ISG15 and ISGylation at the ribosome: mechanistic analysis, innate immune response, and impact on translation
The innate immune response is the first line of defense against all pathogens. The response incorporates the secretion of cytokines like type I interferons (IFN-I). There are hundreds of interferon-stimulated genes (ISGs), with ISG15 being one of the primary ones. ISG15 is a ubiquitin-like protein, which rapidly modifies a vast array of cellular and viral targets via a mechanism known as ISGylation. Like ubiquitination, ISGylation is mediated via an E1-E2-E3 enzymatic cascade. The main ISG15-E3 ligase has been associated with translating ribosomes, suggesting ISG15 preferably modifies newly synthesized proteins. Yet, their effects on the cellular translatome, protein turnover, and feedback to the transcriptome remain largely unknown. Here we provide structural modeling analysis of the known ISG15 ligases: HERC5, HHARI, and TRIM25. This revealed putative ISG15 and substrate interaction sites, as well as putative binding sites near the ribosome exit tunnel. Analyzing HeLa-/-isg15 deletion cells by polysome profiling, we found a cellular translation inhibition phenotype. These cells display lower translation levels, with and without interferon treatment. This is in stark contrast to WT HeLa cells, displaying inhibition of translation, only following IFN treatment. Transfection of HeLa-/-isg15 cells with an IFN-induced vector carrying ISG15 rescued the translation inhibition phenotype. Next, we will perform proteomics and mRNA analysis of translating ribosomes purified from HeLa, HeLa-/-isg15, and U2OS cells, with and without IFN treatment. This will provide mechanistic insight into ISG15 and ribosomal function during infection.
Will be in English