Nobel Hall of Science - Room 105 ) 2 : 30 Effect of ISCU Mutations on Protein Interaction and Iron - sulfur Cluster Assembly in Vitro and i n Vivo

A variety of human diseases, including Friedrich ataxia and mitochondrial myopathy with exercise intolerance, are caused by mutations in the proteins required in iron-sulfur cluster biogenesis. For example, the cause of mitochondrial myopathy with exercise intolerance was recently identified as a G C splice mutation in the gene encoding ISCU, a scaffold protein required in iron-sulfur cluster biogenesis. Iron-sulfur clusters are cofactors necessary for enzymes involved in cellular respiration, DNA synthesis, and other vital cellular processes. The G C splice mutation causes an exon containing a stop codon to be incorporated into the gene sequence, which results in an ISCU protein with a truncated, mutated C-terminus. The objective of this research was to examine how variations of the mutant ISCU produced by the G C splice mutation affected the function of ISCU and iron-sulfur cluster synthesis. PCR mediated site-directed mutagenesis was used to generate three human ISCU mutations: a M141V point mutation; a D144F, A145P, I146A, K147E mutation series; and the splice mutation identified in patients with mitochondrial myopathy. DNA sequencing confirmed the accuracy of each mutation, and the samples were expressed in E. coli. Upon protein purification, the D144F, A145P, I146A, K147E ISCU and the G C splice mutation ISCU both exhibited extensive oligomerization; if this occurs in vivo, the cell may have a reduced level of ISCU, which corresponds to the decreased level of ISCU present in patients with mitochondrial myopathy. Results of an in vitro pull-down assay indicate that the Frataxin interacted in a nonspecific manner with wild type and mutant forms of ISCU. A yeast construct was created by knocking out the yeast homologs of Frataxin and ISCU and inserting human Frataxin and ISCU. This construct provides a system in which the effects of each ISCU mutation on iron-sulfur cluster assembly and cell survival can be examined in vivo.