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+.
Highlights
Propensity of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) to bind single-stranded polynucleotides is an intriguing phenomenon which significance remains enigmatic
Wild type and T99I variant demonstrated different intracellular distribution before and after araC treatment: wild type EGFP-GAPDH fusion 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 EGFPGAPDH, % of total EGFP-GAPDH, p = 0.0007)
Our experiments demonstrated that T99I mutation disrupts NAD+ binding but not RNA binding indicating that RNA interacts with a different site in T99I protein
Summary
Propensity of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) to bind single-stranded polynucleotides is an intriguing phenomenon which significance remains enigmatic. Three types of polynucleotide-GAPDH interaction have been described, so far: (i) GAPDH binding to RNA, mostly represented by AU-Rich Elements (ARE) in the 3’UTR of mRNA (Nagy and Rigby, 1995; Seidler, 2013) (ii) formation of multiprotein-RNA complexes, with GAPDH and RNA being important structural components (Mazurek et al, 1996; Carlile et al, 1998). (iii) GAPDH binding to single-stranded DNA at telomeric sequences near the chromosomal ends (Sundararaj et al, 2004; Demarse et al, 2009). Affinity of GAPDH to ARE results in GAPDH binding to several mRNA coding for c-myc, CSF-1/2, IFN-γ, IL-2, ET1, AT1R and COX-2 (Nagy and Rigby, 1995; Rodriguez-Pascual et al, 2008; Zhou et al, 2008; Backlund et al, 2009; Ikeda et al, 2012). Mutagenesis of residues localized at the GAPDH dimer interface impaired binding of GAPDH to ARE within TNF-α mRNA (White et al, 2015)
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