Regulation of apoptosis by Bcl-2 family proteins.
The Bcl-2 family proteins primarily responsible for drug responses (and the lack thereof) include: i) the anti-apoptotic proteins: Bcl-2, Bcl-XL and Mcl-1, ii) the pro-apoptotic executioner proteins Bax and Bak and iii) the pro-apoptotic regulator BH3-proteins Bid, Bim, Bad, Bik, Puma and Noxa. Our “embedded together” model for how Bcl-2 family proteins regulate mitochondrial permeabilization (MOMP) dominates the field. It shifted attention from unidirectional interactions to dynamic equilibria involving conformational changes induced by binding to membranes. It also introduced the concept of mutual sequestration to explain the biological outcomes resulting from Bcl-2 family protein interactions. Our model has been tested and validated by many independent labs using different experimental strategies. Our 2008 paper in Cell resolved a major controversy by showing that tBid binds to and activates Bax only after both proteins bind membranes. In 2012 in Mol. Cell we reported confirmation of many of the predicted interactions in live cells using FLIM FRET and quantified several important differences in live cells compared to previous in vitro data. We also provided evidence for an additional binding interaction between Bim and Bcl-XL that we have now identified and characterized in vitro and in live cells.
Chemical biology of Bcl-2 family proteins and drug responses in cells.
To provide the impetus for translation of our results to benefit patients we are identifying small molecule inhibitors of pro- and anti-apoptotic proteins. These probes permit us demonstrate to pharmaceutical companies that the targets we identify are pharmaceutically tractable. Using high throughput screening we identified small molecules that target protein-protein interaction sites for Bcl-2 family proteins thereby providing validation directly relevant to pharma. One class of molecules is currently being assessed for full drug development. In preclinical experiments these molecules selectively kill human and murine derived tumours in mice. We are also using high content screening techniques and primary cell cultures of patient cells to identify small molecules targeting Bcl-2 family proteins that elicit apoptosis selectively in leukemia cells of individual patients for personalized medicine applications. In contrast, small molecule inhibitors of pro-apoptotic Bcl-2 family proteins inhibit apoptosis and are being developed for cell based therapies and for treating degenerative diseases. For cell based therapies our goal is to improve the survival of precious cells during implantation. For degenerative diseases, such as macular degeneration, preventing cell death over the long term is key.
Quantifying protein-protein interactions in live cells.
Because of the importance of protein-protein interactions in discovery, translational and pharmaceutical research many methods and instruments have been developed to detect these interactions. However, quantifying protein-protein interactions in live cells remains one of the most difficult tasks in cell biology. To solve this problem we pioneered the use of fluorescence lifetime imaging microscopy fluorescence resonance energy transfer (FLIM FRET). We have used FLIM FRET to examine protein-protein interactions for Bcl-2 family proteins and how these can (and cannot) be modulated by small molecules. FLIM FRET also enabled quantification of an interaction between hexokinase II and PEA-15 that regulates the response of neurons to changes in oxygen and metabolism.
Development of new instrumentation for measuring protein-protein interactions.
Our results highlighted the need for new high content screening instruments custom built for studying protein-protein interactions using FLIM FRET. To generate quantitative data we simultaneously record fluorescence intensity images for the donor and acceptor as well as FLIM data for the donor. To do this we had to improve the hardware, software and the cell biology. Together with industry we build instruments to measure binding constants for protein-protein interactions in live cells. We use these instruments in our own research and to help pharmaceutical companies with their drug development programs.