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

Autor: Anja Basters, Lars Ketscher, Eberhard Krause, Huib Ovaa, Paul P. Geurink, Farid El Oualid, Günter Fritz, Marco S. Casutt, Klaus-Peter Knobeloch
Rok vydání: 2014
Předmět:
Zdroj: FEBS Journal. 281:1918-1928
ISSN: 1742-464X
DOI: 10.1111/febs.12754
Popis: 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 μm. 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 μm 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 ●ISG15 and USP18 bind by microscale thermophoresis (View interaction) ●USP18 cleaves ISG15 by enzymatic study (View interaction)
Databáze: OpenAIRE
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