Fig 2: A working model of a-KG-induced pyroptosis.Under acidic environment, the metabolic enzyme MDH1 promiscuously catalyzes the conversion of a-KG to L-2HG, which then boosts ROS level. This signal is responded by the plasma membrane-localized death receptor DR6 by inducing the oxidation of DR6. Oxidized DR6 internalizes into the cytosol, and then recruits both pro-caspase-8 and GSDMC to the DR6 receptosome, in which active caspase-8 cleaves GSDMC, thereby inducing pyroptosis. This story demonstrates a novel pathway from ROS-initiated DR6 endocytosis to caspase-8-cleaved GSDMC for pyroptosis induction.

Fig 3: a-KG elevates ROS levels through MDH1-catalyzed conversion to L-2HG.HeLa cells were treated with DM-aKG (15 mM) for 6 h to determine the ROS level and L-2HG level, or 24 h to assess DR6 oxidation, pyroptotic features (including cell morphology, GSDMC cleavage, LDH release, and Annv+/PI+ cells), unless specially defined. Cells were treated with Octyl-L-2HG (5 mM) or Octyl-D-2HG (5 mM) for 1 h to determine the ROS level, or 2 h to assess DR6 oxidation and pyroptotic features. a–c Effects of MDH1 on DM-aKG-induced ROS level (a), DR6 oxidation (b), and caspase-8 activation (c). MDH1 was knocked down first in HeLa cells, the cells were then treated with DM-aKG. d, e Effects of MDH1 on DM-aKG-induced pyroptotic morphology, GSDMC cleavage, LDH release (d) and percentage of Annv+/PI+ cells (e). MDH1 was knocked down first in HeLa cells, the cells were then treated with DM-aKG. f, g Effect of MDH1 enzymatic activity on DM-aKG-induced ROS level (f) and pyroptosis (g). MDH1 was knocked down in HeLa cells, and siRNA-resistant MDH1H187Y was then transfected into cells. MDH1WT was used as a positive control. h Effects of MDH1 (left) and its enzymatic activity (right), or OGDH (middle) on the levels of L-2HG. MDH1 (left) or OGDH (middle) was knocked down in HeLa cells as indicated. In MDH1 knockdown cells, siRNA-resistant MDH1H187Y was expressed, and MDH1WT was used as a positive control (right). i?–k Effects of octyl-L-2HG on ROS level (i), DR6 oxidation and caspase-8 activation (j) and pyroptosis (k). HeLa cells were treated with L-2HG at indicated concentrations (j). l??–n Effects of hypoxia on L-2HG level (l), ROS level, DR6 oxidation and caspase-8 activation (m) and pyroptosis (n). Cells were under hypoxia (0.1% O2) for 6 h to determine ROS level, 24 h to analyze L-2HG level, and 48 h to assess DR6 oxidation, caspase-8 activation and pyroptotic features. Tubulin was used to determine the amount of loading proteins. All data are presented as means ± SEM of two or three independent experiments. **P < 0.01, ***P < 0.001; ns, not significant. The data were analyzed using two-tailed Student’s t-test in k–n, and one-way ANOVA followed by Dunnett’s multiple comparison test in f, g, h (right), i or two-way ANOVA followed by the Bonferroni test in a, d, e, h (left and middle).

Fig 4: Schematic of 9 significant differential metabolic pathways and the differentially expressed proteins in them of cholestasis rats with or without melatonin treatment. ACAT2, acetyl-CoA acetyltransferase 2; MDH1, malate dehydrogenase 1; ME1, malic enzyme 1; PPAR, peroxisome proliferator-activated receptors; CYP, cytochrome P450; GSTM3, glutathione S-transferase Mu.

Fig 5: Human papillomavirus E6 inhibits pyroptosis via the p53/MDH1/ROS/GSDMC pathway. (A) Levels of p53 and MDH1 expression following HPV E6 expression detected via western blotting, data were analysed using Student's t-test, **P < 0.01, ***P < 0.001. (B) Western blotting results for p53 and MDH1 levels detected after p53 knockdown, data were analysed using Student's t-test, *P < 0.05, ***P < 0.001. (C) Western blot analysis results for p53 and MDH1 levels following overexpression of p53, data were analysed using Student's t-test, *P < 0.05. (D) MDH1 and GSDMC expression levels detected via western blotting following MDH1 knockdown, data were analysed using Student's t-test, **P < 0.01. (E) Relative levels of L-2HG following MDH1 knockdown at a DM-AKG concentration of 15 mm, data represent the mean ± SD of three independent experiments, data were analysed using Student's t-test, *P < 0.05. (F) Relative levels of LDH released following MDH1 knockdown at a DM-AKG concentration of 15 mm, data represent the mean ± SD of four independent experiments, data were analysed using Student's t-test, *P < 0.05. (G) Levels of ROS produced following MDH1 knockdown at a DM-AKG 15 mm concentration, data represent the mean ± SD of four independent experiments, data were analysed using Student's t-test, *P < 0.05.