title: Nitric oxide donors inhibit formation of the Apaf-1/caspase-9 apoptosome and activation of caspases.
abstract: Caspases are critical for the initiation and execution of apoptosis. Nitric oxide (NO) or derived species can prevent programmed cell death in several cell types, reportedly through S-nitrosation and inactivation of active caspases. Although we find that S-nitrosation of caspases can occur in vitro, our study questions whether this post-translational modification is solely responsible for NO-mediated inhibition of apoptosis. Indeed, using Jurkat cells as a model system, we demonstrate that NO donors block Fas- and etoposide-induced caspase activation and apoptosis (downstream of mitochondrial membrane depolarization) and cytochrome c release. However, caspase activity was not restored by the strong reducing agent dithiothreitol, as predicted for S-nitrosation reactions, thereby excluding active-site-thiol modification of caspases as the only anti-apoptotic mechanism of NO donors in cells. Rather, we observed that processing of procaspases-9, -3 and -8 was decreased due to ineffective formation of the Apaf-1/caspase-9 apoptosome. Gel-filtration and in vitro binding assays indicated that NO donors inhibit correct assembly of Apaf-1 into an active approx. 700 kDa apoptosome complex, and markedly attenuate caspase-recruitment domain (CARD)-CARD interactions between Apaf-1 and procaspase-9. Therefore we suggest that NO or a metabolite acts directly at the level of the apoptosome and inhibits the sequential activation of caspases-9, -3 and -8, which are required for both stress- and receptor-induced death in cells that use the mitochondrial subroute of cell demise.
authors.affiliation: Department of Cell Biology, Faculty of Biology, University of Kaiserslautern, Kaiserslautern, Germany.
authors.forename: Birgit
authors.initials: B
authors.lastname: Zech
authors.forename: Roman
authors.initials: R
authors.lastname: Köhl
authors.forename: Andreas
authors.initials: A
authors.lastname: von Knethen
authors.forename: Bernhard
authors.initials: B
authors.lastname: Brüne
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D053477:Apoptotic Protease-Activating Factor 1; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002474:Cell-Free System; D004789:Enzyme Activation; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D008928:Mitochondria / Q000187:drug effects; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D011506:Proteins / Q000037:antagonists & inhibitors*; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology
chemical_list: C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D061945:Caspase Inhibitors; D020030:Nitric Oxide Donors; D011506:Proteins; D005047:Etoposide; D026423:S-Nitroso-N-Acetylpenicillamine; D020169:Caspases