• Register
X
Forgot Password

If you have forgotten your password you can enter your email here and get a temporary password sent to your email.

X

Leaving Community

Are you sure you want to leave this community? Leaving the community will revoke any permissions you have been granted in this community.

No
Yes

Mutations in the dimer interface of dihydrolipoamide dehydrogenase promote site-specific oxidative damages in yeast and human cells.

Dihydrolipoamide dehydrogenase (DLD) is a multifunctional protein well characterized as the E3 component of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes. Previously, conditions predicted to destabilize the DLD dimer revealed that DLD could also function as a diaphorase and serine protease. However, the relevance of these cryptic activities remained undefined. We analyzed human DLD mutations linked to strikingly different clinical phenotypes, including E340K, D444V, R447G, and R460G in the dimer interface domain that are responsible for severe multisystem disorders of infancy and G194C in the NAD(+)-binding domain that is typically associated with milder presentations. In vitro, all of these mutations decreased to various degrees dihydrolipoamide dehydrogenase activity, whereas dimer interface mutations also enhanced proteolytic and/or diaphorase activity. Human DLD proteins carrying each individual mutation complemented fully the respiratory-deficient phenotype of yeast cells lacking endogenous DLD even when residual dihydrolipoamide dehydrogenase activity was as low as 21% of controls. However, under elevated oxidative stress, expression of DLD proteins with dimer interface mutations greatly accelerated the loss of respiratory function, resulting from enhanced oxidative damage to the lipoic acid cofactor of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase and other mitochondrial targets. This effect was not observed with the G194C mutation or a mutation that disrupts the proteolytic active site of DLD. As in yeast, lipoic acid cofactor was damaged in human D444V-homozygous fibroblasts after exposure to oxidative stress. We conclude that the cryptic activities of DLD promote oxidative damage to neighboring molecules and thus contribute to the clinical severity of DLD mutations.

Pubmed ID: 21930696

Authors

  • Vaubel RA
  • Rustin P
  • Isaya G

Journal

The Journal of biological chemistry

Publication Data

November 18, 2011

Associated Grants

  • Agency: NIA NIH HHS, Id: AG15709
  • Agency: NHLBI NIH HHS, Id: F30 HL099036
  • Agency: NIA NIH HHS, Id: R01 AG015709
  • Agency: NIGMS NIH HHS, Id: T32 GM 65841

Mesh Terms

  • Cells, Cultured
  • Dihydrolipoamide Dehydrogenase
  • Enzyme Stability
  • Female
  • Fibroblasts
  • Humans
  • Male
  • Metabolism, Inborn Errors
  • Mutation, Missense
  • NAD
  • Oxidative Stress
  • Protein Multimerization
  • Protein Structure, Tertiary
  • Proteolysis
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins