Research Article Open Access

Is RNA Binding in the Rossmann Fold Essential for GAPDH Intranuclear Functions?

Natalia Krynetskaia1, Manali Phadke1 and Evgeny Krynetskiy1
  • 1 Temple University School of Pharmacy, United States

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 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+.

American Journal of Biochemistry and Biotechnology
Volume 12 No. 2, 2016, 86-94

DOI: https://doi.org/10.3844/ajbbsp.2016.86.94

Submitted On: 11 February 2016 Published On: 14 June 2016

How to Cite: Krynetskaia, N., Phadke, M. & Krynetskiy, E. (2016). Is RNA Binding in the Rossmann Fold Essential for GAPDH Intranuclear Functions?. American Journal of Biochemistry and Biotechnology, 12(2), 86-94. https://doi.org/10.3844/ajbbsp.2016.86.94

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Keywords

  • Glyceraldehyde-3-Phosphate Dehydrogenase
  • NAD+
  • Fluorescence Recovery after Photobleaching
  • Nuclear Proteins
  • Mutation
  • RNA