well as ISGylated substrates from cell lysates. Moreover, interferon-induced stimulation of ISGylation was increased by depletion of USP16. The USP16-dependent ISG15 interactome indicated that the deISGylating function of USP16 may regulate metabolic pathways. Targeted enzymes include malate dehydrogenase, cytoplasmic superoxide dismutase 1, fructose-bisphosphate aldolase A, and cytoplasmic glutamic-oxaloacetic transaminase 1. USP16 may thus contribute to the regulation of a subset of metabolism-related proteins during type-I interferon responses.

Unveiling the Multifaceted Roles of ISG15: From Immunomodulation to Therapeutic Frontiers

“…There are specific proteases known as deISGylating enzymes that catalyse the removal of ISG15 from its conjugated targets allowing the reversible nature of ISGylation. USP18 is the main human protease that cleaves ISG15 [ 32 ]; however, the deubiquitinase USP16 has recently emerged as a novel ISG15 cross-reactive protease that may regulate metabolic pathways [ 33 ]. This intricate regulatory mechanism enables cells to modulate the potency and duration of ISGylation in response to various cellular and environmental stimuli.…”

The ISG15-Protease USP18 Is a Pleiotropic Enhancer of HIV-1 Replication

“…The ISG15 gene comprises two exons and encodes a 17-kDa precursor. Under physiological conditions, the ISG15 precursor can be cleaved into the mature form of the 15-kDa ISG15 peptide through the removal of eight C-terminal amino acids by USP18 and possibly by USP16, retaining a shared C-terminal amino acid motif, LRLRGG, which allows ISG15 to covalently bind to the lysine residues of the substrate [ 48 , 49 , 50 ]. In addition, ISG15 expression is induced in lipopolysaccharide (LPS)-stimulated and retinoic acid (RA)-treated cells in an IFN-I-dependent manner [ 51 , 52 ].…”

“…USP18 functions as a protease to cleave ISG15 molecules from substrate proteins through isopeptide bonds ( Figure 1 ) and as a negative regulator of IFN-I signaling [ 39 , 49 ] ( Figure 2 ). Recently, USP16 was identified as another ISG15 cross-reactive protease [ 50 ]. USP18 is highly abundant in the liver, spleen, and thymus, with low levels detected in the bone marrow, adipose tissue, and lung tissue.…”