Abstract
Methylmalonic acidemias (MMAs) are inborn errors of metabolism due to the deficient activity of methylmalonyl-CoA mutase (MUT). MUT catalyzes the formation of succinyl-CoA from methylmalonyl-CoA, produced from propionyl-CoA catabolism and derived from odd chain fatty acids β-oxidation, cholesterol, and branched-chain amino acids degradation. Increased methylmalonyl-CoA levels allow for the presymptomatic diagnosis of the disease, even though no approved therapies exist. MMA patients show hyperammonemia, ketoacidosis, lethargy, respiratory distress, cognitive impairment, and hepatomegaly. The long-term consequences concern neurologic damage and terminal kidney failure, with little chance of survival. The cellular pathways affected by MUT deficiency were investigated using a quantitative proteomics approach on a cellular model of MUT knockdown. Currently, a consistent reduction of the MUT protein expression was obtained in the neuroblastoma cell line (SH-SY5Y) by using small-interfering RNA (siRNA) directed against an MUT transcript (MUT siRNA). The MUT absence did not affect the cell viability and apoptotic process in SH-SY5Y. In the present study, we evaluate and quantify the alterations in the protein expression profile as a consequence of MUT-silencing by a mass spectrometry-based label-free quantitative analysis, using two different quantitative strategies. Both quantitative methods allowed us to observe that the expression of the proteins involved in mitochondrial oxido-reductive homeostasis balance was affected by MUT deficiency. The alterated functional mitochondrial activity was observed in siRNA_MUT cells cultured with a propionate-supplemented medium. Finally, alterations in the levels of proteins involved in the metabolic pathways, like carbohydrate metabolism and lipid metabolism, were found.
Highlights
Hereditary methylmalonic acidemias (MMAs) are severe autosomal recessive inborn errors of intermediary metabolism caused by the deficient activity of methylmalonyl-CoA mutase (MUT) or defects in the synthesis of 5-deoxyadenosyl cobalamin, the active form of vitamin B12 and the essential cofactor of MUT
The defect in the MUT protein causes an increase in the level of methylmalonyl-CoA, which is converted into methylmalonic acid (MMA)
The proteomic characterization of a methylmalonyl-CoA mutase-silenced neuroblastoma cell line allowed us to define a dataset of deregulated proteins and relative alterated cellular pathways that may be investigated to highlight the unknown molecular mechanism underlying MMA damage
Summary
Hereditary methylmalonic acidemias (MMAs) are severe autosomal recessive inborn errors of intermediary metabolism caused by the deficient activity of methylmalonyl-CoA mutase (MUT) or defects in the synthesis of 5-deoxyadenosyl cobalamin, the active form of vitamin B12 and the essential cofactor of MUT. MUT converts methylmalonyl-CoA into succinyl-CoA, a Krebs cycle intermediate. The defect in the MUT protein causes an increase in the level of methylmalonyl-CoA, which is converted into methylmalonic acid (MMA). Hereditary MMAs are included in newborn screening panels in several countries [2,3,4,5], allowing for a presymptomatic diagnosis of the disease. To this aim, targeted mass spectrometry-based metabolomics is a powerful tool to profile amino acids and acylcarnitines in a quantitative manner [6,7,8]
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