Bcl-2 protein regulation by conformational flexibility

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Multi-cellular organisms remove damaged or redundant cells with minimized danger to surrounding cells and tissues by apoptosis. The intrinsic apoptosis signaling pathway is controlled by Bcl-2 family protein activities, which are regulated on their part by a complex interplay of Interactions and conformational changes. The changes of Bcl-2 protein conformations determine the sub-cellular localization, affinity for binding partners and apoptotic activity of Bcl-2 family proteins. Despite extensive research only the cellular consequences of Bcl-2 activities and the structures of the inactive states are known, but the true nature of the active Bcl-2 protein conformations and thus potential therapeutic targets remain enigmatic.This project uses the strategy of constraining Bcl-2 proteins not only to study the cellular consequences of conformational changes but to dissect the changes itself. In a previous attempt a set of variants of the pro-apoptotic Bcl-2 family member Bax was generated containing different intramolecular tethers. While Bax activity and its localization from cytosol to mitochondria upon activation is mostly decreased by the intramolecular tethers, one tethered Bax variant localizes constitutively on mitochondria even when inactive. The analysis of this constrained Bax variant showed insensitivity to its regulation and revealed a previously unknown process that accounts for Bax inhibition by prosurvival Bcl-2 proteins. Thus, the use of constrained Bax variants provided novel insight into Bax regulation and will be used to analyze conformational changes in Bcl-2 proteins. This project will also examine the folding dynamics of BH3-only proteins, which regulate Bcl-2 proteins. BH3-only proteins contain a BH3 domain, but are largely unstructured. The project will attempt to investigate the stability of these structures in cells and folding dynamics that might occur upon interactions with Bcl-2 proteins. In a third core area the project will address to what extent and how proteins that assist folding, such as chaperones and peptidyl prolyl cis/trans isomerases (PPIases), accelerate and facilitate folding events in Bcl-2 family members and thus participate in the regulation of apoptosis.