title: Knock-in reconstitution studies reveal an unexpected role of Cys-65 in regulating APE1/Ref-1 subcellular trafficking and function.
abstract: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1) protects cells from oxidative stress via the base excision repair pathway and as a redox transcriptional coactivator. It is required for tumor progression/metastasis, and its up-regulation is associated with cancer resistance. Loss of APE1 expression causes cell growth arrest, mitochondrial impairment, apoptosis, and alterations of the intracellular redox state and cytoskeletal structure. A detailed knowledge of the molecular mechanisms regulating its different activities is required to understand the APE1 function associated with cancer development and for targeting this protein in cancer therapy. To dissect these activities, we performed reconstitution experiments by using wild-type and various APE1 mutants. Our results suggest that the redox function is responsible for cell proliferation through the involvement of Cys-65 in mediating APE1 localization within mitochondria. C65S behaves as a loss-of-function mutation by affecting the in vivo folding of the protein and by causing a reduced accumulation in the intermembrane space of mitochondria, where the import protein Mia40 specifically interacts with APE1. Treatment of cells with (E)-3-(2-[5,6-dimethoxy-3-methyl-1,4-benzoquinonyl])-2-nonyl propenoic acid, a specific inhibitor of APE1 redox function through increased Cys-65 oxidation, confirm that Cys-65 controls APE1 subcellular trafficking and provides the basis for a new role for this residue.
authors.affiliation: Department of Medical and Biological Sciences, University of Udine, 33100 Udine, Italy.
authors.forename: Carlo
authors.initials: C
authors.lastname: Vascotto
authors.forename: Elena
authors.initials: E
authors.lastname: Bisetto
authors.forename: Mengxia
authors.initials: M
authors.lastname: Li
authors.forename: Leo A H
authors.initials: LA
authors.lastname: Zeef
authors.forename: Chiara
authors.initials: C
authors.lastname: D'Ambrosio
authors.forename: Rossana
authors.initials: R
authors.lastname: Domenis
authors.forename: Marina
authors.initials: M
authors.lastname: Comelli
authors.forename: Daniela
authors.initials: D
authors.lastname: Delneri
authors.forename: Andrea
authors.initials: A
authors.lastname: Scaloni
authors.forename: Fabio
authors.initials: F
authors.lastname: Altieri
authors.forename: Irene
authors.initials: I
authors.lastname: Mavelli
authors.forename: Franco
authors.initials: F
authors.lastname: Quadrifoglio
authors.forename: Mark R
authors.initials: MR
authors.lastname: Kelley
authors.forename: Gianluca
authors.initials: G
authors.lastname: Tell
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D016227:Benzoquinones / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055879:Gene Knock-In Techniques; D006801:Humans; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D009154:Mutation; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D011422:Propionates / Q000494:pharmacology; D011485:Protein Binding; D017510:Protein Folding; D021381:Protein Transport / Q000187:drug effects; D015398:Signal Transduction
chemical_list: D016227:Benzoquinones; C507282:CHCHD4 protein, human; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011422:Propionates; C075569:E 3330; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D003545:Cysteine