Journal: PLoS Pathogens

Article Title: A Trigger Enzyme in Mycoplasma pneumoniae: Impact of the Glycerophosphodiesterase GlpQ on Virulence and Gene Expression

doi: 10.1371/journal.ppat.1002263

Figure Lengend Snippet: Examination of M. pneumoniae hydrogen peroxide release. Hydrogen peroxide production of M. pneumoniae mutant strains was measured in the presence of different carbon sources (100 µM) after 2 h. The following strains were used: wild type (wt), glpQ ::Tn, mpn566 ::Tn, and glpD ::Tn, glpQ ::Tn + glpQ (complemented mutant) and glpQ ::Tn + cv (control strain carrying the empty vector used for complementation). Error bars indicate standard deviation (based on three independent experiments). G3P, glycerol-3-phosphate; GPC, glycerophosphocholine; Glc, glucose; Gly, glycerol; w/o, without addition of any carbon source.

Article Snippet: Briefly, 5 µg of glycerophosphodiesterase were incubated with 20 U of rabbit muscle glycerol-3-phosphate dehydrogenase (Sigma) in a 0.9 M glycine-hydrazine buffer containing 0.5 mM glycerophosphodiester and 0.5 mM NAD+ in a volume of 1 ml.

Techniques: Mutagenesis, Plasmid Preparation, Standard Deviation, Gel Permeation Chromatography, Gas Chromatography

Journal: PLoS Pathogens

Article Title: A Trigger Enzyme in Mycoplasma pneumoniae: Impact of the Glycerophosphodiesterase GlpQ on Virulence and Gene Expression

doi: 10.1371/journal.ppat.1002263

Figure Lengend Snippet: Schematic illustration of the machinery for uptake and conversion of carbohydrates leading to the formation of glycerol-3-phosphate in M. pneumoniae . UgpC (MPN134), UgpA (MPN135), and UgpE (MPN136) form a putative ABC transport system for glycerol-3-phosphate, whereas GlpF (MPN043) is the glycerol uptake facilitator. The glycerol kinase GlpK (MPN050) and the glycerol-3-phosphate oxidase GlpD (MPN051) metabolize glycerol to the glycolytic intermediate dihydroxyacetone phosphate. Hydrogen peroxide formation by GlpD is crucial for the cytotoxic effects of M. pneumoniae . GlpQ (MPN420) and MPN566 encode two paralogous glycerophosphodiesterases that may be able to metabolize glycerophosphocholine to glycerol-3-phosphate and choline. The uptake system for glycerophosphocholine is so far unknown. Proteins highlighted in grey seem not to fulfill the predicted function (this work). CM, cell membrane; DHAP, dihydroxyacetone phosphate; G3P, glycerol-3-phosphate; GPC, glycerophosphocholine; Gly, glycerol; Cho, choline.

Article Snippet: Briefly, 5 µg of glycerophosphodiesterase were incubated with 20 U of rabbit muscle glycerol-3-phosphate dehydrogenase (Sigma) in a 0.9 M glycine-hydrazine buffer containing 0.5 mM glycerophosphodiester and 0.5 mM NAD+ in a volume of 1 ml.

Techniques: Gel Permeation Chromatography

Journal: Nature

Article Title: GAPDH inhibits intracellular pathways during starvation for cellular energy homeostasis

doi: 10.1038/s41586-018-0475-6

Figure Lengend Snippet: GAPDH inhibits COPI vesicle fission by targeting the GAP activity of ARFGAP1. a,b, COPI transport in HeLa cells, n=10 fields of cells examined in a representative experiment (out of 3), mean +/− SD, two-tailed t-test: ( a) *p=9.8E-07, **p=9.2E-09, ( b) *p=6.8E-06. c, Vesicle reconstitution system, n=3, GDH (glutamate dehydrogenase), LDH (lactate dehydrogenase), GPDH (glycerol-3-phosphate dehydrogenase). d, Vesicle reconstitution system: EM image of Golgi membrane (left), bar = 50 nm; vesicle quantitation (right), n=10 EM meshes examined from a representative experiment (out of 3), *p=8.9E-07, e, GAP assay using ARF1 and ARFGAP1, and also with metabolic enzymes as indicated, n=3. f, Vesicle reconstitution system: EM image of Golgi membrane (left), bar = 50 nm; vesicle quantitation (right), n=10 EM meshes examined from a representative experiment (out of 3), *p=6.2E-06.

Article Snippet: Purified forms of GAPDH, glutamate dehydrogenase (GDH), glycerol-3-phosphate dehydrogenase (GPDH), and lactate dehydrogenase (LDH) were also obtained (Sigma), as well as purified activated AMPK and the SAMS peptide (HMRSAMSGLHLVKRR) (Promega).

Techniques: Activity Assay, Two Tailed Test, Quantitation Assay, GAP Assay

Journal: Nature

Article Title: Breast cancer cells rely on environmental pyruvate to shape the metastatic niche

doi: 10.1038/s41586-019-0977-x

Figure Lengend Snippet: Pyruvate is required for 3D but not 2D growth of breast cancer cells ( a-b ) Growth curves (2D, left panels) and representative pictures (3D, right panels) of human MCF10A H-Ras V12 and mouse 4T1 cells cultured in media with or without pyruvate or glucose or glutamine. ( c ) Representative pictures of MCF10A H-Ras V12 and MCF10A spheroids in the presence or absence of pyruvate or 0.5% supplemented ECM (Matrigel). Analysis was performed at day 5. Scale bar: 150 µm. ( d ) Cellular pyruvate, α-ketoglutarate and hydroxyproline metabolism. Enzymes are depicted in italics. ALT2 refers to mitochondrial alanine aminotransferase. GDH refers to glutamate dehydrogenase. MCT2 refers to monocarboxylate transporter 2. MPC refers to mitochondrial pyruvate carrier. P4HA refers to collagen prolyl-4-hydroxylase. OH-proline refers to hydroxyproline. Only selected reactions are depicted. The number of biological replicates for each experiment was n=3. Error bars represent SD of mean from biological independent samples. Scale bar: 150 μm.

Article Snippet: Membranes were incubated overnight at 4°C with either MCT2 (LabNed, 0315312; 1:200 dilution), GPT2 (Santa Cruz, 398383; 1:500 dilution), P5CS (Santa Cruz, 515443; 1:500 dilution), GDH (Abcam, 153973; 1:1000 dilution), P4HA1 (Abcam, 59497; 1:2000 dilution), β-Actin (Sigma A5441; 1:10000 dilution) or ERK1/2 (Cell Signaling 4695S; 1:1000 dilution) primary antibodies.

Techniques: Cell Culture

Journal: Nature

Article Title: Breast cancer cells rely on environmental pyruvate to shape the metastatic niche

doi: 10.1038/s41586-019-0977-x

Figure Lengend Snippet: Pyruvate to alanine conversion drives α-ketoglutarate production ( a ) Carbon contribution of 13 C 5 glutamine, 13 C 6 glucose, and 13 C 3 pyruvate to alanine and α-ketoglutarate (α-KG) assessed by 13 C tracer analysis. n=3. ( b ) Alanine uptake/secretion in MCF10A H-Ras V12 spheroids with and without pyruvate measured by the mass spectrometry analysis of the media. n=3. ( c-f ) Intracellular abundance of α-ketoglutarate (α-KG) and hydroxylated collagen in human and mouse breast cancer spheroids upon treatment with the transaminase inhibitor aminooxyacetate (AOA; 0.8 mM), the glutamate dehydrogenase inhibitor epigallocatechin gallate (EGCG; 50 µM), transduced with a lentiviral vector with shRNA for either mitochondrial ALT2 (KD), GDH (KD) or scrambled control sequence in the presence of pyruvate. n=3 for EGCG and AOA treatment (c-d); n=9 for control shRNA, n=6 for GDH shRNA 1 and 2 and n=3 for ALT2 shRNA1 and 2 (MCF10A H-Ras V12 , e-f); n=3 for control shRNA and ALT2 shRNA 1 and 2 (4T1, e-f). In case ALT activity majorly contributs to α-ketoglutarate generation, ECGC (which inhibits the pyruvate independent conversion of glutamate to α-ketoglutarate via the enzyme glutamate dehydrogenase (GDH)), should have a minor effect on α-ketoglutarate abundance and hydroxylated collagen. Indeed, we found that this was the case. Error bars represent SD of mean from biological independent samples. Two-tailed unpaired student’s T-test.

Article Snippet: Membranes were incubated overnight at 4°C with either MCT2 (LabNed, 0315312; 1:200 dilution), GPT2 (Santa Cruz, 398383; 1:500 dilution), P5CS (Santa Cruz, 515443; 1:500 dilution), GDH (Abcam, 153973; 1:1000 dilution), P4HA1 (Abcam, 59497; 1:2000 dilution), β-Actin (Sigma A5441; 1:10000 dilution) or ERK1/2 (Cell Signaling 4695S; 1:1000 dilution) primary antibodies.

Techniques: Mass Spectrometry, Transduction, Plasmid Preparation, shRNA, Sequencing, Activity Assay, Two Tailed Test

Journal: Nature

Article Title: Breast cancer cells rely on environmental pyruvate to shape the metastatic niche

doi: 10.1038/s41586-019-0977-x

Figure Lengend Snippet: Protein and RNA expression of genetically modified breast cancer cells ( a ) Western blot analysis for MCT2 in human (MCF10A H-RAS V12 , MCF7) and mouse (4T1, EMT6.5) breast cancer cells infected with either a control gRNA or two different MCT2 gRNA normalized to control condition. Human positive/negative control: H460/MDA-MB-468; mouse positive/negative control: testis/lung. ( b ) Western blot analysis and relative gene expression for GDH in human MCF10A H-RAS V12 breast cancer cells infected with either a control shRNA or two different GDH shRNA normalized to control condition. ( c ) Western blot analysis and relative gene expression for ALT2 in human (MCF10A H-RAS V12 ) and mouse (4T1) breast cancer cells infected with either a control shRNA or two different ALT2 shRNA normalized to control condition. ( d ) Western blot analysis and relative gene expression for P5CS in human MCF10A H-RAS V12 breast cancer cells infected with either a control shRNA or two different P5CS shRNA. ( e ) Western blot analysis for P4HA in human MCF10A H-RAS V12 breast cancer cells infected with either a control or an overexpressing P4HA vector. ( f-g ) Time resolved contribution of 13 C 6 -glucose, 13 C 5 -glutamine and 13 C 3 -pyruvate to α-ketoglutarate (α-KG) and alanine in human MCF10A H-RAS V12 breast cancer spheroids. .

Article Snippet: Membranes were incubated overnight at 4°C with either MCT2 (LabNed, 0315312; 1:200 dilution), GPT2 (Santa Cruz, 398383; 1:500 dilution), P5CS (Santa Cruz, 515443; 1:500 dilution), GDH (Abcam, 153973; 1:1000 dilution), P4HA1 (Abcam, 59497; 1:2000 dilution), β-Actin (Sigma A5441; 1:10000 dilution) or ERK1/2 (Cell Signaling 4695S; 1:1000 dilution) primary antibodies.

Techniques: RNA Expression, Genetically Modified, Western Blot, Infection, Negative Control, Multiple Displacement Amplification, Expressing, shRNA, Plasmid Preparation

Journal: PLoS ONE

Article Title: Succinate Dehydrogenase Is a Direct Target of Sirtuin 3 Deacetylase Activity

doi: 10.1371/journal.pone.0023295

Figure Lengend Snippet: SIRT3 deacetylates SDHA. Complex II (A) and acetylated proteins (B) were immunoprecipitated from liver mitochondria isolated from SIRT3 WT and KO mice. IPs were immunoblotted with antibodies against acetyl-lysine (AcK), SDHA, SDHB and GDH. (C) Complex II immunoprecipitated from mouse liver mitochondria was incubated with recombinant SIRT3 or catalytically inactive SIRT3 (SIRT3 H248Y) in the presence or absence of NAD and NAM, a sirtuin inhibitor. After deacetylation, IPs were immunoblotted using antibodies against acetylated proteins, SDHA and SIRT3. In all panels, antibodies against GFP were used as negative controls.

Article Snippet: The following antibodies were used: Total OXPHOS antibody cocktail, complex II antibody cocktail and OSCP (Mitosciences); Flag (Sigma); SIRT3 and acetyl-lysine (Cell Signaling); GFP (Santa Cruz), and GDH (USBiological).

Techniques: Immunoprecipitation, Isolation, Mouse Assay, Incubation, Recombinant