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. 2021 Jan 25;42(7):1160–1170. doi: 10.1038/s41401-020-00600-5

Fig. 2. Inhibitory potency of DM10 on DJ-1 deglycase activity by targeting the DJ-1 homodimer.

Fig. 2

a The schematic representation of the N-acetylcysteine deglycation assay. The N-acetylcysteine deglycation assay based on the absorbance at 288 nm indicates the degradation of hemithioacetal formed between N-acetylcysteine and methylglyoxal (MGO) into N-acetylcysteine and lactate in the presence of DJ-1. b Human recombinant wild-type DJ-1 protein was purified and incubated with STK or DMs for 30 min, and then the deglycase activity was assayed. The data are reported as a ratio relative to the initial OD value under test conditions. “1STK” represents the molar ratio of DJ-1:STK = 1:1; “2STK” represents the molar ratio of DJ-1:STK = 1:2; and “1DMs” represents the molar ratio of DJ-1:DMs = 1:1. Independent experiments were repeated three times, and representative data are shown. c The statistical analysis in (b). Data under the test conditions were reported as a ratio relative to the 2STK treatment. Data shown represent the mean ± SD, n = 3; comparisons were made using the two-tailed, unpaired Student’s t test. *P < 0.05 (versus 2STK group). d–f DM10 binds to the DJ-1 homodimer. d The human recombinant wild-type DJ-1 protein was purified and incubated with STK, DM2, or DM10 for 30 min and then analyzed by Western blotting. “2STK” represents the molar ratio of DJ-1:STK = 1:2; “1DM2” represents the molar ratio of DJ-1:DM2 = 1:1; and “1DM10” represents the molar ratio of DJ-1:DM10 = 1:1. Independent experiments were repeated three times, and representative data are shown. e H1299 cells were infected with lentivirus (DJ-1-FLAG) for 72 h. The indicated H1299 cells with DJ-1 re-overexpression were incubated with DM10 (2.5–20 μM) for 48 h and then analyzed by Western blotting. Independent experiments were repeated three times, and representative data are shown. f Recombinant proteins DJ-1-WT (wild type), DJ-1-ΔC2 (deletion of two amino acids from the C-terminus), and DJ-1-ΔC3 (deletion of three amino acids from the C-terminus) were purified and incubated with DM10 (DJ-1:DM10 = 1:1) for 30 min and then analyzed by Western blotting. Independent experiments were repeated three times, and representative data are shown. g A competitive assay between DM10 and STK or DM2. The human recombinant wild-type DJ-1 protein was purified and incubated with STK or DM2 for 30 min, followed by incubation with DM10 for another 30 min, and then analyzed by Western blotting. “2STK” represents the molar ratio of DJ-1:STK = 1:2; “1DM2” represents the molar ratio of DJ-1:DM2 = 1:1; and “1DM10” represents the molar ratio of DJ-1:DM10 = 1:1. Independent experiments were repeated three times, and representative data are shown.