Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Protein-Protein Interaction Inhibitor Reveals a Non-catalytic Role for GAPDH Oligomerization in Cell Death*

Nir Qvit, Amit U Joshi, Anna D Cunningham, Julio C B Ferreira, Daria Mochly-Rosen

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important glycolytic enzyme, has a non-catalytic (thus a non-canonical) role in inducing mitochondrial elimination under oxidative stress. We recently demonstrated that phosphorylation of GAPDH by delta protein kinase C (PKC) inhibits this GAPDH-dependent mitochondrial elimination. deltaPKC phosphorylation of GAPDH correlates with increased cell injury following oxidative stress, suggesting that inhibiting GAPDH phosphorylation should decrease cell injury. Using rational design, we identified pseudoGAPDH peptide, an inhibitor of deltaPKC-mediated GAPDH phosphorylation that does not inhibit the phosphorylation of other deltaPKC substrates. Unexpectedly, pseudoGAPDH decreased mitochondrial elimination and increased cardiac damage in an animal model of heart attack. Either treatment with pseudoGAPDH or direct phosphorylation of GAPDH by deltaPKC decreased GAPDH tetramerization, which corresponded to reduced GAPDH glycolytic activity in vitro and ex vivo. Taken together, our study identified the potential mechanism by which oxidative stress inhibits the protective GAPDH-mediated elimination of damaged mitochondria. Our study also identified a pharmacological tool, pseudoGAPDH peptide, with interesting properties. pseudoGAPDH peptide is an inhibitor of the interaction between deltaPKC and GAPDH, and of the resulting phosphorylation of GAPDH by deltaPKC. pseudoGAPDH peptide is also an inhibitor of GAPDH oligomerization and thus an inhibitor of GAPDH glycolytic activity. Finally, we found that pseudoGAPDH peptide is an inhibitor of the elimination of damaged mitochondria. We discuss how this unique property of increasing cell damage following oxidative stress suggests a potential use for pseudoGAPDH peptide-based therapy.