Cellular and computational models reveal environmental and metabolic interactions in MMUT‐type methylmalonic aciduria

Abstract

Methylmalonyl-coenzyme A (CoA) mutase (MMUT)-type methylmalonic aciduria is a rare inherited metabolic disease caused by the loss of function of the MMUT enzyme. Patients develop symptoms resembling those of primary mitochondrial disorders, but the underlying causes of mitochondrial dysfunction remain unclear. Here, we examined environmental and genetic interactions in MMUT deficiency using a combination of computational modeling and cellular models to decipher pathways interacting with MMUT. Immortalized fibroblast (hTERT BJ5ta) MMUT-KO (MUTKO) clones displayed a mild mitochondrial impairment in standard glucose-based medium, but they did not to show increased reliance on respiratory metabolism nor reduced growth or viability.Consistently, our modeling predicted MUTKO specific growth phenotypes only for lower extracellular glutamine concentrations. Indeed, two of three MMUTdeficient BJ5ta cell lines showed a reduced viability in glutamine-free medium. Further, growth on 183 different carbon and nitrogen substrates identified increased NADH (nicotinamide adenine dinucleotide) metabolism of BJ5ta and HEK293 MUTKO cells compared with controls on purine-and glutamine-based substrates. With this knowledge, our modeling predicted 13 reactions interacting with MMUT that potentiate an effect on growth, primarily those of secondary oxidation of propionyl-CoA, oxidative phosphorylation and oxygen diffusion. Of these, we validated 3-hydroxyisobutytyl-CoA hydrolase (HIBCH) in the secondary propionyl-CoA oxidation pathway. Altogether, these results suggest compensation for the loss of MMUT function by increasing anaplerosis through glutamine or by diverting flux away from MMUT through the secondary propionyl-CoA oxidation pathway, which may have therapeutic relevance.

New insights into the pathophysiology of methylmalonic acidemia

“…Ramon et al treated human BJ5ta‐MMUT fibroblasts with a variety of stressors attempting to induce a phenotype. One regimen studied was the addition of methylmalonic acid at concentrations of 100 mM in the culture medium and like the studies in the MMA mice, no phenotype was observed 55 . Additionally, experiments of toxic metabolite addition have not consistently yielded the same results.…”

“…Both animal and cellular studies using MMUT deficient models illustrate that metabolites alone are insufficient to generate disease pathophysiology 54,55 . Mice engineered to express Mmut in the hepatocytes from a germline transgene ( Mmut −/− ;Tg Ins‐Alb‐Mut ) are fully rescued from the lethality of the full knock out state but manifest significant methylmalonic acidemia, similar to MMA patients who have received an LT 54 .…”

“…One regimen studied was the addition of methylmalonic acid at concentrations of 100 mM in the culture medium and like the studies in the MMA mice, no phenotype was observed. 55 Additionally, experiments of toxic metabolite addition have not consistently yielded the same results. Several studies were unable to confirm direct inhibition of cytochrome oxidase in rat liver mitochondrial following incubation with methylmalonic, tigilic, or propionic acid, inhibition of dicarboxylic carrier protein, or inhibition of electron transport chain complexes.…”

“…Both animal and cellular studies using MMUT deficient models illustrate that metabolites alone are insufficient to generate disease pathophysiology. 54,55 Mice engineered to express Mmut in the hepatocytes from a germline transgene (Mmut À/À ;Tg Ins-Alb-Mut ) are fully rescued from the lethality of the full knock out state but manifest significant methylmalonic acidemia, similar to MMA patients who have received an LT. 54 When control (Mmut +/À ;Tg Ins-Alb-Mut ) and affected mice (Mmut À/À ; Tg Ins-Alb-Mut ) were placed on high protein diet over the course of 6 months, plasma methylmalonic acid levels in affected Mmut À/À ;Tg Ins-Alb-Mut mice reached approximately 2 mM, mirroring what is seen in MMA patients when renal dysfunction is present. 54 Affected mice developed renal pathologies following protein stress including megamitochondria with loss of cristae, however, liver cells which expressed very low levels of MMUT were resistant and displayed totally normal ultrastructure of the mitochondria.…”

Organic acidurias: Ingredients for precision medicine

“…McCorvie et al 5 discuss the complex cooperation of transporters, enzymes and chaperones that are required for the generation and delivery of cobalamin, a cofactor for two human enzymes. This is followed by Ramon et al 6 demonstrating how cellular and computational models help to decipher environmental and metabolic interactions in methylmalonic aciduria, suggesting a compensatory role for increased anaplerosis through glutamine. Next, Head et al 7 outline how precise knowledge about pathophysiology in methylmalonic aciduria has enabled the development of targeted therapy.…”