title: Selective oxidative stress in cell nuclei by nuclear-targeted D-amino acid oxidase.
abstract: The effects of nuclear-localized oxidative stress on both nuclear antioxidant systems, and the processes that they regulate, are not clearly understood. Here, we targeted a hydrogen peroxide (H(2)O(2))-producing enzyme, D-amino acid oxidase (DAAO), to the nucleus (NLS-DAAO) and used this to generate H(2)O(2) in the nuclei of cells. On addition of N-acetyl-D-alanine (NADA), a substrate of DAAO, to NLS-DAAO-transfected HeLa cells, a twofold increase in ROS production relative to untreated, transfected control was observed. Staining of cellular thiols confirmed that NLS-DAAO-induced ROS selectively modified the nuclear thiol pool, whereas the cytoplasmic pool remained unchanged. Furthermore, NLS-DAAO/NADA-induced ROS caused significant oxidation of the nuclear GSH pool, as measured by nuclear protein S-glutathionylation (Pr-SSG), but under the same conditions, nuclear Trx1 redox state was not altered significantly. NF-kappaB reporter activity was diminished by NLS-DAAO/NADA-stimulated nuclear oxidation. We conclude that nuclear GSH is more susceptible to localized oxidation than is nuclear Trx1. Furthermore, the attenuation of NF-kappaB reporter activity in the absence of nuclear Trx1 oxidation suggests that critical nuclear redox proteins are subject to control by S-glutathionylation during oxidative stress in the nucleus.
authors.affiliation: Division of Pulmonary, Allergy, Cystic Fibrosis and Sleep, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Halvey
authors.forename: Jason M
authors.initials: JM
authors.lastname: Hansen
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Johnson
authors.forename: Young-Mi
authors.initials: YM
authors.lastname: Go
authors.forename: Afshin
authors.initials: A
authors.lastname: Samali
authors.forename: Dean P
authors.initials: DP
authors.lastname: Jones
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D003605:D-Amino-Acid Oxidase / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D047468:Immunoprecipitation; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism; D014162:Transfection
chemical_list: D016328:NF-kappa B; D017382:Reactive Oxygen Species; D013879:Thioredoxins; D006861:Hydrogen Peroxide; D003605:D-Amino-Acid Oxidase; D005978:Glutathione; D000409:Alanine; D019803:Glutathione Disulfide