Bid, a BH3-only proapoptotic member of the Bcl-2 family, and the carboxyl terminus of its cleavage product, tBid, are essential regulators of early mitochondrial apoptotic events and release of pro-apoptotic factors into the cytosol.(1) Bid and tBid have a high-affinity binding domain for a mitochondria specific phospholipid, cardiolipin (CL).(2-5) In normal cells, CL is predominantly localized in the inner membrane and contact sites. It plays a very important role in the assembly and maintenance of functionally active respiratory complexes (I, III and IV and F0/F1-ATPase) in the mitochondrial membrane and in normal operation of other mitochondrial multicomponent machines such as adenine nucleotide translocator, as well as tricarboxylate, pyruvate and phosphate carriers.(6-9) Decreased levels of CL caused by siRNA manipulations of CL synthase (CDP-diacylglycerol-phosphatidylglycerol phosphatidyltransferase) in cells were shown to result in disorganization of mitochondria and release of cytochrome c (cyt c) into the cytosol; these cells elicited enhanced sensitivity to proapoptotic stimuli.(10) Recently, interactions of tBid with CL have been implicated as major players in orchestrating apoptotic changes in mitochondrial electron transport as well as activation of pro-apoptotic proteins Bak and Bax.(5,11) During apoptosis, however, CL undergoes significant hydrolysis whose products - mono-lysoCL (MCL) and di-lyso-CL (DCL) - accumulate.(4,12) Cyt c is an important contributor to redox reactions in the intermembrane space of mitochondria where it functions as (1) an electron shuttle between respiratory complexes III and IV and (2) a scavenger of superoxide anion radicals.(13) Recently, we have reported that CL avidly binds cyt c, partially unfolds the protein, and that the complex functions as a peroxidase, catalyzing CL peroxidation essential for the release of proapoptotic factors.(14,15) Notably, CL and MCL display comparable affinities for tBid.(4) Thus, two major components of proapoptotic machinery - tBid and cyt c - interact and possibly compete for the same phospholipid, CL, or its hydrolysis products, MCL and DCL, in apoptotic mitochondria. This potential competition may impede the execution of the apoptotic program. The affinities and role of MCL and DCL in proapoptotic activation of cyt c into a peroxidase and the interactions with tBid have not been characterized. Therefore, we studied MCL and DCL as activators of cyt c into a peroxidase compared to CL in the presence and absence of tBid. Tetraoleoyl-CL (TOCL) and its lyso-derivatives (MCL, DCL), obtained after hydrolysis of TOCL by porcine pancreatic phospholipase A2 (PLA2), were used. The purity of CL-derivatives was confirmed by electrospray ionization mass spectrometry (ESI-MS) (an MS-spectrometer MAT TSQ-700, Thermo Electron Co.) after their separation by two-dimensional high-performance thin-layer chromatography (2D-HPTLC). Characteristic signals with m/z values of 727.7, 595.9 and 463.4 for doubly charged ions of TOCL and its hydrolysis products, mono-lyso-tri-oleo-CL and di-lyso-dioleoyl-CL, respectively, were detected (data not shown). To characterize the peroxidase reaction, we evaluated luminol oxidation monitored by the chemiluminescence response. Expectedly, CL was very effective in activating cyt c into a peroxidase and caused a 25.0-fold higher response than cyt c alone or cyt c in the presence of dioleoyl-phosphatidylcholine (DOPC), which does not effectively bind cyt c.(16) MCL and DCL enhanced cyt c peroxidase activity less effectively than CL (by a factor of 6.7 and 3.7, respectively). tBid almost completely inhibited the peroxidase activity of CL/cyt c complex only at ratios exceeding 20 : 1 (versus cyt c). Similarly, MCL- and DCL-stimulated peroxidase activity of cyt c could be eliminated by a large excess (versus cyt c) of tBid.
VE Kagan, Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh, Bridgeside Point 100 Technology Drive, Suite 350, Pittsburgh, PA 15260