Project P6

"Deciphering the BH3 code for the neutralization of pro-survival Bcl-2 proteins"

People involved: Ana Garcia Saez

Abstract:
The BH3-only proteins have evolved to sense apoptotic stimuli and initiate apoptosis through direct interactions with other Bcl-2 proteins via their BH3 domain. Small variations within this domain determine the specificity of interactions with other family members, and thus their power to induce cell death. However, the sequence code that specifies complex formation is not well understood. Despite the fact that pro-survival Bcl-2 members inhibit BH3-only proteins at the mitochondrial outer membrane, most studies so far were limited to the analysis of complexes in solution.
In this project we aim to decipher the sequence details that determine the pattern of binding affinities between the BH3-only proteins and pro-survival Bcl-2 family members in membranes. For this purpose, we plan to use two-colour fluorescence correlation spectroscopy to compare quantitatively the ability of a library of BH3-like peptides to interact with Bcl-xL and A1 in giant unilamellar vesicles. The development of predictive power to design BH3-like molecules with specific binding patterns will be of great interest for drug development.
Recent studies suggest a role of the phosphorylation state of the Bcl-2 proteins on apoptosis signalling, but the molecular consequences remain unclear. By exploiting the same biophysical approach, we will analyse the effect of phosphorylation on complex formation between BH3-only proteins and Bcl-xL and A1. In collaboration with the Brunner/Frickey group, we will incorporate this data into a mathematical model of Bcl-2 signalling.
Additionally, we will investigate the molecular mechanism of Bok in reconstituted systems. Bok is a poorly understood Bcl-2 homolog that has been proposed to act similarly to Bax/Bak, but almost nothing is know about its molecular mechanism. In collaboration with the Kaufmann group, we will study the membrane activity of recombinant Bok at the single vesicle level using confocal microscopy. We will also analyse quantitatively the interactions of Bok with other Bcl-2 family members in solution and in giant unilamellar vesicles using two-colour fluorescence correlation spectroscopy.

Co-operations within the FOR2036 consortium:
Andreas Villunger, Innsbruck
Christoph Borner, Freiburg
Georg Haecker, Freiburg
Thomas Kaufmann, Bern