Characterisation of the Bax-nucleophosmin interaction: the importance of the Bax C-terminus

Jane Thompson, Keith Finlayson, Eliane Salvo-Chirnside, David MacDonald, James McCulloch, Lorraine Kerr, John Sharkey

    Research output: Contribution to journalArticlepeer-review

    16 Citations (Scopus)

    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.
    Original languageEnglish
    Pages (from-to)394-403
    Number of pages10
    JournalApoptosis: an International Journal on Programmed Cell Death
    Volume13
    Issue number3
    DOIs
    Publication statusPublished - 2008

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