MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it http://mint.bio.uniroma2.it/mint anonymous-2014b-1 Molecular characterization of USP18 reveals substrate specificity for ISG15 in vitro In vitro mst microscale thermophoresis mst predetermined predetermined participant predetermined guenter.fritz@uniklinik-freiburg.de 2014 imex curation Basters A., Geurink P.P., Oualid F.E., Ketscher L., Casutt M.S., Krause E., Ovaa H., Knobeloch K.P., Fritz G. FEBS J. (1742-464X) Only protein-protein interactions anonymous-2014c-2 Molecular characterization of USP18 reveals substrate specificity for ISG15 in vitro In vitro enzymatic study enzymatic study predetermined predetermined participant predetermined guenter.fritz@uniklinik-freiburg.de 2014 imex curation Basters A., Geurink P.P., Oualid F.E., Ketscher L., Casutt M.S., Krause E., Ovaa H., Knobeloch K.P., Fritz G. FEBS J. (1742-464X) Only protein-protein interactions isg15_mouse Ubiquitin-like protein ISG15 Isg15 G1p2 Ucrp Interferon-induced 15 kDa protein Interferon-induced 17 kDa protein Ubiquitin cross-reactive protein protein protein mouse Mus musculus Mouse MAWDLKVKMLGGNDFLVSVTNSMTVSELKKQIAQKIGVPAFQQRLAHQTAVLQDGLTLSSLGLGPSSTVMLVVQNCSEPLSILVRNERGHSNIYEVFLTQTVDTLKKKVSQREQVHEDQFWLSFEGRPMEDKELLGEYGLKPQCTVIKHLRLRGGGGDQCA In response to IFN-tau secreted by the conceptus, may ligate to and regulate proteins involved in the release of prostaglandin F2-alpha (PGF), and thus prevent lysis of the corpus luteum and maintain the pregnancy mint Ubiquitin-like protein that is conjugated to intracellular target proteins after IFN-alpha or IFN-beta stimulation. Its enzymatic pathway is partially distinct from that of ubiquitin, differing in substrate specificity and interaction with ligating enzymes. ISG15 conjugation pathway uses a dedicated E1 enzyme, but seems to converge with the Ub conjugation pathway at the level of a specific E2 enzyme. Targets include STAT1, SERPINA3G/SPI2A, JAK1, MAPK3/ERK1, PLCG1, EIF2AK2/PKR, MX1/MxA, and RIG-1. Deconjugated by USP18/UBP43. Shows specific chemotactic activity towards neutrophils and activates them to induce release of eosinophil chemotactic factors. May serve as a trans-acting binding factor directing the association of ligated target proteins to intermediate filaments. May also be involved in autocrine, paracrine and endocrine mechanisms, as in cell-to-cell signaling, possibly partly by inducing IFN-gamma secretion by monocytes and macrophages. Seems to display antiviral activity during viral infections ubp18_mouse Ubl carboxyl-terminal hydrolase 18 Usp18 Ubp43 43 kDa ISG15-specific protease ISG15-specific-processing protease Ubl thioesterase 18 protein protein mouse Mus musculus Mouse MGKGFGLLRKPCQSVVAEPQQYSALEEERTMKRKRVLSRDLCSAWDSPHGLVGLHNIGQTCCLNSLLQVFMMNMDFRMILKRITVPRSAEERKRSVPFQLLLLLEKMQDSRQKAVLPTELVQCLQKYNVPLFVQHDAAQLYLTIWNLTKDQITDTDLTERLQGLFTIWTQESLICVGCTAESSRRSKLLTLSLPLFDKDAKPLKTLEDALRCFVQPKELASSDMCCESCGEKTPWKQVLKLTHLPQTLTIHLMRFSARNSRTEKICHSVNFPQSLDFSQVLPTEEDLGDTKEQSEIHYELFAVIAHVGMADFGHYCAYIRNPVDGKWFCFNDSHVCWVTWKDVQCTYGNHRYRWRETAYLLVYTKTGS isg15-usp18 1497264 n/a ISG15 1497266 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified n/a USP18 1497267 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction false false false 1497264 f3a Purified ISG15-C76S and fluorescence-labelled USP18 were used for microscale thermophoresis to determine the binding affinity. The binding data could be fitted assuming a 1:1 stoichiometry of USP18 and ISG15-C76S in the formed complex, showing that ISG15 bound specifically to one site. The USP18-ISG15 complex displayed a Kd of 1.3±0.2 FM (Fig. 3). isg15-usp18-1 1497265 n/a ISG15 1497266 tag fluorescence tag visualisation by fluorescence tag fluorescence enzyme target enzyme target substrate substrate neutral component neutral component purified purified n/a USP18 1497267 predetermined predetermined participant predetermined enzyme enzyme neutral component neutral component purified purified cleavage cleavage reaction cleavage false false false 1497265 f4b f4c f4e The activity of USP18 was quantified by using a newly developed ISG15-based fluorescence polarization (FP) assay reagent (Fig. 4A). The advantage of this reagent over conventional ISG15-based reagents, such as ISG15-amidomethylcoumarin (ISG15-AMC), is the presence of a native isopeptide linkage between the C-terminal Gly of ISG15 and a Lys side chain resembling natural ISG15-linked substrates more closely [13]. The substrate was synthesized from murine ISG15-C76S and TAMRA-(γ-thioK)-G peptide using the reported general method for the synthesis of analogous Ub and Ub-like protein-based FP reagents [30]. Incubation of a fixed concentration of 100 nM of this substrate with different concentrations of USP18 resulted in a dose-dependent decrease of the polarization value (in mP) indicating proteolytic cleavage of the substrate (Fig. 4C). Using the assay described above, we determined the Michaelis-Menten kinetic parameters of USP18 in a buffer with physiological pH and salt concentration. The parameters were obtained by incubating different amounts of the substrate with a fixed concentration of USP18 (Fig. 4B). The measurements revealed a Km=4.6±0.2 μM, a Vmax=0.14±0.003 FM min-1, a kcat=of 0.23±0.004 sec-1, and catalytic efficiency kcat/Km=5 104±2 104 M-1 sec-1 for USP18