title: Characterization of the redox activity and disulfide bond formation in apurinic/apyrimidinic endonuclease.
abstract: Apurinic/apyrimidinic endonuclease (APE1) is an unusual nuclear redox factor in which the redox-active cysteines identified to date, C65 and C93, are surface inaccessible residues whose activities may be influenced by partial unfolding of APE1. To assess the role of the five remaining cysteines in APE1's redox activity, double-cysteine mutants were analyzed, excluding C65A, which is redox-inactive as a single mutant. C93A/C99A APE1 was found to be redox-inactive, whereas other double-cysteine mutants retained the same redox activity as that observed for C93A APE1. To determine whether these three cysteines, C65, C93, and C99, were sufficient for redox activity, all other cysteines were substituted with alanine, and this protein was shown to be fully redox-active. Mutants with impaired redox activity failed to stimulate cell proliferation, establishing an important role for APE1's redox activity in cell growth. Disulfide bond formation upon oxidation of APE1 was analyzed by proteolysis of the protein followed by mass spectrometry analysis. Within 5 min of exposure to hydrogen peroxide, a single disulfide bond formed between C65 and C138 followed by the formation of three additional disulfide bonds within 15 min; 10 total disulfide bonds formed within 1 h. A single mixed-disulfide bond involving C99 of APE1 was observed for the reaction of oxidized APE1 with thioredoxin (TRX). Disulfide-bonded APE1 or APE1-TRX species were further characterized by size exclusion chromatography and found to form large complexes. Taken together, our data suggest that APE1 is a unique redox factor with properties distinct from those of other redox factors.
authors.affiliation: Section of Pediatric Hematology and Oncology, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana 46202-3082, United States.
authors.forename: Meihua
authors.initials: M
authors.lastname: Luo
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Hongzhen
authors.initials: H
authors.lastname: He
authors.forename: Dian
authors.initials: D
authors.lastname: Su
authors.forename: Qiujia
authors.initials: Q
authors.lastname: Chen
authors.forename: Michael L
authors.initials: ML
authors.lastname: Gross
authors.forename: Mark R
authors.initials: MR
authors.lastname: Kelley
authors.forename: Millie M
authors.initials: MM
authors.lastname: Georgiadis
mesh_terms: D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003545:Cysteine; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction / Q000187:drug effects; D011422:Propionates / Q000494:pharmacology; D011487:Protein Conformation; D013879:Thioredoxins / Q000378:metabolism
chemical_list: D016227:Benzoquinones; D004220:Disulfides; D011422:Propionates; C075569:E 3330; D013879:Thioredoxins; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D003545:Cysteine