Permeabilization of the mitochondrial outer membrane by Bax/truncated Bid (tBid) proteins as sensitized by cardiolipin hydroperoxide translocation: mechanistic implications for the intrinsic pathway of oxidative apoptosis

Abstract

Cytochrome c (cyt c) release upon oxidation of cardiolipin (CL) in the mitochondrial inner membrane (IM) under oxidative stress occurs early in the intrinsic apoptotic pathway. We postulated that CL oxidation mobilizes not only cyt c but also CL itself in the form of hydroperoxide (CLOOH) species. Relatively hydrophilic CLOOHs could assist in apoptotic signaling by translocating to the outer membrane (OM), thus promoting recruitment of the pro-apoptotic proteins truncated Bid (tBid) and Bax for generation of cyt c-traversable pores. Initial testing of these possibilities showed that CLOOH-containing liposomes were permeabilized more readily by tBid plus Ca(2+) than CL-containing counterparts. Moreover, CLOOH translocated more rapidly from IM-mimetic to OM-mimetic liposomes than CL and permitted more extensive OM permeabilization. We found that tBid bound more avidly to CLOOH-containing membranes than to CL counterparts, and binding increased with increasing CLOOH content. Permeabilization of CLOOH-containing liposomes in the presence of tBid could be triggered by monomeric Bax, consistent with tBid/Bax cooperation in pore formation. Using CL-null mitochondria from a yeast mutant, we found that tBid binding and cyt c release were dramatically enhanced by transfer acquisition of CLOOH. Additionally, we observed a pre-apoptotic IM-to-OM transfer of oxidized CL in cardiomyocytes treated with the Complex III blocker, antimycin A. These findings provide new mechanistic insights into the role of CL oxidation in the intrinsic pathway of oxidative apoptosis.

FIGURE 1.

Effect of preexisting CL or CLOOH on LUV susceptibility to tBid permeabilization. shown is ANTS/DPX leakage from POPC/CL (95:5 mol/mol) (A), POPC/CLOOH (95:5 (mol/mol) (B), and POPC/POPC-OOH (95:5 mol/mol) (C) LUVs. Each reaction mixture, 2.0 ml in a fluorescence cuvette at 37 °C, contained 50 μm total lipid and 10 mm CaCl₂ in 25 mm HEPES buffer (pH 7.4). Mixtures were subjected to gentle magnetic stirring throughout. tBid (40 nm) in a negligible volume was introduced at the indicated time points (arrows). Fluorescence emission was monitored at 530 nm using 360-nm excitation. Maximal fluorescence was determined by lysing the LUVs with 0.1% Triton X-100. Traces shown are representative of three separate experiments with identical results.

FIGURE 2.

Effect of CL or CLOOH transfer uptake on LUV susceptibility to tBid permeabilization. POPC LUV acceptors were mixed with SUV donors, each at a final concentration of 50 μm total lipid in 25 mm HEPES buffer containing 10 mm CaCl₂ (pH 7.4). The SUVs consisted of POPC/CL (8:2, mol/mol) (A and B) and POPC/CLOOH (8:2, mol/mol) (C and D). Reaction mixtures were incubated in the absence (A and C) or presence (B and D) of nsLTP (50 μg/ml) for 18 h at 37 °C. tBid (40 nm) was introduced at the indicated times during incubation (arrows). Other details were as described in Fig. 1. Data from this and a duplicate experiment are also represented in Table 1.

FIGURE 3.

Comparative kinetics of CL and CLOOH transfer between liposomes; spontaneous versus nsLTP-facilitated transfer. A, spontaneous transfer of CL (○) and CLOOH (△) from SUVs to LUVs is shown. Reaction mixtures at 37 °C contained POPC/CL/LacPE (7:2:1 by mol) or POPC/CL/CLOOH/LacPE (7:1:1:1 by mol) SUV donors and POPC LUV acceptors (1:5 lipid mol ratio). At the indicated incubation times, the membranes were separated by agglutination, and CL/CLOOH in the LUV fraction was analyzed by HPLC using A₂₀₅ and A₂₃₂ for CL and CLOOH detection, respectively. B, nsLTP-enhanced CL (○) and CLOOH (△) transfer. Reaction mixtures were the same as in A except for the inclusion of nsLTP (50 μg/ml). Timed samples were quenched on ice and analyzed for LUV-acquired CL or CLOOH as described in A. Incubations in the absence and presence of nsLTP were carried out simultaneously. Plotted values in A and B are the means ± S.E. of values from three separate experiments. C_d(t_o) and C_a(t) denote analyte concentration in donor SUVs at time 0 and in acceptor LUVs at time t, respectively. Calculated apparent first-order rate constants for transfer are as follows: A, 2.2 ± 0.3 × 10⁻³ h⁻¹ (CL), 7.3 ± 1.5 × 10⁻³ h⁻¹ (CLOOH); B, 1.3 ± 0.2 × 10⁻² h⁻¹ (CL), 9.8 ± 0.2 × 10⁻² h⁻¹ (CLOOH).

FIGURE 4.

CLOOH sensitization of LUVs to cooperative permeabilization by tBid and Bax. Reaction mixtures contained FD-10-bearing DOPC/CL (95:5, mol/mol), DOPC/CLOOH (95:5, mol/mol), or DOPC-only LUVs in 20 mm HEPES, 150 mm KCl, 1 mm EDTA, 0.05 mm DFO (pH 7.4). Each mixture (20 μm in total lipid) was gently stirred at 37 °C. At the indicated time points (arrows), tBid (40 nm) and monomeric Bax (100 nm) were added sequentially. FD-10 fluorescence emission was monitored at 520 nm using 490-nm excitation. Maximal fluorescence was determined by including 0.1% Triton X-100.

FIGURE 5.

tBid binding by CLOOH-containing versus CL-containing liposomes. [¹⁴C]phosphocholine (PC)-only and [¹⁴C]PC/PCOOH, [¹⁴C]PC/CL, and [¹⁴C]PC/CLOOH MLVs of the indicated mol % CL, PCOOH, or CLOOH (∼1 -OOH/P_i for PCOOH; ∼1 -OOH/2 P_i for CLOOH; 1 mm total lipid; 0.1 μCi/ml) were prepared by sonication in 10 mm HEPES, 20 mm KCl, 5 mm MgCl₂, 0.1 mm DFO (pH 7.4). The MLVs were incubated with 1 μm tBid for 10 min at 30 °C and then sedimented by centrifugation; lipid recovery was monitored by scintillation counting. Bound tBid was determined by immunoblotting using the same amount of total MLV lipid for each sample lane. tBid bands and densitometrically measured band intensities for each condition are shown. Values are the means ± S.E. (n = 3). *, p < 0.01 versus 10% CL; **, p < 0.001 versus 30% CL.

FIGURE 6.

Transfer-acquired CLOOH sensitization of YZD5 mitochondria to tBid/Bax-dependent poration. Suspensions containing POPC/POPE/PI/[¹⁴C]CLOOH/Ch (41:27:9:16:7 by mol) SUVs (1 mm total lipid; ∼10 nCi/ml), YZD5 mitochondria (3.3 mg of protein/ml), and nsLTP (50 μg/ml) in MS buffer were incubated for 1 h at 30 °C, then centrifuged and washed once. A control system without CLOOH, i.e. with POPC/POPE/PI/Ch (57:27:9:7 by mol), SUVs was run alongside. Mitochondria were recovered, resuspended in protease inhibitor-containing MS buffer to 0.6 mg of protein/ml, then incubated in the presence of 40 nm tBid alone, 100 nm monomeric Bax alone, or tBid plus Bax for 1 h at 30 °C. After centrifugation, mitochondrial fractions were recovered and analyzed for protein content and the extent of [¹⁴C]CLOOH translocation uptake, as determined by scintillation counting. Western blots for released cyt c in the supernatant fractions and remaining cyt c and bound tBid in the pellet fractions are shown. Numbers below the released cyt c bands for each system represent integrated band intensities relative to the control and normalized to cytochrome c oxidase (Cox) as a loading standard. Data from one experiment representative of three are shown.