Year: 2016
Type: article
Abstract: In addition to its prominent enzymatic activity, GAPDH is an enigmatic component of multiple unrelated biochemical entities.In this study, we explored a series of mutated GAPDH and fusion EGFP-GAPDH polypeptides and compared nuclear accumulation, intranuclear mobility and RNA binding properties of the wild type and variant GAPDH proteins.Our results revealed that RNA binding to T99I-mutated GAPDH with nonfunctional NAD + binding center occurred outside the Rossmann fold.At the cellular level, wild type and mutated EGFP-GAPDH demonstrated distinct intranuclear localization in unstressed cells versus cells exposed to genotoxic stress.Wild type EGFP-GAPDH protein localized in the cytoplasm of untreated cells and accumulated in the nucleus following araC treatment (11.8±2.72% vs. 27.4±4.28%nuclear EGFP-GAPDH, % of total EGFP-GAPDH, p = 0.0007).Mutated T99I EGFP-GAPDH accumulated at high level in the nuclei of untreated and araC-treated cells (34.3±8.49% vs. 41.3±16.0%nuclear EGFP-GAPDH, % of total EGFP-GAPDH, p = 0.21).Mutated T99I EGFP-GAPDH lost its ability to form tight interactions with intranuclear macromolecules.After araC treatment, immobile fraction (1-Mf) of wild type EGFP-GAPDH in the nuclei of SW48-297 cells was three times higher (0.75±0.127 vs. 0.26±0.133%,p<0.0001), recovery half-time was three times higher (0.84±0.075 vs. 0.3±0.085s, p<0.0001) and diffusion coefficient D was five times lower (4.6±0.85 vs. 23.3±13.79µm 2 /s, p = 0.0001) compared to cells expressing T99I variant.Our results suggest that the switch between RNA and NAD + binding to GAPDH could be a regulatory mechanism governing participation of GAPDH in NAD +dependent complexes and could serve a depot to supply diverse biochemical processes with NAD + .
Institution: Temple University
Language: English
Topic: RNA Research and Splicing
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: diamond
Funder: Temple University
OpenAlex ID: W2432090808