Cellular Targets and the Lysine Selectivity of the HERC5 ISG15 Ligase

ISG15 is a type I interferon-induced ubiquitin-like protein modifier that functions in innateimmune responses. The single major human ISG15 ligase is HERC5 (hHERC5), a ribosome-associated HECT E3s that broadly ISGylates proteins cotranslationally. Here, we characterized the hHERC5-dependent ISGylome and identified over 5,000 modified lysines in over 2,000 proteins. In parallel, we characterized the substrate selectivity of the major mouse ISG15 ligase, mHERC6, and analysis of sequences surrounding ISGylation sites revealed that hHERC5 and mHERC6 have distinct preferences for amino acid sequence context. Several features of the datasets were consistent with ISGylation of ribosome-tethered nascent chains, and mHERC6, like hHERC5, associated with polysomes and cotranslationally modified nascent polypeptides. Like many genes encoding innate immune effectors, the hHERC5 and mHERC6 genes are under positive genetic selection and we propose that the differing lysine selectivities of these ligases may reflect species-specific adaptations to pathogen challenges experienced over evolutionary timeframes.