Functional characterization of novel genotypes and cellular oxidative stress studies in propionic acidemia.
Propionic acidemia (PA), caused by a deficiency of the mitochondrial biotin dependent enzyme propionyl-CoA carboxylase (PCC) is one of the most frequent organic acidurias in humans. PA is caused by mutations in either the PCCA or PCCB genes encoding the α- and β-subunits of the PCC enzyme which are assembled as an α6β6 dodecamer. In this study we have investigated the molecular basis of the defect in ten fibroblast samples from PA patients. Using homology modeling with the recently solved crystal structure of the PCC holoenzyme and a eukaryotic expression system we have analyzed the structural and functional effect of novel point mutations, also revealing a novel splice defect by minigene analysis. In addition, we have investigated the contribution of oxidative stress to cellular damage measuring reactive oxygen species (ROS) levels and apoptosis parameters in patient fibroblasts, as recent studies point to a secondary mitochondrial dysfunction as pathophysiological mechanism in this disorder. The results show an increase in intracellular ROS content compared to controls, correlating with the activation of the JNK and p38 signaling pathways. Highest ROS levels were present in cells harboring functionally null mutations, including one severe missense mutation. This work provides molecular insight into the pathogenicity of PA variants and indicates that oxidative stress may be a major contributing factor to the cellular damage, supporting the proposal of antioxidant strategies as novel supplementary therapy in this rare disease.
Centro de Diagnóstico de Enfermedades Moleculares, Centro de Biología Molecular Severo Ochoa, UAM-CSIC, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), IdiPAZ, Madrid, Spain.
Lorena
L
Gallego-Villar
Celia
C
Pérez-Cerdá
Belén
B
Pérez
David
D
Abia
Magdalena
M
Ugarte
Eva
E
Richard
Lourdes R
LR
Desviat
D017209:Apoptosis / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D056726:Genetic Association Studies; D005838:Genotype; D006801:Humans; D020935:MAP Kinase Signaling System / Q000235:genetics; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D018384:Oxidative Stress / Q000235:genetics*; D017354:Point Mutation; D056693:Propionic Acidemia / Q000235:genetics* / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism
D017382:Reactive Oxygen Species