Fig 1: Schematic model of the phosphoenolpyruvate carboxykinase 2 (PCK2)–lactate–polyglutamine-binding protein 1 (PQBP1)–protein arginine methyltransferase 5 (PRMT5) signaling axis driving airway inflammation. The diagram illustrates that in the asthmatic microenvironment (right), lactate accumulation induces the stabilization of PCK2 via succinylation (K100), establishing a lactate positive feedback loop. The amplified lactate signal then induces the lactylation of PQBP1 (K223), which in turn inhibits PRMT5 activity, ultimately lifting the H4R3me2s-dependent transcriptional repression of downstream proinflammatory genes (e.g., the mitogen-activated protein kinase [MAPK] pathway). In the basal state (left), this pathway is suppressed.
Fig 2: Polyglutamine-binding protein 1 (PQBP1)-K223 lactylation mediates inflammation and metabolic reprogramming. (A) Immunoprecipitation (IP) of endogenous PQBP1 to detect the effect of lactate stimulation on its lactylation (Kla). (B) Evolutionary conservation analysis of PQBP1, with K189 and K223 sites highlighted. (C) Detection of exogenous PQBP1 lactylation induced by lactate in cells expressing wild-type (WT) or mutant (K223R/T) PQBP1. (D to F) Molecular dynamics simulation analysis of the effect of K223 lactylation on PQBP1 protein conformation, including backbone structure (D), RMSF (E), and Gibbs free energy landscape (F). (G and H) Measurement of extracellular acidification rate (ECAR) (G) and lactate production (H) in cells expressing WT, K223R, or K223T mutant PQBP1. (I) Time-course analysis of Effect of interleukin-1β (IL-1β) on intracellular lactate levels in BEAS-2B cells. Note: Multiple bands present in the pan-lactylation blots correspond to ubiquitinated species and partial degradation fragments of PQBP1, rather than nonspecific interacting proteins. All Western blots are representative of ≥3 independent experiments. Data are presented as means ± standard error of the mean (SEM) from ≥3 independent experiments. Analyzed by analysis of variance (ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 3: Polyglutamine-binding protein 1 (PQBP1)-K223 lactylation drives airway inflammation in vivo via the protein arginine methyltransferase 5 (PRMT5)/mitogen-activated protein kinase (MAPK) axis. House dust mite (HDM)-challenged mice were treated with adeno-associated viruses (AAVs) delivering PQBP1-NC, PQBP1-K223R, or Prmt5-short hairpin RNA (shRNA), followed by: (A) Pathological staining (hematoxylin and eosin [H&E], Masson, and periodic acid-Schiff [PAS]) of lung tissues. Scale bar, 50 μm. (B) Flow cytometric quantification of eosinophils (CD45.2+ CD11c− Siglec-F+) in bronchoalveolar lavage fluid (BALF). (C)The concentrations of interleukin-4 (IL-4), IL-5, IL-13, and IL-17 in the BALF supernatant were measured by enzyme-linked immunosorbent assay (ELISA) in HDM-challenged mice transduced with AAV-PQBP1-NC, AAV-PQBP1-K223R, or AAV-shPrmt5. Data are presented as means ± standard error of the mean (SEM). P < 0.001 versus PQBP1-NC group; ###P < 0.001 versus PQBP1-NC group (indicating exacerbation by shPrmt5***). (D) Assessment of airway hyperresponsiveness (AHR) regulated by the PQBP1–PRMT5 axis. Lung resistance (% change from phosphate-buffered saline [PBS]) was measured in HDM-challenged mice transduced with AAV-PQBP1-NC, AAV-PQBP1-K223R, or AAV-shPrmt5 in response to increasing concentrations of methacholine (MCh). Data are presented as means ± SEM. Symbols indicate statistical significance compared to the PQBP1-NC group (* indicates PQBP1-K223R versus NC; # indicates Prmt5-shRNA versus NC). (E) Western blot analysis of PRMT5, H4R3me2s, and MAPK pathway phosphorylation in lung tissues. (F and G) Immunohistochemistry (IHC) staining for PRMT5/H4R3me2s (F) and immunofluorescence (IF) staining for p-P38 (G) in lung sections. Scale bar, 200 μm. (H) Quantitative polymerase chain reaction (qPCR) analysis of T helper 2 (Th2) (Il-4, Il-5, and Il-13) and proinflammatory cytokine (Il-1β and Tnf-α) mRNA in lung tissues. (I) Endogenous coimmunoprecipitation (Co-IP) validating the PQBP1–PRMT5-WD repeat domain 77 (WDR77) interaction in lung tissues. All Western blots are representative of ≥3 independent experiments. Data are presented as means ± SEM (n = 6 to 8 per group). Analyzed by Student t test, 1 or 2-way analysis of variance (ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.
Fig 4: Lactylated polyglutamine-binding protein 1 (PQBP1) reprograms the epigenome and transcriptome by inhibiting the protein arginine methyltransferase 5 (PRMT5) complex. (A) Coimmunoprecipitation (Co-IP) analysis of the interaction between wild-type (WT) or K223R mutant PQBP1 and PRMT5/WD repeat domain 77 (WDR77). (B) Immunofluorescence showing the colocalization of H4R3me2S and PRMT5 in lung tissues of house dust mite (HDM)-challenged mice. Scale bar, 100 μm. (C) Molecular docking model illustrating the interaction interface between PRMT5 (light cyan) and PQBP1 (gray). (D) Schematic diagram of the PRMT5 and PQBP1 mutants designed based on the docking results. (E) Co-IP assays showing the binding affinity between the PRMT5 and PQBP1 mutants. (F) Western blot analysis of global H4R3me2s levels in cells overexpressing PQBP1-WT versus a negative control (NC). (G to J) H4R3me2s chromatin immunoprecipitation (ChIP)-seq analysis in cells overexpressing PQBP1, showing an enrichment heatmap at promoter regions genome-wide (G) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of target genes (H). (I and J) Quantitative polymerase chain reaction (qPCR) analysis of representative signaling pathway target genes and the metabolic regulatory gene (J) mRNA transcription in cells overexpressing PQBP1. (K) In vitro assay showing the direct effect of PQBP1 on the methyltransferase activity of PRMT5. (L to N) Assessment of epithelial-immune crosstalk via conditioned media (CM) transfer. THP-1 differentiated macrophages were incubated for 24 h with CM collected from BEAS-2B cells (transfected with si-NC or si-PCK2 and stimulated with HDM). Bar graphs show the relative mRNA expression levels of the proinflammatory cytokines IL-1β (L), IL-6 (M), and TNF-α (N) in macrophages. All Western blots are representative of ≥3 independent experiments. Data are presented as means ± standard error of the mean (SEM) (n = 3 independent experiments). Analyzed by Student t test or 1-way analysis of variance (ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.
Fig 5: HDAC1 regulates polyglutamine-binding protein 1(PQBP1) lactylation and protein stability. (A) Endogenous coimmunoprecipitation (Co-IP) showing the interaction of PQBP1 with HDAC1/2 in BEAS-2B cells. (B and C) Detection of lactate-induced PQBP1 lactylation following overexpression (B) or knockdown (C) of HDAC1/2. (D) Comparison of the signal abundance of HDAC1 interacting with PQBP1 from immunoprecipitation-mass spectrometry (IP-MS) data. (E) In vitro HDAC1 delactylation assay. Purified hyper-lactylated Flag-PQBP1 substrate was incubated with recombinant HDAC1-WT (wild-type) or the catalytically inactive mutant (H141A), and PQBP1 lactylation levels were assessed by Western blot (Pan-Kla). (F) Cycloheximide (CHX) chase assay to determine the effect of overexpressing HDAC1 (WT or inactive mutant) on the degradation rate of PQBP1. (G) Co-IP experiment to assess the interaction of HDAC1 with WT or K223R mutant PQBP1. (H) Immunoprecipitation (IP) and Western blot analysis of PQBP1 lactylation (Pan-Kla) in BEAS-2B cells. Cells were pretreated with the EP300-specific catalytic inhibitor A-485 (or dimethyl sulfoxide [DMSO] vehicle) for 2 h prior to lactate (L-Lac) stimulation. Total EP300 and PQBP1 in the input, as well as the immunoprecipitated PQBP1, were verified. The black arrowhead indicates the target lactylated PQBP1 band. All Western blots are representative of ≥3 independent experiments. Data are presented as means ± standard error of the mean (SEM) (n = 3 independent experiments). Analyzed by 1 or 2-way analysis of variance (ANOVA). *P < 0.05, ****P < 0.0001.
Supplier Page from Abcam for Recombinant Human PQBP1 protein (His tag N-Terminus)