Humanin binds and nullifies Bid activity by blocking its activation of Bax and Bak

Recently, we discovered that Humanin (HN), a small endogenous peptide of 24 amino acids, binds to and inhibits the proapoptotic protein Bax. We show here that HN also interacts with the BH3-only Bcl-2/Bax family protein, Bid, as well as a truncated form of Bid (tBid) associated with protease-mediated activation of this proapoptotic protein. Synthetic HN peptide binds purified Bid and tBid in vitro and blocks tBid-induced release of cytochrome c and SMAC from isolated mitochondria, whereas mutant peptides that fail to bind Bid or tBid lack this activity. Moreover, HN peptide also retained protective activity on bax-/-mitochondria, indicating that HN can block tBid-induced release of apoptogenic proteins from these organelles in a Bax-independent manner. HN peptide inhibits tBid-induced oligomerization of Bax and Bak in mitochondrial membranes, as shown by experiments with chemical cross-linkers or gel filtration. Gene transfection experiments showed that HN (but not an inactive mutant of HN) also protects intact cells from apoptosis induced by overexpression of tBid. We conclude that Bid represents an additional cellular target of HN, and we propose that HN-mediated suppression of Bid contributes to the antiapoptotic activity of this endogenous peptide.     

Bid, a widely expressed proapoptotic protein of the Bcl-2 family, displays lipid transfer activity

Bid is an abundant proapoptotic protein of the Bcl-2 family that is crucial for the induction of death receptor-mediated apoptosis in primary tissues such as liver. Bid action has been proposed to involve the relocation of its truncated form, tBid, to mitochondria to facilitate the release of apoptogenic cytochrome c. The mechanism of Bid relocation to mitochondria was unclear. We report here novel biochemical evidence indicating that Bid has lipid transfer activity between mitochondria and other intracellular membranes, thereby explaining its dynamic relocation to mitochondria. First, physiological concentrations of phospholipids such as phosphatidic acid and phosphatidylglycerol induced an accumulation of full-length Bid in mitochondria when incubated with light membranes enriched in endoplasmic reticulum. Secondly, native and recombinant Bid, as well as tBid, displayed lipid transfer activity under the same conditions and at the same nanomolar concentrations leading to mitochondrial relocation and release of cytochrome c. Thus, Bid is likely to be involved in the transport and recycling of mitochondrial phospholipids. We discuss how this new role of Bid may relate to its proapoptotic action.     

Proapoptotic Bid binds to monolysocardiolipin,a new molecular connection between mitochondrial membranes and cell death

Recent evidence indicates that the mitochondrial lipid cardiolipin may be instrumental in the proapoptotic action of Bcl-2 family proteins on mitochondrial membranes, leading to the release of apoptogenic factors. However, contrasting evidence indicates that progressive loss of cardiolipin occurs during apoptosis. Here we show that Bid, a crucial proapoptotic protein that integrates the action of other Bcl-2 family members, exhibits discrete specificity for metabolites of cardiolipin, especially monolysocardiolipin (MCL). MCL, normally present in the remodelling of mitochondrial lipids, progressively increases in mitochondria during Fas-mediated apoptosis as a by-product of cardiolipin degradation, and also enhances Bid binding to membranes. MCL may thus play a crucial role in connecting lipid metabolism, relocation of Bid to mitochondria and integrated action of Bcl-2 proteins on mitochondrial membranes. We propose that Bid interaction with MCL 'primes' the mitochondrial outer membrane via segregation of lipid domains, facilitating membrane discontinuity and leakage of apoptogenic factors.     

Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells

Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.     

Lysosomal membrane permeabilization as apoptogenic factor

Lysosomal membrane labilizing agents (incl. proapoptotic proteins of Bcl-2 family, LAPF, p53), estimation of lysosomal membrane permeabilization in living cells, the new data on differential permeabilization of lysosomal membranes, membrane stabilizing factors (incl. Hsp70), relations between lysosomal membrane damage, and initiation of apoptosis were considered. Signal effect of lysosomal membrane permeabilization is caused preferentially by release of cathepsin B and D in cytosol. Subsequent numerous pathways of apoptogenic signalization include proteolytic attack/activation on signal cytosolic proteins, mitochondria, procaspases, cell nuclei. The mainstream of the cell damage is connected with activation pf proapoptotic Bid and Bax, leading to permeabilization of the outer mitochondrial membrane, release of cytochrome c into cytosol and activation of caspase cascade. Translocation of the lysosoma enzymes in cytosol is capable to induce both the caspase-dependent and caspase-independent paths of apoptosis.     

Three-dimensional structure of Bax-mediated pores in membrane bilayers

B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) is a member of the Bcl-2 protein family having a pivotal role in triggering cell commitment to apoptosis. Bax is latent and monomeric in the cytosol but transforms into its lethal, mitochondria-embedded oligomeric form in response to cell stress, leading to the release of apoptogenic factors such as cytochrome C. Here, we dissected the structural correlates of Bax membrane insertion while oligomerization is halted. This strategy was enabled through the use of nanometer-scale phospholipid bilayer islands (nanodiscs) the size of which restricts the reconstituted system to single Bax-molecule activity. Using this minimal reconstituted system, we captured structural correlates that precede Bax homo-oligomerization elucidating previously inaccessible steps of the core molecular mechanism by which Bcl-2 family proteins regulate membrane permeabilization. We observe that, in the presence of BH3 interacting domain death agonist (Bid) BH3 peptide, Bax monomers induce the formation of ∼3.5-nm diameter pores and significantly distort the phospholipid bilayer. These pores are compatible with promoting release of ions as well as proteinaceous components, suggesting that membrane-integrated Bax monomers in the presence of Bid BH3 peptides are key functional units for the activation of the cell demolition machinery.     

Bid truncation, bid/bax targeting to the mitochondria, and caspase activation associated with neutrophil apoptosis are inhibited by granulocyte colony-stimulating factor

Neutrophil apoptosis constitutes a way of managing neutrophil-mediated reactions. It allows coping with infections, but avoiding overt bystander tissue damage. Using digitonin-based subcellular fractionation and Western blotting, we found that spontaneous apoptosis of human neutrophils (after approximately 20 h of culture) was associated with translocation of two proapoptotic Bcl-2 homologues, Bid and Bax, to the mitochondria and truncation of Bid, with subsequent release of Omi/HtrA2 and Smac/DIABLO into the cytosol. These events were accompanied by processing and increased enzymatic activity of caspase-8, -9, and -3. A G-CSF-mediated reduction in apoptosis coincided with inhibition of all these reactions. The G-CSF-induced effects were differentially dependent on newly synthesized mediators. Whereas inhibition of Bax targeting to the mitochondria and inhibition of caspase activation by G-CSF were dependent on protein synthesis, Bid truncation and redistribution were prevented by G-CSF regardless of the presence of the protein synthesis inhibitor cycloheximide. Apparently, the observed Bid changes were dispensable for neutrophil apoptosis. Although the regulators of the inhibitor of apoptosis proteins (IAPs), Omi/HtrA2 and Smac/DIABLO, were released into the cytosol during apoptosis, we did not observe cleavage of X-linked IAP, which suggests that another mechanism of IAP deactivation is involved. Together our results support an integrative role of the mitochondria in induction and/or amplification of caspase activity and show that G-CSF may act by blocking Bid/Bax redistribution and inhibiting caspase activation.     

Cleavage of Bax enhances its cell death function

Members of the Bcl-2 family of proteins are key regulators of apoptosis. Some of these proteins undergo posttranslational modification, such as phosphorylation or proteolysis, that serves to alter their function. Caspases are known to cleave Bid, a proapoptotic family member, as well as Bcl-2 and Bcl-X(L), two prosurvival family members, which activate their cytotoxic activity resulting in the release of cytochrome c from mitochondria. Previously we showed that Bax was cleaved by calpain rather than by caspases from full-length 21 kDa to generate a cleavage fragment of 18 kDa. Since cleavage of Bid serves to activate its cytotoxic activity, we wanted to determine if the p18 form of Bax exhibited increased cytotoxicity compared to p21 Bax. Using a transient transfection system in human embryonic kidney 293T cells we show that the p18 form of Bax displays a more potent ability to induce cell death. The pancaspase inhibitor Z-VAD-fmk completely blocked apoptosis induced by p21 Bax but only partially inhibited apoptosis induced by p18 Bax. Cyclosporin A, an inhibitor of the mitochondrial permeability transition (PT) pore, had no effect on Bax-mediated apoptosis of 293T cells suggesting that apoptosis was independent of the PT. Thus cleavage of p21 Bax during apoptosis to the p18 form may serve to increase the intrinsic cytotoxic properties of this proapoptotic molecule and enhance its cell death function at the mitochondria.     

Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane

In many types of apoptosis, the proapoptotic protein Bax undergoes a change in conformation at the level of the mitochondria. This event always precedes the release of mitochondrial cytochrome c, which, in the cytosol, activates caspases through binding to Apaf-1. The mechanisms by which Bax triggers cytochrome c release are unknown. Here we show that following binding to the BH3-domain-only proapoptotic protein Bid, Bax oligomerizes and then integrates in the outer mitochondrial membrane, where it triggers cytochrome c release. Bax mitochondrial membrane insertion triggered by Bid may represent a key step in pathways leading to apoptosis.     

BH3-only protein Bim inhibits activity of antiapoptotic members of Bcl-2 family when expressed in yeast

Proteins of the Bcl-2 family regulate programmed cell death in mammals by promoting the release of cytochrome c from mitochondria in response to various proapoptotic stimuli. The mechanism by which BH3-only members of the family activate multidomain proapoptotic proteins Bax and Bak to form a pore in mitochondrial membranes remains under dispute. We report that cell death promoting activity of BH3-only protein Bim can be reconstituted in yeast when both Bax and antiapoptotic protein Bcl-X(L) are present, suggesting that Bim likely activates Bax indirectly by inhibiting antiapoptotic proteins.     

Essential roles of the Bcl-2 family of proteins in caspase-2-induced apoptosis

Caspase-2 is an initiating caspase required for stress-induced apoptosis in various human cancer cells. Recent studies suggest that it can mediate the death function of tumor suppressor p53 and is activated by a multimeric protein complex, PIDDosome. However, it is not clear how caspase-2 exerts its apoptotic function in cells and whether its enzymatic activity is required for the apoptotic function. In this study, we used both in vitro mitochondrial cytochrome c release assays and cell culture apoptosis analyses to investigate the mechanism by which caspase-2 induces apoptosis. We show that active caspase-2, but neither a catalytically mutated caspase-2 nor active caspase-2 with its inhibitor, can cause cytochrome c release. Caspase-2 failed to induce cytochrome c release from mitochondria with Bid(-/-) background, and the release could be restored by addition of the wild-type Bid protein, but not by Bid with the caspase-2 cleavage site mutated. Caspase-2 was not able to induce cytochrome c release from Bax(-/-)Bak(-/-) mitochondria either. In cultured cells, gene deletion of Bax/Bak or Bid abrogated apoptosis induced by overexpression of caspase-2. Collectively, these results indicate that proteolytic activation of Bid and the subsequent induction of the mitochondrial apoptotic pathway through Bax/Bak is essential for apoptosis triggered by caspase-2.     

Peptides derived from apoptotic Bax and Bid reproduce the poration activity of the parent full-length proteins

Bax and Bid are proapoptotic proteins of the Bcl-2 family that regulate the release of apoptogenic factors from mitochondria. Although they localize constitutively in the cytoplasm, their apoptotic function is exerted at the mitochondrial outer membrane, and is related to their ability to form transbilayer pores. Here we report the poration activity of fragments from these two proteins, containing the first alpha-helix of a colicinlike hydrophobic hairpin (alpha-helix 5 of Bax and alpha-helix 6 of Bid). Both peptides readily bind to synthetic lipid vesicles, where they adopt predominantly alpha-helical structures and induce the release of entrapped calcein. In planar lipid membranes they form ion conducting channels, which in the case of the Bax-derived peptide are characterized by a two-stage pattern, a large conductivity and lipid-charge-dependent ionic selectivity. These features, together with the influence of intrinsic lipid curvature on the poration activity and the existence of two helical stretches of different orientations for the membrane-bound peptide, suggest that it forms mixed lipidic/peptidic pores of toroidal structure. In contrast, the assayed Bid fragment shows a markedly different behavior, characterized by the formation of discrete, steplike channels in planar lipid bilayers, as expected for a peptidic pore lined by a bundle of helices.     

Preservation of mitochondrial structure and function after Bid- or Bax-mediated cytochrome c release

Proapoptotic members of the Bcl-2 protein family, including Bid and Bax, can activate apoptosis by directly interacting with mitochondria to cause cytochrome c translocation from the intermembrane space into the cytoplasm, thereby triggering Apaf-1-mediated caspase activation. Under some circumstances, when caspase activation is blocked, cells can recover from cytochrome c translocation; this suggests that apoptotic mitochondria may not always suffer catastrophic damage arising from the process of cytochrome c release. We now show that recombinant Bid and Bax cause complete cytochrome c loss from isolated mitochondria in vitro, but preserve the ultrastructure and protein import function of mitochondria, which depend on inner membrane polarization. We also demonstrate that, if caspases are inhibited, mitochondrial protein import function is retained in UV-irradiated or staurosporine-treated cells, despite the complete translocation of cytochrome c. Thus, Bid and Bax act only on the outer membrane, and lesions in the inner membrane occurring during apoptosis are shown to be secondary caspase-dependent events.     

Phosphorylation of rat brain mitochondrial voltage-dependent anion as a potential tool to control leakage of cytochrome c

Apoptosis is a controlled form of cell death that participates in development, elimination of damaged cells and maintenance of cell homeostasis. Also, it plays a role in neurodegenerative disorders like Alzheimer's disease. Recently, mitochondria have emerged as being pivotal in controlling apoptosis. They house a number of apoptogenic molecules, such as cytochrome c, which are released into the cytoplasm at the onset of apoptosis. When rat brain mitochondrial voltage-dependent anion channel (VDAC), an outer mitochondrial membrane protein, interacts with Bcl-2 family proteins Bax and tBid, its pore size increases, leading to the release of cytochrome c and other apoptogenic molecules into the cytosol and causing cell death. Regulation of this tBid- and Bax-induced increase in pore size of VDAC is a significant step to control cell death induced by cytochrome c. In this work, we have shown, through bilayer electrophysiological experiments, that the increase in VDAC conductance as a result of its interaction with Bax and tBid is reduced because of the action of cyclic AMP-dependent protein kinase A (PKA) in the presence of ATP. This indicates that the increase in the pore size of VDAC after its interaction with Bax and tBid is controlled via phosphorylation of this channel by PKA. This, we believe, could be a mechanism of controlling cytochrome c-mediated cell death in living cells.     

Apop-1, a novel protein inducing cyclophilin D-dependent but Bax/Bak-related channel-independent apoptosis

In the intrinsic pathway of apoptosis, mitochondria play a crucial role by releasing cytochrome c from the intermembrane space into the cytoplasm. Cytochrome c release through Bax/Bak-dependent channels in mitochondria has been well documented. In contrast, cyclophilin D (CypD), an important component of permeability transition pore-dependent protein release, remains largely undefined, and no apoptogenic proteins that act specifically in a CypD-dependent manner have been reported to date. Here, we describe a novel and evolutionarily conserved protein, apoptogenic protein (Apop). Mouse Apop-1 expression induces apoptotic death by releasing cytochrome c from mitochondria into the cytosolic space followed by activation of caspase-9 and -3. Apop-1-induced apoptosis is not blocked by Bcl-2 or Bcl-xL, inhibitors of Bax/Bak-dependent channels, whereas it is completely blocked by cyclosporin A, an inhibitor of permeability transition pore. Cells lacking CypD were resistant to Apop-induced apoptosis. Moreover, inhibition of Apop expression prevented the cell death induced by apoptosis-inducing substances. Our findings, thus, indicate that the expression of Apop-1 induces apoptosis though CypD-dependent pathway and that Apop-1 plays roles in cell death under physiological conditions.     

Curcumin induces Apaf-1-dependent,p21-mediated caspase activation and apoptosis

Previous studies have demonstrated that curcumin induces mitochondria-mediated apoptosis. However, understanding of the molecular mechanisms underlying curcumin-induced cell death remains limited. In this study, we demonstrate that curcumin treatment of cancer cells caused dose- and time-dependent caspase-3 activation, which is required for apoptosis as confirmed using the pan caspase inhibitor, z-VAD. Knockdown experiments and knockout cells excluded a role of caspase-8 in curcumin-induced caspase-3 activation. In contrast, Apaf-1 deficiency or silencing inhibited the activity of caspase-3, pointing to a requisite role of Apaf-1 in curcumin-induced apoptotic cell death. Curcumin treatment led to Apaf-1 upregulation both at the protein and mRNA levels. Cytochrome c release from mitochondria to the cytosol in curcumin-treated cells was associated with upregulation of proapoptotic proteins such as Bax, Bak, Bid, and Bim. Crosslinking experiments demonstrated Bax oligomerization during curcumin-induced apoptosis, suggesting that induced expression of Bax, Bid, and Bim causes Bax-channel formation on the mitochondrial membrane. The release of cytochrome c was unaltered in p53-deficient cells, whereas absence of p21 blocked cytochrome c release, caspase activation, and apoptosis. Importantly, p21-deficiency resulted in reduced expression of Apaf-1 during curcumin treatment, indicating a requirement of p21 in Apaf-1 dependent caspase activation and apoptosis. Together, our findings demonstrate that Apaf-1, Bax, and p21 as novel potential targets for curcumin or curcumin-based anticancer agents.     

Translocation of Bax and Bid to Mitochondria, Endoplasmic Reticulum and Nuclear Envelope: Possible Control Points in Apoptosis

The cross-talk between endoplasmic reticulum (ER) and mitochondria was investigated during apoptosis in a breast cancer cell line (MCF-7) in culture. The effect of camptothecin, an inducer of apoptosis and a specific inhibitor of topoisomerase I, was investigated by morphological, immunocytochemical and histochemical techniques for electron microscopy. Our ultrastructural morphological data demonstrate alterations in ER configuration and communication with neighbouring mitochondria early after stimulation by camptothecin. Immunoelectron studies have demonstrated that Bax and Bid translocate from cytoplasm to mitochondria where they initiate mitochondrial dysfunction and cytochrome c release. Bax and Bid were also localized in ER and nuclear envelope. Since ER and mitochondria function as intracellular Ca2+ storage, we hypothesize that Bax and Bid are involved in the emptying of ER Ca2+ pool, triggers secondary changes in mitochondrial Ca2+ levels that contribute to cytochrome c release and cell death.     

Bid-induced release of AIF from mitochondria causes immediate neuronal cell death

Mitochondrial dysfunction and release of pro-apoptotic factors such as cytochrome c or apoptosis-inducing factor (AIF) from mitochondria are key features of neuronal cell death. The precise mechanisms of how these proteins are released from mitochondria and their particular role in neuronal cell death signaling are however largely unknown. Here, we demonstrate by fluorescence video microscopy that 8-10 h after induction of glutamate toxicity, AIF rapidly translocates from mitochondria to the nucleus and induces nuclear fragmentation and cell death within only a few minutes. This markedly fast translocation of AIF to the nucleus is preceded by increasing translocation of the pro-apoptotic bcl-2 family member Bid (BH3-interacting domain death agonist) to mitochondria, perinuclear accumulation of Bid-loaded mitochondria, and loss of mitochondrial membrane integrity. A small molecule Bid inhibitor preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated glutamate-induced neuronal cell death, as shown by experiments using Bid small interfering RNA (siRNA). Cell death induced by truncated Bid was inhibited by AIF siRNA, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that although caspase-3 was activated, specific caspase-3 inhibition did not protect neuronal cells against glutamate toxicity. In conclusion, Bid-mediated mitochondrial release of AIF followed by rapid nuclear translocation is a major mechanism of glutamate-induced neuronal death.     

Tumor necrosis factor-alpha induces Bax-Bak interaction and apoptosis, which is inhibited by adenovirus E1B 19K

Tumor necrosis factor (TNF)-alpha-mediated death signaling induces oligomerization of proapoptotic Bcl-2 family member Bax into a high molecular mass protein complex in mitochondrial membranes. Bax complex formation is associated with the release of cytochrome c, which propagates death signaling by acting as a cofactor for caspase-9 activation. The adenovirus Bcl-2 homologue E1B 19K blocks TNF-alpha-mediated apoptosis by preventing cytochrome c release, caspase-9 activation, and apoptosis of virus-infected cells. TNF-alpha induces E1B 19K-Bax interaction and inhibits Bax oligomerization. Oligomerized Bax may form a pore to release mitochondrial proteins, analogous to the homologous pore-forming domains of bacterial toxins. E1B 19K can also bind to proapoptotic Bak, but the functional significance is not known. TNF-alpha signaling induced Bak-Bax interaction and both Bak and Bax oligomerization. E1B 19K was constitutively in a complex with Bak, and blocked the Bak-Bax interaction and oligomerization of both. The TNF-alpha-mediated cytochrome c and Smac/DIABLO release from mitochondria was inhibited by E1B 19K expression in adenovirus-infected cells. Since either Bax or Bak is essential for death signaling by TNF-alpha, the interaction between E1B 19K and both Bak and Bax may be required to inhibit their cooperative or independent oligomerization to release proteins from mitochondria which promote caspase activation and cell death.     

Translocation of full-length Bid to mitochondria during anoikis

Epithelial cells require adhesion to the extracellular matrix for survival, and in the absence of adhesion they undergo apoptosis (anoikis). This is distinct from apoptosis induced by extracellular death ligands, such as tumor necrosis factor, which result in direct activation of caspase 8. Bid is a member of the BH3-only subfamily of the Bcl-2 proteins and is important for most cell types to apoptose in response to Fas and tumor necrosis factor receptor activation. Caspase 8 cleaves full-length Bid, resulting in truncated p15 tBid. p15 tBid is potently apoptotic and activates the multidomain Bcl-2 protein, Bax, resulting in release of cytochrome c from mitochondria. We have previously shown that Bax rapidly translocates from the cytosol to mitochondria following loss of adhesion and that this is required for anoikis. We have now examined the role of Bid in anoikis. Bid translocates to mitochondria with identical kinetics as Bax. Although Bid is required for anoikis, it does not require proteolytic cleavage by caspase 8. Furthermore, it does not require Bid to interact directly with other Bcl-2 family proteins, such as Bax. Our data indicate that Bid is important for regulating apoptosis via the intrinsic pathway and has implications for how Bid may fulfill that role.