Enzymatic activity of TF
-USP18 (A) Cell lysates of USP18 knockout mouse embryonic fibroblasts were stimulated with IFN β resulting in elevated ISGylation or left untreated. Cell lysates were incubated with and without TFAAA-USP18 for the indicated times at 37°C. TFAAA-USP18-mediated ISG15 deconjugation was monitored by Western blot with an ISG15-specific antibody. A decrease of protein ISGylation with a concomitant increase of free ISG15 was observed. (B) Quantification of ISGylation and free ISG15 from (A) revealed a 3-fold decrease of conjugated and a corresponding increase of free ISG15 upon incubation with TFAAA-USP18. Densitometric values of free ISG15 and ISGylated proteins were normalized to β-Actin and to the protein levels in IFN β-treated cells at 0 hours without addition of TFAAA-USP18. (C) TFAAA-USP18 or the catalytically inactive mutant TFAAA-USP18-C61A were incubated with substoichiometric amounts of either ISG15 vinyl sulfone (ISG15-VS) or ubiquitin vinyl sulfone (Ub-VS) for 1 h at 37°C. The covalent adduct of USP18 with ISG15 is shown by a shift to higher molecular mass visualized on a Coomassie-stained SDS PAGE. (D) Catalytic activity of TFAAA-USP18 monitored by cleavage of ISG15-AMC: different amounts of TFAAA-USP18 were incubated with 600 nM ISG15-AMC. Release of AMC was monitored by its specific fluorescence at 460 nm over a period of 30 minutes. RFU: Relative fluorescence units.