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Bcl2 Network

Our work is focused on mapping the interaction network and the conformational changes of Bcl-2 proteins during cell death
The intrinsic apoptosis pathway is orchestrated by the Bcl-2 (B-cell lymphoma-2) protein family: i) pro-survival members (e.g. Bcl-XL) inhibit apoptosis; ii) pro-apoptotic members (Bax, Bak) permeabilize the mitochondrial outer membrane; iii) the BH3-only proteins (e.g. Bid, PUMA) sense apoptotic stimuli and regulate the other two members of the family. Up to now, a satisfactory description of the interaction network between pro-apoptotic and pro-survival players at the membrane is missing.

Site-directed spin labeling EPR has been applied up to now to membrane proteins inserted in liposomes, membrane bilayers or nanodiscs. The idea is to mimic the native environment. However, the molecular crowding and the lipid composition of each membrane differ tremendously, and may introduce additional still unknown triggering factors which modify the conformaional changes of the proteins under investigation. In cell EPR of site-directed spin labeled proteins is an emerging area, which faces challenges in terms of sensitivity, selectivity and labels' lifetime. In this project we will explore the use of isolated organelles instead, to follow the conformational changes of the Bcl-2 proteins during apoptosis. Intra- and inter-protein distances in the Bcl-2 network will be obtained in organelle using isolated mitochondria from rat, to directly address the conformational changes under conditions close to the physiological ones. We will test the advantages of gadolinium labels and different types of nitroxide labels in the presence of organelles. Orthogonal labeling strategies will be adopted to follow simultaneously different players at the onset of apoptosis.

Collaboration partners:
- Ana Garcia-Saez, University of Tübingen, Germany
- Tudor Moldoveanu, St. Jude Children's Research Hospital , Memphis, TN, US