Helena La Vieira

Bcl-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator.

Bcl-2 family protein including anti-apoptotic (Bcl-2) or pro-apoptotic (Bax) members can form ion channels when incorporated into synthetic lipid bilayers. This contrasts with the observation that Bcl-2 stabilizes the mitochondrial membrane barrier function and inhibits the permeability transition pore complex (PTPC). Here we provide experimental data which may explain this apparent paradox. Bax and adenine nucleotide translocator (ANT), the most abundant inner mitochondrial membrane protein, can interact in artificial lipid bilayers to yield an efficient composite channel whose electrophysiological properties differ quantitatively and qualitatively from the channels formed by Bax or ANT alone. The formation of this composite channel can be observed in conditions in which Bax protein alone has no detectable channel activity. Cooperative channel formation by Bax and ANT is stimulated by the ANT ligand atractyloside (Atr) but inhibited by ATP, indicating that it depends on the conformation of ANT. In contrast to the combination of Bax and ANT, ANT does not form active channels when incorporated into membranes with Bcl-2. Rather, ANT and Bcl-2 exhibit mutual inhibition of channel formation. Bcl-2 prevents channel formation by Atr-treated ANT and neutralizes the cooperation between Bax and ANT. Our data are compatible with a ménage à trois model of mitochondrial apoptosis regulation in which ANT, the likely pore forming protein within the PTPC, interacts with Bax or Bcl-2 which influence its pore forming potential in opposing manners.

Mitochondrial membrane permeabilization is a critical step of lysosome-initiated apoptosis induced by hydroxychloroquine.

Hydroxychloroquine (HCQ) is a lysosomotropic amine with cytotoxic properties. Here, we show that HCQ induces signs of lysosomal membrane permeabilization (LMP), such as the decrease in the lysosomal pH gradient and the release of cathepsin B from the lysosomal lumen, followed by signs of apoptosis including caspase activation, phosphatidylserine exposure, and chromatin condensation with DNA loss. HCQ also induces mitochondrial membrane permeabilization (MMP), as indicated by the insertion of Bax into mitochondrial membranes, the conformational activation of Bax within mitochondria, the release of cytochrome c from mitochondria, and the loss of the mitochondrial transmembrane potential. To determine the molecular order among these events, we introduced inhibitors of LMP (bafilomycin A1), MMP (Bcl-XL, wild-type Bcl-2, mitochondrion-targeted Bcl-2, or viral mitochondrial inhibitor of apoptosis from cytomegalovirus), and caspases (Z-VAD.fmk) into the system. Our data indicate that caspase-independent MMP is rate-limiting for LMP-mediated caspase activation. Mouse embryonic fibroblasts lacking the expression of both Bax and Bak are resistant against hydroxychloroquine-induced apoptosis. Such Bax−/− Bak−/− cells manifest normal LMP, yet fail to undergo MMP and subsequent cell death. The data reported herein indicate that LMP does not suffice to trigger caspase activation and that Bax/Bak-dependent MMP is a critical step of LMP-induced cell death.

Oxidation of a critical thiol residue of the adenine nucleotide translocator enforces Bcl-2 independent permeability transition pore opening and apoptosis.

Mitochondrial membrane permeabilization is a critical event in the process leading to physiological or chemotherapy-induced apoptosis. This permeabilization event is at least in part under the control of the permeability transition pore complex (PTPC), which interacts with oncoproteins from the Bcl-2 family as well as with tumor suppressor proteins from the Bax family, which inhibit or facilitate membrane permeabilization, respectively. Here we show that thiol crosslinking agents including diazenedicarboxylic acid bis 5N,N-dimethylamide (diamide), dithiodipyridine (DTDP), or bis-maleimido-hexane (BMH) can act on the adenine nucleotide translocator (ANT), one of the proteins within the PTPC. ANT alone reconstituted into artificial lipid bilayers suffices to confer a membrane permeabilization response to thiol crosslinking agents. Diamide, DTDP, and BMH but not tert-butylhydroperoxide or arsenite cause the oxidation of a critical cysteine residue (Cys 56) of ANT. Thiol modification within ANT is observed in intact cells, isolated mitochondria, and purified ANT. Recombinant Bcl-2 fails to prevent thiol modification of ANT. Concomitantly, a series of different thiol crosslinking agents (diamide, DTDP, and BMH, phenylarsine oxide) but not tert-butylhydroperoxide or arsenite induce mitochondrial membrane permeabilization and cell death irrespective of the expression level of Bcl-2. These data indicate that thiol crosslinkers cause a covalent modification of ANT which, beyond any control by Bcl-2, leads to mitochondrial membrane permeabilization and cell death.

The adenine nucleotide translocator: a target of nitric oxide, peroxynitrite, and 4-hydroxynonenal

Nitric oxide (NO), peroxynitrite, and 4-hydroxynonenal (HNE) may be involved in the pathological demise of cells via apoptosis. Apoptosis induced by these agents is inhibited by Bcl-2, suggesting the involvement of mitochondria in the death pathway. In vitro, NO, peroxynitrite and HNE can cause direct permeabilization of mitochondrial membranes, and this effect is inhibited by cyclosporin A, indicating involvement of the permeability transition pore complex (PTPC) in the permeabilization event. NO, peroxynitrite and HNE also permeabilize proteoliposomes containing the adenine nucleotide translocator (ANT), one of the key components of the PTPC, yet have no or little effects on protein-free control liposomes. ANT-dependent, NO-, peroxynitrite- or HNE-induced permeabilization is at least partially inhibited by recombinant Bcl-2 protein, as well as the antioxidants trolox and butylated hydroxytoluene. In vitro, none of the tested agents (NO, peroxynitrite, HNE, and tert-butylhydroperoxide) causes preferential carbonylation HNE adduction, or nitrotyrosylation of ANT. However, all these agents induced ANT to undergo thiol oxidation/derivatization. Peroxynitrite and HNE also caused significant lipid peroxidation, which was antagonized by butylated hydroxytoluene but not by recombinant Bcl-2. Transfection-enforced expression of vMIA, a viral apoptosis inhibitor specifically targeted to ANT, largely reduces the mitochondrial and nuclear signs of apoptosis induced by NO, peroxynitrite and HNE in intact cells. Taken together these data suggest that NO, peroxynitrite, and HNE may directly act on ANT to induce mitochondrial membrane permeabilization and apoptosis.

Permeabilization of the mitochondrial inner membrane during apoptosis: impact of the adenine nucleotide translocator.

Mitochondrial membrane permeabilization can be a rate limiting step of apoptotic as well as necrotic cell death. Permeabilization of the outer mitochondrial membrane (OM) and/or inner membrane (IM) is, at least in part, mediated by the permeability transition pore complex (PTPC). The PTPC is formed in the IM/OM contact site and contains the two most abundant IM and OM proteins, adenine nucleotide translocator (ANT, in the IM) and voltage-dependent anion channel (VDAC, in the OM), the matrix protein cyclophilin D, which can interact with ANT, as well as apoptosis-regulatory proteins from the Bax/Bcl-2 family. Here we discuss that ANT has two opposite functions. On the one hand, ANT is a vital, specific antiporter which accounts for the exchange of ATP and ADP on IM. On the other hand, ANT can form a non-specific pore, as this has been shown by electrophysiological characterization of purified ANT reconstituted into synthetic lipid bilayers or by measuring the permeabilization of proteoliposomes containing ANT. Pore formation by ANT is induced by a variety of different agents (e.g. Ca2+, atractyloside, thiol oxidation, the pro-apoptotic HIV-1 protein Vpr, etc.) and is enhanced by Bax and inhibited by Bcl-2, as well as by ADP. In isolated mitochondria, pore formation by ANT leads to an increase in IM permeability to solutes up to 1500 Da, swelling of the mitochondrial matrix, and OM permeabilization, presumably due to physical rupture of OM. Although alternative mechanisms of mitochondrial membrane permeabilization may exist, ANT emerges as a major player in the regulation of cell death. Cell Death and Differentiation (2000) 7, 1146–1154

Bid acts on the permeability transition pore complex to induce apoptosis

Similar to most if not all pro-apoptotic members of the Bcl-2 family, Bid (and its truncated product t-Bid) triggers cell death via mitochondrial membrane permeabilization (MMP). This effect can be monitored in intact cells, upon microinjection of recombinant Bid protein into the cytoplasm, as well as in purified mitochondria, upon addition of Bid protein. Here we show that Bid-induced MMP can be inhibited, both in cells and in the cell-free system, by three pharmacological inhibitors of the permeability transiton pore complex (PTPC), namely cyclosporin A, N-methyl-4-Val-cyclosporin A, and bongkrekic acid (a ligand of the adenine nucleotide translocase, ANT, one of the PTPC components). Bid effects on synthetic membranes were studied either in proteoliposomes or in synthetic bilayers subjected to electrophysiological measurements. Full length Bid preferentially permeabilizes membranes and induces the formation of large conductance channels at neutral pH, when added to liposomes or bilayers containing both purified ANT and Bax, yet has no or little effect combined with ANT or Bax alone. t-Bid acts on membranes containing ANT alone with the same efficiency as on those containing both ANT and Bax. These results suggest that the proapoptotic effects of Bid are mediated, at least in part, by its functional interaction with ANT, one of the major components of PTPC.

Adenine nucleotide translocator mediates the mitochondrial membrane permeabilization induced by lonidamine, arsenite and CD437.

An increasing number of experimental chemotherapeutic agents induce apoptosis by directly triggering mitochondrial membrane permeabilization (MMP). Here we examined MMP induced by lonidamine, arsenite, and the retinoid derivative CD437. Cells overexpressing the cytomegalovirus-encoded protein vMIA, a protein which interacts with the adenine nucleotide translocator, were strongly protected against the MMP-inducing and apoptogenic effects of lonidamine, arsenite, and CD437. In a cell-free system, lonidamine, arsenite, and CD437 induced the permeabilization of ANT proteoliposomes, yet had no effect on protein-free liposomes. The ANT-dependent membrane permeabilization was inhibited by the two ANT ligands ATP and ADP, as well as by recombinant Bcl-2 protein. Lonidamine, arsenite, and CD437, added to synthetic planar lipid bilayers containing ANT, elicited ANT channel activities with clearly distinct conductance levels of 20±7, 100±30, and 47±7 pS, respectively. Altering the ATP/ADP gradient built up on the inner mitochondrial membrane by inhibition of glycolysis and/or oxidative phosphorylation differentially modulated the cytocidal potential of lonidamine, arsenite, and CD437. Inhibition of F0F1ATPase without glycolysis inhibition sensitized to lonidamine-induced cell death. In contrast, only the combined inhibition of glycolysis plus F0F1ATPase sensitized to arsenite-induced cell death. No sensitization to cell death induction by CD437 was achieved by glucose depletion and/or oligomycin addition. These results indicate that ANT is a target of lonidamine, arsenite, and CD437 and unravel an unexpected heterogeneity in the mode of action of these three compounds.

Endoplasmic reticulum stress-induced cell death requires mitochondrial membrane permeabilization

Accumulating evidence suggests that mitochondrial membrane permeabilization (MMP) is a rate-limiting step of programmed (developmental) cell death as well as stressinduced cell death, including in the context of anti-cancer chemotherapy or viral infection. This notion is re-enforced by the observation that the knock-out of proteins involved in the pathways leading to MMP or closely linked to MMP (Bax, Bak, Bim, AIF, cytochrome c, Apaf-1, caspase-9 etc.) results in a major phenotype. Recently, we have launched the working hypothesis that MMP may be a rate-limiting event of apoptosis induction even when cell death is initiated through a primary stimulus affecting other organelles than mitochondria such as nuclei (via p53 activation), lysosomes (via activation of cathepsins) or the endoplasmic reticulum (ER). The proapoptotic agents that specifically act on the ER include tunicamycin (TM, which inhibits N-linked glycosylation), brefeldin A (BFA, which inhibits ER-Golgi transport) and thapsigargin (TG, which inhibits the sarcoplasmic/endoplasmic Ca-ATPase SERCA). Whereas BFA and TG elicit a local unfolded protein response, TG depletes ER Ca and thus impinges on Ca signaling. Mouse embryonic fibroblasts lacking both Bax and Bak become resistant to apoptosis induction by TM and BFA, an observation that may be attributed to the obligatory participation of Bax and Bak in MMP induction, or alternatively, suggests an as yet poorly characterized function of Bax and Bak at the ER level. Indeed, Bax redistributes both to mitochondria and to ER upon apoptosis induction, and overexpression of Bax reportedly causes a loss of ER Ca content. To probe the importance of MMP for ER stress-induced cell death, we assessed the effects of two local MMP inhibitors, Bcl-XL and vMIA on cellular alterations provoked by TM, BFA, and TG (Figure 1). Bcl-XL is found inserted in intracellular membranes (in particular the outer mitochondrial membrane) and is known to stabilize the mitochondrial membrane barrier function by local interactions with pore forming proteins contained in the permeability transition pore complex, namely the voltage-dependent anion channel (VDAC), the adenine nucleotide translocase (ANT), and pro-apoptotic Bcl-2 family members. In contrast to Bcl-2, ± 14 no local ER effects have been described for Bcl-XL. 15 Viral mitochondrial inhibitor of apoptosis (vMIA) is encoded by the Cytomegalovirus UL37 gene, has no obvious structural similarity with Bcl-2-like molecules, and is exclusively found in mitochondrial membranes, where it specifically interacts with ANT but not with VDAC, as shown by mass spectroscopic identification of vMIAinteracting proteins and confirmed by co-immunoprecipitation assays. ± 18 TM, BFA, and TG induced signs of MMP in several different cell lines including B cell lymphoma BJAB (Figure 1a) and cervical carcinoma HeLa cells (Figure 1b). Such signs consist in the loss of the mitochondrial transmembrane potential (DCm), as determined by means of the DCm-sensitive fluorochrome, DiOC6 3 (Figure 1a), and the release of cytochrome c from mitochondria, as determined by immunofluorescence analysis (Figure 1b,c). Thus, both the permeability of the inner membrane (on which the DCm builds up) and that of the outer membrane (which retains cytochrome c) were compromised by the three different ER-targeted toxins. ER stress-induced MMP was an early event since it was detectable in a fraction of cells that still lack signs of chromatin condensation (not shown). Stable transfection with vMIA and Bcl-XL prevented ER stressinduced MMP (Figure 1a,b,c), both in BJAB and in HeLa cells. It has been reported that, in determined circumstances, for instance in type I cells stimulated by CD95 ligation, Bcl-2-mediated MMP inhibition is not sufficient for the prevention of apoptosis. Therefore, we assessed whether MMP inhibition would suffice to suppress apoptosis induction by TM, BFA, or TG. Clearly, vMIA and Bcl-XL overexpression did reduce the frequency of cells which manifest caspase activation (not shown), nuclear chromatin condensation (Figure 1b), phosphatidylserine exposure on the outer leaflet of the plasma membrane (as determined with an Annexin V-FITC conjugate, Figure 1d), and loss of viability (as determined by staining with propidium iodide, PI, Figure 1e). In conclusion, it appears that MMP inhibition by Bxl-XL or vMIA can protect cells against apoptosis induction by ERspecific toxins. Using a monoclonal antibody specific for the apoptogenic conformation of Bax (6A7), we determined the putative link between vMIA-induced (presumably ANTmediated ± 18 apoptosis inhibition and the Bax/Bak mediated MMP induced by ER stress. ER stress did induce an apoptosis-associated change in Bax conformation, linked to its aggregation in cytoplasmic spots (Figure 2f), some of which coincide with mitochondria (as determined by confocal microscopy, not shown). vMIA did prevent the apoptosis associated conformational change of Bax, as well as its aggregation, a finding that may link our previous observation that Bax and ANT can interact to induce MMP. In conclusion, it appears that ER stress can induce apoptosis through a reaction that depends on pro-apoptotic members of the Bcl-2 family (Bax, Bak) and which involves MMP as a critical step towards cellular demise. Rather than Cell Death and Differentiation (2002) 9, 465 ± 467 ã 2002 Nature Publishing Group All rights reserved 1350-9047/02

The adenine nucleotide translocator in apoptosis

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Cell permeable BH3-peptides overcome the cytoprotective effect of Bcl-2 and Bcl-X L

Alteration of mitochondrial membrane permeability is a central mechanism leading invariably to cell death, which results, at least in part, from the opening of the permeability transition pore complex (PTPC). Indeed, extended PTPC opening is sufficient to trigger an increase in mitochondrial membrane permeability and apoptosis. Among the various PTPC components, the adenine nucleotide translocator (ANT) appears to act as a bi-functional protein which, on the one hand, contributes to a crucial step of aerobic energy metabolism, the ADP/ATP translocation, and on the other hand, can be converted into a pro-apoptotic pore under the control of onco- and anti-oncoproteins from the Bax/Bcl-2 family. In this review, we will discuss recent advances in the cooperation between ANT and Bax/Bcl-2 family members, the multiplicity of agents affecting ANT pore function and the putative role of ANT isoforms in apoptosis control.