Improving the diagnosis of mitochondrial diseases: Application of monoclonal antibody technologies to NADH:ubiquinone oxidoreductase and cytochrome c oxidase defects

Identifying mitochondrial oxidative phosphorylation (OXPHOS) defects is complicated by a non-Mendelian mode of inheritance, heterogeneous tissue expression, and difficult enzyme assays. Mutations in five OXPHOS complexes are predominant causes of mitochondrial disease. Recent studies also associate the development of neurodegenerative disorders, such as Parkinson's and Alzheimer's disease, with mitochondrial defects. We sought to advance the diagnosis of mitochondrial defects by expanding an immunological approach applicable to the analysis of the first and fourth complex of the OXPHOS chain, NADH:ubiquinone oxidoreductase (complex I) and cytochrome c oxidase (complex IV).; Herein, we describe a procedure for isolating complex I from small tissue amounts and show that it can be characterized by proteomic and mass spectrometric methods. Human homologues to 42 bovine polypeptides are identified, including GRIM-19, an apoptotic protein, NP17.2, a neuronal protein, plus a new subunit, NDUFA11, which contains homology to peptide import machinery. This system has also proven useful for characterizing post-translational modifications of complex I.; Additionally, a procedure to isolate complex IV from small tissue amounts using antibodies is described. The isolate is active and analyzable by proteomic and mass spectrometric methods. It is also possible to identify 12 of 13 components acknowledged as genuine subunits and to detect tissue isoforms. Moreover, we distinguish sites of hydroxyl carbonylation and 3-nitrotyrosine formation on complex IV with in vitro-generated oxidative damage. We anticipate using this procedure for analyzing the role of complex IV in neurodegenerative diseases.; A monoclonal antibody array is also described that is invaluable for diagnosing complex I deficiencies and its utility is demonstrated with patient fibroblasts. Using this method, it is observed that complex I assembly/stability is affected by mutations in 4 of 7 mitochondrial DNA encoded genes, ND1, ND3, ND5, and ND6 . Furthermore, the profile of complex I resolved by gradient fractionation indicates that mutations in ND5 have a dominant effect on enzyme assembly. Likewise, a GRIM-19 subcomplex is detected within ND1 patient mitochondria. We conclude that complex I biogenesis in humans is dependent on critical core subunits.