title: Fas-mediated apoptosome formation is dependent on reactive oxygen species derived from mitochondrial permeability transition in Jurkat cells.
abstract: Generation of reactive oxygen species (ROS) and activation of caspase cascade are both indispensable in Fas-mediated apoptotic signaling. Although ROS was presumed to affect the activity of the caspase cascade on the basis of findings that antioxidants inhibited the activation of caspases and that the stimulation of ROS by itself activated caspases, the mechanism by which these cellular events are integrated in Fas signaling is presently unclear. In this study, using human T cell leukemia Jurkat cells as well as an in vitro reconstitution system, we demonstrate that ROS are required for the formation of apoptosome. We first showed that ROS derived from mitochondrial permeability transition positively regulated the apoptotic events downstream of mitochondrial permeability transition. Then, we revealed that apoptosome formation in Fas-stimulated Jurkat cells was clearly inhibited by N-acetyl-L-cysteine and manganese superoxide dismutase by using both the immunoprecipitation and size-exclusion chromatography methods. To confirm these in vivo findings, we next used an in vitro reconstitution system in which in vitro-translated apoptotic protease-activating factor 1 (Apaf-1), procaspase-9, and cytochrome c purified from human placenta were activated by dATP to form apoptosome; the formation of apoptosome was markedly inhibited by reducing reagents such as DTT or reduced glutathione (GSH), whereas hydrogen peroxide prevented this inhibition. We also found that apoptosome formation was substantially impaired by GSH-pretreated Apaf-1, but not GSH-pretreated procaspase-9 or GSH-pretreated cytochrome c. Collectively, these results suggest that ROS plays an essential role in apoptosome formation by oxidizing Apaf-1 and the subsequent activation of caspase-9 and -3.
authors.affiliation: Department of Internal Medicine (Section 4), Sapporo Medical University, School of Medicine, South-1 West-16, Chuo-ku, Sapporo 060-8543, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Sato
authors.forename: Takuro
authors.initials: T
authors.lastname: Machida
authors.forename: Sho
authors.initials: S
authors.lastname: Takahashi
authors.forename: Satoshi
authors.initials: S
authors.lastname: Iyama
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Sato
authors.forename: Kageaki
authors.initials: K
authors.lastname: Kuribayashi
authors.forename: Kohichi
authors.initials: K
authors.lastname: Takada
authors.forename: Takatomi
authors.initials: T
authors.lastname: Oku
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kawano
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Okamoto
authors.forename: Rishu
authors.initials: R
authors.lastname: Takimoto
authors.forename: Takuya
authors.initials: T
authors.lastname: Matsunaga
authors.forename: Tetsuji
authors.initials: T
authors.lastname: Takayama
authors.forename: Minoru
authors.initials: M
authors.lastname: Takahashi
authors.forename: Junji
authors.initials: J
authors.lastname: Kato
authors.forename: Yoshiro
authors.initials: Y
authors.lastname: Niitsu
mesh_terms: D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D019169:Jurkat Cells; D008928:Mitochondria / Q000378:metabolism*; D010539:Permeability; D011506:Proteins / Q000378:metabolism; D017382:Reactive Oxygen Species; D019014:fas Receptor / Q000502:physiology*
chemical_list: C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D011506:Proteins; D017382:Reactive Oxygen Species; D019014:fas Receptor; D045304:Cytochromes c; D006861:Hydrogen Peroxide; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases