Journal: The Journal of Biological Chemistry

Article Title: Cardiolipin-induced activation of pyruvate dehydrogenase links mitochondrial lipid biosynthesis to TCA cycle function

doi: 10.1074/jbc.RA119.009037

Figure Lengend Snippet: Decreased activity of PDH in tafazzin-deficient cells. A, PDH activity was assayed in isolated mitochondria as described under “Experimental procedures.” Data shown are mean ± S.D. (n = 3). *, p < 0.05. B, levels of PDH-E1α in mitochondrial extracts were determined by Western blot analysis with anti-PDH-E1α antibody. 50 μg of total protein from each sample was loaded onto an SDS gel under reducing conditions, and the mitochondrial protein Ndufb6 was used as a loading control. C, PDH activity was assayed in heart tissue extracted from WT and TAZ-KO mice. Each column represents a separate mouse. Data shown are mean ± S.D. (n = 2). D, WT and TAZ-KO cells were seeded in DMEM (without glucose, pyruvate, or glutamine/1% FCS), followed by 3-h incubation in Dulbecco's PBS (Life Technologies) supplemented with 100 μm [1-14C]pyruvate, and the PDH flux assay was performed in the presence or absence of 5 mm DCA. PDH activity was assayed by measuring the release of 14CO2 from [1-14C]pyruvate as described under “Experimental procedures.” Data shown are mean ± S.D. (n = 3). *, p < 0.05.

Article Snippet: PDH bound to lipids on the membrane was detected by immunoblotting using primary antibody (Assaypro) against the PDH complex (1:1000) and secondary antibody conjugated to horseradish peroxidase (1:3000).

Techniques: Activity Assay, Isolation, Western Blot, SDS-Gel, Incubation, Flux Assay

Journal: The Journal of Biological Chemistry

Article Title: Cardiolipin-induced activation of pyruvate dehydrogenase links mitochondrial lipid biosynthesis to TCA cycle function

doi: 10.1074/jbc.RA119.009037

Figure Lengend Snippet: CL binds to PDH and increases its activity. A, digitonin-solubilized mitochondria from WT and TAZ-KO cells were treated with or without CL. PDH activity was assayed as described under “Experimental procedures.” B, purified PDH complex (Sigma) was incubated with exogenous CL or phosphatidic acid (PA). PDH activity was assayed as described under “Experimental procedures”. Data shown are mean ± S.D. (n = 3). *, p < 0.05. C, the indicated lipids CL, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 3,4,5-trisphosphate (PIP3), and phosphatidic acid were serially diluted and spotted onto a nitrocellulose membrane, which was incubated overnight in buffer containing 10 μg of PDH complex. Interactions were detected by immunoblotting with an antibody against the PDH complex.

Article Snippet: PDH bound to lipids on the membrane was detected by immunoblotting using primary antibody (Assaypro) against the PDH complex (1:1000) and secondary antibody conjugated to horseradish peroxidase (1:3000).

Techniques: Activity Assay, Purification, Incubation, Western Blot

Journal: Nature Communications

Article Title: Non-canonical dihydrolipoyl transacetylase promotes chemotherapy resistance via mitochondrial tetrahydrofolate signaling

doi: 10.1038/s41467-025-63892-3

Figure Lengend Snippet: a Cisplatin response of cancer cells with endogenous DLAT knockdown and rescue expression of human DLAT wildtype (WT) or enzyme-inactive mutant (ΔB; catalytic domain B deleted). DLAT knockdown A549 cisR and KB-3-1 cisR cells expressing DLAT variants were under cisplatin-treated conditions (A549 cisR 2 μg/ml and KB-3-1 cisR 5 μg/ml) for 48 h. Apoptosis and cell viability were assessed by annexin V staining and ATP measurement, respectively. Total DLAT levels in cells with DLAT variants are shown by immunoblotting. b, c DLAT knockdown effect on PDC activity. A549 cisR and KB-3-1 cisR cells with DLAT knockdown were treated with cisplatin for 24 h. PDC activity was determined by monitoring NADH production from PDH reaction ( b ) and phosphorylation of PDHA1 at S293 ( c ). d DLAT knockdown effect on PDC assembly. Interaction between E1 (PDH), E3 (DLD), and E2 (DLAT) was determined by E1 co-immunoprecipitation in cells with or without DLAT knockdown and cisplatin treatment. e Effect of PDC inhibition on cisplatin sensitivity. Cells were treated with 5 mM PDK inhibitor dichloroacetate (DCA) and cisplatin for 48 h. Apoptotic cell death and cell viability were measured as described in ( a ). f Overexpression of ACSS1 in cells lacking DLAT. Acetyl-CoA levels were measured by LC-MS. Apoptotic cell death and cell viability were measured as described in ( a ). Effect of PDH ( g ) or DLD ( h ) knockdown on cisplatin-induced apoptotic cell death and cell viability. Data are mean ± SD from 4 independent biological replicates for apoptosis rates of ( a ) and cell viability of ( g , h left) and 3 independent biological replicates for ( b , e , f ), cell viability of ( a , h right), apoptosis rates of ( g , h ). P values were determined by one-way ANOVA for all panels. Source data are provided as a Source Data file.

Article Snippet: Antibodies against PDH1-E1α (E1) (sc-377092/D-6), α-tubulin (sc-23948/B-5-1-2), Bax (sc-493), Tom40 (sc-11414/H-300), p21 (sc-397/C-19), and POLRMT/MtRPOL (sc-365082/B-1) were purchased from Santa Cruz Biotechnology.

Techniques: Knockdown, Expressing, Mutagenesis, Staining, Western Blot, Activity Assay, Phospho-proteomics, Immunoprecipitation, Inhibition, Over Expression, Liquid Chromatography with Mass Spectroscopy

Journal: International journal of molecular sciences

Article Title: Association of Phosphorylated Pyruvate Dehydrogenase with Pyruvate Kinase M2 Promotes PKM2 Stability in Response to Insulin.

doi: 10.3390/ijms241813697

Figure Lengend Snippet: Figure 1. In silico analysis of interaction between p-PDHA1 and PKM2. (A) HADDOK2.4 pro- vided the statistic of 10 clusters from 164 possible complexes. Among them, the most favourable model, which superimposed with other models, aligned perfectly, and occupied similar binding regions, was selected. Conformations of binding proteins of p-PDHA1 (violet) and PKM2 (green: N-terminal domain, pink: A1 and A2 domains; blue: B domain; and sky blue: C-terminal domain) are shown (left panel). Detailed amino acids residues involved in the protein interaction are revealed, and the p-Ser264 residue is shown in arrow (right panel). (B) The detailed conformations of Ser264 (pink) and p-Ser264 (grey) are shown. (C) Amino acids participating in interaction between p-PDHA1 and PKM2 with H-bond, electrostatic, π-alky, and carbon–hydrogen bonds are summarized. (D) The RMSF (root-mean-square fluctuation), which indicates specific amino acid structurally contributing the most to molecular motion, is shown. (E) The RMSD (root-mean-square deviation), which means a numeric measurement representing the difference between two structures is presented.

Article Snippet: Small interfering RNAs (si-RNA) PDHA1 (sc-91064 and 5160), si-PKM2 (sc62820 and 5315), and control si-RNA-A (sc-37007) were purchased from Santa Cruz and Bioneer.

Techniques: In Silico, Binding Assay, Residue

Journal: International journal of molecular sciences

Article Title: Association of Phosphorylated Pyruvate Dehydrogenase with Pyruvate Kinase M2 Promotes PKM2 Stability in Response to Insulin.

doi: 10.3390/ijms241813697

Figure Lengend Snippet: Figure 2. DCA reduces the cell proliferation and p-PDHA1 level upon insulin simulation. (A,B) HepG2 and Huh7 cells were transfected with si-IR-A and stimulated with insulin (100 nM) for 24 h. p-PDHA1 and PKM2 levels were determined by Western blotting. (C,D) HepG2 and Huh7 cells were stimulated with 100 nM insulin in the absence or presence of DCA (25 mM). PKM2 and p-PDHA1 levels were detected by Western blotting. Statistical data are presented in a line graph. (E,F) Human HepG2 and Huh7 cells were stimulated with 100 nM insulin, 25 mM DCA, with or without insulin for 48 h, and finally, cell proliferation was measured by using an MTT assay. The relative growth was quantified as a percentage of control cells. Data are presented in a bar graph and the data represent the mean ± S.E. of three independent experiments (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Small interfering RNAs (si-RNA) PDHA1 (sc-91064 and 5160), si-PKM2 (sc62820 and 5315), and control si-RNA-A (sc-37007) were purchased from Santa Cruz and Bioneer.

Techniques: Transfection, Western Blot, MTT Assay, Control

Journal: International journal of molecular sciences

Article Title: Association of Phosphorylated Pyruvate Dehydrogenase with Pyruvate Kinase M2 Promotes PKM2 Stability in Response to Insulin.

doi: 10.3390/ijms241813697

Figure Lengend Snippet: Figure 3. Effects of p-PDHA1 on PKM2 upon insulin stimulation. (A) HepG2 cells were transfected with control si-RNA or si-PDHA1 (30–100 nM) for 48 h, and PKM2 and p-PDHA1 levels in transfected HepG2 cells were determined by Western blotting. PKM2 levels were quantified and are presented as the mean ± S.E. of three independent experiments (** p < 0.01). (B) HepG2 cells were transfected with si-PDHA1 and reconstituted with PDHA1 WT, S293D, or S293A, then activated by insulin (100 nM) for 48 h. Target proteins band were determined by Western blotting. (C) si-PDHA1 (50 nM) was transfected to HepG2 cells, cyclohexamide (CHX) (20 mM) and insulin (100 nM) were administered for a defined time, then p-PDHA1 and PKM2 were determined with Western blotting. (D) HepG2 cells transfected with si-PDHA1 and reconstituted with PDHA1 S293A were stimulated with 100 nM insulin in the presence of MG132 (1 µM) or leupeptin (20 µM). PDHA1 and PKM2 level changes were determined by Western blotting. (E) HepG2 cells were transfected with si-PDHA1 and reconstituted with PDHA1 WT, S293D, or S293A mutants. Then, cells were stimulated by 100 nM insulin, and PKM2 enzyme activity levels were measured by using the BioVision enzyme colorimetric activity assay kit (K709-100, BioVision, Milpitas, CA, USA). (F) HepG2 cells were transfected first with si- PDHA1 and reconstituted with PDHA1 WT, S293D, or S293A, then cells were activated with insulin (100 nM) for 48 h. Cells proliferation were measured by a CCK-8 assay. The levels of knockdown or reconstitution of target constructs were checked by Western blotting. The data represent the mean ± S.E. of three independent experiments (* p < 0.05, ** p < 0.01; *** p < 0.001).

Article Snippet: Small interfering RNAs (si-RNA) PDHA1 (sc-91064 and 5160), si-PKM2 (sc62820 and 5315), and control si-RNA-A (sc-37007) were purchased from Santa Cruz and Bioneer.

Techniques: Transfection, Control, Western Blot, Activity Assay, CCK-8 Assay, Knockdown, Construct

Journal: International journal of molecular sciences

Article Title: Association of Phosphorylated Pyruvate Dehydrogenase with Pyruvate Kinase M2 Promotes PKM2 Stability in Response to Insulin.

doi: 10.3390/ijms241813697

Figure Lengend Snippet: Figure 4. Effects of PKM2 on p-PDHA1 upon insulin stimulation. (A) HepG2 cells were transfected with si-PKM2 and p-PDHA1, and PDHA1 levels were measured by immunoblotting after a 100 nM insulin treatment for 48 h. (B) Si-PDHA1 (50 nM) was transfected and cyclohexamide (CHX) (20 mM) and insulin (100 nM) were administered; then, p-PDHA1 and PKM2 levels were determined with Western blotting depending on time. (C) HepG2 cells were transfected with si-PKM2 and stimulated by 100 nM insulin. PDH enzyme activity was then measured by a PDH activity colorimetric assay protocol (Novagen, Darmstadt, Germany). (D,E) Purified recombinant GST-PDHA1 was first treated with alkaline phosphatase to remove endogenous phospho-groups in PDHA1. GST-PDHA1 was then incubated with recombinant PKM2 (0.2 µg) and PEP (20 µM) or ATP (30 µM), and p-Ser293 PDHA1 was detected by Western blotting. Purified GST-PDH-WT protein band was stained with Coomassie blue. (F) HepG2 cells were transfected with si-PDHA1 and then PKM2 K305R and K305Q mutants, and cells were stimulated with 100 nM insulin. The PDHA1 and PKM2 levels were determined by Western blotting. (G) HepG2 cells were transfected with PKM2 WT, K305R, and K305Q and then stimulated with insulin (100 nM). P-PDHA1 immunoprecipitated and coimmunoprecipitated PKM2 was detected by Western blotting. (H) HepG2 cells were transfected with si-PKM2 and reconstituted with PKM-WT and stimulated by insulin (100 nM) for 48 h. Cell proliferation was assessed with a CCK-8 assay, and PKM2 levels were determined by Western blotting. The statistical data represent the mean ± S.E. of three independent experiments (* p < 0.05, ** p < 0.01; *** p < 0.001, ns, non-significant).

Article Snippet: Small interfering RNAs (si-RNA) PDHA1 (sc-91064 and 5160), si-PKM2 (sc62820 and 5315), and control si-RNA-A (sc-37007) were purchased from Santa Cruz and Bioneer.

Techniques: Transfection, Western Blot, Activity Assay, Colorimetric Assay, Recombinant, Incubation, Staining, Immunoprecipitation, CCK-8 Assay