Specific Detection of Cellular Glutamine Hydrolysis in Live Cells Using HNCO Triple Resonance NMR

Glutamine plays key roles as a biosynthetic precursor or an energy source in cancers, and interest in its metabolism is rapidly growing. However, the proper evaluation of glutamine hydrolysis, the very first reaction in the entire glutaminolysis, has been difficult. Here, we report a triple resonance NMR-based assay for specific detection of glutaminase activity carrying out this reaction using stable-isotope labeled glutamine. Compared to conventional methods involving coupled enzyme assays, the proposed approach is direct because it detects the presence of the H–N–CO amide spin system. In addition, the method is unique in enabling the measurement of glutamine hydrolysis reaction in real-time in live cells. The approach was applied to investigating the effects of a glutaminase inhibitor and the inhibitory effects of glucose on glutamine metabolism in live cells. It can be easily applied to studying other signals that affect cellular glutamine metabolism

Metabotyping of the <i>C. elegans sir-2.1</i> Mutant Using <i>in Vivo</i> Labeling and ¹³C‑Heteronuclear Multidimensional NMR Metabolomics

The roles of <i>sir-2.1</i> in <i>C. elegans</i> lifespan extension have been subjects of recent public and academic debates. We applied an efficient workflow for <i>in vivo</i> ¹³C-labeling of <i>C. elegans</i> and ¹³C-heteronuclear NMR metabolomics to characterizing the metabolic phenotypes of the <i>sir-2.1</i> mutant. Our method delivered sensitivity 2 orders of magnitude higher than that of the unlabeled approach, enabling 2D and 3D NMR experiments. Multivariate analysis of the NMR data showed distinct metabolic profiles of the mutant, represented by increases in glycolysis, nitrogen catabolism, and initial lipolysis. The metabolomic analysis defined the <i>sir-2.1</i> mutant metabotype as the decoupling between enhanced catabolic pathways and ATP generation. We also suggest the relationship between the metabotypes, especially the branched chain amino acids, and the roles of <i>sir-2.1</i> in the worm lifespan. Our results should contribute to solidifying the roles of <i>sir-2.1</i>, and the described workflow can be applied to studying many other proteins in metabolic perspectives

Metabotyping of the <i>C. elegans sir-2.1</i> Mutant Using <i>in Vivo</i> Labeling and ¹³C‑Heteronuclear Multidimensional NMR Metabolomics

The roles of <i>sir-2.1</i> in <i>C. elegans</i> lifespan extension have been subjects of recent public and academic debates. We applied an efficient workflow for <i>in vivo</i> ¹³C-labeling of <i>C. elegans</i> and ¹³C-heteronuclear NMR metabolomics to characterizing the metabolic phenotypes of the <i>sir-2.1</i> mutant. Our method delivered sensitivity 2 orders of magnitude higher than that of the unlabeled approach, enabling 2D and 3D NMR experiments. Multivariate analysis of the NMR data showed distinct metabolic profiles of the mutant, represented by increases in glycolysis, nitrogen catabolism, and initial lipolysis. The metabolomic analysis defined the <i>sir-2.1</i> mutant metabotype as the decoupling between enhanced catabolic pathways and ATP generation. We also suggest the relationship between the metabotypes, especially the branched chain amino acids, and the roles of <i>sir-2.1</i> in the worm lifespan. Our results should contribute to solidifying the roles of <i>sir-2.1</i>, and the described workflow can be applied to studying many other proteins in metabolic perspectives

A Highly Facile and Specific Assay for Cancer-Causing Isocitrate Dehydrogenase Mutant Using ¹³C₄‑Labeled α‑Ketoglutarate and Heteronuclear NMR

Isocitrate dehydrogenase mutations with neomorphic activity of converting α-ketoglutarate to 2-hydroxyglutarate have been found in many types of cancers. We report an NMR-based assay specific for the mutant using ¹³C₄-labeled α-ketoglutarate. It can be done in a complex mixture without extraction, give time-dependent absolute quantitation, and be applied to enzyme inhibition studies. Its merits over conventional assays should facilitate inhibitor developments for a new class of target-oriented anticancer agents

Alanine-Metabolizing Enzyme Alt1 Is Critical in Determining Yeast Life Span, As Revealed by Combined Metabolomic and Genetic Studies

Alterations in metabolic pathways are gaining attention as important environmental factors affecting life span, but the determination of specific metabolic pathways and enzymes involved in life span remains largely unexplored. By applying an NMR-based metabolomics approach to a calorie-restricted yeast (<i>Saccharomyces cerevisiae</i>) model, we found that alanine level is inversely correlated with yeast chronological life span. The involvement of the alanine-metabolizing pathway in the life span was tested using a deletion mutant of <i>ALT1</i>, the gene for a key alanine-metabolizing enzyme. The mutant exhibited increased endogenous alanine level and much shorter life span, demonstrating the importance of <i>ALT1</i> and alanine metabolic pathways in the life span. <i>ALT1</i>’s effect on life span was independent of the TOR pathway, as revealed by a <i>tor1</i> deletion mutant. Further mechanistic studies showed that <i>alt1</i> deletion suppresses cytochrome <i>c</i> oxidase subunit 2 expression, ultimately generating reactive oxygen species. Overall, <i>ALT1</i> seems critical in determining yeast life span, and our approach should be useful for the mechanistic studies of life span determinations

Alanine-Metabolizing Enzyme Alt1 Is Critical in Determining Yeast Life Span, As Revealed by Combined Metabolomic and Genetic Studies

Alterations in metabolic pathways are gaining attention as important environmental factors affecting life span, but the determination of specific metabolic pathways and enzymes involved in life span remains largely unexplored. By applying an NMR-based metabolomics approach to a calorie-restricted yeast (<i>Saccharomyces cerevisiae</i>) model, we found that alanine level is inversely correlated with yeast chronological life span. The involvement of the alanine-metabolizing pathway in the life span was tested using a deletion mutant of <i>ALT1</i>, the gene for a key alanine-metabolizing enzyme. The mutant exhibited increased endogenous alanine level and much shorter life span, demonstrating the importance of <i>ALT1</i> and alanine metabolic pathways in the life span. <i>ALT1</i>’s effect on life span was independent of the TOR pathway, as revealed by a <i>tor1</i> deletion mutant. Further mechanistic studies showed that <i>alt1</i> deletion suppresses cytochrome <i>c</i> oxidase subunit 2 expression, ultimately generating reactive oxygen species. Overall, <i>ALT1</i> seems critical in determining yeast life span, and our approach should be useful for the mechanistic studies of life span determinations