ISGylation: is our genome yearning for such a modification?

Abstract

ISGylation is the post-translational modification of protein substrates covalently conjugated with the ubiquitin-like protein, interferon-stimulated gene 15 (ISG15). Although initially linked to antiviral immunity, recent evidence highlights important roles for ISGylation in various biological processes, such as maintaining genomic stability, promoting tumourigenesis, and being involved in other pathological conditions. In this review, we examine the molecular mechanisms underlying ISGylation, its interplay with other post-translational modifications, and its involvement in diverse biological and pathological processes. We propose future research directions to advance the field and discuss how ISGylation might be harnessed to ensure human health, particularly genome instability-associated diseases.

1. Introduction

Host cells release interferons (IFNs) in response to foreign pathogens or abnormal endogenous signals. These IFNs bind to the interferon-stimulated response element (ISRE) on specific promoters, thereby activating the expression of interferon-stimulated genes (ISGs) [1,2]. Among these, ISG15 is a 15 kDa protein initially discovered in IFN-treated Ehrlich ascites tumor (EAT) cells [3] and was previously referred to as ubiquitin cross-reactive protein (UCRP) [4]. ISG15 is typically expressed at low levels under physiological conditions but is one of the most highly expressed ISGs when activated by type I interferons (IFN-Is), lipopolysaccharide (LPS), DNA damage, viral and bacterial infections, or other pathogenic stimuli [1,5,6]. ISG15 comprises two tandem repeats of ubiquitin-like (UBL) domains, each playing distinct roles in ISGylation [7,8]. It was also identified as the first member of the Class 1 UBL proteins superfamily [9,10]. Initially synthesized as a 165-amino-acid precursor, pro-ISG15 undergoes processing by a human ortholog of yeast ubiquitin-specific protease Ubp1 or a related protein (Figure 1) [10]. This protease specifically cleaves the Gly157-Gly158 peptide bond, in addition to the removal of NH2-terminal methionine to yield a mature ISG15 protein of 156 amino acids [9,10]. This maturation process is essential for ISG15’s functionality within the ubiquitin (Ub)-like modification system. A conserved motif consisting of lysine, arginine, and glycine residues (151-LRLRGG-156) at the end of the C-terminal is vital for ISG15’s binding to specific target proteins by sequence similarity [8,11,12].

The process of ISGylation (Figure 2) involves a series of enzymatic steps analogous to ubiquitination. First, the E1-activating enzyme, such as Ub-like modifier-activating enzyme 7 (UBA7, also known as UBE1L), binds to ISG15’s C terminus via a high-energy thioester bond, facilitated by the hydrolysis of ATP [13]. Next, active ISG15 is transferred to the E2-conjugating enzyme, such as ubiquitin-conjugating enzyme E2 UbcH8 (also known as UBE2E2, the primary E2 enzyme for ISG15 conjugation) [14,15], or UbcH6 (also known as UBE2E1) [16], through a transesterification reaction. These steps rely on the UBL2 domain of ISG15. Subsequently, UbcH8 transfers ISG15 to E3 ligases such as HECT and RLD domain contained E3 ubiquitin-protein ligase 5/6 (HERC5/6) [17–20], tripartite motif containing 25 (TRIM25) [21], SCF (Skp1-Cul1-F-box) protein E3 ligase SCFFBXL19 [22], human homolog of Ariadne (HHARI) [23], and DTX3L [24]. Finally, E3 ligases catalyze the covalent attachment of ISG15 to target proteins on lysine residues (Table 1), a process facilitated by the UBL1 domain of ISG15.

Similar to ubiquitination, ISGylation is reversible due to the activity of deubiquitinases (DUBs) via a mechanism referred to as deISGylation. We will discuss this intriguing overlap between ISGylation and ubiquitination in the next section. Ubiquitin-specific peptidase 18 (USP18, also known as UBP43) is the primary ISG15-specific dUB that removes ISG15 from target proteins [13,66,67]. In addition, other members of the USP protease family, including USP2, USP5, USP13, USP14, USP16, and USP21, have been identified as ISG15-reactive proteases with similar functionalities [68–70].