Cardiomyopathies are known to have contributing genetic factors, including significant contributions from mitochondrial disorders. Mitochondrial mutations are found at an incidence of 1 in 5-10,000 births. Since mitochondria are built from proteins that derive from two genetic systems, clinical mutations are found in both nuclear and mitochondrial genes. More than half are found in mitochondrial DNA, which encodes only 13 polypeptides, along with genes for rRNA and tRNA. The primary role of these polypeptides is in oxidative phosphorylation, where they contribute to the ATP synthase, and to 3 of the electron transport complexes, Complex I, III, and IV. In this project about 25 sites of clinically-identified mutations that may impact the NADH-arm of Complex I will be examined biochemically. Structurally, Complex I (NADH:ubiquinone oxidoreductase) consists of a membrane arm including 7 core subunits that are encoded by the mitochondrial genes, and an NADH-arm including another 7 core subunits that are encoded by nuclear genes. The peripheral arm contains the NADH binding site, and the ubiquinone binding site is found at the junction of the two domains. In the first aim, mutations that map to interfaces of individual subunits within the NADH-arm will be analyzed. It will be determined whether the mutations generally disrupt the structure of that polypeptide chain, if they prevent the binding of one of the partner subunits, or if they do not impact assembly at all, but rather affect enzyme activity more directly. Or, it is possible that no phenotype can be detected. In the second aim, mutations that map to the interface of the NADH-module with the membrane arm of Complex I will be analyzed. These clinical mutations have not been confirmed to cause to disease, but are known to be associated with disease. It will be determined whether these mutations disrupt Complex I activity in our model system. If so, possible effects on enzyme assembly will be examined. All mutations will be constructed at homologous sites in the E. coli Complex I genes. Enzyme activities will be measured in preparations of membrane vesicles. Assembly will be verified by native gel electrophoresis and western blotting. The results will provide insights into the assembly pathways of Complex I, and the nature of disruptions by clinically-identified mutations in the core subunits. Results will provide guidance in the diagnosis of mutations that continue to be discovered during DNA sequencing.
|Program type||AHA Institutional Research Enhancement Award (AIRE|
|Effective start/end date||07/01/2017 → 06/30/2019|