Journal: bioRxiv

Article Title: The transaminase-ω-amidase pathway is a redox switch in glutamine metabolism that generates α-ketoglutarate

doi: 10.1101/2024.08.28.610061

Figure Lengend Snippet: Mean of log 2 fold change in TCA cycle metabolites (untargeted metabolomics) of human umbilical endothelial cells (HUVEC) exposed to either menadione (5 µM) (A) or H 2 O 2 (300 µM) (B). C: Superoxide oxidizes the iron-sulphur cluster in aconitase, however, the glutaminase I pathway can replenish α-ketoglutarate (αKG) into the TCA cycle through the action of glutaminase 1 (GLS1). D-E Changes in αKGM and αKG in HUVEC after exposure to menadione or H 2 O 2 . F: Reactions of the glutamine transaminase-ω-amidase (GTωA) pathway. ACO: Aconitase GLUD1: Glutamate Dehydrogenase 1 KYAT1: Kynurenine aminotransferase 1 or Glutamine transaminase of kidney KYAT3: Kynurenine aminotransferase 3 or Glutamine transaminase of liver NIT2: Nitrilase-like 2, ω-amidase

Article Snippet: The human 10x His-Tag NIT2 plasmid was obtained from Sino Biological (#HG23517-CH, Wayne, USA).

Techniques:

Journal: bioRxiv

Article Title: The transaminase-ω-amidase pathway is a redox switch in glutamine metabolism that generates α-ketoglutarate

doi: 10.1101/2024.08.28.610061

Figure Lengend Snippet: A: Colocalization analyses and regional association plots of αKGM mQTL (CLSA metabolite, n = 8203) with cis-eQTL for NIT2 in artery tibial (n = 475) and aortic tissue (n= 329) (GTEx v8 study). The sentinel variant rs3830303 and rs277627 are indicated. B: strategy for CRISPR/cas9 deletion of the regulatory element containing rs277627 in HEK 293 cells. C: Genomic PCR showing a 437 bp deletion of the rs277627 containing locus. NIT2 expression in NTC and rs27767 −/− HEK 293 cells by RT-qPCR (D) and Western blot (E). TSS: transcription start site. RT-qPCR: Real-time polymerase chain reaction. FP: forward primer. RP: reverse primer. NTC: non-targeting control.

Article Snippet: The human 10x His-Tag NIT2 plasmid was obtained from Sino Biological (#HG23517-CH, Wayne, USA).

Techniques: Variant Assay, CRISPR, Expressing, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: bioRxiv

Article Title: The transaminase-ω-amidase pathway is a redox switch in glutamine metabolism that generates α-ketoglutarate

doi: 10.1101/2024.08.28.610061

Figure Lengend Snippet: A: rs277627 is located in a regulatory element (REM, analyzed with EpiRegioDB) with binding sites for the transcription factors ZNF320 VEZF1 KLF16 GLIS2 MAZ ZNF740 ZNF148 ZNF467 GLIS3 that are expressed in HUVEC and have a gain of function and likely repress transcription. B: A two gRNA approach was employed to delete a 437 bp region where the SNV rs277627 (chr3:100336428-100336429) in NIT2 (intron 1) is located. gRNA1 targets the chr3:100,336,234-100,336,263 region whereas gRNA2 the chr3:100,336,702-100,336,725. A successful deletion was obtained between 100,336,264-100,336,701 as shown by sanger sequencing. The SNV G is labeled in red. C: Data from GTex for the expression of wild type (GG) rs277627 and its variants GA and AA on NIT2 expression in human aorta and tibial artery.

Article Snippet: The human 10x His-Tag NIT2 plasmid was obtained from Sino Biological (#HG23517-CH, Wayne, USA).

Techniques: Binding Assay, Sequencing, Labeling, Expressing

Journal: bioRxiv

Article Title: The transaminase-ω-amidase pathway is a redox switch in glutamine metabolism that generates α-ketoglutarate

doi: 10.1101/2024.08.28.610061

Figure Lengend Snippet: Targeted LC-MS/MS for αKGM and αKG in plasma (A) and urine (B) of mice treated with lipopolysaccharide (LPS, 4 mg/kg, 4 h). C: Schematic representation of biotinylated iodoacetamide (BIAM) switch assay. D-F: BIAM switch assay followed by immunoblotting for NIT2 in HUVEC (D) exposed to H 2 O 2 or human granulocytes (E) incubated with or without zymosan opsonized by human plasma. *p<0.05, without vs. with stimulation, t-test. Microscopy images show aggregation of granulocytes in response to zymosan. F: BIAM switch assay with a H 2 O 2 concentration and time (G) response curve. H: Cartoon representation of the predicted structure of human NIT2 (AlphaFold2). The 7 cysteine residues are depicted. The insert shows a zoom in view of Cys153 and the catalytic centre (C153-L112-E43). I: Schematic representation of overexpression, affinity purification, redox LC-MS and activity assay for NIT2. J: Heatmap summarizing reversible modifications of Cys in NIT2. K: NIT2 activity assay using competitive amine substitution of succinic acid by hydroxylamine. *p<0.05 as compared to wild type NIT2 without H 2 O 2 , one-way ANOVA with Bonferroni correction. L: Solvent exposed surface of the predicted NIT2 model depicted in purple. Cys153 and Cys44 (yellow spheres) are 11 Å apart. Cys44 localizes at the bottom of the open substrate binding channel capped by the flexible loop 2 (L2). Arrow shows a tunnel from the protein surface down to the Cys153. IP: immunoprecipitation. IB: immunoblotting.

Article Snippet: The human 10x His-Tag NIT2 plasmid was obtained from Sino Biological (#HG23517-CH, Wayne, USA).

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Incubation, Microscopy, Concentration Assay, Over Expression, Affinity Purification, Activity Assay, Solvent, Binding Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: The transaminase-ω-amidase pathway is a redox switch in glutamine metabolism that generates α-ketoglutarate

doi: 10.1101/2024.08.28.610061

Figure Lengend Snippet: Multiple sequence alignment and structural features of human NIT2 predicted by AlphaFold2. Abreviations: Hs, Homo sapiens (Q9NQR4); Rn, Rattus norvegicus (Q497B0); Mm, Mouse musculus (Q9JHW2); Dr, Danio rerio (Q4VBV9); Sc, Saccharomyces cerevisiae (P47016); Pa, Pyrococcus abyssi (Q9UYV8); Rg, Rhodococcus qingshengii (A0AA46MND0); At, Arabidopsis thaliana (P32962). The human cysteine residues are indicated with yellow triangles, the catalytic residues as red triangles, and the loops involved in the formation of the substrate channel and active site are colored in blue, green, and pink squares.

Article Snippet: The human 10x His-Tag NIT2 plasmid was obtained from Sino Biological (#HG23517-CH, Wayne, USA).

Techniques: Sequencing

Journal: bioRxiv

Article Title: The transaminase-ω-amidase pathway is a redox switch in glutamine metabolism that generates α-ketoglutarate

doi: 10.1101/2024.08.28.610061

Figure Lengend Snippet: A: Western blot analysis (using an anti-His-tag antibody) for the His-tagged mutants of NIT2 C146 expressed in HEK 293 cells. B: Aggregation points of NIT2 and NIT2 C146A as determined by thermal shift assay. n=3; ** p<0.01, Welchs’ correction. C: Melting curve of purified NIT2 and NIT2 C146A protein.

Article Snippet: The human 10x His-Tag NIT2 plasmid was obtained from Sino Biological (#HG23517-CH, Wayne, USA).

Techniques: Western Blot, Thermal Shift Assay, Purification

Journal: bioRxiv

Article Title: The transaminase-ω-amidase pathway is a redox switch in glutamine metabolism that generates α-ketoglutarate

doi: 10.1101/2024.08.28.610061

Figure Lengend Snippet: The AlfaFold2 structure prediction of NIT2 is depicted as the solvent-exposed surface in purple, and the substrate channel entry is shown from the top view (A) and the cut view to the bottom (B). The surface charge representation (red negative, white hydrophobic and blue positive) is shown on the side cut view (C) and top cut (D). Cys153 and Cys44 depicted as yellow spheres. The black arrow indicates the channel entry.

Article Snippet: The human 10x His-Tag NIT2 plasmid was obtained from Sino Biological (#HG23517-CH, Wayne, USA).

Techniques: Solvent

Journal: bioRxiv

Article Title: The transaminase-ω-amidase pathway is a redox switch in glutamine metabolism that generates α-ketoglutarate

doi: 10.1101/2024.08.28.610061

Figure Lengend Snippet: NIT2 −/− , GLS1 −/− and NIT2/GLS1 −/− HUVEC were generated by CRISPR/cas9 and knockout efficiency was validated by Western blot (A). Cellular distribution of NIT2 and GLS1 as shown by immunofluorescence (B). C-D: Targeted LC-MS/MS measurements for αKGM and αKG in CRISPR/cas9 HUVEC. *p<0.05 as compared to NTC, ANOVA with Bonferroni correction. E: Scheme for isotopic tracing of fully labelled glutamine that generates m+5, m+1 αKGM and m+5 αKG. F: m+5, m+1 αKGM and m+5 αKG (G) in HUVEC. *p<0.05 as compared to NTC. H: Volcano plots of significantly altered metabolites as measured by untargeted metabolomics. NTC: non-targeting control.

Article Snippet: The human 10x His-Tag NIT2 plasmid was obtained from Sino Biological (#HG23517-CH, Wayne, USA).

Techniques: Generated, CRISPR, Knock-Out, Western Blot, Immunofluorescence, Liquid Chromatography with Mass Spectroscopy, Control

Journal: bioRxiv

Article Title: The transaminase-ω-amidase pathway is a redox switch in glutamine metabolism that generates α-ketoglutarate

doi: 10.1101/2024.08.28.610061

Figure Lengend Snippet: A: Volcano plots for each knockout as indicated. B: Number of differentially expressed genes. C: Annotation pathway. D: Heat map of top differentially expressed genes in HUVEC NTC, NIT2 −/− , GLS1 −/− and NIT2/GLS1 −/− .

Article Snippet: The human 10x His-Tag NIT2 plasmid was obtained from Sino Biological (#HG23517-CH, Wayne, USA).

Techniques: Knock-Out

Journal: bioRxiv

Article Title: The transaminase-ω-amidase pathway is a redox switch in glutamine metabolism that generates α-ketoglutarate

doi: 10.1101/2024.08.28.610061

Figure Lengend Snippet: A: LoxP sites flanking exon 2 of the NIT2 gene were inserted by CRISPR/cas9. B: Nit2 flox/flox mice were crossed with Tg(Cdh5-Cre-ERT2) 1Rha to generate endothelial-specific, tamoxifen-inducible knockout mice of Nit2. C: Validation of knockout efficiency by Western blot analysis for Nit2 in global, constitutive knockout mice of Nit2 that were generated by CRISPR/cas9 deletion of ∼500bp. Nit2 wt/wt and Nit2 ko/ko are littermates in a heterozygous breeding of Nit2 wt/ko with Nit2 wt/ko mice. SMC: aortic smooth muscle cells.

Article Snippet: The human 10x His-Tag NIT2 plasmid was obtained from Sino Biological (#HG23517-CH, Wayne, USA).

Techniques: CRISPR, Knock-Out, Western Blot, Generated

Journal: bioRxiv

Article Title: The transaminase-ω-amidase pathway is a redox switch in glutamine metabolism that generates α-ketoglutarate

doi: 10.1101/2024.08.28.610061

Figure Lengend Snippet: Generation of a tamoxifen-inducible, endothelial cell-specific knockout mouse of Nit2 (ecNit2 −/− ). A: Validation of the knockout efficiency by Western blot of aortic endothelial cells and immunofluorescence in retina vessels (B). C-D: LC-MS/MS for αKGM and αKG in plasma of CTL and ecNit2 −/− mice. *p<0.05 as compared to CTL, Mann Whitney test. E-F: Untargeted metabolomics from CTL and ecNit2 −/− mice from lung tissue (E) and plasma (F). G: Retina angiogenesis in neonatal mice as indicated (post natal day 7), isolectin staining from CTL and ecNit2 −/− mice with quantification of vascularization (H). I: Ex vivo endothelial cell outsprouting from aortic segments isolated from CTL and ecNit2 −/− mice with quantification, normalized to CTL –VEGF (J). *p<0.05, Mann Whitney test (-VEGF versus +VEGF). K: Validation of NIT2 deletion by Western blot in aortic endothelial cells of a global, constitutive knockout mouse of Nit2 (Nit2 wt/wt and Nit2 ko/ko ). L: Retina angiogenesis in neonatal mice as in G with quantification (M). *p<0.05 Nit2 wt/wt as compared to Nit2 ko/ko , Mann Whitney test.

Article Snippet: The human 10x His-Tag NIT2 plasmid was obtained from Sino Biological (#HG23517-CH, Wayne, USA).

Techniques: Knock-Out, Western Blot, Immunofluorescence, Liquid Chromatography with Mass Spectroscopy, MANN-WHITNEY, Staining, Ex Vivo, Isolation

Journal: bioRxiv

Article Title: The transaminase-ω-amidase pathway is a redox switch in glutamine metabolism that generates α-ketoglutarate

doi: 10.1101/2024.08.28.610061

Figure Lengend Snippet: A-C: sprout outgrowth assay of HUVEC with and without VEGF-A (10 ng/mL) as indicated. $ p<0.05 as compared to NTC without VEGF-A and #p<0.05 as compared to NTC with VEGF-A, one-way ANOVA, Bonferroni correction. D: migration and (E) proliferation assays. F-G: senescence assay with β-galactosidase staining in HUVEC as indicated. Nucl: nucleosides. * p<0.05 as compared to NTC, one-way ANOVA with Bonferroni correction. H: NIT2 is a redox switch in the glutamine transaminase-ω-amidase pathway.

Article Snippet: The human 10x His-Tag NIT2 plasmid was obtained from Sino Biological (#HG23517-CH, Wayne, USA).

Techniques: Migration, Staining