Medical Biology - Theses
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ItemStructural investigations of pro‑apoptotic Bcl‑2 family proteins( 2017)The Bcl‑2 protein family regulates the intrinsic apoptotic pathway through an intricate network of protein:protein and protein:membrane interactions. The pathway culminates in the permeabilisation of the mitochondrial outer membrane by the pro‑apoptotic effector proteins Bak and Bax, an event that irreversibly commits a cell to death. To facilitate membrane permeabilisation, Bak and Bax undergo a series of conformational changes to convert from inert monomers to membrane‑embedded homodimers that nucleate and propagate apoptotic oligomers. While great strides have been made in structurally characterising these conformational changes, questions remain surrounding homodimer interactions with the membrane, oligomerisation, and membrane pore formation. This thesis addresses these questions by providing structures of lipids bound to Bak BH3:groove core homodimers (Chapter 2). These are the first structures of any Bcl‑2 family protein in complex with lipid. They reveal symmetric binding sites for phospholipid headgroups and acyl chains. In one structure, adjacent Bak homodimers are cross‑linked by the acyl chains of single phospholipids, suggesting homodimer oligomerisation could be mediated by lipid. Bak oligomers could be dissociated with phospholipase A2, supporting a role for lipid in oligomer stability. Collectively, the structures presented here indicate that lipids may play a direct role in Bak oligomerisation. Like Bak, Bax homodimerises and oligomerises on the mitochondrial outer membrane. The original Bax BH3:groove core homodimer structure was solved as a GFP fusion at low resolution. Here, a tetrameric structure consisting of two Bax BH3:groove core homodimers alone was solved at high resolution (Chapter 3), providing details for canonical interactions in atomic detail. A crystal structure of Bax BH3:groove core homodimers containing lipid was also solved, although the structure could not be refined due to severe twinning. This result demonstrates that Bax core domains also associate with lipid, and provides a starting point for crystal optimisation. Pro‑survival Bcl‑2 family proteins antagonise the apoptotic function of Bak and Bax by preventing their activation and sequestering their activated forms. Sequestration of activated Bak and Bax in heterodimeric Mode 2 complexes involves binding of the Bak/Bax BH3 domain to a conserved hydrophobic groove. Beyond this, little is known regarding the topology of these complexes. The pro‑survival protein Bcl‑XL can undergo similar conformational changes to Bak and Bax, but whether it forms BH3:groove heterodimers with Bak/Bax was unknown. Using cysteine cross‑linking on mitochondria, I show that Bcl‑XL can form reciprocal BH3:groove heterodimers with Bax, and possibly Bak (Chapter 4). These results challenge a simplistic view of Mode 2 complexes, implicating more extensive interactions beyond the canonical BH3 in groove interface. Bok is a third potential pro‑apoptotic effector protein that shares sequence similarity with Bak and Bax, but its role in apoptosis remains unresolved. To investigate the structure and function of Bok, I developed a recombinant expression system to produce human, rat, and chicken Bok. The first crystal structure of Bok, from the chicken, reveals the canonical Bcl‑2 family fold, with deviations that may explain its proposed constitutive activity (Chapter 5). The structure paves the way for mutagenesis studies that will further our understanding of this enigmatic protein.
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ItemBak (and Bax) activation and apoptotic pore formation: roles for the N and C terminus( 2015)Bak and Bax are members of the Bcl-2 family that regulate the mitochondrial pathway of apoptotic cell death. This type of cell death requires either Bak or Bax to form pores in the mitochondrial outer membrane. Both Bak and Bax are globular alpha-helical proteins containing nine α-helices including a C-terminal transmembrane domain (α9) that acts as a membrane anchor. The binding of pro-apoptotic BH3-only relatives at a hydrophobic surface groove (α3-α5) triggers Bak and Bax activation (i.e. conversion into pore-forming proteins). Activation involves a series of conformational changes that result in the formation of symmetric homodimers of Bak (and of Bax), which then associate to form the apoptotic pore. This thesis investigates the role of the C-terminal membrane anchor (α9) in Bak and Bax activation and pore formation. Cysteine mutagenesis and linkage studies were employed to: (1) determine the topology of the Bak C terminus before and after pore formation; and (2) investigate viral protein mediated inhibition of Bak. Studies on the topology of the Bak C-terminal membrane anchor (α9) revealed that although it formed a transmembrane domain as expected, the C terminus did not line the apoptotic pore after apoptosis. However, the C termini from adjacent Bak (or Bax) molecules could come into proximity to form an α9:α9 protein interface after an apoptotic stimulus. Combining linkage at the α9:α9 interface with previously characterized BH3:groove or α6:α6 protein interfaces yielded higher order oligomers, demonstrating the flexible nature of the α6-α9 region in oligomerized Bak. Additional studies on a viral protein that inhibits Bak revealed that the viral protein blocked Bak apoptotic function at the step of activation by binding at a non-canonical site on Bak. Thus, this thesis provides insights into the regulation of Bak and Bax apoptotic function mediated through novel sites. As each site is specific in Bak or Bax, these findings enhance the prospects of developing agents that can directly and specifically activate or inhibit Bak or Bax.
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ItemThe Bcl-2 apoptotic switch – clarifying the functions of proapoptotic and prosurvival players( 2015)The Bcl-2 apoptotic switch is the key decision point in the intrinsic pathway of apoptosis. The study of Bcl-2 family protein function in vitro has been hampered by the lack of full-length recombinant BH3-only proteins, the ‘stimuli’ of the Bcl-2 family. Therefore, a panel of chimeras of Bid with the BH3 domains of other BH3-only proteins was generated to dissect the layers of regulation governing the Bcl-2 effector proteins Bak and Bax. Using Bid BH3 chimeras and BH3 peptides on isolated mitochondria and liposomes, we concluded that most BH3-only proteins could directly activate the effector Bcl-2 family proteins Bak and Bax. BH3 sequences from Bid and Bim were the strongest activators, followed by Puma, Hrk, Bmf and Bik, while Bad and Noxa were not activators. Moreover, Bak and Bax were activated by the same stimuli, in contrast to a recent report. The Bid chimeras were also used to investigate a mechanism of prosurvival Bcl-2 protein function in which a prosurvival protein causes resistance to apoptosis by binding activated Bak (MODE 2) rather than BH3-only proteins (MODE 1). The findings were validated using full-length proteins on isolated mitochondria and in cultured cells. In response to BH3-only proteins, the prosurvival proteins Mcl-1 and Bcl-xL were able to prevent cytochrome c release or apoptosis via MODE 2. In particular, MODE 2 interactions between Bak and Mcl-1 caused profound resistance to Bid. The studies presented here are the first direct evidence that MODE 2 can protect cultured cells without over-expressed prosurvival proteins, and