Dysfunctions of Cellular Oxidative Metabolism in Patients with Mutations in the NDUFS1 and NDUFS4 Genes of Complex I

Arcangela Iuso,Salvatore Scacco,Claudia Piccoli,Francesco Bellomo,Vittoria Petruzzella,Raffaella Trentadue,Michele Minuto,Maria Ripoli,Nazzareno Capitanio,Massimo Zeviani,Sergio Papa

doi:10.1074/jbc.m513387200

Abstract

The pathogenic mechanism of a G44A nonsense mutation in the NDUFS4 gene and a C1564A mutation in the NDUFS1 gene of respiratory chain complex I was investigated in fibroblasts from human patients. As previously observed the NDUFS4 mutation prevented complete assembly of the complex and caused full suppression of the activity. The mutation (Q522K replacement) in NDUFS1 gene, coding for the 75-kDa Fe-S subunit of the complex, was associated with (a) reduced level of the mature complex, (b) marked, albeit not complete, inhibition of the activity, (c) accumulation of H(2)O(2) and O(2)(.-) in mitochondria, (d) decreased cellular content of glutathione, (e) enhanced expression and activity of glutathione peroxidase, and (f) decrease of the mitochondrial potential and enhanced mitochondrial susceptibility to reactive oxygen species (ROS) damage. No ROS increase was observed in the NDUFS4 mutation. Exposure of the NDUFS1 mutant fibroblasts to dibutyryl-cAMP stimulated the residual NADH-ubiquinone oxidoreductase activity, induced disappearance of ROS, and restored the mitochondrial potential. These are relevant observations for a possible therapeutical strategy in NDUFS1 mutant patients.

Highlights

Complex I is the largest of the respiratory chain enzymes, being composed of seven mitochondrial DNA and at least 39 nuclear DNA-encoded subunits [11]
And Catalytic Activities of Complex I—The assembly of complex I in the inner mitochondrial membrane was examined by Western blot of several complex I subunits on two-dimension blue native/SDS-PAGE of the mitoplast fraction isolated from the patient’s fibroblasts
The NDUFS4 mutation was associated with complete suppression of the forward, rotenone-sensitive NADH-ubiquinone oxidoreductase activity

Summary

EXPERIMENTAL PROCEDURES

Reagents and Cell Lines—DMEM, PBS, trypsin (0.05%), EDTA (0.02%), penicillin, streptomycin, calf serum, and fetal bovine serum were from EuroClone. Dibutyryl cyclic AMP, diphenylene iodonium, glutathione reductase, reduced glutathione, and rotenone were from Sigma. NADH was from Boehringer, decylubiquinone and 3-isobutyl-1-methylxanthine (IBMX) from Calbiochem, and 2Ј,7Јdichlorofluorescein diacetate, MitoCapture, from Biovision. Mito Tracker Red and MitoSOX was from Molecular Probes; TRIzol, Super-

Complex I Genetic Dysfunction

NS oxidase

RESULTS

DISCUSSION