Is RNA Binding in the Rossmann Fold Essential for GAPDH Intranuclear Functions?
Natalia Krynetskaia, Manali Phadke and Evgeny Krynetskiy
DOI : 10.3844/ajbbsp.2016.86.94
American Journal of Biochemistry and Biotechnology
Volume 12, Issue 2
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+.
© 2016 Natalia Krynetskaia, Manali Phadke and Evgeny Krynetskiy. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.