Reactions of Glyceraldehyde 3-Phosphate Dehydrogenase Sulfhydryl Groups with Bis-Electrophiles Produce DNA–Protein Cross-Links but Not Mutations

Abstract

The environmental contaminant 1,2-dibromoethane and diepoxybutane, an oxidation product of the important industrial chemical butadiene, are bis-functional electrophiles and known to be mutagenic and carcinogenic. One mechanism by which bis-electrophiles can exert their toxic effects is through the induction of genotoxic and mutagenic DNA-peptide crosslinks. This mechanism has been shown in systems overexpressing the DNA repair protein O 6 -alkylguanine DNA-alkyltransferase (AGT) or glutathione S-transferase and involves reactions with nucleophilic cysteine residues. The hypothesis that DNA-protein crosslink formation is a more general mechanism for genotoxicity by bis-electrophiles was investigated by screening nuclear proteins for reactivity with model monofunctional electrophiles. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was identified as a candidate due to the nucleophilicity of two cysteine residues (Cys 152 and Cys 246 ) in reaction screens with model electrophiles (Dennehy, M. K. et al. (2006) Chem. Res. Toxicol. 19,[20][21][22][23][24][25][26][27][28][29]. Incubation of GAPDH with bis-electrophiles resulted in inhibition of its catalytic activity, but only at high concentrations of diepoxybutane. In vitro assays indicated DNA-GAPDH crosslink formation in the presence of diepoxybutane, and biselectrophile reactivity at Cys 246 was confirmed using mass spectral analysis. In contrast to AGT, overexpression of human GAPDH in Escherichia coli did not enhance mutagenesis by diepoxybutane. We propose that the lack of mutational enhancement is in part due to the inherently lower reactivity of GAPDH toward bis-electrophiles as well as the reduced DNA binding ability relative to AGT, preventing the in vivo formation of DNA-protein crosslinks and enhanced mutagenesis.

Replication past the Butadiene Diepoxide-Derived DNA Adduct S-[4-(N6-Deoxyadenosinyl)-2,3-dihydroxybutyl]glutathione by DNA Polymerases

“…replication, transcription, and recombination. 37 DNA-protein cross-links induced by DEB were first observed in livers of B6C3F1 mice exposure to BD 38 and have been reported in vitro including O 6 -alkylguanine DNA alkyltransferase (AGT), 49 glyceradehyde 3-phosphate dehydrogenase, 40 and histone H3. 41 However, there is no evidence that DEB-derived DNA-protein cross-links generate mutations, aside from the case of these formed with AGT.…”

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with apurinic/apyrimidinic sites in DNA

“…Although the main GAPDH function is not related with DNA recognition, this enzyme was shown to interact with various DNA damages including structural analogues of nucleotides used for anticancer therapy, such as thioguanine, mercaptopurine, cytosine arabinoside and 5-fluorouracil [28,29]; covalent DNA adducts with the alkylating agents saframycin [30] and S23906-1 (benzoacronicine derivative) [31]; covalent DNA adducts with bis-electrophiles, e.g. 1,2-dibromoethane and diepoxybutane [53] that allows to propose the involvement of GAPDH in DNA repair. In addition, direct interaction of GAPDH with some proteins participating in DNA repair has been reported [32,29].…”

Conjugation of Butadiene Diepoxide with Glutathione Yields DNA Adducts in Vitro and in Vivo

“…23,24 DNA-protein cross-links induced by DEB were first reported in liver tissue of B6C3F1 mice exposure to butadiene. 41 Recently, cross-linking studies induced by DEB with purified recombinant proteins have been reported in vitro including O 6 -alkylguanine DNA alkyltransferase (AGT), 42 glyceradehyde 3-phosphate dehydrogenase (GAPDH), 43 and histone H3. 44 Although GAPDH and histone H3 with DEB form DNA-protein cross-links, the adducts are not mutagenic.…”