TY  - JOUR
AU  - Krynetskaia, Natalia 
AU  - Phadke, Manali 
AU  - Krynetskiy, Evgeny 
PY  - 2016
TI  - Is RNA Binding in the Rossmann Fold Essential for GAPDH Intranuclear Functions?
JF  - American Journal of Biochemistry and Biotechnology
VL  - 12
IS  - 2
DO  - 10.3844/ajbbsp.2016.86.94
UR  - https://thescipub.com/abstract/ajbbsp.2016.86.94
AB  - 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 non-functional 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±0.085 s, p<0.0001) and diffusion coefficient D was five times lower (4.6±0.85 vs. 23.3±13.79 μm2/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+.