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

• Published in: Biochimica et biophysica acta. General subjects Vol. 1862; no. 12; pp. 2555 - 2563
• Main Authors: Kunjithapatham, Rani, Ganapathy-Kanniappan, Shanmugasundaram
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2018
• Subjects: Amino Acid Sequence; Animals; arginine; Binding Sites; bioinformatics; cancer metabolism; Catalysis; cell free system; Cell Line, Tumor; competitive inhibition; disease course; energy; energy metabolism; GAPDH; glucose; Glucose - metabolism; glutamine; glyceraldehyde-3-phosphate dehydrogenase; Glyceraldehyde-3-Phosphate Dehydrogenases - chemistry; Glyceraldehyde-3-Phosphate Dehydrogenases - genetics; Glyceraldehyde-3-Phosphate Dehydrogenases - metabolism; glycolysis; Humans; isoleucine; Male; Mice; Mice, Nude; Mutation; NAD - metabolism; NAD+-binding; neoplasm cells; neoplasms; Neoplasms - metabolism; Neoplasms - pathology; phenotype; phenylalanine; site-directed mutagenesis; Stress, Physiological; therapeutics; cancer metabolism; GAPDH; NAD+-binding; glycolysis; competitive inhibition; NAD(+)-binding
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2018.08.001

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. 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. 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. Mutant-GAPDH affects the enzymatic function of cellular-GAPDH and disrupts energy metabolism. 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. [Display omitted] •Catalytically dysfunctional mutant-GAPDH competitively inhibits the wild-type.•Mutant-GAPDH disrupts glucose metabolism and induces metabolic stress.•Altered solvent accessibility underlies the loss of function in mutant-GAPDH.