Abstract
The expression of the ubiquitin-like molecule ISG15 (UCRP) and protein modification by ISG15 (ISGylation) are strongly activated by interferon, genotoxic stress, and pathogen infection, suggesting that ISG15 plays an important role in innate immune responses. Inducible nitric-oxide synthase (iNOS) is induced by the similar stimuli as ISG15 and enhances the production of nitric oxide (NO), a pleiotropic free radical with antipathogen activity. Here, we report that cysteine residues (Cys-76 and -143 in mouse, Cys-78 in human) of ISG15 can be modified by NO, and the NO modification of ISG15 decreases the dimerization of ISG15. The mutation of the cysteine residue of ISG15 to serine improves total ISGylation. The NO synthase inhibitor S-ethylisothiourea reduces endogenous ISGylation. Furthermore, ectopic expression of iNOS enhanced total ISGylation. Together, these results suggest that nitrosylation of ISG15 enhances target protein ISGylation. This is the first report of a relationship between ISGylation and nitrosylation.
Highlights
The ISG15 E1 (UBE1L/UBA7), E2s (UbcH6 and UbcH8), and E3s [5,6,7,8,9,10,11]
The Vmax of Inducible nitric-oxide synthase (iNOS) is ϳ10-fold greater than that of NOS1 and NOS3 [16]. iNOS is not expressed under normal conditions; it is induced in response to cytokines, microbes, or microbial products, resulting in the sustained production of nitric oxide (NO) [16]
We found that ISG15 (C76S and C76S/C143S) mutants did not show dimerization, which means that Cys-76 but not Cys-143 contributes to stable dimer formation, which is detectable by nonreducing SDS-PAGE
Summary
Rified proteins were treated with SNOC (100 M) in the dark at room temperature for up to 1 h. Human IFN␣-2a (5000 units/ml) and/or ETU (1 mM) were added to the culture medium of HeLa cells and incubated for 36 h. Immunoprecipitation, Ni-NTA Pulldown, and Western Blot Analysis—Immunoprecipitation, NiNTA pulldown, and Western blot analysis were done as reported previously [20]. Antibodies—Antibodies against FLAG (Sigma), HA (Covance), iNOS (BD Transduction LaboratoriesTM), and His (Clontech) were purchased from the respective manufacturers. Rabbit anti-mouse ISG15 polyclonal antibodies have been described previously [23]. Rabbit anti-mUbcH8 antibodies were reported previously [7]. Anti-biotin antibody and the specific second antibody were from NitroGlo nitrosylation detection kit (PerkinElmer Life Sciences)
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