Emiko Bare

Research: Understanding Interaction Specificity Among Bcl-2 Proteins

The Bcl-2 protein family is important in regulating apoptosis. Bcl-2 family members can be pro-apoptotic or anti-apoptotic and classified into three subfamilies: the anti-apoptotic Bcl-2-like, the pro-apoptotic Bax-like, and the pro-apoptotic BH3-only subfamilies. These proteins regulate apoptosis through protein-protein interactions in which the BH3 region is central. The interactions between BH3s and anti-apoptotic proteins, like Bcl-xL, are mediated by helical BH3s binding hydrophobic grooves on anti-apoptotic proteins, seen in NMR and crystal structures. The interactions between BH3 regions and Bax-like proteins are much more speculative, though recent work indicates the first helix of Bax may directly associate with BH3 regions. The BH3 regions have varied specificities for Bcl-2 proteins, where some BH3s bind promiscuously and others bind a limited number of proteins. In one model proposed to explain the specificity differences, the BH3-onlies function as “activators” activating pro-apoptotic Bax-like proteins or as “enablers” inhibiting the anti-apoptotic proteins. Another model uses the promiscuity of the BH3 regions for anti-apoptotic proteins to explain difference in ability to induce apoptosis, such that BH3s that bind more pro-survival members are better able to promote cell death.

Our goals are to further understand interactions among Bcl-2 family members: the specificity of those interactions and the determinants of that specificity. With the binding specificities of BH3 peptides, we can speculate about interaction models and identify important residues that determine interaction specificity. With this information, we can begin dissecting the Bcl-2 network/pathway, designing BH3 sequences with altered specificities. We are using three techniques to study interactions between BH3 regions and Bcl-2 proteins. A pull-down assay with FLAG-tagged BH3 peptides is a quick, easy test to detect interactions with Bcl-2 proteins. A fluorescence polarization assay is a quantitative method to determine binding affinities. And a protein microarray technique developed in this lab will identify the comprehensive Bcl-2 protein-BH3 peptide interaction network with relative binding affinities. I will also use X-ray crystallography of Bcl-2 protein/BH3 peptide complexes to examine the molecular mechanism of the specificity.

Collaborators within the Keating lab:

Xiaoran Stowell, Jeremy Fisher, and Shaun Deignan