title: Molecular characterization of ubiquitin-specific protease 18 reveals substrate specificity for interferon-stimulated gene 15.
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 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.
  ●ISG15 and USP18 bind by microscale thermophoresis (View interaction) ●USP18 cleaves ISG15 by enzymatic study (View interaction).
authors.affiliation: Institute of Neuropathology, University of Freiburg, Germany; Faculty of Biology, University of Freiburg, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Basters
authors.forename: Paul P
authors.initials: PP
authors.lastname: Geurink
authors.forename: Farid
authors.initials: F
authors.lastname: El Oualid
authors.forename: Lars
authors.initials: L
authors.lastname: Ketscher
authors.forename: Marco S
authors.initials: MS
authors.lastname: Casutt
authors.forename: Eberhard
authors.initials: E
authors.lastname: Krause
authors.forename: Huib
authors.initials: H
authors.lastname: Ovaa
authors.forename: Klaus-Peter
authors.initials: KP
authors.lastname: Knobeloch
authors.forename: Günter
authors.initials: G
authors.lastname: Fritz
mesh_terms: D000818:Animals; D016207:Cytokines / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D061987:Sf9 Cells; D018411:Spodoptera; D013379:Substrate Specificity; D043222:Ubiquitin Thiolesterase / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D016207:Cytokines; C489550:G1p2 protein, mouse; D014452:Ubiquitins; C118306:Usp18 protein, mouse; D043222:Ubiquitin Thiolesterase
keywords: ISG15; USP18; fluorescence probe; interferon; ubiquitin