title: Metal-catalyzed oxidation of the Werner syndrome protein causes loss of catalytic activities and impaired protein-protein interactions.
abstract: Metal-catalyzed oxidation reactions target amino acids in the metal binding pocket of proteins. Such oxidation reactions generally result in either preferential degradation of the protein or accumulation of a catalytically inactive pool of protein with age. Consistently, levels of oxidized proteins have been shown to increase with age. The segmental, progeroid disorder Werner syndrome results from loss of the Werner syndrome protein (WRN). WRN is a member of the RecQ family of DNA helicases and possesses exonuclease and ATP-dependent helicase activities. Furthermore, each of the helicase and exonuclease domains of WRN contains a metal binding pocket. In this report we examined for metal-catalyzed oxidation of WRN in the presence of iron or copper. We found that WRN was oxidized in vitro by iron but not by copper. Iron-mediated oxidation resulted in the inhibition of both WRN helicase and exonuclease activities. Oxidation of WRN also inhibited binding to several known protein partners. In addition, we did not observe degradation of oxidized WRN by the 20 S proteasome in vitro. Finally, exposure of cells to hydrogen peroxide resulted in oxidation of WRN in vivo. Therefore, our results demonstrate that WRN undergoes metal-catalyzed oxidation in the presence of iron, and iron-mediated oxidation of WRN likely results in the accumulation of a catalytically inactive form of the protein, which may contribute to age-related phenotypes.
authors.affiliation: Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Baltimore, Maryland 21224, USA.
authors.forename: Jeanine A
authors.initials: JA
authors.lastname: Harrigan
authors.forename: Jason
authors.initials: J
authors.lastname: Piotrowski
authors.forename: Luca
authors.initials: L
authors.lastname: Di Noto
authors.forename: Rodney L
authors.initials: RL
authors.lastname: Levine
authors.forename: Vilhelm A
authors.initials: VA
authors.lastname: Bohr
mesh_terms: D001665:Binding Sites / Q000502:physiology; D002384:Catalysis; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D005090:Exodeoxyribonucleases; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D010641:Phenotype; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D053484:RecQ Helicases / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014898:Werner Syndrome / Q000201:enzymology*; D000071657:Werner Syndrome Helicase
chemical_list: D016877:Oxidants; D011994:Recombinant Proteins; D003300:Copper; D006861:Hydrogen Peroxide; D007501:Iron; D005090:Exodeoxyribonucleases; D046988:Proteasome Endopeptidase Complex; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase