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Gallic acid binds USP2. 28 / 28
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"These findings collectively suggested the specific interaction between GA and USP2."
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"Next, we performed a Cellular Thermal Shift Assay (CETSA) to determine whether GA directly binds to USP2 in cells."
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"Considering that USP2 functions as a ubiquitin-specific protease, we hypothesized that the binding of GA to USP2 may inhibit its enzymatic activity."
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"Cysteine 284 is critical for the covalent binding of GA to USP2."
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"We then investigated the mode of interaction between GA and USP2."
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"Interestingly, preincubation of iodoacetic acid (IAA), a cysteine alkylating agent, with USP2 completely inhibited the binding of biotin-GA to USP2 ( xref ), suggesting that GA may covalently bind to the cysteine(s) of USP2, which is consistent with previous reports that GA can covalently bind to its targets through cysteine residues xref ."
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"It can be observed from xref C that the binding of GA to USP2 was dependent on the incubation time, following the pattern of an irreversible binding mechanism."
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"These data suggest that GA covalently binds to USP2 in a dose- and time-dependent manner."
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"GA binds to USP2 through an allosteric pocket."
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"As shown in xref F, molecular docking indicated that P565 and R289 are critical for forming the pocket for GA to bind to USP2."
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"Specifically, we employed GA as a chemical probe and demonstrated that GA could covalently bind to and inhibit USP2 through an allosteric pocket."
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"Molecular docking simulations further revealed a potential allosteric pocket for GA binding to USP2."
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"That is, USP2 first forms an initial encounter complex with GA, USP2:GA, then irreversibly forms a covalently linked complex USP2-GA."
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"The mutation of Cys284 does eliminate the covalent binding of GA to USP2, but it doesn't affect the binding of GA to the allosteric pocket."
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"We then discovered that GA could covalently bind to USP2 in an allosteric manner, thereby inhibiting its activity."
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"(1):(1)USP2+GA⇄USP2:GA→USP2–GATo determine the values of k , the rate constant for USP2–GA formation at saturating [GA], and K , the apparent dissociation constant for the initial USP2: GA complex (USP2: GA), USP2 was incubated with a large excess of GA for different periods and the covalent linked USP2–GA complexes [USP2–GA] were analyzed by SDS gel and subsequent Western blot using streptavidin-HRP."
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"Under the pseudo-first-order experimental conditions, the concentrations of USP2 and USP2–GA complex follows the equations below:(2)USP2t=USP20·"
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"The interaction between USP2 and GA was further confirmed by a competition experiment: the binding of biotin-GA to recombinant USP2 was competitively inhibited by higher concentrations of unlabeled GA (Fig. 2D)."
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"These findings collectively suggested the specific interaction between GA and USP2.Next, we performed a Cellular Thermal Shift Assay (CETSA) to determine whether GA directly binds to USP2 in cells."
39807309
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"Considering that USP2 functions as a ubiquitin-specific protease, we hypothesized that the binding of GA to USP2 may inhibit its enzymatic activity."
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"These findings collectively suggest that GA could relatively specifically inhibit the deubiquitinating activity of USP2 in vitro.3.4 Cysteine 284 is critical for the covalent binding of GA to USP2."
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"We then investigated the mode of interaction between GA and USP2."
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"It can be observed from Fig. 3C that the binding of GA to USP2 was dependent on the incubation time, following the pattern of an irreversible binding mechanism."
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"These data suggest that GA covalently binds to USP2 in a dose- and time-dependent manner.3.5 GA binds to USP2 through an allosteric pocket."
39807309
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"Specifically, we employed GA as a chemical probe and demonstrated that GA could covalently bind to and inhibit USP2 through an allosteric pocket."
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"Molecular docking simulations further revealed a potential allosteric pocket for GA binding to USP2."
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"The interaction between USP2 and GA follows a two-step mode for covalent inhibitors."
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"The mutation of Cys284 does eliminate the covalent binding of GA to USP2, but it doesn't affect the binding of GA to the allosteric pocket."
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