S-Glutathionylation of Cysteine 99 in the APE1 Protein Impairs Abasic Endonuclease Activity

Abstract

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a central participant in the base excision repair pathway, exhibiting AP endonuclease activity that incises the DNA backbone 5′ to an abasic site. Besides its prominent role as a DNA repair enzyme, APE1 was separately identified as a protein called redox effector factor 1, which is able to enhance the DNA binding activity of several transcription factors through a thiol-exchange-based reduction–oxidation mechanism. In the present study, we found that human APE1 is S-glutathionylated under conditions of oxidative stress both in the presence of glutathione in vitro and in cells. S-glutathionylated APE1 displayed significantly reduced AP endonuclease activity on abasic-site-containing oligonucleotide substrates, a result stemming from impaired DNA binding capacity. The combination of site-directed mutagenesis, biochemical assays, and mass spectrometric analysis identified Cys99 in human APE1 as the critical residue for the S-glutathionylation that leads to reduced AP endonuclease activity. This modification is reversible by reducing agents, which restore APE1 incision function. Our studies describe a novel posttranslational modification of APE1 that regulates the DNA repair function of the protein.

Biological chemistry and functionality of protein sulfenic acids and related thiol modifications

“…Site-directed mutagenesis of Cys residues within the redox regulatory portion of the protein (Cys65Ala, Cys93Ala and Cys99Ala) revealed that mutants containing Cys99Ala were not affected by oxidation, indicating that the oxidative inactivation of APE1 activity requires Cys99. The redox active Cys99 site was further confirmed by MS analysis, which identified glutathionylation with GSH/diamide treatment and –SOH, -SO 2 H and disulfides with H 2 O 2 treatment [214,215]. …”

Base Excision Repair: Contribution to Tumorigenesis and Target in Anticancer Treatment Paradigms

“…As will be discussed below, specific portions of the protein are still being assigned to particular function(s), but currently, the redox activity has been associated with the N-terminus of the protein, whereas the majority of the protein, the C-terminus, is essential for its DNA repair activities. Post-translational modifications of APE1, consisting of phosphorylation, acetylation, glutathionylation and ubiquitination, can regulate protein function, although the biological significance of these modifications is not yet fully understood [34,35,36]. …”

BERing the burden of damage: Pathway crosstalk and posttranslational modification of base excision repair proteins regulate DNA damage management

“…Documented PTMs detected in APEX1 include phosphorylation 96,97 , acetylation 98–100 , ubiquitination 101 , and S-nitrosylation (Table 1) 96,102 . However, S-glutathionylation also occurs in response to the altered redox state of the cell 103 . Glutathionylation, addition of a glutathione group, occurs at cysteine residues and three candidate cysteines are located in the redox responsive domain of APEX1 103 .…”

“…However, S-glutathionylation also occurs in response to the altered redox state of the cell 103 . Glutathionylation, addition of a glutathione group, occurs at cysteine residues and three candidate cysteines are located in the redox responsive domain of APEX1 103 . As cysteine modifications include disulfide bridges, S-nitrosylation, and S-glutathionylation, understanding the competition between different PTMs and how they impact APEX1 function is crucial.…”

“…As cysteine modifications include disulfide bridges, S-nitrosylation, and S-glutathionylation, understanding the competition between different PTMs and how they impact APEX1 function is crucial. APEX1 undergoes reversible glutathionylation in vitro 103 . Glutathionylation of APEX1 occurs on C99, and inhibits APEX1-mediated AP site cleavage by 90%.…”