Abstract 1131: Disruption of the NAD+- binding site affects GAPDH interactions in the nuclei of cancer cells outside glucose metabolism
Authors: Krynetskiy Evgeny , Phadke Manali , Krynetskaia Natalia , Mishra Anurag , Barrero Carlos , Merali Salim , Gothe Scott A. 
Published: August 2015

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a pivotal enzyme of the glycolytic pathway in cancer cells (the Warburg effect) which also performs signaling/regulatory functions in several cellular pathways including apoptosis and accelerated senescence. Multiple post-translational modifications, e.g., phosphorylation, acetylation, and nitrosylation, modulate functions and protein interactions of GAPDH in the cell. To assess the potential role of GAPDH as a molecular target within the cancer energy metabolism network, we (1) characterized structural elements of human GAPDH polypeptide pertinent to its intranuclear functions, and (2) evaluated mobility of nuclear GAPDH in cancer cells treated with chemotherapeutic agents. Using MALDI-TOF analysis, we identified novel phosphorylation sites within the NAD+-binding center of GAPDH at Y94, Ser98, and Thr99. We generated polyclonal antibody specific to phosphoThr99-containing peptide within GAPDH, and demonstrated accumulation of T99-phosphoGAPDH in the nuclear fractions of A549 cancer cells following the cytotoxic stress using 2D-Western blot analysis. Analysis of the extant crystal structures for human GAPDH and molecular modeling experiments indicated that T99 is adjacent to the NAD+-binding site. Molecular mechanics and dynamics experiments suggested that mutation of Thr99 may adversely affect the local geometry of the NAD+-binding cleft, and is likely to decrease affinity for NAD+. We performed site-mutagenesis of the functional domains in GAPDH, and estimated the enzymatic properties, intranuclear distribution, and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+-binding center abrogated enzymatic activity of GAPDH due to decreased affinity to NAD+ (Km = 0.41±0.17 mM in T99I vs. 0.04±0.01 mM in wild type GAPDH). FRAP analysis showed that mutation in NAD+-binding center of GAPDH didn't disrupt its nuclear translocation after chemotherapeutic agents, but prevented intranuclear interactions. Molecular dynamics parameters of nuclear EGFP_GAPDH_WT before vs. after araC treatment were: t(1/2) = 0.12 vs. 1.09 sec, and diffusion coefficient D = 28.88 vs. 3.2 μm2/s. The same parameters for EGFP-GAPDH_T99I were: t(1/2) 0.39 vs. 0.68 sec, and D = 9.1 vs. 5.1 μm2/s. The studies on the functional consequences and a mechanism of Thr99 phosphorylation will open avenues to the rational design of modulators of GAPDH activity, an important target in anticancer therapy.

Citation Format: Evgeny Krynetskiy, Manali Phadke, Natalia Krynetskaia, Anurag Mishra, Carlos Barrero, Salim Merali, Scott A. Gothe. Disruption of the NAD+- binding site affects GAPDH interactions in the nuclei of cancer cells outside glucose metabolism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1131. doi:10.1158/1538-7445.AM2015-1131