Permeabilization of the Mitochondrial Outer Membrane by Bax/Truncated Bid (tBid) Proteins as Sensitized by Cardiolipin Hydroperoxide Translocation

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.Cytochrome c (cyt c) 2 dissociation from the mitochondrial inner membrane (IM), movement into the intermembrane space, and then release into cytosol is recognized as a key early event in the intrinsic (mitochondrion-initiated) pathway of oxidative stress-induced apoptosis (1-3). How this occurs is still not completely understood, but accumulating evidence suggests that oxidative modification of cardiolipin (CL), e.g. conversion to hydroperoxide species (CLOOHs), plays an important role (4 -7). CL (diphosphatidylglycerol) is located primarily in the mitochondrial IM of normal eukaryotic cells, where it interacts with and supports the functions of cyt c, Complex III (cytochromes b and c 1 ), and Complex IV (cytochrome c oxidase) (8). Unlike most other phospholipids in which only the sn-2 fatty acyl group is unsaturated, both sn-1 and sn-2 groups of natural CL are typically unsaturated. In mammalian heart, for example, tetra-linoleoyl CL composes 75-80% of overall CL molecular species (9). Consequently, CL oxidizability and -OOH content per average oxidized molecule are typically much higher than for more conventional phospholipids such as those in the phosphatidylcholine and phosphatidylethanolamine families. Model studies with bovine heart CL in thin film or liposomal form have shown that its normally strong interaction with some fraction of IM cyt c is progressively weakened with increasing -OOH content (4), suggesting possible involvement in pro-apoptotic cyt c releas...

Spatiotemporal autophagic degradation of oxidatively damaged organelles after photodynamic stress is amplified by mitochondrial reactive oxygen species

“…Antibodies against TOMM20 (ab56783) and calreticulin (ab92516) 70 Therefore, our results support the hypothesis proposed by Kagan et al 45 that CLOOH could act as a 'molecular switch' that initiates the development of pre-apoptotic events when oxidative pressure inside the mitochondria is too high and mitophagy fails to effectively eliminate oxidatively damaged cellular components. In line with this, Korytowski et al 71 have recently published that CLOOHs and other CL oxidation products can translocate via the aqueous intermembrane space from the inner mitochondrial membrane to the outer mitochondrial membrane of oxidatively stressed mitochondria in order to sensitize the outer membrane for pro-apoptotic recruitment of tBID and BAX. This is an interesting possibility which deserves to be explored in further studies.…”

Oxidative stress and lipotoxicity

“…Moreover, initial studies showed that CL specifically is essential for Bcl2 mediated membrane permeabilization through Bax and Bid (40). More recent work has indicated that oxidation of CL results not only in cytochrome C mobilization in the membrane, but also in migration of CL from the inner mitochondrial membrane to the outer mitochondrial membrane (41,42). Here, CLox recruits and interacts with Bax to initiate formation of the mitochondrial transition pore.…”

Cardiolipin and Its Different Properties in Mitophagy and Apoptosis

“…The Fe•••S (Met80) bond was found disrupted upon CL binding, which gave H 2 O 2 access to the heme (Kagan et al 2009b;Kapralov et al 2011). Korytowski et al (2011) showed that CLox species were significantly increased in mitochondria when exposed to apoptotic stress. This might sensitize the mitochondrial membrane for the recruitment of tBid and Bax.…”

“…This is the earliest report of an association between CL and Cyt c in apoptosis. In fact, oxidation and peroxidation of CL are the reasons for decreased NAO fluorescence (Mileykovskaya et al 2001) Korytowski et al (2011) showed that CLox species were significantly increased in mitochondria when exposed to apoptotic stress. This might sensitize the mitochondrial membrane for the recruitment of tBid and Bax.…”