Human Glyceraldehyde-3-phosphate Dehydrogenase Plays a Direct Role in Reactivating Oxidized Forms of the DNA Repair Enzyme APE1

Sonish Azam,Nathalie Jouvet,Arshad Jilani,Ratsavarinh Vongsamphanh,Xiaoming Yang,Stephen Yang,Dindial Ramotar

doi:10.1074/jbc.m801401200

Sonish Azam, Nathalie Jouvet + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m801401200

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Journal: Journal of Biological Chemistry	Publication Date: Nov 1, 2008
Citations: 134	License type: cc-by

Affiliation: Université de Montréal

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has diverse biological functions including its nuclear translocation in response to oxidative stress. We show that GAPDH physically associates with APE1, an essential enzyme involved in the repair of abasic sites in damaged DNA, as well as in the redox regulation of several transcription factors. This interaction allows GAPDH to convert the oxidized species of APE1 to the reduced form, thereby reactivating its endonuclease activity to cleave abasic sites. The GAPDH variants C152G and C156G retain the ability to interact with but are unable to reactivate APE1, implicating these cysteines in catalyzing the reduction of APE1. Interestingly, GAPDH-small interfering RNA knockdown sensitized the cells to methyl methane sulfonate and bleomycin, which generate lesions that are repaired by APE1, but showed normal sensitivity to 254-nm UV. Moreover, the GAPDH knockdown cells exhibited an increased level of spontaneous abasic sites in the genomic DNA as a result of diminished APE1 endonuclease activity. Thus, the nuclear translocation of GAPDH during oxidative stress constitutes a protective mechanism to safeguard the genome by preventing structural inactivation of APE1.

Highlights

Dative stress, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rapidly undergoes disulfide bond formation leading to reduction in its enzymatic activity (2, 3)
A Minor AP Endonuclease Activity Is Associated with the Glycolytic Enzyme GAPDH—We initially set out to examine whether mammalian cells contained a Mg2ϩ-independent AP endonuclease(s) belonging to the E. coli endonuclease IV family, because such an activity has not been previously identified
We show for the first time that GAPDH interacts with a key DNA repair enzyme, APE1, which functions in the base excision repair pathway

Summary

EXPERIMENTAL PROCEDURES

Bacteria Strains—The Escherichia coli strains used in this work were BW528 [⌬(xth-pnc), nfo1::kan] Purification of GST and His-tagged Proteins—GST-GAPDH fusion proteins were overexpressed in E. coli BW528 strain and purified by glutathione-Sepharose 4B mini columns as previously described (23), and the His6-APE1 was purified using Talon affinity beads according to the manufacturer’s protocol (Clontech). GST Pulldown Assays—Glutathione-Sepharose 4B beads (100 ␮l) alone or bound to 10 ␮g of the indicated GST-tagged proteins were mixed with purified N-terminal His-tagged APE1 (1 ␮g) in 0.5 ml of buffer A and incubated for 30 min at room temperature with gentle rotation. Oxidation of APE1 Protein with H2O2—The His-APE1 protein bound to the TALON metal affinity column (1 ml; BD Biosciences) was treated with H2O2 (5 ␮M for 5 min), washed three times with buffer B (50 mM sodium phosphate, pH 7.0, and 300 mM NaCl), and eluted using buffer B containing 150 mM imidazole. Was measured by using the DNA damage quantification kit according to the manufacturer’s protocol (Biovison Inc.)

RESULTS

DISCUSSION

GAPDH could form aggregates following incubation with oxidized