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The FEBS journal

Molecular characterization of ubiquitin-specific protease 18 reveals substrate specificity for interferon-stimulated gene 15.


PMID 24533902

Abstract

Protein modification by interferon-stimulated gene 15 (ISG15), an ubiquitin-like modifier, affects multiple cellular functions and represents one of the major antiviral effector systems. Covalent linkage of ISG15 to proteins was previously reported to be counteracted by ubiquitin-specific protease 18 (USP18). To date, analysis of the molecular properties of USP18 was hampered by low expression yields and impaired solubility. We established high-yield expression of USP18 in insect cells and purified the protease to homogeneity. USP18 binds with high affinity to ISG15, as shown by microscale thermophoresis with a Kd of 1.3xa0±xa00.2xa0μm. The catalytic properties of USP18 were characterized by a novel assay using ISG15 fused to a fluorophore via an isopeptide bond, giving a Km of 4.6xa0±xa00.2xa0μm and a kcat of 0.23xa0±xa00.004xa0s(-1) , respectively, at pHxa07.5. Furthermore, the recombinant enzyme cleaves efficiently ISG15 but not ubiquitin from endogenous cellular substrates. In line with these data, USP18 exhibited neither cross-reactivity with an ubiquitin isopeptide fluorophore substrate, nor with a ubiquitin vinyl sulfone, showing that the enzyme is specific for ISG15. ●ISG15xa0andxa0USP18xa0bindxa0byxa0microscale thermophoresisxa0(View interaction) ●USP18xa0cleavesxa0ISG15xa0byxa0enzymatic studyxa0(View interaction).