Intermembrane transfer of oxidized cardiolipin and recognition by proapoptotic Bcl‐2 family member tBid

Abstract

Release of cytochrome c due to cardiolipin (CL) peroxidation in the inner mitochondrial membrane is recognized as an early event in the intrinsic pathway of oxidant-induced apoptosis. Like other oxidized lipids, CL hydroperoxides (CLOOHs) are capable of translocating from one membrane to another and this could disseminate their redox and signaling activities. We postulated that in oxidatively stressed mitochondria, peroxidation mobilizes CL and fosters its translocation to the outer membrane, where it enhances recruitment of pro-apoptotic proteins tBid and Bax for formation of cytochrome c-transversible pores. This possibility was initially tested by incubating donor liposomes composed of phosphatidylcholine (PC) and CL or CLOOH with PC-only acceptor liposomes bearing a quenched fluorophore (ANTS). We found that CLOOH donors sensitized acceptors to tBid permeabilization (dequenching of ANTS fluorescence) and this was strongly enhanced by lipid transfer protein (nsLTP). CL donors also sensitized acceptors, but leakage rate was <10% that observed with CLOOHs. Analysis of extracted acceptor lipids provided direct evidence for CLOOH transfer uptake, its rate being substantially greater than that of CL. A CLOOH dose-dependent increase in immunodetectable tBid association with acceptor vesicles was demonstrated. Using isolated mitochondria, we found that tBid binding was sharply elevated following transfer acquisition of liposomal CLOOHs, this mimicking the tBid response to doxorubicin treatment. These findings provide new mechanistic insight into the role of CL in oxidative apoptosis. Supported by USPHS grant CA72630.