Funktionelle Defekte der humanen Proteine ISCA1, ISCA2 und IBA57 beeinträchtigen die Reifung mitochondrialer Fe/S-Proteine und führen zu mitochondrialen Erkrankungen

Iron-sulfur- (Fe/S-) clusters are inorganic protein cofactors and are essential for life. These cofactors perform various tasks within a cell. For instance, Fe/S clusters of some proteins are required for the transport of electrons, whereas others mediate enzyme catalysis. Additionally, Fe/S proteins can act as oxygen sensors or participate in the regulation of gene expression. Finally, Fe/S clusters are necessary for the stability of several proteins. Synthesis of all cellular Fe/S proteins requires a complex and highly conserved mechanism, and is dependent on mitochondria. In this organelle the so-called Fe/S-cluster- (ISC) assembly machinery is localised. The core part of the ISC-assembly machinery mediates biogenesis of both mitochondrial [2Fe-2S]-proteins and all cytosolic/nuclear Fe/S proteins. Maturation of mitochondrial [4Fe-4S]-proteins requires additional ISC members. These late-acting ISC factors include the so-called A-type proteins and Iba57. The function of these proteins was elucidated via several studies in bacteria and yeast. However, functional data on the human homologues ISCA1, ISCA2 and IBA57 were missing at the beginning of this work. The first part of the present work aimed to clarify the role of human ISCA1, ISCA2 and IBA57 in cellular Fe/S protein biogenesis. To this end, all three proteins were depleted in human cell culture in order to analyse the depletion phenotype via various biochemical and molecular biological methods. These analyses showed that ISCA1, ISCA2 and IBA57 exclusively participate in the maturation of mitochondrial [4Fe-4S]-proteins. In contrast, biogenesis of mitochondrial [2Fe-2S]-proteins and cytosolic/nuclear Fe/S proteins was independent of these three ISC proteins. Collectively, these data showed the conserved role of the human ISCA proteins and IBA57 in mitochondrial [4Fe-4S]-protein biogenesis. In the second part of this work, the biochemical consequences of two different pathogenic mutations in IBA57 were investigated. These mutations were linked to two distinct human diseases. The first case was characterized by severe myopathy and encephalopathy, and was lethal in early infancy. In contrast, patients suffering from the second mutation developed a rare form of hereditary spastic paraplegia combined with optic atrophy and peripheral neuropathy, called SPOAN. Initially, the impact of these mutations on the expression of IBA57 protein was measured. In the first case a point mutation in IBA57 caused a decrease in protein below physiologically critical levels, whereas in the second case a splice mutation generated an aberrant truncated splice product which was not translated. However, in that case a minor portion of normally spliced IBA57 mRNA remained in the patients’ cells leading to the production of minute amounts of wild-type IBA57. Finally, it was evaluated if mutations in IBA57 were causative for the disease phenotypes. Analysis of cell material derived from both patient groups revealed an impaired maturation of mitochondrial [4Fe-4S]-proteins. In contrast, formation of mitochondrial [2Fe-2S]-proteins and cytosolic/nuclear Fe/S proteins was normal. Interestingly, the effect on Fe/S protein biogenesis was stronger in patient cells of the first group lacking IBA57 protein. The biochemical phenotype of the patient cells reflects functional data achieved by IBA57 depletion in yeast and human cell culture. Thus, the identified mutations in IBA57 are likely causative for the two presented diseases. The biochemical variations in both patient groups are probably a result of the differences in IBA57 protein expression. In all likelihood, these expression differences contribute to the development of the two completely different disease phenotypes. In summary, the human ISC factors ISCA1, ISCA2 and IBA57 are essential for the formation of mitochondrial [4Fe-4S]-proteins. The function as late-acting ISC assembly factors is conserved from Bacteria to man. The hypothesized role of IBA57 in mitochondrial [4Fe-4S]-protein biogenesis was verified by analysis of IBA57-deficient patient cells.