Fig. 4.

Integration of identified metabolites and transcriptional level changes of MAPK/ERK deficiency. (A-I) Quantification of metabolite levels (shown in boxes with solid lines) and gene expression levels (shown in boxes with dashed lines) from metabolomics and RNA-seq analyses, respectively, were used to integrate transcriptional and metabolite level changes. Metabolite levels were measured by LC/MS from four independent control (DMSO-treated) and four MEK-inhibited (U0126-treated) mK4 cell samples. Gene expression level changes were retrieved from RNA-seq performed in isolated populations of three control (WT) and three Mek1/2 double-knockout (dko) NP populations (GSE174229; Kurtzeborn et al., 2022). For the box plots shown, boxes indicate the 25-75th percentiles, whiskers show the s.d., the central line indicates the average, and each dot represents an individual data point. Dot plots show each datapoint as distinct spots. The y-axes indicate detected metabolite levels and relative mRNA expression. (A) These analyses identified diminished levels of the amino acid L-leucine and the related mitochondrially expressed branched-chain aminotransferase 2 (Bcat2), which catalyzes the first reaction in the catabolism of essential branched-chain amino acids (including leucine); L-tryptophan and its producer tryptophanyl-tRNA synthetase (Wars or Wars1); L-tyrosine and acyl-CoA thioesterase 7 (Acot7) – acetyl-CoA derived from tyrosine via acetoacetate is catabolized by ACOT7; L-methionine and methionine-tRNA synthetase (Mars or Mars1); and L-phenylalanine and phenylalanyl-tRNA synthetase subunit α (Farsa). (B) Glycolysis-related changes include those in the levels of glucose itself and the levels of the direct downstream modulators hexokinase 1 (Hk1) and glucose 6-phosphate (G-6-P); 3-phosphoglyceric acid (3PG) and its upstream regulator phosphoglycerate kinase 1 (Pgk1) and downstream metabolic modulators 3-phosphoglycerate dehydrogenase (Phgdh) and phosphoglycerate mutase 1 (Pgam1); and phosphoenolpyruvate (PEP), the levels of which depends on the activity of enolase 3 (Eno3), which also exhibits diminished levels. (C) Reduced glycine levels, together with reduced levels of the enzymes serine hydroxymethyltransferase 2 (Shmt2), which metabolizes glycine from serine, and glutathione synthetase (Gss) and sarcosine dehydrogenase (Sardh), which further metabolize glycine to glutathione and sarcosine, were seen. (D) Glutathione synthesis derives changes not only from glycine shortage (C) but also from reduced levels of glutathione peroxidases 1 (Gpx1), 3 (Gpx3), 4 (Gpx4) and 7 (Gpx7). (E) Reduced citrate metabolite levels are associated with reduced levels of aconitate hydratase 1 (Aco1), which balances citrate and isocitrate levels. (F) Reduced aspartate levels are associated with lower citrate levels and diminished expression of glutamatic-oxaloacetic transaminase 2 (Got2) and asparagine synthetase (Asns). (G) Proline metabolism is related to both aspartate metabolism (F) and glutaminolysis (H), and reduced proline levels associate with reduced expression of proline-producing enzymes pyrroline-5-carboxylate reductase family, member 2 (Pyrc2) and pyrroline-5-carboxylate reductase-like (Pyrl). (H) The levels of glutamate and glutamine metabolites together with glutaminase (Gls) and pyrimidine biosynthesis-related carbamoyl-phosphate synthetase 2 (Cad), are diminished. (I) Reduced ATP levels associate with several transcriptional changes in ATP synthase subunits [H+ transporting, mitochondrial F1 complex, δ subunit (Atp5d) and H+ transporting, mitochondrial F0 complex, subunit D (Atp5h)], in RNA polymerase subunits [polymerase I polypeptide C (Polr1c), polymerase II polypeptide E (Polr2e), polymerase II polypeptide I (Polr2i) and polymerase III polypeptide D (Polr3d)] and in nucleoside diphosphate kinases (Nme4 and Nme6).