title: Protein disulfide isomerase as a novel target for cyclopentenone prostaglandins: implications for hypoxic ischemic injury.
abstract: Cyclooxygenase-2 (COX-2) is an important contributor to ischemic brain injury. Identification of the downstream mediators of COX-2 toxicity may allow the development of targeted therapies. Of particular interest is the cyclopentenone family of prostaglandin metabolites. Cyclopentenone prostaglandins (CyPGs) are highly reactive molecules that form covalent bonds with cellular thiols. Protein disulfide isomerase (PDI) is an important molecule for the restoration of denatured proteins following ischemia. Because PDI has several thiols, including thiols within the active thioredoxin-like domain, we hypothesized that PDI is a target of CyPGs and that CyPG binding of PDI is detrimental. CyPG-PDI binding was detected in vitro via immunoprecipitation and MS. CyPG-PDI binding decreased PDI enzymatic activity in recombinant PDI treated with CyPG, and PDI immunoprecipitated from neuronal culture treated with CyPG or anoxia. Toxic effects of binding were demonstrated in experiments showing that: (a) pharmacologic inhibition of PDI increased cell death in anoxic neurons, (b) PDI overexpression protected neurons exposed to anoxia and SH-SY5Y cells exposed to CyPG, and (c) PDI overexpression in SH-SY5Y cells attenuated ubiquitination of proteins and decreased activation of pro-apoptotic caspases. In conclusion, CyPG production and subsequent binding of PDI is a novel and potentially important mechanism of ischemic brain injury. We show that CyPGs bind to PDI, cyclopentenones inhibit PDI activity, and CyPG-PDI binding is associated with increased neuronal susceptibility to anoxia. Additional studies are necessary to determine the relative role of CyPG-dependent inhibition of PDI activity in ischemia and other neurodegenerative disorders.
authors.affiliation: Geriatric Research Education and Clinical Center, V.A. Pittsburgh Healthcare System, PA, USA.|Department of Neurology, University of Pittsburgh School of Medicine, PA, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Liu
authors.affiliation: Department of Neurology, University of Pittsburgh School of Medicine, PA, USA.|Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh, PA, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.affiliation: Geriatric Research Education and Clinical Center, V.A. Pittsburgh Healthcare System, PA, USA.|Department of Neurology, University of Pittsburgh School of Medicine, PA, USA.
authors.forename: Wenjin
authors.initials: W
authors.lastname: Li
authors.affiliation: Geriatric Research Education and Clinical Center, V.A. Pittsburgh Healthcare System, PA, USA.|Department of Neurology, University of Pittsburgh School of Medicine, PA, USA.
authors.forename: Marie E
authors.initials: ME
authors.lastname: Rose
authors.affiliation: Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh, PA, USA.
authors.forename: Sunita N
authors.initials: SN
authors.lastname: Shinde
authors.affiliation: Biomedical Mass Spectrometry Center, University of Pittsburgh, PA, USA.
authors.forename: Manimalha
authors.initials: M
authors.lastname: Balasubramani
authors.affiliation: Biomedical Mass Spectrometry Center, University of Pittsburgh, PA, USA.
authors.forename: Guy T
authors.initials: GT
authors.lastname: Uechi
authors.affiliation: Department of Chemistry & Biochemistry, University of Windsor, ON, Canada.
authors.forename: Bülent
authors.initials: B
authors.lastname: Mutus
authors.affiliation: Geriatric Research Education and Clinical Center, V.A. Pittsburgh Healthcare System, PA, USA.|Department of Neurology, University of Pittsburgh School of Medicine, PA, USA.
authors.forename: Steven H
authors.initials: SH
authors.lastname: Graham
authors.affiliation: Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh, PA, USA.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Hickey
mesh_terms: D002460:Cell Line; D003517:Cyclopentanes / Q000494:pharmacology*; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D015151:Immunoblotting; D011453:Prostaglandins / Q000494:pharmacology*; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*
chemical_list: D003517:Cyclopentanes; D011453:Prostaglandins; D019704:Protein Disulfide-Isomerases; C013905:cyclopentenone
keywords: brain; cyclooxygenase; cyclopentenone; ischemia; protein disulfide isomerase