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Molecular characterization of ubiquitin-specific protease 18 reveals substrate specificity for interferon-stimulated gene 15.
- Source :
-
FEBS Journal . Apr2014, Vol. 281 Issue 7, p1918-1928. 11p. - Publication Year :
- 2014
-
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 1.3 ± 0.2 μ m. 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 4.6 ± 0.2 μ m and a kcat of 0.23 ± 0.004 s−1, respectively, at pH 7.5. 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. Structured digital abstract ● and by () ● by () [ABSTRACT FROM AUTHOR]
- Subjects :
- *UBIQUITIN
*PROTEINS
*ANTINEOPLASTIC agents
*ANTIVIRAL agents
*ENZYMES
Subjects
Details
- Language :
- English
- ISSN :
- 1742464X
- Volume :
- 281
- Issue :
- 7
- Database :
- Academic Search Index
- Journal :
- FEBS Journal
- Publication Type :
- Academic Journal
- Accession number :
- 95299528
- Full Text :
- https://doi.org/10.1111/febs.12754