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

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2555-2563
Number of pages9
JournalBiochimica et Biophysica Acta - General Subjects
Volume1862
Issue number12
DOIs
StatePublished - Dec 2018

Keywords

  • GAPDH
  • NAD-binding
  • cancer metabolism
  • competitive inhibition
  • glycolysis

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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