Medical Biology - Theses
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ItemStructural transitions during cell death: bak activation and oligomerisation( 2015)Apoptotic stimuli activate and oligomerise the pro-apoptotic proteins Bak and Bax resulting in mitochondrial outer membrane permeabilisation and subsequent cell death. This thesis investigates structural transitions occurring to Bak during apoptosis. I present crystal structures of a Bak core/latch dimer and demonstrate the dissociation of the core and latch domains upon Bak activation. I provide the first high-resolution details for the core domain dimer, a subunit upon which the larger Bak oligomer builds. Cellular assays, guided by the presented crystal structures, confirm the physiological relevance of these key events in the intrinsic apoptotic pathway (Chapter 2). I also describe the first crystal structures of Bak in complex with the BH3-domain of Bim (Chapter 3). These studies complement previous work performed on Bax and support an analogous mechanism of activation and oligomerisation. Certain detergents have been reported to activate Bak in vitro. Here I demonstrate that some detergents can oligomerise Bak and/or promote hetero-complexes between Bak and the pro-survival protein Mcl-1. I describe the production of homo-oligomeric and hetero-oligomeric complexes of Bak, which may be amenable to structural studies (Chapter 4). The literature on apoptosis assumes that mouse and human Bak are analogous in structure and therefore function. Here I report structural differences between Bak homologs from these two species (Chapter 5). These differences exist at the site of ligand binding, yet it remains unclear whether they result in functional variations. These data may aid in the development of novel agonists and antagonists of Bak, and could prove fundamental to designing murine-based pre-clinical trials. The BH3-only protein from the Schistosoma mansoni worm has been identified as a potential direct activator of Bak and Bax. This direct activator BH3-only protein is unique as it only binds to one of the pro-survival proteins (Mcl-1). Here I describe the crystal structure of the Schistosoma BH3-domain (sBH3) in complex with Bax. Liposome release assays demonstrate that sBH3 can directly activate Bak and Bax and induce the formation of membrane permeabilising oligomers (Chapter 6). These studies support current models for the activation and oligomerisation of Bax. Defining the structural characteristics of the intrinsic apoptotic pathway provides novel opportunities for drug design. Organic agonists of Bak may prove useful for the treatment of cancers, while antagonists could provide therapy for diseases characterised by excessive cell death.