title: Characterisation of the Bax-nucleophosmin interaction: the importance of the Bax C-terminus.
abstract: The molecular chaperone nucleophosmin has been identified as a novel Bax binding protein with this interaction proposed to be a key event in the activation and translocation of Bax in mitochondrial dysfunction and apoptotic cell death. Using a proximity assay, we have quantitatively defined the high affinity and saturable interaction between Bax and nucleophosmin indicative of a competitive and specific mechanism. Binding of full length Bax to nucleophosmin was only observed after conformational change was induced using non-ionic detergents (e.g., NP-40). The Bax-nucleophosmin interaction was inhibited by a Bax C-terminal antibody (IC(50) = 1 nM) but minimally affected by antibodies directed against either the N-terminus or alpha-helices 4 and 5. Bcl-2 and p53 inhibited the interaction between full length activated Bax and nucleophosmin. The proximity assay based on the Bax-nucleophosmin interaction was robust and reproducible (Z' = 0.50) facilitating its use for screening a small chemical library. A low molecular weight non-peptide compound, 2-(5-methyl-2-phenyl-1,3-thiazol-4-yl)ethanohydrazide, partially inhibited the Bax-nucleophosmin interaction (IC(50) = 100 nM) and also attenuated UV-induced cell death of HEK293 cells. The present investigations demonstrate the importance of exposure of the C-terminus of Bax for its interaction with nucleophosmin. These protein-protein interaction assays provide a technical approach both for the study of Bax-interacting proteins and for the discovery of novel anti-apoptotic agents.
authors.affiliation: Astellas CNS Research in Edinburgh, The University of Edinburgh, The Chancellors Building, 49 Little France Crescent, Edinburgh, EH16 4SB, UK. jthompson@cyclacel.com
authors.forename: Jane
authors.initials: J
authors.lastname: Thompson
authors.forename: Keith
authors.initials: K
authors.lastname: Finlayson
authors.forename: Eliane
authors.initials: E
authors.lastname: Salvo-Chirnside
authors.forename: David
authors.initials: D
authors.lastname: MacDonald
authors.forename: James
authors.initials: J
authors.lastname: McCulloch
authors.forename: Lorraine
authors.initials: L
authors.lastname: Kerr
authors.forename: John
authors.initials: J
authors.lastname: Sharkey
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000528:radiation effects; D002478:Cells, Cultured; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology; D018832:Molecular Chaperones / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D000090243:Nucleophosmin; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D025941:Protein Interaction Mapping; D013844:Thiazoles / Q000494:pharmacology; D014466:Ultraviolet Rays; D051028:bcl-2-Associated X Protein / Q000276:immunology / Q000378:metabolism*
chemical_list: C530011:2-(5-methyl-2-phenyl-1,3-thiazol-4-yl)ethanohydrazide; D000906:Antibodies; D006834:Hydrazines; D018832:Molecular Chaperones; C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D013844:Thiazoles; D051028:bcl-2-Associated X Protein; D000090243:Nucleophosmin