The Effect of Oxidized Lipids on the Interplay of Bcl-2 and Bax Proteins at Mitochondrial Membranes

Abstract

Mitochondria play a crucial role in the intrinsic apoptotic pathway. The Bcl-2 family proteins, which interact with the mitochondrial outer membrane to modulate membrane permeability, are key regulators of this pathway. Intracellular oxidative stress is one major factor leading to apoptosis accompanied by the permeabilization of the mitochondrial outer membrane; a process causing release of apoptotic factors such as cytochrome c. Upon onset of intracellular stress, phospholipids can become oxidized in their unsaturated fatty acid region. These oxidized phospholipids (OxPls) can severely alter the properties of these mitochondrial membranes, and can therefore have i) a direct effect on the membrane properties and its perforation and ii) can have an indirect effect by altering the function of membrane-associated Bcl-2 proteins (such as the anti-apoptotic Bcl-2 and the apoptotic Bax). We therefore devised a model system that mimics oxidative stress conditions by incorporating oxidized phospholipids (OxPls) into mitochondria-mimicking vesicles, and studied the OxPls’ impact on Bax-membrane interactions. Conformational changes in the protein upon contact with the lipid membranes were monitored using far-UV circular dichroism (CD) spectroscopy. Differential scanning calorimetry (DSC) and solid-state magic angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy was used to study membrane organization. In a biophysical study combining CD with surface plasmon resonance techniques we also investigated the putative interaction of solubilized full-length human Bcl-2 with Bax. There, we found a direct Bcl-2 interaction with Bax in the presence of sub-CMC concentration of detergent and could observe a high affinity between both partners (KD 35.8 nM).