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Proteomic analysis of methylmalonic acidemia fibroblasts

ABSTRACT: Methylmalonic academia (MMA) is a rare inborn error of the metabolism due to defects of the methylmalonyl-CoA mutase (MUT) enzyme that result in methylmalonic acid accumulation in the blood and increased excretion in urine. Affected patients can manifest vomiting, dehydration, hypotonia, developmental delay and failure to thrive. The emergency treatment of the newborns with MMA mainly comprises rehydration and promotion of anabolism, followed by long-term dietary management that mostly includes restriction of propiogenic amino acids by a low protein diet and carnitine and vitamin B12 supplementation. The effects of MUT deficiency on the cellular damage in patients is still unknown. Fibroblasts obtained from two distinct MMA patients were analyzed by shotgun label-free quantitative (LFQ) proteomics.

INSTRUMENT(S): Orbitrap Exploris 240

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Fibroblast

DISEASE(S): Methylmalonic Aciduria Due To Methylmalonyl-coa Mutase Deficiency

SUBMITTER: Michele Costanzo

LAB HEAD: Michele Costanzo

PROVIDER: PXD044025 | Pride | 2024-06-14

REPOSITORIES: Pride

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Project description:Methylmalonic acidemias (MMA) consist in a group of autosomal recessive inherited metabolic disorders whose pathogenesis involves the catabolism of propionyl-CoA. The propionyl-CoA produced from the degradation of cholesterol, branched-chain amino acids (valine, isoleucine, methionine, threonine) and the -oxidation of odd-chain fatty acids is converted into methylmalonyl-CoA, which is further converted in succinyl-CoA, required for energy production in the Krebs cycle. In mitochondria of MMA patients, the conversion of methylmalonyl-CoA into succinyl-CoA, catalysed by the vitamin B12-dependent methylmalonyl-CoA mutase (MUT) enzyme, is altered. This can be due to: 1) defective activity of MUT apoenzyme or 2) a defect in the synthesis or transport of adenosylcobalamin (MUT cofactor). In the first case it is referred to as isolated MMA (mut0 or mut- phenotypes if the deficiency is total or partial, respectively), while in the second case as cbl-related MMA (there is a specific disorder according to which gene is mutated) []. The blockage of this enzymatic reaction leads to increase in the levels of methylmalonic acid, hallmark of this group of diseases []. Independently from the pathogenesis, along with methylmalonic acid, other MMA-associated metabolites like acylcarnitines, amino acids and organic acids result altered in MMA [REF]. These metabolites are easily detectable and quantifiable by metabolomics approaches, such as liquid chromatography - tandem mass spectrometry (LC-MS/MS) or gas chromatography - mass spectrometry (GC-MS), in dried blood spots or urine or plasma to make diagnosis. Because of the early onset of MMA in patients, MMA is included in the panel of diseases for the expanded newborn screening in several countries, including Italy [ref]. Our work aims at understanding the mechanisms of cellular damage in MMA disease. The majority of MMA complications arise from brain damage, on which depend neurological alterations and movement disorders. In the brain, the astrocytes are damaged by hyperammonemia, causing edema and hypoperfusion. (REF lavoro) Renal function is commonly compromised in MMA patients, even the pathogenesis of kidney injury is not clear. Renal insufficiency and chronic kidney disease are partially resolved after kidney transplantation. Hyperammonemia and metabolic acidosis are considered as key mechanisms acting in the proximal tubule to lead to renal damage. In fact, general organ metabolic alterations are thought to be caused by the breakdown of branched‐chain amino acids that lead to the accumulation of circulating toxic acidic metabolites. Thus, metabolic acidosis is induced with diminishing of bicarbonate levels. Lactic acidosis and hyperammonemia result as the main metabolic alterations of MMA. (REF lavoro) Results of a previous work on a MUT-knockdown neuroblastoma cell line [Costanzo, 2018] were coherent with the disease, but did not completely elucidate such mechanisms, possibly due to limitation of the cellular model. We presumed that the transient silencing of MUT gene was not sufficient to input long-term decompensation due to absence of this protein. For this reason, we have developed a new cellular model for isolated MMA by stably knocking out MUT gene in HEK293 cell line using CRISPR/CAS9 genome editing technology. We also performed a global proteomic analysis to describe protein changes strictly connected to MUT absence and related altered pathways. Altogether, the results obtained shed new light on the molecular mechanisms of cellular damage, including alterations of cell architecture in combination with the acquisition of a higher sensitivity to stress.

			Action	DRS
	Fibroblasts_CTRL1.raw	Raw
	Fibroblasts_CTRL2.raw	Raw
	Fibroblasts_CTRL3.raw	Raw
	Fibroblasts_CTRL4.raw	Raw
	Fibroblasts_M01_1.raw	Raw

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Proteomic analysis of methylmalonic acidemia fibroblasts

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