Tandem UIMs confer Lys48 ubiquitin chain substrate preference to deubiquitinase USP25.
Ubiquitin-specific protease (USP) 25, belonging to the USP family of deubiquitinases, harbors two tandem ubiquitin-interacting motifs (UIMs), a ~20-amino-acid α-helical stretch that binds to ubiquitin. However, the role of the UIMs in USP25 remains unclear. Here we show that the tandem UIM region binds to Lys48-, but not Lys63-, linked ubiquitin chains, where the two UIMs played a critical and cooperative role. Purified USP25 exhibited higher ubiquitin isopeptidase activity to Lys48-, than to Lys63-, linked ubiquitin chains. Mutations that disrupted the ubiquitin-binding ability of the tandem UIMs resulted in a reduced ubiquitin isopeptidase activity of USP25, suggesting a role for the UIMs in exerting the full catalytic activity of USP25. Moreover, when mutations that convert the binding preference from Lys48- to Lys63-linked ubiquitin chains were introduced into the tandem UIM region, the USP25 mutants acquired elevated and reduced isopeptidase activity toward Lys63- and Lys48-linked ubiquitin chains, respectively. These results suggested that the binding preference of the tandem UIMs toward Lys48-linked ubiquitin chains contributes not only to the full catalytic activity but also to the ubiquitin chain substrate preference of USP25, possibly by selectively holding the Lys48-linked ubiquitin chain substrates in the proximity of the catalytic core.
Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama 226-8501, Japan.|School of Life Science and Technology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.
Kohei
K
Kawaguchi
School of Life Science and Technology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.
Kazune
K
Uo
School of Life Science and Technology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.
Toshiaki
T
Tanaka
Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama 226-8501, Japan.|School of Life Science and Technology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.
Masayuki
M
Komada
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008954:Models, Biological; D010447:Peptide Hydrolases / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D013379:Substrate Specificity; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C404488:USP25 protein, human; D025801:Ubiquitin; D010447:Peptide Hydrolases; D043222:Ubiquitin Thiolesterase; D008239:Lysine