title: Mechanism of oxidative inactivation of human presequence protease by hydrogen peroxide.
abstract: The mitochondrial presequence protease (PreP) is a member of the pitrilysin class of metalloproteases. It degrades the mitochondrial targeting presequences of mitochondria-localized proteins as well as unstructured peptides such as amyloid-β peptide. The specific activity of PreP is reduced in Alzheimer patients and animal models of Alzheimer disease. The loss of activity can be mimicked in vitro by exposure to oxidizing conditions, and indirect evidence suggested that inactivation was due to methionine oxidation. We performed peptide mapping analyses to elucidate the mechanism of inactivation. None of the 24 methionine residues in recombinant human PreP was oxidized. We present evidence that inactivation is due to oxidation of cysteine residues and consequent oligomerization through intermolecular disulfide bonds. The most susceptible cysteine residues to oxidation are Cys34, Cys112, and Cys119. Most, but not all, of the activity loss is restored by the reducing agent dithiothreitol. These findings elucidate a redox mechanism for regulation of PreP and also provide a rational basis for therapeutic intervention in conditions characterized by excessive oxidation of PreP.
authors.affiliation: Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, Bethesda, MD 20892, USA.
authors.forename: Jue
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Pedro Filipe
authors.initials: PF
authors.lastname: Teixeira
authors.affiliation: Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Elzbieta
authors.initials: E
authors.lastname: Glaser
authors.affiliation: Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, Bethesda, MD 20892, USA. Electronic address: rlevine@nih.gov.
authors.forename: Rodney L
authors.initials: RL
authors.lastname: Levine
mesh_terms: D003553:Cystine / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry*; D007700:Kinetics; D008715:Methionine; D024101:Mitochondrial Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D055503:Protein Multimerization; D012697:Serine Endopeptidases / Q000737:chemistry*
chemical_list: D024101:Mitochondrial Proteins; D003553:Cystine; D008715:Methionine; D006861:Hydrogen Peroxide; C514899:PREP protein, human; D012697:Serine Endopeptidases
keywords: Cysteine oxidation; Free radicals; Methionine sulfoxide; Peptide degradation; Presequence protease; Protein oxidation