Medical Biology - Theses

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Subject: cancer × Subject: BH3-only × Subject: Bcl-G × Type: PhD thesis ×

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    Functional characterisation of Bcl-G
    GIAM, MAYBELLINE ( 2011)
    Apoptosis is a controlled and precise form of cell death necessary for maintaining tissue homeostasis and normal development. Perturbations of this cell death process contribute to a wide range of diseases such as cancer and autoimmunity. The Bcl-2 family regulates the intrinsic pathway of apoptosis and members share homology at one or more of the four different Bcl-2 Homology (BH) domains. They can be subdivided into the pro-survival proteins, the multi-domain pro-apoptotic proteins and the BH3-only proteins. The physiological functions and mechanism of action of the main players of the Bcl-2-regulated apoptotic pathway have been studied extensively using mouse models. However, the roles played by less prominent Bcl-2 family members are not as well characterised. In this thesis, we described the characterisation of Bcl-G, an evolutionarily conserved novel Bcl-2 family member implicated in cancer. Human BCL-G produces two major isoforms, BCL-GL and BCL-GS. BCL-GS only contains a BH3 domain while BCL-GL also contains a BH2 domain. While BCL-GL exhibited little killing ability, in vitro over-expression studies suggested that BCL-GS kills cells by binding BCL-XL. The mouse Bcl-G gene only produces one isoform, which contains both the BH2 and BH3 domains. Little is known about its functions and roles in apoptosis. To characterise the tissue distribution and subcellular localisation of mBcl-G, we produced monoclonal antibodies specific for this protein. Bcl-G was found to be predominantly cytoplasmic when over-expressed in HeLa cells and is present in a wide range of mouse tissues including spleen, thymus, lung, intestine and testis. Anti-Bcl-G immunohistochemistry revealed that it is expressed highly by some dendritic cell (DC) subtypes and certain epithelial cell types including those lining the gastrointestinal tract. To study the functions of mBcl-G and gain clues on its human orthologue, I generated and characterised Bcl-G-deficient mice. Bcl-G-1- mice were born viable, developed normally and had similar numbers of DCs and other immune cell subtypes when compared to their wild-type counterparts. Notably, Bcl-G-deficient cells were normally sensitive to the range of apoptotic stimuli tested. I also analysed the gastrointestinal tract of the Bcl-G-1- mice but did not observe any abnormalities in the gross morphology of the stomach, small intestine and colon. However, Bcl-G loss attenuated the colitis response to acute dextran sodium sulphate (DSS) administration, resulting in reduced immune infiltration and destruction of crypt architecture. The role mBcl-G plays in DSS-induced colitis is unclear and currently under investigation. To gain clues on the processes in which Bcl-G is involved in, I conducted a search for binding partners using two different approaches: co-immunoprecipitation coupled with mass spectrometric analysis (IP-MS) and yeast two-hybrid screening. Importantly, no Bcl-2 family member, whether it is pro-survival or pro-apoptotic, was pulled-down in either screen. In fact, in vitro over-expression studies revealed that Bcl-G’s BH3 domain was incapable of binding Bcl-XL and activating apoptosis when placed in the context of BH3-only protein Bim. These results raise doubts that mBcl-G interacts with the pro-survival proteins and is involved in the Bcl-2-regulated apoptotic pathway. Instead, Trappc6b, a subunit of the Trafficking protein particle (Trapp) complex, was identified as a potential Bcl-G binding partner. Mammalian Trapp is a multi-subunit protein complex involved in intracellular membrane traffic. This finding proposes a novel role of this Bcl-2 family member in protein trafficking and vesicle transport. In a second project, I have studied how BH3-only members activate the essential effectors Bax and Bak. Two models have been proposed, but the issue remains controversial. The indirect activation model suggests that BH3-only proteins simply neutralize all of the pro-survival proteins, whereas the direct activation model proposes that Bim and Bid must activate Bax and Bak by direct binding. As numerous in vitro studies have not resolved this issue, we have investigated Bim's activity in vivo by a genetic approach. Because the BH3 domain determines binding specificity for Bcl-2 relatives, we generated mice having the Bim BH3 domain replaced by that of Bad, Noxa, or Puma. The mutants bound the expected subsets of pro-survival relatives but lost interaction with Bax. Analysis of these mice showed that Bim's pro-apoptotic activity is not solely determined by its ability to engage its pro-survival relatives or solely to its binding to Bax. Thus, initiation of apoptosis in vivo appears to require features of both models.