| Resumo: | Pyruvate dehydrogenase complex (PDC) occupies an essential position in cellular energy production. The oxidative decarboxylation of pyruvate to acetyl-coenzyme A is conducted by a highly organized multienzyme system. Impaired PDC activity leads to a metabolic disorder affecting mainly the tissues with a high demand for ATP. This work is dedicated to a comprehensive study of PDC and PDC deficiency at diverse levels. We described and discussed the clinical, biochemical and genotypic findings from thirteen Portuguese PDC deficient patients, we submitted the clinically relevant variants to the detailed structural and functional analyses, and we evaluated the ability of arginine and/or thiamine to restore PDC function in patient-derived cell lines. All patients from our cohort had the clinical onset between the neonatal period and infancy, manifested different degrees of neurological involvement and, interestingly, most of them reached adulthood. The mutational spectrum revealed ten different mutations in the following genes coding for the respective subunits PDHA1 (PDC-E1α), PDHX (PDC-E3BP), and DLD (PDC-E3). The most striking evidence was a relatively high incidence of E3BP deficiency, nevertheless, PDHA1 mutations were predominant. The selected pathogenic PDC-E1α variants with amino acid substitutions in different structural regions were analyzed by biochemical and biophysical methodologies, combined with molecular dynamics simulations, revealing a limited impact of the mutations on the conformational stability and presenting a significant functional impairment in terms of reduced residual PDC-E1 enzymatic activity and lower affinity for the thiamine pyrophosphate cofactor. Applying identical methodologies, we evaluated the effect of arginine supplementation on recombinant PDC-E1 variants. Furthermore, we conducted a study assessing the impact of arginine and/or thiamine treatment in patient-derived cell lines. The rescue efficacy of these stabilizing molecules is clearly determined by the mutation per se. Remarkably, the major effect of arginine was observed in p.R253G variant, at all levels of this study. The beneficial effect of arginine upon the recombinant protein and the considerable response to arginine and thiamine supplementation in the patient´s fibroblast, confirmed previous observations demonstrating the efficacy of arginine treatment in improving the clinical phenotype of the patient carrying the p.R253G mutation.
|
|---|