ISG15 conjugation : cellular targets and the lysine selectivity of HERC5
ISG15 is a 17 kDa ubiquitin-like protein that functions in the innate immune response to pathogens. While human ISG15 is a multifunctional protein, its canonical function is that of an intracellular ubiquitin-like protein modifier. Like ISG15, the human E1 (UBE1L), E2 (UBE2L6) and E3 (HERC5) enzymes for ISG15 are all induced at the transcriptional level by type I IFN. ISGylation of pathogen and host proteins is a component of cellular defense against viruses. Consistent with this, many viruses have evolved mechanisms to counteract the effects of ISGylation. SARS-CoV-2, for example, encodes an ISG15-deconjugating activity (PLpro) that reverses ISGylation. Similarly, foot and mouth disease virus (FMDV) expresses a protease that not only deconjugates ISG15 but renders it incapable of being re-conjugated, while the Influenza B NS1 protein binds and sequesters ISGylated proteins to block the anti-viral effect of ISG15 conjugation. HERC5 is the major ISG15 ligase in humans and targets hundreds of cellular and viral proteins for ISGylation. HERC5 associates with polysomes and appears to target nascent polypeptides in a near stochastic manner, with limited substrate selectivity. A model for HERC5 function is that, in the context of a type I interferon response, newly translated viral proteins are the biologically relevant targets of ISGylation. This would be expected to exert selective pressure on the evolution of viral protein sequences, which in turn would influence the evolution of HERC5 target protein and lysine selectivity. Consistent with a host-pathogen “arms race”, HERC5 been shown to be under positive selection. Here, I have used DiGly proteomics, a bottom-up proteomics methodology that employs a general motif antibody to enrich ISGylation site-containing peptides, to characterize HERC5 activities and specificities with respect to residues flanking modified lysines, local physiochemical and structural properties of modified sites and exploring the possible biological implications of ISGylation. With 1164 HERC5-dependent ISGylation sites from 615 proteins identified, it was determined human HERC5 exhibits a preference for modifying lysines based on primary sequence context. Several residues were found significantly enriched (e.g., glycine flanking the modification sites, arginine at specific sites C-terminal to the modified lysines), while negatively charged amino acids were generally underrepresented on both sides of modified lysines. Analysis of the local amino acid environment of the sites showed that properties like overall polarity, side chain volume, secondary structure and whether it is structured or intrinsically disordered regions might affect the selectivity of lysine residues for ISGylation. Many enzymes involved in different metabolic pathways were targeted for ISGylation. Among these, glycolytic enzymes (nine out of eleven) and TCA cycle enzymes (six out of eight) were heavily ISGylated, with the number of modified sites identified within these enzymes ranging from one to 15. Interestingly, it was recently shown that glycolytic enzymes in mouse adipocytes were heavily ISGylated, and that ISGylation of LDHA suppressed thermogenesis in beige adipocytes. This study also showed that ISG15-null mice were resistant to diet-induced obesity, which has important implications for development of HERC5-based therapeutics. Interestingly, while HERC5 is the major ISG15 E3 ligase in humans, mice do not have a HERC5 gene. Instead, a closely related gene, mouse HERC6, encodes the major ISG15 ligase. Protein sequence alignment of all 28 HECT ligases (including HERC5 and HERC6) across species showed HERC5 and HERC6 were more divergent and less conserved (with a median of sequence identity at 73.8% and 68% for HERC5 and HERC6, respectively) than other HECT ligases (almost all of which had median of sequence identity greater than 90%). Like hHERC5, mHERC6 was shown to be a rapidly evolving gene and plays important roles in the innate immune system to viral infection. Analysis of the modification sites targeted by the two ligases may provide insight into any preferences or biases in recognizing lysine residues. By examining the lysine selectivity of the two ligases, it may be possible to elucidate how viral or bacterial infections have influenced the evolution of HERC5 and HERC6, and how they have adapted to challenges posed by pathogens. In this study, sites of human HERC5- and mouse HERC6-depedent ISGylome were identified with DiGly proteomics in the HEK293T-HERC5KO cells transfected UBA7, human or mouse UBE2L6, HERC5 or HERC6, and FLAG-ISG15. Only 30% of the sites targeted by human HERC5 were also modified by mouse HERC6. Sequence analysis revealed that human HERC5 showed a relative preference for lysine residues surrounded by positively charged amino acids, whereas negatively charged residues were overrepresented flanking the targets modified by mouse HERC6. The divergence of their lysine selectivity might reflect the specific pathogen-mediated evolutionary pressures that shaped the innate immune systems of specific mammalian clades and species. We have also shown that mouse HERC6, like hHERC5, co-fractionates with polyribosomes, and ISGylates nascent polypeptides within active translation complexes. Several aspects of the proteomics data were consistent with a cotranslational model. We noted a positive correlation between ISGylation and translational activity (based on ribosome profiling), as well as a positive correlation between “dwell time” of nascent chains on polysomes and ISGylation. Further, ISGylation was shown to occur on lysines that were in some cases predicted to be buried or partially buried in fully folded proteins, consistent with a model where such lysines were modified before nascent polypeptides attained a fully folded state. These analyses have contributed to our knowledge of the lysine selectivity of the major ISG15 ligase in human and mouse cells and represent the most detailed analysis of lysine selectivity of any ubiquitin or Ubl ligase. This work will provide a basis for further probing the biological functions of ISGylation in innate immune responses.
DiGly proteomic, Sequence preference, Human HERC5, Mouse HERC6, Cotranslational, ISG15, Conjugation
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