| Popis: |
Cytochrome c oxidase (COX) is a component of the mitochondrial oxidative phosphorylation system (OXPHOS) which is the principal source of ATP for the majority of human tissues. The COX holo-complex is a homodimer of 13 different subunits, encoded by both the nuclear and mitochondrial genomes (mtDNA), and contains metal ions and haem A prosthetic groups. Disrupted biogenesis of the holo-complex is the most common cause of COX deficiency. As genetic manipulation of human cells is difficult, cells derived from patients with COX deficiency provide a valuable resource for improving our understanding of COX biogenesis and COX deficiency. This thesis is a comparative study of COX deficient fibroblasts derived from seven patients with mitochondrial disorders of undetermined genetic origin, normal controls and disease controls carrying mutations in mitochondrial tRNA genes or the COX assembly factors SCO1 and COX10. The project was designed to answer two questions: What are the molecular mechanisms behind the enzyme deficiency in the patient cells What can this tell us about the biogenesis of the COX holo-complex Phenotyping of the cultures revealed distinct patterns of OXPHOS subunit expression and confirmed that the COX defects were caused by disrupted biogenesis of the holo-complex. Genotyping excluded the involvement of mtDNA and sequencing of the COX assembly factor SURF1 identified mutations in four of the patients. Blue-native polyacrylamide gel electrophoresis found that COX sub-complexes, which resembled known assembly intermediates, accumulated in SURF1 and SCO1 mutant cells but not those with COX10 mutations. This suggests that SURF1 and SCO1 function at a similar point in COX biogenesis and supports the view that COX10 functions early in COX assembly. The results are discussed in the context of our current understanding of COX biogenesis and the causes of COX deficiency with emphasis on the molecular pathology of SURF1 mutant cells. |
