title: Nucleophosmin is a novel Bax chaperone that regulates apoptotic cell death.
abstract: The proapoptotic B-cell lymphoma-2 family protein Bax is a key regulatory point in the intrinsic apoptotic pathway. However, the factors controlling the process of Bax activation and translocation to mitochondria have yet to be fully identified and characterized. We performed affinity chromatography using peptides corresponding to the mitochondrial-targeting region of Bax, which is normally sequestered within the inactive structure. The molecular chaperone nucleophosmin was identified as a novel Bax-binding protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Reciprocal co-immunoprecipitation and proximity assays confirmed the Bax-nucleophosmin protein-protein interaction and verified that nucleophosmin only bound to activated conformationally altered Bax. Confocal microscopy in a cell-based apoptosis model, demonstrated that nucleophosmin translocation from nucleolus to cytosol preceded Bax movement. Specific knockdown of nucleophosmin expression using RNAi attenuated apoptosis as measured by mitochondrial cytochrome c release and activation of the caspase cascade. In a mouse model of ischaemic stroke, subcellular fractionation studies verified that nucleophosmin translocation occurred within 3 h, at a time before Bax translocation but after Bax conformational changes have occurred. Thus, we have elucidated a novel molecular mechanism whereby Bax becomes activated and translocates to the mitochondria to orchestrate mitochondrial dysfunction and apoptotic cell death, which opens new avenues for therapeutic intervention.
authors.affiliation: Astellas CNS Research in Edinburgh, The University of Edinburgh, Edinburgh, UK. Lorraine.Kerr@ed.ac.uk
authors.forename: L E
authors.initials: LE
authors.lastname: Kerr
authors.forename: J-L A
authors.initials: JL
authors.lastname: Birse-Archbold
authors.forename: D M
authors.initials: DM
authors.lastname: Short
authors.forename: A L
authors.initials: AL
authors.lastname: McGregor
authors.forename: I
authors.initials: I
authors.lastname: Heron
authors.forename: D C
authors.initials: DC
authors.lastname: Macdonald
authors.forename: J
authors.initials: J
authors.lastname: Thompson
authors.forename: G J
authors.initials: GJ
authors.lastname: Carlson
authors.forename: J S
authors.initials: JS
authors.lastname: Kelly
authors.forename: J
authors.initials: J
authors.lastname: McCulloch
authors.forename: J
authors.initials: J
authors.lastname: Sharkey
mesh_terms: D000818:Animals; D017209:Apoptosis; D002545:Brain Ischemia / Q000378:metabolism* / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D002466:Cell Nucleolus; D002846:Chromatography, Affinity; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008297:Male; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D009687:Nuclear Proteins / Q000502:physiology*; D000090243:Nucleophosmin; D021381:Protein Transport; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D018832:Molecular Chaperones; C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D051028:bcl-2-Associated X Protein; D000090243:Nucleophosmin; D045304:Cytochromes c; D020169:Caspases