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

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important glycolytic enzyme, has a non-catalytic (thus a noncanonical) role in inducing mitochondrial elimination under oxidative stress. We recently demonstrated that phosphorylation of GAPDH by ␦ protein kinase C (␦PKC) inhibits this GAPDH-dependent mitochondrial elimination. ␦PKC 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 pseudo-GAPDH (GAPDH) peptide, an inhibitor of ␦PKC-mediated GAPDH phosphorylation that does not inhibit the phosphorylation of other ␦PKC substrates. Unexpectedly, GAPDH decreased mitochondrial elimination and increased cardiac damage in an animal model of heart attack. Either treatment with GAPDH or direct phosphorylation of GAPDH by ␦PKC 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 GAPDHmediated elimination of damaged mitochondria. Our study also identified a pharmacological tool, GAPDH peptide, with interesting properties. GAPDH peptide is an inhibitor of the interaction between ␦PKC and GAPDH and of the resulting phosphorylation of GAPDH by ␦PKC. GAPDH peptide is also an inhibitor of GAPDH oligomerization and thus an inhibitor of GAPDH glycolytic activity. Finally, we found that GAPDH 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 GAPDH peptide-based therapy.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that catalyzes the conversion of glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate. We previously found that in addition to its role as a glycolytic enzyme, GAPDH is also involved in mitochondrial elimination by mitophagy (1). GAPDH binds to damaged mitochondria, and its phosphorylation by ␦ protein kinase C (␦PKC) under oxidative stress inhibits the removal of the damaged mitochondria through mitophagy (1). We set out to determine whether ␦PKC-mediated GAPDH phosphorylation alone is sufficient to mediate cell death following oxidative stress, using Langendorff, an ex vivo model of heart attack.We first designed a novel inhibitor that selectively inhibits GAPDH phosphorylation without affecting the phosphorylation of other ␦PKC substrates. To this end, we relied on the rationale that, similar to many other protein kinases, ␦PKC contains a highly conserved catalytic domain and a regulatory domain that keeps the enzyme in an inactive conformation. This inactive conformation is stabilized by several intramolecular auto-inhibitory interactions (2). The first intramolecular interaction identified was the one mediated by the pseudosubstrate site located at the N terminus of the kinase, which mimics the phospho-acceptor sequence in PKC substrate...

Identification of Bacterial Protein Interaction Partners Points to New Intracellular Functions of Francisella tularensis Glyceraldehyde-3-Phosphate Dehydrogenase

“…Identification of protein interaction partners thus provides valuable clues to identify new functions for such a protein (Rao et al, 2014). The interaction of eukaryotic GAPDH with various proteins has been studied for many decades (Kumagai and Sakai, 1983;Glaser et al, 2002;Andrade et al, 2004;Azam et al, 2008;Qvit et al, 2016). On the other hand, similar knowledge on bacterial GAPDH is still very limited.…”

Targeting Mitochondrial Fission-Fusion Imbalance in Heart Failure

“…Selective short peptide inhibitors are designed based on the sequence homology, structure, and sites of interaction between protein-protein intermolecular interactions [82]. Indeed, rationale designed peptides have been widely used to inhibit different protein-protein interactions and treat degenerative diseases in both pre-clinical [22,25] and clinical [45,46,83] studies.…”

Quantitative Proteomic Analyses Reveal That Energy Metabolism and Protein Biosynthesis Reinitiation are Responsible for the Initiation of Bolting Induced by High Temperature in Lettuce (Lactuca sativa L.)

“…With the increase of pyruvate kinase 1 (PK1) expression, the transformation from phoehoenolpyruvate to pyruvate was also promoted.With the increase of pyruvate decarboxylase 1 like (PD1L) and aldehyde dehydrogenase family 2 member B4 (ADF2MB4) expression and the decrease of alcohol dehydrogenase (AD) expression, pyruvate was nally transformed into acetaldehyde and acetate instead of ethanol. Among these proteins, G-3-PD is the key enzyme in the glycolytic metabolic pathway and is closely associated with energy generation [32]. G-3-PD can catalyse the conversion of glyceraldehyde 3-phosphate to 1,3diphosphoglycerate in glycolysis, and in this oxidation process, energy is generated and stored as ATP.…”