title: Thioredoxin as a molecular target of cyclopentenone prostaglandins.
abstract: Prostaglandin (PG) D2, a major cyclooxygenase product in a variety of tissues and cells, readily undergoes dehydration to yield the bioactive cyclopentenone-type PGs of the J2 series, such as 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2). We have shown previously that 15d-PGJ2 is a potent electrophile that causes intracellular oxidative stress and redox alteration in human neuroblastoma SH-SY5Y cells. In the present study, based on the observation that the electrophilic center of 15d-PGJ2 was involved in the pro-oxidant effect, we investigated the role of thioredoxin 1 (Trx), an endogenous redox regulator, against 15d-PGJ2-induced oxidative cell injury. It was observed that the 15d-PGJ2-induced oxidative stress was significantly suppressed by the Trx overexpression. In addition, the treatment of SH-SY5Y cells with biotinylated 15d-PGJ2 resulted in the formation of a 15d-PGJ2-Trx adduct, indicating that 15d-PGJ2 directly modified the endogenous Trx in the cells. To further examine the mechanism of the 15d-PGJ2 modification of Trx, human recombinant Trx treated with 15d-PGJ2 was analyzed by mass spectrometry. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the 15d-PGJ2-treated human recombinant Trx demonstrated the addition of one molecule of 15d-PGJ2 per protein molecule. Moreover, the electrospray ionization-liquid chromatography/mass spectrometry/mass spectrometry analysis identified two cysteine residues, Cys-35 and Cys-69, as the targets of 15d-PGJ2. These residues may represent the direct sensors of the electrophilic PGs that induce the intracellular redox alteration and neuronal cell death.
authors.affiliation: Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Shibata
authors.forename: Takaaki
authors.initials: T
authors.lastname: Yamada
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ishii
authors.forename: Shigenori
authors.initials: S
authors.lastname: Kumazawa
authors.forename: Hajime
authors.initials: H
authors.lastname: Nakamura
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Masutani
authors.forename: Junji
authors.initials: J
authors.lastname: Yodoi
authors.forename: Koji
authors.initials: K
authors.lastname: Uchida
mesh_terms: D000595:Amino Acid Sequence; D019921:Biotinylation; D016923:Cell Death; D003517:Cyclopentanes / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009474:Neurons / Q000473:pathology; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D015230:Prostaglandin D2 / Q000031:analogs & derivatives / Q000378:metabolism; D011453:Prostaglandins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013879:Thioredoxins / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: C097240:15-deoxy-delta(12,14)-prostaglandin J2; D003517:Cyclopentanes; D016877:Oxidants; D010455:Peptides; D011453:Prostaglandins; D011994:Recombinant Proteins; D013879:Thioredoxins; C013905:cyclopentenone; D015230:Prostaglandin D2; D010100:Oxygen