Bcl-XL Inhibits Membrane Permeabilization by Competing with Bax

Although Bcl-XL and Bax are structurally similar, activated Bax forms large oligomers that permeabilize the outer mitochondrial membrane, thereby committing cells to apoptosis, whereas Bcl-XL inhibits this process. Two different models of Bcl-XL function have been proposed. In one, Bcl-XL binds to an activator, thereby preventing Bax activation. In the other, Bcl-XL binds directly to activated Bax. It has been difficult to sort out which interaction is important in cells, as all three proteins are present simultaneously. We examined the mechanism of Bax activation by tBid and its inhibition by Bcl-XL using full-length recombinant proteins and measuring permeabilization of liposomes and mitochondria in vitro. Our results demonstrate that Bcl-XL and Bax are functionally similar. Neither protein bound to membranes alone. However, the addition of tBid recruited molar excesses of either protein to membranes, indicating that tBid activates both pro- and antiapoptotic members of the Bcl-2 family. Bcl-XL competes with Bax for the activation of soluble, monomeric Bax through interaction with membranes, tBid, or t-Bid-activated Bax, thereby inhibiting Bax binding to membranes, oligomerization, and membrane permeabilization. Experiments in which individual interactions were abolished by mutagenesis indicate that both Bcl-XL–tBid and Bcl-XL–Bax binding contribute to the antiapoptotic function of Bcl-XL. By out-competing Bax for the interactions leading to membrane permeabilization, Bcl-XL ties up both tBid and Bax in nonproductive interactions and inhibits Bax binding to membranes. We propose that because Bcl-XL does not oligomerize it functions like a dominant-negative Bax in the membrane permeabilization process.     

Mitochondrial membrane permeabilization produced by PTP, Bax and apoptosis: a 1H-NMR relaxation study

To analyze the involvement of structured water (bound to macromolecules) in apoptosis-induced mitochondrial outer-membrane permeability, we compared the dynamics of water protons from nuclear magnetic resonance (NMR) data in apoptotic liver mitochondria with that of control mitochondria incubated in vitro with free Ca(2+) (opening of the permeability transition pore, PTP) or with Bax alpha. Our results demonstrate that water molecules in apoptotic mitochondria exhibit an accelerated translational motion of structured water common with that induced by the opening of the PTP, but limited in amplitude. On the other hand, no significant quantitative change in structured water was observed in apoptotic mitochondria, a phenomenon also observed with Bax alpha-induced permeability. We conclude that the changes observed in the different water phases differ both quantitatively and qualitatively during the opening of the PTP and the Bax alpha-induced permeability, and that the apoptotic mitochondria exhibit mixed properties between these model situations.     

Bax, Bid and the permeabilization of the mitochondrial outer membrane in apoptosis

Mitochondria provide a key amplification step in the apoptotic pathway of many cells by releasing apoptogenic proteins into the cytosol. Recent studies have provided insights into how Bax and Bid may operate synergistically to recruit mitochondria into the pathway and how GD3 ganglioside, a metabolite of the sphingomyelin pathway, may also be used. In ischaemic disease, activation of the mitochondrial permeability transition pore may bypass the requirement for these factors.     

PUMA Promotes Bax Translocation by Both Directly Interacting with Bax and by Competitive Binding to Bcl-XL during UV-induced Apoptosis

Cell apoptosis induced by UV irradiation is a highly complex process in which different molecular signaling pathways are involved. p53 up-regulated modulator of apoptosis (PUMA) has been proposed as an important regulator in UV irradiation-induced apoptosis. However, the molecular mechanism through which PUMA regulates apoptosis, especially how PUMA activates Bcl-2-associated X protein (Bax) in response to UV irradiation is still controversial. In this study, by using real-time single-cell analysis and fluorescence resonance energy transfer, we investigated the tripartite nexus among PUMA, Bax, and Bcl-X_L in living human lung adenocarcinoma cells (ASTC-a-1) to illustrate how PUMA promotes Bax translocation to initiate apoptosis. Our results show that the interaction between PUMA and Bax increased gradually, with Bax translocating to mitochondria and colocalizing with PUMA after UV irradiation, indicating PUMA promotes Bax translocation directly. Simultaneously, the interaction increased markedly between PUMA and Bcl-X_L and decreased significantly between Bcl-X_L and Bax after UV treatment, suggesting PUMA competitively binds to Bcl-X_L to activate Bax indirectly. The above-mentioned results were further confirmed by coimmunoprecipitation experiments. In addition, pifithrin-α (a p53 inhibitor) and cycloheximide (a protein synthesis inhibitor) could inhibit PUMA-mediated Bax translocation and cell apoptosis. Together, these studies create an important conclusion that PUMA promotes Bax translocation by both by directly interacting with Bax and by competitive binding to Bcl-X_L in UV-induced apoptosis.     

Clusterin inhibits apoptosis by interacting with activated Bax

Clusterin is an enigmatic glycoprotein that is overexpressed in several human cancers such as prostate and breast cancers, and squamous cell carcinoma. Because the suppression of clusterin expression renders human cancer cells sensitive to chemotherapeutic drug-mediated apoptosis, it is currently an antisense target in clinical trials for prostate cancer. However, the molecular mechanisms by which clusterin inhibits apoptosis in human cancer cells are unknown. Here we report that intracellular clusterin inhibits apoptosis by interfering with Bax activation in mitochondria. Intriguingly, in contrast to other inhibitors of Bax, clusterin specifically interacts with conformation-altered Bax in response to chemotherapeutic drugs. This interaction impedes Bax oligomerization, which leads to the release of cytochrome c from mitochondria and caspase activation. Moreover, we also find that clusterin inhibits oncogenic c-Myc-mediated apoptosis by interacting with conformation-altered Bax. Clusterin promotes c-Myc-mediated transformation in vitro and tumour progression in vivo. Taken together, our results suggest that the elevated level of clusterin in human cancers may promote oncogenic transformation and tumour progression by interfering with Bax pro-apoptotic activities.     

Mitochondrial membrane permeabilization by HIV-1 Vpr

The mitochondrion is a privileged target for apoptosis-modulatory proteins of viral origin. Thus, viral protein R (Vpr) can target mitochondria and induce apoptosis via a specific interaction with the permeability transition pore complex (PTPC). Vpr cooperates with the adenine nucleotide translocator (ANT) to form large conductance channels and to trigger all the hallmarks of mitochondrial membrane permeabilization (MMP). The Vpr/ANT interaction is direct, since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT, ADP, ATP, or by Bcl-2. Accordingly, Vpr modulates MMP through direct structural and functional interactions with PTPC proteins.     

RACK1 promotes Bax oligomerization and dissociates the interaction of Bax and Bcl-XL

Bax, a member of Bcl-2 family, plays an essential role in apoptotic pathways induced by a number of apoptotic stimulus. In a search for new potential binding partners of Bax, we identified the receptor for activated C-kinase 1 (RACK1) by a yeast two-hybrid assay. We demonstrated that RACK1 interacts with Bax through its BH3 domain both in vitro and in vivo. Using immunostaining and immunoprecipitation experiments, we found that RACK1 colocalizes with Bax oligomers and promotes Bax oligomerization both in vitro and in vivo. Furthermore, we observed that RACK1 also interacts with Bcl-XL, an anti-apoptotic protein associated with Bax. Interestingly, the Bcl-XL/Bax interaction is decreased when RACK1 is overexpressed, but is increased when RACK1 is depleted, suggesting RACK1 disrupts the association of Bax and Bcl-XL. In addition, we found that overexpression of RACK1 promotes UV-induced apoptosis, while knocking down RACK1 inhibits the effects. Together, these results indicate that RACK1 promotes apoptosis by promoting Bax oligomerization and dissociating the complex of Bax and Bcl-XL.     

Bcl-XL protects BimEL-induced Bax conformational change and cytochrome C release independent of interacting with Bax or BimEL

The Bcl-2 homology (BH) 3-only pro-apoptotic Bcl-2 family protein Bim plays an essential role in the mitochondrial pathway of apoptosis through activation of the BH1-3 multidomain protein Bax or Bak. To further understand how the BH3-only protein activates Bax, we provide evidence here that BimEL induces Bax conformational change and apoptosis through a Bcl-XL-suppressible but heterodimerization-independent mechanism. Substitution of the conserved leucine residue in the BH3 domain of BimEL for alanine (M1) inhibits the interaction of BimEL with Bcl-XL but does not abolish the ability of BimEL to induce Bax conformational change and apoptosis. However, removal of the C-terminal hydrophobic region from the M1 mutant (M1DeltaC) abolishes its ability to activate Bax and to induce apoptosis, although deletion of the C-terminal domain (DeltaC) alone has little if any effect on the pro-apoptotic activity of BimEL. Subcellular fractionation experiments show that the Bim mutant M1DeltaC is localized in the cytosol, indicating that both the C-terminal hydrophobic region and the BH3 domain are required for the mitochondrial targeting and pro-apoptotic activity of BimEL. Moreover, the Bcl-XL mutant (mt1), which is unable to interact with Bax and BimEL, blocks Bax conformational change and cytochrome c release induced by BimEL in intact cells and isolated mitochondria. BimEL or Bak-BH3 peptide induces Bax conformational change in vitro only under the presence of mitochondria, and the outer mitochondrial membrane fraction is sufficient for induction of Bax conformational change. Interestingly, native Bax is attached loosely on the surface of isolated mitochondria, which undergoes conformational change and insertion into mitochondrial membrane upon stimulation by BimEL, Bak-BH3 peptide, or freeze/thaw damage. Taken together, these findings indicate that BimEL may activate Bax by damaging the mitochondrial membrane structure directly, in addition to its binding and antagonizing Bcl-2/Bcl-XL function.     

Zinc inhibits calcineurin activity in vitro by competing with nickel

Calcineurin (CN) is a Ca(2+)/calmodulin (CaM)-dependent protein serine/threonine phosphatase that contains Zn(2+) in its catalytic domain and can be stimulated by divalent ions such as Mn(2+) and Ni(2+). In this study, the role of exogenous Zn(2+) in the regulation of CN activity and its relevance to the role of Ni(2+) was investigated. Zn(2+) at a concentration range of 10nM-10 micro M inhibited Ni(2+)-stimulated CN-activity in vitro in a dose-dependent manner and approximately 50% inhibition was attained with 0.25 micro M Zn(2+). Kinetic analysis showed that Zn(2+) inhibited the activity of CN by competing with Ni(2+). Interaction of CN and CaM was not inhibited with Zn(2+) at 10 micro M. Zn(2+) never affected the activity of cAMP phosphodiesterase 1 or myosin light-chain kinase (CaM-dependent enzymes) and rather activated alkaline phosphatase. The present results indicate that Zn(2+) should be a potent inhibitor for CN activity although this ion is essential for CN.     

An antisense RNA inhibits translation by competing with standby ribosomes

Most antisense RNAs in bacteria inhibit translation by competing with ribosomes for translation initiation regions (TIRs) on nascent mRNA. We propose a mechanism by which an antisense RNA inhibits translation without binding directly to a TIR. The tisAB locus encodes an SOS-induced toxin, and IstR-1 is the antisense RNA that counteracts toxicity. We show that full-length tisAB mRNA (+1) is translationally inactive and endonucleolytic processing produces an active mRNA (+42). IstR-1 binding inhibits translation of this mRNA, and subsequent RNase III cleavage generates a truncated, inactive mRNA (+106). In vitro translation, toeprinting, and structure mapping suggest that active, but not inactive, tisAB mRNAs contain an upstream ribosome loading or "standby" site. Standby binding is required for initiation at the highly structured tisB TIR. This may involve ribosome sliding to a transiently open tisB TIR. IstR-1 competes with ribosomes by base pairing to the standby site located approximately 100 nucleotides upstream.     

Control of electric field induced cell membrane permeabilization by membrane order

Cells can be made temporarily permeable if pulsed by high-intensity short-duration electric fields. The molecular mechanisms underlying this electropermeabilization are still unknown. The kinetic events may be described by four successive steps: induction, expansion, stabilization, and resealing. On one hand, cell electropermeabilization is detected only under more stringent conditions when cells have been treated by ethanol. On the other hand, lysolecithin is observed to facilitate cell electropermeabilization. More precisely, these molecules that modify membrane order, when used in concentrations compatible with cell viability, are shown to affect only the expansion and resealing steps. Electropermeabilization is inducing a transition in the membrane organization. Membrane order is modulating the energy barrier needed to evoke this membrane transition which occurs when cells are submitted to a field larger than a characteristic threshold (expansion step). Less order would increase the magnitude of this energy barrier; more order would decrease it.     

Bax inhibition protects against free fatty acid-induced lysosomal permeabilization

Lysosomal permeabilization is a key feature of hepatocyte lipotoxicity, yet the mechanisms mediating this critical cellular event are unclear. This study examined the mechanisms involved in free fatty acid (FFA)-induced lysosomal permeabilization and the role of Bax, a Bcl-2 family member, in this event. Exposure of liver cells to palmitate induced Bax activation and translocation to lysosomes. Studies to suppress Bax activation either by pharmacological approaches or small interfering-RNA-mediated inhibition of Bax expression showed that lysosomal permeabilization is Bax dependent. In addition, palmitate treatment resulted in a significant decrease in Bcl-X(L), a Bax antagonist. Moreover, forced Bcl-X(L) expression blocked lysosomal permeabilization. Lysosomal permeabilization by FFA was ceramide and caspase independent. Finally, paradigms that inhibit lysosomal permeabilization also reduced apoptosis. In conclusion, these data strongly support a regulatory role for Bax in FFA-mediated lysosomal permeabilization and subsequent cell death.     

The murine cytomegalovirus cell death suppressor m38.5 binds Bax and blocks Bax-mediated mitochondrial outer membrane permeabilization

Apoptosis is increasingly implicated as an early line of defense against viral infections. Viruses have devised numerous strategies to delay apoptosis of infected cells. Many viruses encode cell death suppressors that target mitochondrial apoptotic signaling pathway, indicating the importance of this pathway in the anti-viral response. Human and primate cytomegaloviruses encode the viral mitochondria-localized inhibitor of apoptosis vMIA, but no overt homologue of vMIA was identified in any non-primate cytomegalovirus. Here we report that m38.5 protein encoded by murine cytomegalovirus, which is unrelated to vMIA in its amino acid sequence, delays death receptor ligation-induced cell death, and that m38.5 associates with Bax, recruits it to mitochondria, and blocks Bax-mediated but not Bak-mediated mitochondrial outer membrane permeabilization. Thus, primate and murine cytomegaloviruses have evolved non-homologous but functionally similar cell death suppressors selectively targeting the Bax-mediated branch of the mitochondrial apoptotic signaling pathway, indicating the importance of this branch in the response of diverse host organisms against cytomegalovirus infections.     

Roles of integral protein in membrane permeabilization by amphidinols

Amphidinols (AMs) are a group of dinoflagellate metabolites with potent antifungal activity. As is the case with polyene macrolide antibiotics, the mode of action of AMs is accounted for by direct interaction with lipid bilayers, which leads to formation of pores or lesions in biomembranes. However, it was revealed that AMs induce hemolysis with significantly lower concentrations than those necessary to permeabilize artificial liposomes, suggesting that a certain factor(s) in erythrocyte membrane potentiates AM activity. Glycophorin A (GpA), a major erythrocyte protein, was chosen as a model protein to investigate interaction between peptides and AMs such as AM2, AM3 and AM6 by using SDS-PAGE, surface plasmon resonance, and fluorescent-dye leakages from GpA-reconstituted liposomes. The results unambiguously demonstrated that AMs have an affinity to the transmembrane domain of GpA, and their membrane-permeabilizing activity is significantly potentiated by GpA. Surface plasmon resonance experiments revealed that their interaction has a dissociation constant of the order of 10 microM, which is significantly larger than efficacious concentrations of hemolysis by AMs. These results imply that the potentiation action by GpA or membrane integral peptides may be due to a higher affinity of AMs to protein-containing membranes than that to pure lipid bilayers.     

Roles of integral protein in membrane permeabilization by amphidinols

Amphidinols (AMs) are a group of dinoflagellate metabolites with potent antifungal activity. As is the case with polyene macrolide antibiotics, the mode of action of AMs is accounted for by direct interaction with lipid bilayers, which leads to formation of pores or lesions in biomembranes. However, it was revealed that AMs induce hemolysis with significantly lower concentrations than those necessary to permeabilize artificial liposomes, suggesting that a certain factor(s) in erythrocyte membrane potentiates AM activity. Glycophorin A (GpA), a major erythrocyte protein, was chosen as a model protein to investigate interaction between peptides and AMs such as AM2, AM3 and AM6 by using SDS-PAGE, surface plasmon resonance, and fluorescent-dye leakages from GpA-reconstituted liposomes. The results unambiguously demonstrated that AMs have an affinity to the transmembrane domain of GpA, and their membrane-permeabilizing activity is significantly potentiated by GpA. Surface plasmon resonance experiments revealed that their interaction has a dissociation constant of the order of 10 μM, which is significantly larger than efficacious concentrations of hemolysis by AMs. These results imply that the potentiation action by GpA or membrane integral peptides may be due to a higher affinity of AMs to protein-containing membranes than that to pure lipid bilayers.     

Mechanisms of membrane permeabilization by picornavirus 2B viroporin

Cell infection by picornaviruses leads to membrane permeabilization. Recent evidence suggests the involvement of the non-structural protein 2B in this process. We have recently reported the detection of 2B porin-like activity in isolated membrane-protein systems that lack other cell components. According to data derived from these model membranes, four self-aggregated 2B monomers (i.e. tetramers) would be sufficient to permeabilize a single lipid vesicle, allowing the free diffusion of solutes under ca. 1000 Da. Our findings also support a role for lipids in protein oligomerization and subsequent pore opening. The lipid dependence of these processes points to negatively charged cytofacial surfaces as 2B cell membrane targets.     

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.     

Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization

Mitochondrial fusion and division play important roles in the regulation of apoptosis. Mitochondrial fusion proteins attenuate apoptosis by inhibiting release of cytochrome c from mitochondria, in part by controlling cristae structures. Mitochondrial division promotes apoptosis by an unknown mechanism. We addressed how division proteins regulate apoptosis using inhibitors of mitochondrial division identified in a chemical screen. The most efficacious inhibitor, mdivi-1 (for mitochondrial division inhibitor) attenuates mitochondrial division in yeast and mammalian cells by selectively inhibiting the mitochondrial division dynamin. In cells, mdivi-1 retards apoptosis by inhibiting mitochondrial outer membrane permeabilization. In vitro, mdivi-1 potently blocks Bid-activated Bax/Bak-dependent cytochrome c release from mitochondria. These data indicate the mitochondrial division dynamin directly regulates mitochondrial outer membrane permeabilization independent of Drp1-mediated division. Our findings raise the interesting possibility that mdivi-1 represents a class of therapeutics for stroke, myocardial infarction, and neurodegenerative diseases.