The Role of Lipids in Regulation of Programmed Cell Death

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In the mitochondrial apoptotic pathway; a major regulator in mammalian cells death, meet pro- and anti-apoptotic Bcl-2 proteins at the mitochondrial outer membrane (MOM) and tightly regulate the fate of a cell. In case of a death sentence the membrane becomes permeable via the action of the apoptotic Bax protein and apoptotic factors like cytochrome c get released. Recently it was found, that the mitochondrial membrane system seems to play an active and crucial role with its lipids being directly involved. Under oxidative stress conditions oxidized lipids can be generated with direct implication on mitochondrial apoptosis via direct modification of membrane properties and/or lipid-protein interactions with Bcl-2 family members. We therefore devised an oxidative stress emulating MOM model system. By embedding OxPls into membranes we were able to study their impact on the recruitment of pro-apoptotic Bax protein and its membrane action. By using a biophysical approach ranging from calorimetry and fluorescence leakage to solid state NMR and labelled proteins we investigated following three key steps: i) Bax mediated pore formation in membranes containing oxidized lipids (OxPls) without the requirement of further mediator proteins such as tBid. ii) specificity of the pore formation on the type of OxPls species used and iii) concentration dependence of the membrane pore formation on OxPls and Bax concentrations. Using fluorescence leakage studies of GUVs and LUVs we found that Bax induced leakage which was sensitive to the type and concentration of OxPL in the membrane. And the leakage rate was correlated to the Bax level. Additional solid state NMR studies confirmed that the incorporation of OxPls distorts the membrane organization, which is essential for the Bax to penetrate into the membrane without a high energy penalty. Further calorimetric experiments support these NMR results.