Dihydrolipoamide dehydrogenase (DID) is a highly conserved multifunctional mitochondrial enzyme. The main function of DLD is to regenerate oxidized lipoamide cofactor in the context of the pyruvate- and alpha-ketoglutarate-dehydrogenase complexes. In this capacity, DLD generates NADH and plays a central role in ATP production. However, DLD can also function as a diaphorase, using NADH to generate reactive oxygen species (ROS). Moreover, my lab discovered that DLD has a cryptic proteolytic activity by which DLD cleaves the iron chaperone and storage protein frataxin to shorter products unable to detoxify iron. The dehydrogenase activity is present in the DLD dimer while diaphorase and protease functions are predicted to be most active in monomeric DLD. The diaphorase and proteolytic activities of DLD have been studied largely in vitro, and their roles in vivo are still undefined. Based on my Preliminary Results, I hypothesize that DLD contributes to ROS production within mitochondria, both directly through its diaphorase activity, and indirectly by affecting the function of frataxin. To test this hypothesis, I will study human DLD mutations that in vitro have variable effects on the diaphorase and/or proteolytic activity of the enzyme. These mutations cause a fatal metabolic disorder of infancy associated with hypertrophic cardiomyopathy, suggesting that they alter the balance between ATP production and ROS generation, which is particularly critical for tissue with high energy metabolism such as heart. I will utilize yeast and human cell models to study the effects of these mutations on mitochondrial function, frataxin function, and oxidative stress. I will further utilize x-ray crystallography to understand the structural changes that lead to activation of DLD diaphorase and/or proteolytic activity.

Public Health Relevance

TO HUMAN HEALTH: The research proposed in this application will elucidate a potentially important mechanism responsible for oxidative damage in heart and other tissues with high energy consumption. In the long term, this new knowledge could lead to strategies to enhance the metabolic activity of DLD and to prevent activation of its diaphorase and/or proteolytic activity, which could be useful to enhance or improve cardiac function.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
Project #
5F30HL099036-03
Application #
8209057
Study Section
Special Emphasis Panel (ZRG1-F10A-S (20))
Program Officer
Carlson, Drew E
Project Start
2010-01-11
Project End
2012-11-10
Budget Start
2012-01-11
Budget End
2012-11-10
Support Year
3
Fiscal Year
2012
Total Cost
$32,600
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905