Fig. 2
From: Malate initiates a proton-sensing pathway essential for pH regulation of inflammation
BiP binds L-malate and is required for its anti-inflammatory effect. (a) Coomassie blue staining of SDS-PAGE gel for Raw264.7 cell lysates which were digested with protease with or without L-malate (1 mM, pH 7.0) according to DARTS assay. The bands protected by L-malate (1 mM, pH 7.0) was indicated by red frame. Representative data from three experiments. (b) Mass spectrometry analysis revealed the enrichment of BiP protein (shown as orange dots) in the protected bands from the L-malate-treated cell lysate in DARTS assays. (c and d) Raw264.7 cell lysates were digested with protease with or without L-malate (1 mM, pH 7.0) according to DARTS assay, then the levels of BiP protein was assayed using Western blot (c) and quantified (d). (e and f) BIAcore diagram showing responses measured in resonance unit (RU) of E. coli-expressed BiP protein (chip-coupled) to ATP or L-malate (pH 7.4). Representative data from three experiments (e); KD for each interaction of BiP with ATP and L-malate is indicated (f). (g) Crystal structure of BiP (PDB: 5E84) with different regions colored by palecyan (aa 1-125), orange (aa 126-499), and palepink (aa 500-654). (h and i) Il1b mRNA expressions quantified by qPCR (h) and pro-IL-1β protein abundance visualized by immunoblotting (i) in BMDMs isolated from Hspa5f/f or Hspa5f/f; Lyz2-Cre mice. These BMDMs were treated with or without L-malate (6 mM, pH 6.7), under stimulation of LPS for 24 h. (j, k and l) Il1b mRNA expressions quantified by qPCR (j) and pro-IL-1β protein abundance visualized (k) and quantified (l) by immunoblotting in BMDMs treated with or without L-malate (6 mM) in medium pH 6.9, under stimulation of LPS for 16 h. In addition, BMDMs were isolated from Hspa5f/f or Hspa5f/f; Lyz2-Cre mice. (m) pHi of in BMDMs treated with or without L-malate (6 mM) in medium pH 6.9, under stimulation of LPS for 3-6 h. (n) Survival rate in Hspa5f/f or Hspa5f/f; Lyz2-Cre mice (n = 6) administrated intragastrically with L-malate (400 mg kg−1) and injected intraperitoneally LPS. n ≥ 3. The data are shown as the mean ± SEM. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001. (two-sided Mann‒Whitney U test, unpaired Student’s t-test, Mantel‒Cox survival analysis). See also Supplementary Fig. 6–8
