TY - JOUR
T1 - GAPDH with NAD+-binding site mutation competitively inhibits the wild-type and affects glucose metabolism in cancer
AU - Kunjithapatham, Rani
AU - Ganapathy-Kanniappan, Shanmugasundaram
N1 - Funding Information:
This work was supported by the funding from the Society of Interventional Radiology (SIR) Foundation. We gratefully acknowledge the support by Charles Wallace Pratt Research Fund . We thank Dr. Scott Kominsky for allowing us to use the UV-Vis Spectrophotometer. While the study was in progress Dr. Kunjithapatham passed away. Her exemplary contribution, meticulous approach and unparalleled enthusiasm for science made this manuscript possible. Rani was a great source of inspiration to us and will be greatly missed.
Publisher Copyright:
© 2018 Elsevier B.V.
PY - 2018/12
Y1 - 2018/12
N2 - 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.
AB - 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.
KW - GAPDH
KW - NAD-binding
KW - cancer metabolism
KW - competitive inhibition
KW - glycolysis
UR - http://www.scopus.com/inward/record.url?scp=85051628355&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85051628355&partnerID=8YFLogxK
U2 - 10.1016/j.bbagen.2018.08.001
DO - 10.1016/j.bbagen.2018.08.001
M3 - Article
C2 - 30077773
AN - SCOPUS:85051628355
SN - 0304-4165
VL - 1862
SP - 2555
EP - 2563
JO - Biochimica et Biophysica Acta - General Subjects
JF - Biochimica et Biophysica Acta - General Subjects
IS - 12
ER -