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Title: The identification and characterisation of Bax chaperone proteins within a neuronal cell model of instrinsic apoptotic cell death
Author: MacDonald, David C.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2007
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This thesis aimed to identify Bax-binding proteins using a proteomics-based approach in an in vitro model of the intrinsic apoptotic pathway and to characterise their involvement in Bax-mediated apoptosis using molecular techniques.  An in vitro neuronal cell model was established using human neuroblastoma SH-SY5Y cells treated with the intrinsic apoptotic pathway inducer, staurosporine. Endogenous Bax was immunoprecipitated from SH-SY5Y whole cell lysates and bound proteins, separated by 2D gel electrophoresis, were identified by peptide mass fingerprinting to be the heat-shock proteins, Hsc71, GRP78 and Hsp60, and the cytoskeletal proteins, β-actin, tubulin and vimentin. affinity chromatography with a wild type 21 amino acid C-terminal Bax peptide and a mutant peptide containing a serine to valine substitution at position 184 (S184V), which mimics the membrane-targeting capacity of activated Bax, also identified Hsp60, β-actin and the molecular chaperone nucleophosmin, with the latter binding exclusively to the mutant peptide. Together, the data suggest that Bax is retained in the cytosol of quiescent cells through association with heat shock and cytoskeletal proteins, and that exposure of the C-terminal domain and subsequent translocation of Bax during apoptosis may be regulated by Hsp60, β-actin and nucleophosmin. The role of nuceophosmin in regulating the translocation of Bax was investigated further. Confocal microscopy revealed that nucleophosmin underwent translocation from the nucleolus to the cytosol during a 5h time course of staurosporine treatment and preceded the translocation of Bax to mitochondria. Applying RNA interference against endogenous nucleophosmin mRNA within SH-SY5Y cells attenuated mitochondrial cytochrome c release and pro-caspase 3 cleavage during staurosporine-induced apoptosis. Data demonstrate that nucleophosmin is a novel regulator of Bax-mediated apoptosis and represents a novel target for therapeutic intervention in diseases associated with dysfunctional apoptosis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available