Divergent Biochemical Effects on Mitochondrial Complex I Define the Clinical Presentation of LHON and MELAS

Abstract

Purpose: : We set out to study the molecular mechanisms of disease pathogenesis in these diseases.

Methods: : We have built a system to model mitochondrial disease mutations in Escherichia coli NDH-1. Here we present the modeling of the human LHON/MELAS overlap syndrome mtDNA mutation 3376, which causes the ND1 subunit substitution E24K. We compare the results to the previously modeled LHON mutations 14459, 14484 and 14498 in the ND6 gene, and in addition, to MELAS missense mutations 3946 and 3949 in the ND1 gene. The modeled protein domains are well conserved and the mutated amino acids are the same in human and E. coli enzymes (except that ND6-Ala72 is Met in E. coli). The corresponding NDH-1 mutants were generated and analyzed, using deamino-NADH, decylubiquinone and hexammineruthenium as substrates.

Results: : The modeled LHON mutations 14459 and 14484 decreased NDH-1 activity by 17-47 % and induced a slight decrease in the affinity for decylubiquinone. The 14498 mutation lowered activity by 27 % and, strikingly, induced a marked substrate inhibition at higher decylubiquinone concentrations. The MELAS mutations decreased NDH-1 turnover and the amount of membrane bound enzyme, but unlike the LHON mutations in the ND6 subunit, did not alter ubiquinone binding. The modeled 3376 mutation, i.e. E36K in E. coli, decreased the amount of membrane bound enzyme and the assembled enzyme was inactive. We also introduced E36Q mutation to this position and the mutant enzyme was assembled to the membrane and active, but presented a marked decrease in the ubiquinone binding affinity.

Conclusions: : The 3376 LHON/MELAS overlap syndrome mutation combines the biochemical hallmarks of the individually presented diseases. To our knowledge, this is the first time that biochemical differences between separate mitochondrial diseases or clinical entities affecting mitochondrial complex I can be shown in the same model system.

Keywords: mitochondria • proteins encoded by disease genes • genetics