Inhibition of apurinic/apyrimidinic endonuclease I's redox activity revisited

Jun Zhang, Meihua Luo, Daniela Marasco, Derek Logsdon, Kaice A. Lafavers, Qiujia Chen, April Reed, Mark Kelley, Michael L. Gross, Millie Georgiadis

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

The essential base excision repair protein, apurinic/apyrimidinic endonuclease 1 (APE1), plays an important role in redox regulation in cells and is currently targeted for the development of cancer therapeutics. One compound that binds APE1 directly is (E)-3-[2-(5,6-dimethoxy-3-methyl-1,4-benzoquinonyl)] -2-nonylpropenoic acid (E3330). Here, we revisit the mechanism by which this negatively charged compound interacts with APE1 and inhibits its redox activity. At high concentrations (millimolar), E3330 interacts with two regions in the endonuclease active site of APE1, as mapped by hydrogen-deuterium exchange mass spectrometry. However, this interaction lowers the melting temperature of APE1, which is consistent with a loss of structure in APE1, as measured by both differential scanning fluorimetry and circular dichroism. These results are consistent with other findings that E3330 concentrations of >100 μM are required to inhibit APE1's endonuclease activity. To determine the role of E3330's negatively charged carboxylate in redox inhibition, we converted the carboxylate to an amide by synthesizing (E)-2-[(4,5-dimethoxy-2-methyl-3,6- dioxocyclohexa-1,4-dien-1-yl)methylene]-N-methoxy-undecanamide (E3330-amide), a novel uncharged derivative. E3330-amide has no effect on the melting temperature of APE1, suggesting that it does not interact with the fully folded protein. However, E3330-amide inhibits APE1's redox activity in in vitro electrophoretic mobility shift redox and cell-based transactivation assays, producing IC 50 values (8.5 and 7 μM) lower than those produced with E3330 (20 and 55 μM, respectively). Thus, E3330's negatively charged carboxylate is not required for redox inhibition. Collectively, our results provide additional support for a mechanism of redox inhibition involving interaction of E3330 or E3330-amide with partially unfolded APE1.

Original languageEnglish
Pages (from-to)2955-2966
Number of pages12
JournalBiochemistry
Volume52
Issue number17
DOIs
StatePublished - Apr 30 2013

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DNA-(Apurinic or Apyrimidinic Site) Lyase
Endonucleases
Oxidation-Reduction
Amides
Freezing
Melting point
E 3330
Fluorometry
Electrophoretic mobility
Temperature
Deuterium
Circular Dichroism
DNA Repair
Transcriptional Activation
Mass spectrometry
Hydrogen
Assays
Mass Spectrometry
Catalytic Domain
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Zhang, J., Luo, M., Marasco, D., Logsdon, D., Lafavers, K. A., Chen, Q., ... Georgiadis, M. (2013). Inhibition of apurinic/apyrimidinic endonuclease I's redox activity revisited. Biochemistry, 52(17), 2955-2966. https://doi.org/10.1021/bi400179m

Inhibition of apurinic/apyrimidinic endonuclease I's redox activity revisited. / Zhang, Jun; Luo, Meihua; Marasco, Daniela; Logsdon, Derek; Lafavers, Kaice A.; Chen, Qiujia; Reed, April; Kelley, Mark; Gross, Michael L.; Georgiadis, Millie.

In: Biochemistry, Vol. 52, No. 17, 30.04.2013, p. 2955-2966.

Research output: Contribution to journalArticle

Zhang, J, Luo, M, Marasco, D, Logsdon, D, Lafavers, KA, Chen, Q, Reed, A, Kelley, M, Gross, ML & Georgiadis, M 2013, 'Inhibition of apurinic/apyrimidinic endonuclease I's redox activity revisited', Biochemistry, vol. 52, no. 17, pp. 2955-2966. https://doi.org/10.1021/bi400179m
Zhang J, Luo M, Marasco D, Logsdon D, Lafavers KA, Chen Q et al. Inhibition of apurinic/apyrimidinic endonuclease I's redox activity revisited. Biochemistry. 2013 Apr 30;52(17):2955-2966. https://doi.org/10.1021/bi400179m
Zhang, Jun ; Luo, Meihua ; Marasco, Daniela ; Logsdon, Derek ; Lafavers, Kaice A. ; Chen, Qiujia ; Reed, April ; Kelley, Mark ; Gross, Michael L. ; Georgiadis, Millie. / Inhibition of apurinic/apyrimidinic endonuclease I's redox activity revisited. In: Biochemistry. 2013 ; Vol. 52, No. 17. pp. 2955-2966.
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