102 - Abnormal Methylation and Redox Homeostasis in the Onset and Progression of Combined Homocystinuria and Methylmalonic Aciduria (MMACHC)

Abstract

Nutritional and genetic deficiencies of cobalamin (vitamin B12) lead to severe neurological and hematological dysfunction, and in severe cases, to death. Intracellular processing of dietary cobalamins is catalyzed by the glutathione-dependent MMACHC protein, which primes the cobalamin molecule prior to its delivery to cytosolic methionine synthase (MS) and mitochondrial methylmalonyl-CoA mutase (MM). Dysfunction or absence of MMACHC lead to combined homocystinuria and methylmalonic aciduria (OMIM 609831) by inactivation of MS and MM, respectively. We examined the proteome of cytosolic and nuclear fractions of cultured skin fibroblasts from five healthy neonates and five patients with early onset MMACHC disease. Stable isotope labeling by amino acids in cell culture (SILAC) and mass spectrometry permitted the identification of 3450 proteins. Methylated Arg, Lys and Glu residues were found in 11 histones and 126 non-histone proteins. Significant changes in protein expression levels were confirmed for enzymes of the methionine cycle, the trans-sulfuration pathway, mitochondrial respiration and the elimination of superoxide and peroxides. Targeted metabolomics confirmed functional disturbances in relevant endogenous aminothiols. A direct interaction of MMACHC with heat shock protein 90 was also demonstrated. This is the first study to investigate redox homeostasis in an inherited metabolic disorder without external induction of oxidative stress. Our data suggest that natural mutations in the MMACHC gene impair cellular methylation and redox homeostasis, which may contribute to the vascular and neurological phenotype observed in these patients.