ISG15 - Proteins and Enzymes

The protein contains 2 tandem Ub homology domains, similar to FAT10. In contrast to other UBLs, ISG15 has not been identified in lower eukaryotes (yeast, nematode, insects, plants) indicating its role in specialized functions in higher eukaryotes. ISG15 was identified as an interferon stimulated gene (ISG) since its expression is induced in response to type I interferons and lipopolysaccharide treatment. ISG15 pathway components are induced upon bacterial and viral infection and thus appears to be an important modulator of immunity if vertebrate animals. Upon interferon treatment, ISG15 can be detected in both free and conjugated forms, and is secreted from monocytes and lymphocytes where it possibly functions as a cytokine. In the cell, ISG15 co-localizes with intermediate filaments and ISGylation may modulate critical signal transduction processes eg. JAK-STAT pathway.

The mechanism of ISGylation and deISGylation is similar to that of ubiquitin, although the complete system components have not yet been identified.ISG15 is also translated as a precursor protein with additional C-terminal residues that have to be removed to expose the conserved di-glycine motif that is characteristic of the mature and active form. The specific protease that generates the mature form has not yet been identified.

The activating E1 enzyme (UBE1L) activates ISG15 by forming a high-energy thioester intermediate and then transfers it to the UbcH8 E2 protein. UbcH8 has been identified as the major E2 for ISGlyation, although it also functions in ubiquitination. The E2 protein subsequently transfers the ISG15 to specific E3 ligases and relevant intracellular substrates. A small number of ISG15-specific E3 ligases have been identified including Efp, HERC5 and HHARI. The C-terminal UBL domain of ISG15 is sufficient for thioester formation with UbcH8, but both UBL domain are required for conjugation to substrate proteins. Proteins that are known to be modified by ISG15 include serpin 2a, ERK1/2, phospholipase Cy1, Stat1 and Jak1. The functional consequences of these specific protein modifications are currently unknown. Only one deconjugating protease with specificity to ISG15 has been identified to date: UBP43 (USP18, a member of the USP family) cleaves ISG15-peptide fusions and also removes ISG15 (deISGylation) from native conjugates.

The chemical or enzymatic modification of ISG15 proteins at important functional sites results in several useful derivatives. Modifying the C-terminal glycine carboxyl of ISG15 to an aldehyde (ISG15-H) or vinylsulfone (ISG15-VS) groups results in highly potent inhibitors of ISG15-specific hydrolases. Alternatively, substrates for the kinetic analysis of enzymes like UBP43 are generated by synthetically conjugating fluorophores to the C-terminus of ISG15 (ISG15-AMC). ISG15 can also be coupled to agarose via its primary amines, leaving the C-terminus free and available to purify ubiquitin binding proteins. The N-terminus or lysine groups of ISG15 can be modified by small other haptens (fluorescein/rhodamine or biotin) for the sensitive detection by direct fluorescence or secondary reagents (eg. avidin or streptavidin reagents) respectively. These groups can also be used for the affinity purification of ISG15 conjugates or ISG15 binding proteins. Since the chemistry of ISG15 conjugation involves its C-terminus, these modifications result in a fully functional molecule that is viable in conjugation reactions.