Mass Spectrometric Approaches to Probing the Redox Function of Ape1

Abstract

Human apurinic/apyrimidinic endonuclease 1 (hApe1) is a multi-functional protein having two major functions: apurinic/apyrimidinic endonuclease activity for DNA damage repair and redox activity for gene regulation. Many studies have shown the action of Ape1 in the base excision repair pathway leading to cell survival. It has also been reported that Ape1 reduces a number of important transcription factors that are involved in cancer promotion and progression. Though the repair activity is well understood, the redox mechanism is not yet clear. What is known about Ape1 is its structure and that it contains seven cysteines (C65, C93, C99, C138, C208, C296, and C310), none of which are disulfide bonded. Two of these cysteines, C99 and C138, are solvent-accessible, and C65, C93, and C99 are located in the redox domain. It is believed that one or more cysteines are involved in the redox function and is hypothesized that hApe1 reduces the down-stream transcription factors by a disulfide exchange mechanism. E3330, (2E)-3-[5-(2,3-dimethoxy-6-methyl-1,4-benzoquninoyl)]2-nonyl-2-propenoic acid, is a specific inhibitor for the redox function of hApe1. The interaction mechanism is not known. Using N-Ethylmaleimide (NEM) chemical footprinting, combined with Hydrogen/Deuterium Exchange (HDX) data, we propose that a locally unfolded form coexists with the folded form in an equilibrium that is driven by irreversible NEM labeling, and that E3330 interacts with and stabilizes this locally unfolded form. This locally unfolded form is thereby proposed to be the redox-active form. We further support this claim with LC-MS/MS analysis showing an increase of disulfide bonds induced by E3330 among the cysteines in the redox domain, which would be too far apart from each other in the folded form to form a disulfide bond. We also studied three analogs of E3330. The need for an E3330 analog is to develop a more efficient and effective compound that would allow for sub-micromolar levels of activity (E3330 requires a micromolar amount). Study of the analogs will also allow us to gain perspective of the mechanism or mechanisms of E3330’s activity in Ape1’s redox function.