death executioner Bcl-2 homologue

EVOLUTIONARY HOMOLOGS

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Bcl-2 family members as regulators of the cell death hierarchy: Bcl-2 proteins as ion channels and regulators of mitochondrial membrane permiability

The crystal and solution structures of a Bcl-2 family member, Bcl-xL, have been solved. The structures consist of two central, primarily hydrophobic alpha-helices, which are surrounded by amphipathic helices. A 60-residue loop connecting helices alpha1 and alpha2 was found to be flexible and non-essential for anti-apoptotic activity. The three functionally important Bcl-2 homology regions (BH1, BH2 and BH3) are in close spatial proximity and form an elongated hydrophobic cleft that may represent the binding site for other Bcl-2 family members. The arrangement of the alpha-helices in Bcl-xL is reminiscent of the membrane translocation domain of bacterial toxins, in particular diphtheria toxin and the colicins. The structural similarity may provide a clue to the mechanism of action of the Bcl-2 family of proteins (Muchmore, 1996).

The BCL-2 family of proteins is composed of both pro- and antiapoptotic regulators, although its most critical biochemical functions remain uncertain. The structural similarity between the BCL-XL monomer and several ion-pore-forming bacterial toxins has prompted electrophysiologic studies. Both BAX and BCL-2 insert into KCl-loaded vesicles in a pH-dependent fashion and demonstrate macroscopic ion efflux. Release is maximum at approximately pH 4.0 for both proteins; however, BAX demonstrates a broader pH range of activity. Both purified proteins also insert into planar lipid bilayers at pH 4.0. Single-channel recordings reveal a minimal channel conductance for BAX of 22 pS that evolve to channel currents with at least three subconductance levels. The final, apparently stable BAX channel has a conductance of 0.731 nS at pH 4. 0, which changes to 0.329 nS when shifted to pH 7.0, but which remains mildly Cl- selective and predominantly open. When BAX-incorporated lipid vesicles are fused to planar lipid bilayers at pH 7.0, a Cl- selective (PK/PCl = 0.3) 1.5-nS channel displaying mild inward rectification is noted. In contrast, BCL-2 forms mildly K+-selective (PK/PCl = 3.9) channels with a most prominent initial conductance of 80 pS, which increases to 1.90 nS. Fusion of BCL-2-incorporated lipid vesicles into planar bilayers at pH 7.0 also reveals mild K+ selectivity (PK/PCl = 2.4) with a maximum conductance of 1.08 nS. BAX and BCL-2 each form channels in artificial membranes that have distinct characteristics, including ion selectivity, conductance, voltage dependence, and rectification. Thus, one role of these molecules may include pore activity at selected membrane sites (Schlesinger, 1997).

Mitochondrial physiology is disrupted in either apoptosis or necrosis. A wide variety of apoptotic and necrotic stimuli induce progressive mitochondrial swelling and outer mitochondrial membrane rupture. Discontinuity of the outer mitochondrial membrane results in cytochrome c redistribution from the intermembrane space to the cytosol, followed by subsequent inner mitochondrial membrane depolarization. The mitochondrial membrane protein Bcl-xL can inhibit these changes in cells treated with apoptotic stimuli. Bcl-xL-expressing cells adapt to growth factor withdrawal or staurosporine treatment by maintaining a decreased mitochondrial membrane potential. Bcl-xL expression also prevents mitochondrial swelling in response to agents that inhibit oxidative phosphorylation, in particular, oligomycin and antimycin A. Oligomycin functions to block the inner membrane F1F0-ATPase, which utilizes the H+ ion gradient to generate ATP. If H+ ions are not consumed and electron transport continues, the mitochondria become acutely hyperpolarized and subsequently undergo the osmotic swelling associated with necrosis. Antimycin A inhibits complex III of the electron transport chain. Cells treated with antimycin A are unable to maintain mitochondrial osmotic homeostasis following the inhibition of electron transport. These data suggest that Bcl-xL promotes cell survival by regulating the electrical and osmotic homeostasis of mitochondria (Vander Heiden, 1997).

Bcl-2-related proteins are critical regulators of cell survival that are localized to the outer mitochondrial, outer nuclear and endoplasmic reticulum membranes. Despite their physiological importance, the biochemical function of Bcl-2-related proteins has remained elusive. The three-dimensional structure of Bcl-xL, an inhibitor of apoptosis, is similar to the structures of the pore-forming domains of bacterial toxins. A key feature of these pore-forming domains is the ability to form ion channels in biological membranes. Bcl-xL shares this functional feature. Like the bacterial toxins, Bcl-xL can insert into either synthetic lipid vesicles or planar lipid bilayers and form an ion-conducting channel. This channel is pH-sensitive and becomes cation-selective at physiological pH. The ion-conducting channel(s) formed by Bcl-xL displays multiple conductance states that have identical ion selectivity. Together, these data suggest that Bcl-xL may maintain cell survival by regulating the permeability of the intracellular membranes to which it is distributed (Minn, 1997).

Bax is a pro-apoptotic member of the Bcl-2 protein family that resides in the outer mitochondrial membrane. It is controversial whether Bax promotes cell death directly through its putative function as a channel protein versus indirectly by inhibiting cellular regulators of the cell death proteases (caspases). Addition of submicromolar amounts of recombinant Bax protein to isolated mitochondria can induce cytochrome c (Cyt c) release, whereas a peptide representing the Bax BH3 domain is inactive. When placed into purified cytosol, neither mitochondria nor Bax individually induce proteolytic processing and activation of caspases. In contrast, the combination of Bax and mitochondria triggered release of Cyt c from mitochondria and induce caspase activation in cytosols. Supernatants from Bax-treated mitochondria also induce caspase processing and activation. Recombinant Bcl-XL protein abrogates Bax-induced release of Cyt c from isolated mitochondria and prevented caspase activation. In contrast, the broad-specificity caspase inhibitor benzyloxycarbonyl-valinyl-alaninyl-aspartyl-(0-methyl)- fluoromethylketone (zVAD-fmk) and the caspase-inhibiting protein X-IAP have no effect on Bax-induced release of Cyt c from mitochondria in vitro but prevent the subsequent activation of caspases in cytosolic extracts. Unlike Ca2+, a classical inducer of mitochondrial permeability transition, Bax does not induce swelling of mitochondria in vitro. Because the organellar swelling caused by permeability transition causes outer membrane rupture, the findings, therefore, dissociate these two events, implying that Bax uses an alternative mechanism for triggering release of Cyt c from mitochondria (Jurgensmeier, 1998).

A hydrophobic cleft formed by the BH1, BH2 and BH3 domains of Bcl-xL is responsible for interactions between Bcl-xL and BH3-containing death agonists. Bcl-xL primarily counters the proapoptotic effect of Bax by forming an inactivating heterodimer with Bax in solution. Mutants of Bcl-xL were constructed that do not bind to Bax but retain anti-apoptotic activity. Since Bcl-xL can form an ion channel in synthetic lipid membranes, the possibility that this property has a role in heterodimerization-independent cell survival was tested by replacing amino acids within the predicted channel-forming domain, the region surrounding the Bcl-xL helix 5 and helix 6 hairpin, with the corresponding amino acids from Bax. This chimera is referred to as XB. The resulting XB chimera shows a reduced ability to adopt an open conductance state over a wide range of membrane potentials. Although this construct retains the ability to heterodimerize with Bax and to inhibit apoptosis, when a mutation is introduced that renders the chimera incapable of heterodimerization, the resulting protein fails to prevent both apoptosis in mammalian cells and Bax-mediated growth defect in yeast. Similar to mammalian cells undergoing apoptosis, yeast cells expressing Bax exhibit changes in mitochondrial properties that are inhibited by Bcl-xL through heterodimerization-dependent and -independent mechanisms. These data suggest that Bcl-xL regulates cell survival by at least two distinct mechanisms; one is associated with heterodimerization and the other with the ability to form a sustained ion channel. Bax is able to disrupt mitochondrial function, leading to apoptosis in mammalian cells and growth inhibition in yeast. Bcl-xL primarily counters this effect by forming an inactivating heterodimer with Bax in solution. However, when Bcl-xL is prevented from interacting with Bax, it may form an ion channel that establishes a permeability pathway that counters the effects of the Bax ion channel. The establishment and/or properties of this Bcl-xL-mediated permeability pathway may be disrupted in the XB mutants. Alternatively, this counter mechanism may involve interactions within the membrane between the central hydrophobic helices of membrane-inserted Bcl-xL and membrane-inserted Bax, resulting in Bax channel inactivation or the formation of a hybrid, non-toxic channel. In this scenario, the XB mutation prevents proper intermembrane helical interactions. Interactions involving membrane-inserted helices are thought to be the mechanism by which immunity proteins prevent the toxicity of bactericidal colicins, which contain a pore-forming domain structurally similar to that of Bcl-xL (Minn, 1999).

Loss of the mitochondrial membrane potential (Deltapsi) precedes apoptosis and chemical-hypoxia-induced necrosis; this is prevented by the expression of Bcl-2. An examination was carried out of the biochemical mechanism involved when Bcl-2 prevents Deltapsi loss. Mitochondria were isolated either from a cell line overexpressing human Bcl-2 or from livers of Bcl-2 transgenic mice. Although Bcl-2 has no effect on the respiration rate of isolated mitochondria, it prevents both Deltapsi loss and the permeability transition (PT) induced by various reagents, including Ca2+, H2O2, and tert-butyl hydroperoxide. Even under conditions that did not allow PT, Bcl-2 maintains Deltapsi, suggesting that the functional target of Bcl-2 is regulation of Deltapsi but not PT. Bcl-2 also maintains Deltapsi in the presence of the protonophore SF6847, which induces proton influx, suggesting that Bcl-2 regulates ion transport to maintain Deltapsi. Although treatment with SF6847 in the absence of Ca2+ causes massive H+ influx in control mitochondria, the presence of Bcl-2 induces H+ efflux after transient H+ influx. In this case, Bcl-2 does not enhance K+ efflux. Bcl-2 enhances H+ efflux but not K+ flux after treatment of mitochondria with Ca2+ or tert-butyl hydroperoxide. These results suggest that Bcl-2 maintains Deltapsi by enhancing H+ efflux in the presence of Deltapsi-loss-inducing stimuli (Shimizu, 1998).

During transduction of an apoptotic (death) signal into the cell, there is an alteration in the permeability of the membranes of the cell's mitochondria that causes the translocation of the apoptogenic protein cytochrome c into the cytoplasm. In turn, this activates death-driving proteolytic proteins known as caspases. The Bcl-2 family of proteins, whose members may be anti-apoptotic or pro-apoptotic, regulates cell death by controlling this mitochondrial membrane permeability during apoptosis, but how that is achieved is unclear. Liposomes that carry the mitochondrial porin channel (also called the voltage-dependent anion channel, or VDAC) have been created to show that the recombinant pro-apoptotic proteins Bax and Bak accelerate the opening of VDAC, whereas the anti-apoptotic protein Bcl-x(L) closes VDAC by binding to it directly. Bax and Bak allow cytochrome c to pass through VDAC out of liposomes, but passage is prevented by Bcl-x(L). In agreement with this, VDAC1-deficient mitochondria from a mutant yeast do not exhibit a Bax/Bak-induced loss in membrane potential and cytochrome c release, both of which are inhibited by Bcl-x(L). These results indicate that the Bcl-2 family of proteins bind to the VDAC in order to regulate the mitochondrial membrane potential and the release of cytochrome c during apoptosis (Shimizu, 1999).

Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic Bcl-2 family members such as Bax and Bak induce apoptogenic mitochondrial cytochrome c release and membrane potential (Deltapsi) loss in isolated mitochondria. Using isolated mitochondria, it has been shown that Bid and Bik, BH3-only proteins from the Bcl-2 family, induce cytochrome c release but not Deltapsi loss. Unlike Bax/Bak, the cytochrome c release induced by Bid/Bik is Ca(2+)-independent, cyclosporin A-insensitive, and respiration-independent. Furthermore, in contrast to Bax/Bak, Bid/Bik neither interacte with VDAC nor directly affected the VDAC activity in liposomes. Consistently, Bid/Bik induces apoptosis without Deltapsi loss, whereas Bax induces apoptosis with Deltapsi loss. These findings indicate the involvement of a different mechanism in BH3-only, protein-induced apoptogenic cytochrome c release (Shimizu, 2000).

The proapoptotic protein BAX contains a single predicted transmembrane domain at its COOH terminus. In unstimulated cells, BAX is located in the cytosol and in peripheral association with intracellular membranes including mitochondria, but inserts into mitochondrial membranes after a death signal. This failure to insert into mitochondrial membrane in the absence of a death signal correlates with repression of the transmembrane signal-anchor function of BAX by the NH2-terminal domain. Targeting can be instated by deleting the domain or by replacing the BAX transmembrane segment with that of BCL-2. In stimulated cells, the contribution of the NH2 terminus of BAX correlates with further exposure of this domain after membrane insertion of the protein. The peptidyl caspase inhibitor zVAD-fmk partly blocks the stimulated mitochondrial membrane insertion of BAX in vivo, which is consistent with the ability of apoptotic cell extracts to support mitochondrial targeting of BAX in vitro, dependent on activation of caspase(s). Taken together, these results suggest that regulated targeting of BAX to mitochondria in response to a death signal is mediated by discrete domains within the BAX polypeptide. The contribution of one or more caspases may reflect an initiation and/or amplification of this regulated targeting (Goping, 1998).

Release of proteins through the outer mitochondrial membrane can be a critical step in apoptosis, and the localization of apoptosis-regulating Bcl-2 family members in this membrane suggests that Bcl-2 proteins control this process. Planar phospholipid membranes were used to test the effect of full-length Bax and Bcl-xL synthesized in vitro and native Bax purified from bovine thymocytes. Instead of forming pores with reproducible conductance levels expected for ionic channels, Bax, but not Bcl-xL, creates arbitrary and continuously variable changes in membrane permeability and decreases the stability of the membrane, regardless of whether the source of the protein is synthetic or native. This breakdown of the membrane permeability barrier and destabilization of the bilayer was quantified by using membrane lifetime measurements. Bax decreases membrane lifetime in a voltage- and concentration-dependent manner. Bcl-xL does not protect against Bax-induced membrane destabilization, supporting the idea that these two proteins function independently of one another. Corresponding to a physical theory for lipidic pore formation, Bax potently diminishes the linear tension of the membrane (i.e., the energy required to form the edge of a new pore). It is suggested that Bax acts directly by destabilizing the lipid bilayer structure of the outer mitochondrial membrane, promoting the formation of a pore (the apoptotic pore) large enough to allow mitochondrial proteins such as cytochrome c to be released into the cytosol. Bax could then enter and permeabilize the inner mitochondrial membrane through the same hole (Basanez, 1999).

Bcl-2 family members either promote or repress programmed cell death. Bax, a death-promoting member, is a pore-forming, mitochondria-associated protein whose mechanism of action is still unknown. During apoptosis, cytochrome C is released from the mitochondria into the cytosol where it binds to APAF-1, a mammalian homolog of Ced-4, and participates in the activation of caspases. The release of cytochrome C has been postulated to be a consequence of the opening of the mitochondrial permeability transition pore (PTP). Bax is reported to be sufficient to trigger the release of cytochrome C from isolated mitochondria. This pathway is distinct from the previously described calcium-inducible, cyclosporin A-sensitive PTP. Rather, the cytochrome C release induced by Bax is facilitated by Mg2+ and cannot be blocked by PTP inhibitors. These results strongly suggest the existence of two distinct mechanisms leading to cytochrome C release: one stimulated by calcium and inhibited by cyclosporin A; the other Bax dependent, Mg2+ sensitive but cyclosporin insensitive (Eskes, 1998).

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. This study shows 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 (Eskes, 2000).

Members of the BCL-2 family of proteins either promote or repress programmed cell death. Neonatal sympathetic neurons undergoing apoptosis after nerve growth factor (NGF) deprivation exhibit a protein synthesis-dependent, caspase-independent subcellular redistribution of BAX from cytosol to mitochondria, followed by a loss of mitochondrial cytochrome c and cell death. Treatment with elevated concentrations of the neuroprotectants KCl or cAMP at the time of deprivation prevents BAX translocation and cytochrome c release. However, administration of KCl or cAMP 12 hr after NGF withdrawal prevents acute loss of mitochondrial cytochrome c, but not redistribution of BAX; rescue with NGF prevents both events. Overexpression of Bcl-2 neither alters the normal subcellular localization of BAX nor prevents its redistribution with deprivation but does inhibit the subsequent release of cytochrome c, caspase activation, and cell death. Bcl-2 overexpression does not prevent cell death induced by cytoplasmic microinjection of cytochrome c into NGF-deprived competent-to-die neurons. These observations suggest that the subcellular redistribution of BAX is a critical event in neuronal apoptosis induced by trophic factor deprivation. BCL-2 acts primarily, if not exclusively, at the level of mitochondria to prevent BAX-mediated cytochrome c release, whereas NGF, KCl, or cAMP may abort the apoptotic program at multiple checkpoints (Putcha, 1999).

Unlike apoptosis induced by NGF deprivation in sympathetic neurons, which requires de novo protein synthesis, Fas- or TNF-mediated cell death is facilitated by the inhibition of macromolecular synthesis. It has been reported that activation of the Fas and TNF-R1 receptors causes receptor oligomerization, recruitment and activation of procaspase-8, and N-terminal cleavage of BID; this is followed by translocation of the C-terminal fragment to mitochondria, cytochrome c release, and cell death It is proposed that a critical difference between macromolecular synthesis-dependent (e.g., NGF deprivation in sympathetic neurons) and independent (e.g., Fas or TNF treatment in non-neuronal cells) paradigms of cell death may be that the former require the expression of new gene products to induce the translocation of a BH3-domain-containing, proapoptotic BCL-2 family member to mitochondria, whereas the latter do not. In NGF-deprived sympathetic neurons, BAX alone appears to serve the role of this proapoptotic, BH3-containing protein that mediates the loss of cytochrome c, caspase activation, and cell death. In other models of cell death the identity of the proapoptotic, BH3-containing protein responsible for the loss of mitochondrial cytochrome c may vary according to cell type and apoptotic stimulus (Putcha, 1999 and references).

The mechanism of cytochrome c release in response to apoptotic stimuli and its regulation by the Bcl2 family of proteins is unclear. Inasmuch as the structure of Bcl-xL is reminiscent of pore-forming proteins of bacterial toxins such as diphtheria toxin and colicins, it has been hypothesized that Bcl-xL may function as an ion channel that regulates the permeability of mitochondria. Such an ion channel could minimize osmotic stress, and in doing so, the release of cytochrome c would be prevented due to mitochondrial matrix swelling and outer membrane disruption. Indeed, both swelling of the mitochondrial matrix and bursting of the outer membrane were observed in cells treated with agonistic antibody against Fas. However, whether such a phenomenon is the cause of cytochrome c release or an effect of the apoptotic program is unclear. Activation of cell surface receptor Fas leads to rapid inactivation of the electron transfer activity of cytochrome c and subsequent release of cytochrome c from mitochondria. The inactivation and release of cytochrome c induced by Fas activation is sensitive to z-VAD-fmk, a broad range caspase inhibitor. Since activation of cell surface death receptor leads to rapid activation of caspase-8, the apical caspase in the Fas-induced apoptotic pathway, the loss of cytochrome c from mitochondria is likely a result of caspase-8 activation. Indeed, addition of active caspase-8 to a Xenopus cell-free system induces rapid cytochrome c release from mitochondria. The activation of caspase-8, therefore, initiates two pathways leading to the activation of downstream caspases. Caspase-8 can activate downstream caspases (like caspase-3, caspase-6, and caspase-7) by directly cleaving them. Caspase-8 activates these downstream caspases indirectly by causing cytochrome c release from mitochondria that triggers caspase activation through Apaf1. The latter pathway is regulated by Bcl2 or Bcl-xL while a caspase-8 inhibitor like CrmA blocks both pathways. The contributions of these two pathways to Fas-induced cell death vary between different cell types, presumably due to different levels of activity (Luo, 1998 and references).

The target of Caspase-8, the apical caspase activated by cell surface death receptors such as Fas and TNF, has now been identifed. A cytosolic protein has been purified that induces cytochrome c release from mitochondria in response to caspase-8. Peptide mass fingerprinting identified this protein as Bid, a BH3 domain-containing protein known to interact with both Bcl2 and Bax. Caspase-8 cleaves Bid, and the COOH-terminal part translocates to mitochondria where it triggers cytochrome c release. Immunodepletion of Bid from cell extracts eliminates the cytochrome c releasing activity. The cytochrome c releasing activity of Bid is antagonized by Bcl2. A mutation at the BH3 domain diminishes its cytochrome c releasing activity. Bid, therefore, relays an apoptotic signal from the cell surface to mitochondria (Luo, 1998). The role of mitochondrial Ca²⁺ [Ca(m)] homeostasis in cell survival has been investigated. Disruption of Ca(m) homeostasis via depletion of the mitochondrial Ca(2+) store is the earliest event that occurs during staurosporine-induced apoptosis in neuroblastoma cells (SH-SY5Y). The decrease of Ca(m) precedes activation of the caspase cascade and DNA fragmentation. Overexpression of the anti-apoptosis protein Bcl-2 leads to increased Ca(m) load, increased mitochondrial membrane potential (DeltaPsi(m)), and inhibition of staurosporine-induced apoptosis. On the other hand, ectopic expression of the pro-apoptotic protein Bik leads to decreased Ca(m) load and decreased DeltaPsi(m). Inhibition of calcium uptake into mitochondria by ruthenium red induces a dose-dependent apoptosis as determined by nuclear staining and DNA ladder assay. Similarly, reducing the Ca(m) load by lowering the extracellular calcium concentration also leads to apoptosis. It is suggested that the anti-apoptotic effect of Bcl-2 is related to its ability to maintain a threshold level of Ca(m) and DeltaPsi(m) while the pro-apoptotic protein Bik has the opposite effect. Furthermore, both ER and mitochondrial Ca(2+) stores are important, and the depletion of either one will result in apoptosis. Thus, these results, for the first time, provide evidence that the maintenance of Ca(m) homeostasis is essential for cell survival (Zhu, 1999).

Bcl-2 family proteins regulate the release of proteins like cytochrome c from mitochondria during apoptosis. Cell-free systems and ultimately a vesicular reconstitution from defined molecules have been used to show that outer membrane permeabilization by Bcl-2 family proteins requires neither the mitochondrial matrix, the inner membrane, nor other proteins. Bid, or its BH3-domain peptide, activates monomeric Bax to produce membrane openings that allow the passage of very large (2 megadalton) dextran molecules. This explains the translocation of large mitochondrial proteins during apoptosis. This process requires cardiolipin and is inhibited by antiapoptotic Bcl-xL. It is concluded that mitochondrial protein release in apoptosis can be mediated by supramolecular openings in the outer mitochondrial membrane, promoted by BH3/Bax/lipid interaction and directly inhibited by Bcl-xL (Kuwana, 2002).

The data show that both pro- and anti-apoptotic Bcl-2 family proteins can regulate macromolecular efflux directly at the mitochondrial outer membrane. Bax forms supramolecular openings in mitochondrial outer membranes and liposomes, suggesting that the ion channel activity of Bcl-2 family proteins may be irrelevant for mitochondrial protein release in apoptosis. Furthermore, these results show that neither swelling of the mitochondrial matrix and inner membrane nor, indeed, any other process requiring ANT or the inner membrane is required for Bid/Bax-induced membrane permeabilization. Similarly, prior studies on cells and mitochondria concluded that cytochrome c release can take place in the absence of permeability transition, matrix swelling, or outer membrane rupture. It is concluded that permeabilization requires only the interaction of Bcl-2 family proteins such as Bax and Bid with the outer membrane. Other mitochondrial proteins, including VDAC, are not required for protein efflux. However, in principle other proteins could modulate the function or membrane localization of Bax, for example by altering lipid microdomains in the outer membrane or by modifying Bax postsynthetically (Kuwana, 2002).

Signaling downstream of Bcl-2 family members

The anti-apoptosis protein Bcl-2 potently inhibits p53-dependent transcriptional activation of various p53-responsive promoters in reporter gene co-transfection assays in human embryonic kidney 293 and MCF7 cells, without affecting nuclear accumulation of p53 protein. In contrast, Bcl-2(Deltatransmembrane (TM)), which lacks a hydrophobic membrane-anchoring domain, has no effect on p53 activity. Similarly, in MCF7 cells stably expressing either Bcl-2 or Bcl-2(DeltaTM), nuclear levels of p53 protein are up-regulated upon treatment with the DNA-damaging agents doxorubicin and UV radiation, whereas p53-responsive promoter activity and expression of p21(CIP1/WAF1) are strongly reduced in MCF7-Bcl-2 cells but not in MCF7-Bcl-2(DeltaTM) or control MCF7 cells. The issue of membrane anchoring was further explored by testing the effects of Bcl-2 chimeric proteins that contained heterologous transmembrane domains from the mitochondrial protein ActA or the endoplasmic reticulum protein cytochrome b5. Both Bcl-2(ActA) and Bcl-2(Cytob5) suppresses p53-mediated transactivation of reporter gene plasmids with efficiencies comparable to wild-type Bcl-2. These results suggest that (a) Bcl-2 not only suppresses p53-mediated apoptosis but also interferes with the transcriptional activation of p53 target genes at least in some cell lines, and (b) membrane anchoring is required for this function of Bcl-2. It is speculated that membrane-anchored Bcl-2 may sequester an unknown factor necessary for p53 transcriptional activity (Froesch, 1999).

Cytolytic granule-mediated target cell killing is effected in part through the synergistic action of the membrane-acting protein perforin and serine proteases such as granzymes (Gr) A and B. In this study, the subcellular distribution of granzymes in the presence of perforin and the induction of apoptosis in mouse FDC-P1 myeloid and YAC-1 lymphoma cells that express the proto-oncogene bcl2 were examined. Using confocal laser scanning microscopy to visualize and quantitate subcellular transport of fluoresceinated granzyme, it was found that granzyme entry into the cytoplasm in the absence of perforin is not impaired in the bcl2-expressing lines. However, perforin-dependent enhancement of granzyme cellular uptake and, importantly, granzyme redistribution to the nucleus are strongly inhibited in the bcl2-expressing lines, concomitant with greatly increased resistance to granzyme/perforin-induced cell death. DNA fragmentation induced by granzyme/perforin is severely reduced in the bcl2-expressing lines, implying that prevention of granzyme nuclear translocation blocks the nuclear events of apoptosis. The kinetics of GrB nuclear uptake and induction of apoptosis are faster than for GrA, whereas YAC-1 cells show greater resistance to granzyme nuclear uptake and apoptosis than FDC-P1 cells. In all cases, granzyme nuclear accumulation in the presence of perforin correlates precisely with ensuing apoptosis. All results supported the idea that GrA and GrB share a common, specific nuclear targeting pathway that contributes significantly to the nuclear changes of apoptosis (Jans, 1999).

Apoptosis and autophagy are both tightly regulated biological processes that play a central role in tissue homeostasis, development, and disease. The anti-apoptotic protein, Bcl-2, interacts with the evolutionarily conserved autophagy protein, Beclin 1 (Coiled-coil myosin-like BCL2-interacting protein: Drosophila homolog - CG5429). However, little is known about the functional significance of this interaction. Wild-type Bcl-2 antiapoptotic proteins, but not Beclin 1 binding defective mutants of Bcl-2, inhibit Beclin 1-dependent autophagy in yeast and mammalian cells and cardiac Bcl-2 transgenic expression inhibits autophagy in mouse heart muscle. Furthermore, Beclin 1 mutants that cannot bind to Bcl-2 induce more autophagy than wild-type Beclin 1 and, unlike wild-type Beclin 1, promote cell death. Thus, Bcl-2 not only functions as an antiapoptotic protein, but also as an antiautophagy protein via its inhibitory interaction with Beclin 1. This antiautophagy function of Bcl-2 may help maintain autophagy at levels that are compatible with cell survival, rather than cell death (Pattingre, 2005).

Interaction between mutant superoxide dismutase and Bcl-2

Familial amyotrophic lateral sclerosis (ALS)-linked mutations in the copper-zinc superoxide dismutase (SOD1) gene cause motor neuron death in about 3% of ALS cases. While the wild-type (wt) protein is anti-apoptotic, mutant SOD1 promotes apoptosis. Both wt and mutant SOD1 bind the anti-apoptotic protein Bcl-2, providing evidence of a direct link between SOD1 and an apoptotic pathway. This interaction is evident in vitro and in vivo in mouse and human spinal cord. In mice and humans, Bcl-2 binds to high molecular weight SDS-resistant mutant SOD1 containing aggregates that are present in mitochondria from spinal cord but not liver. These findings provide new insights into the anti-apoptotic function of SOD1 and suggest that entrapment of Bcl-2 by large SOD1 aggregates may deplete motor neurons of this anti-apoptotic protein (Belford, 2004).

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