GAPDH with NAD+-binding site mutation competitively inhibits the wild-type and affects glucose metabolism in cancer

Background

Rapid utilization of glucose is a metabolic signature of majority of cancers, hence enzymes of the glycolytic pathway remain attractive therapeutic targets. Recent reports have shown that targeting the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an abundant, ubiquitous multifunctional protein frequently upregulated in cancer, affects cancer progression. Here, we report that a catalytically-deficient mutant-GAPDH competitively inhibits the wild-type, and disrupts glucose metabolism in cancer cells.

Methods

Using site-directed mutagenesis, the human GAPDH clone was mutated at one of the NAD⁺-binding sites, (i.e.) arginine (R13) and isoleucine (I14) to glutamine (Q13) and phenylalanine (F14), respectively. The inhibitory role of the mutant-GAPDH, and its effect on energy metabolism and cancer phenotype was determined using in vitro and in vivo models of cancer.

Results

The enzymatically-dysfunctional mutant-GAPDH competitively inhibited the wild-type GAPDH in a cell-free system. In cancer cells, ectopic expression of the mutant-GAPDH, but not the wild-type, inhibited the glycolytic capacity of cellular-GAPDH, and led to the induction of metabolic stress accompanied by a sharp decline in glucose-uptake. Furthermore, expression of mutant-GAPDH affected cancer growth in vitro and in vivo. Mechanistically, structural analysis by bioinformatics revealed that the mutations at the NAD⁺-binding site altered the solvent-accessibility that perhaps affected the functionality of mutant-GAPDH.

Conclusion

Mutant-GAPDH affects the enzymatic function of cellular-GAPDH and disrupts energy metabolism.

General significance

Our findings demonstrate that a minimal mutation at the NAD⁺-binding site is sufficient to generate a competitive but dysfunctional GAPDH, and its ectopic expression inhibits the wild-type to disrupt glycolysis.