Pro-apoptotic cleavage products of Bcl-xL form cytochrome c-conducting pores in pure lipid membranes

During apoptotic cell death, cells usually release apoptogenic proteins such as cytochrome c from the mitochondrial intermembrane space. If Bcl-2 family proteins induce such release by increasing outer mitochondrial membrane permeability, then the pro-apoptotic, but not anti-apoptotic activity of these proteins should correlate with their permeabilization of membranes to cytochrome c. Here, we tested this hypothesis using pro-survival full-length Bcl-x(L) and pro-death Bcl-x(L) cleavage products (DeltaN61Bcl-x(L) and DeltaN76Bcl-x(L)). Unlike Bcl-x(L), DeltaN61Bcl-x(L) and DeltaN76Bcl-x(L) caused the release of cytochrome c from mitochondria in vivo and in vitro. Recombinant DeltaN61Bcl-x(L) and DeltaN76Bcl-x(L), as well as Bcl-x(L), cleaved in situ by caspase 3-possessed intrinsic pore-forming activity as demonstrated by their ability to efficiently permeabilize pure lipid vesicles. Furthermore, only DeltaN61Bcl-x(L) and DeltaN76Bcl-x(L), but not Bcl-x(L), formed pores large enough to release cytochrome c and to destabilize planar lipid bilayer membranes through reduction of pore line tension. Because Bcl-x(L) and its C-terminal cleavage products bound similarly to lipid membranes and formed oligomers of the same size, neither lipid affinity nor protein-protein interactions appear to be solely responsible for the increased membrane-perturbing activity elicited by Bcl-x(L) cleavage. Taken together, these data are consistent with the hypothesis that Bax-like proteins oligomerize to form lipid-containing pores in the outer mitochondrial membrane, thereby releasing intermembrane apoptogenic factors into the cytosol.     

Peptides corresponding to helices 5 and 6 of Bax can independently form large lipid pores

Proteins of the B-cell lymphoma protein 2 (Bcl2) family are key regulators of the apoptotic cascade, controlling the release of apoptotic factors from the mitochondrial intermembrane space. A helical hairpin found in the core of water-soluble folds of these proteins has been reported to be the pore-forming domain. Here we show that peptides including any of the two alpha-helix fragments of the hairpin of Bcl2 associated protein X (Bax) can independently induce release of large labelled dextrans from synthetic lipid vesicles. The permeability promoted by these peptides is influenced by intrinsic monolayer curvature and accompanied by fast transbilayer redistribution of lipids, supporting a toroidal pore mechanism as in the case of the full-length protein. However, compared with the pores made by complete Bax, the pores made by the Bax peptides are smaller and do not need the concerted action of tBid. These data indicate that the sequences of both fragments of the hairpin contain the principal physicochemical requirements for pore formation, showing a parallel between the permeabilization mechanism of a complex regulated protein system, such as Bax, and the much simpler pore-forming antibiotic peptides.     

Transmembrane pore formation by the carboxyl terminus of Bax protein

Bax is a cytosolic protein that responds to various apoptotic signals by binding to the outer mitochondrial membrane, resulting in membrane permeabilization, release of cytochrome c, and caspase-mediated cell death. Currently discussed mechanisms of membrane perforation include formation of hetero-oligomeric complexes of Bax with other pro-apoptotic proteins such as Bak, or membrane insertion of multiple hydrophobic helices of Bax, or formation of lipidic pores physically aided by mitochondrial membrane-inserted proteins. There is compelling evidence provided by our and other groups indicating that the C-terminal “helix 9” of Bax mediates membrane binding and pore formation, yet the mechanism of pore forming capability of Bax C-terminus remains unclear. Here we show that a 20-amino acid peptide corresponding to Bax C-terminus (VTIFVAGVLTASLTIWKKMG) and two mutants where the two lysines are replaced with glutamate or leucine have potent membrane pore forming activities in zwitterionic and anionic phospholipid membranes. Analysis of the kinetics of calcein release from lipid vesicles allows determination of rate constants of pore formation, peptide–peptide affinities within the membrane, the oligomeric state of transmembrane pores, and the importance of the lysine residues. These data provide insight into the molecular details of membrane pore formation by a Bax-derived peptide and open new opportunities for design of peptide-based cytotoxic agents.     

Mitochondrial Outer Membrane Proteins Assist Bid in Bax-mediated Lipidic Pore Formation

Mitochondrial outer membrane permeabilization (MOMP) is a critical step in apoptosis and is regulated by Bcl-2 family proteins. In vitro systems using cardiolipin-containing liposomes have demonstrated the key features of MOMP induced by Bax and cleaved Bid; however, the nature of the “pores” and how they are formed remain obscure. We found that mitochondrial outer membranes contained very little cardiolipin, far less than that required for liposome permeabilization, despite their responsiveness to Bcl-2 family proteins. Strikingly, the incorporation of isolated mitochondrial outer membrane (MOM) proteins into liposomes lacking cardiolipin conferred responsiveness to cleaved Bid and Bax. Cardiolipin dependence was observed only when permeabilization was induced with cleaved Bid but not with Bid or Bim BH3 peptide or oligomerized Bax. Therefore, we conclude that MOM proteins specifically assist cleaved Bid in Bax-mediated permeabilization. Cryoelectron microscopy of cardiolipin-liposomes revealed that cleaved Bid and Bax produced large round holes with diameters of 25–100 nm, suggestive of lipidic pores. In sum, we propose that activated Bax induces lipidic pore formation and that MOM proteins assist cleaved Bid in this process in the absence of cardiolipin.     

Interaction of hagfish cathelicidin antimicrobial peptides with model lipid membranes

Hagfish intestinal antimicrobial peptides (HFIAPs) are a family of polycationic peptides exhibiting potent, broad-spectrum bactericidal activity. In an attempt to unravel the mechanism of action of HFIAPs, we have studied their interaction with model membranes. Synthetic HFIAPs selectively bound to liposomes mimicking bacterial membranes, and caused the release of vesicle-encapsulated fluorescent markers in a size-dependent manner. In planar lipid bilayer membranes, HFIAPs induced erratic current fluctuations and reduced membrane line tension according to a general theory for lipidic pores, suggesting that HFIAP pores contain lipid molecules. Consistent with this notion, lipid transbilayer redistribution accompanied HFIAP pore formation, and membrane monolayer curvature regulated HFIAP pore formation. Based on these studies, we propose that HFIAPs kill target cells, at least in part, by interacting with their plasma membrane to induce formation of lipid-containing pores. Such a membrane-permeabilizing function appears to be an evolutionarily conserved host-defense mechanism of antimicrobial peptides.     

Protein Arcs May Form Stable Pores in Lipid Membranes

Electron microscopy and atomic force microscopy images of cholesterol-dependent cytolysins and related proteins that form large pores in lipid membranes have revealed the presence of incomplete rings, or arcs. Some evidence indicates that these arcs are inserted into the membrane and induce membrane leakage, but other experiments seem to refute that. Could such pores, only partially lined by protein, be kinetically and thermodynamically stable? How would the lipids be structured in such a pore? Using the antimicrobial peptide protegrin-1 as a model, we test the stability of pores only partially lined by peptide using all-atom molecular dynamics simulations in POPC and POPE/POPG membranes. The data show that, whereas pure lipid pores close rapidly, pores partially lined by protegrin arcs are stable for at least 300 ns. Estimates of the thermodynamic stability of these arcs using line tension data and implicit solvent calculations show that these arcs can be marginally stable in both zwitterionic and anionic membranes. Arcs provide an explanation for the observed ion selectivity in protegrin electrophysiology experiments and could possibly be involved in other membrane permeabilization processes where lipids are thought to participate, such as those induced by antimicrobial peptides and colicins, as well as the Bax apoptotic pore.     

Protein Arcs May Form Stable Pores in Lipid Membranes

Electron microscopy and atomic force microscopy images of cholesterol-dependent cytolysins and related proteins that form large pores in lipid membranes have revealed the presence of incomplete rings, or arcs. Some evidence indicates that these arcs are inserted into the membrane and induce membrane leakage, but other experiments seem to refute that. Could such pores, only partially lined by protein, be kinetically and thermodynamically stable? How would the lipids be structured in such a pore? Using the antimicrobial peptide protegrin-1 as a model, we test the stability of pores only partially lined by peptide using all-atom molecular dynamics simulations in POPC and POPE/POPG membranes. The data show that, whereas pure lipid pores close rapidly, pores partially lined by protegrin arcs are stable for at least 300 ns. Estimates of the thermodynamic stability of these arcs using line tension data and implicit solvent calculations show that these arcs can be marginally stable in both zwitterionic and anionic membranes. Arcs provide an explanation for the observed ion selectivity in protegrin electrophysiology experiments and could possibly be involved in other membrane permeabilization processes where lipids are thought to participate, such as those induced by antimicrobial peptides and colicins, as well as the Bax apoptotic pore.     

Relationships between the orientation and the structural properties of peptides and their membrane interactions

Physical properties of membranes, such as fluidity, charge or curvature influence their function. Proteins and peptides can modulate those properties and conversely, the lipids can affect the activity and/or the structure of the former. Tilted peptides are short hydrophobic protein fragments characterized by an asymmetric distribution of their hydrophobic residues when helical. They were detected in viral fusion proteins and in proteins involved in different biological processes that need membrane destabilization. Those peptides and non lamellar lipids such as PE or PA appear to cooperate in the lipid destabilization process by enhancing the formation of negatively-curved domains. Such highly bent lipidic structures could favour the formation of the viral fusion pore intermediates or that of toroidal pores. Structural flexibility appears as another crucial property for the interaction of peptides with membranes. Computational analysis on another kind of lipid-interacting peptides, i.e. cell penetrating peptides (CPP) suggests that peptides being conformationally polymorphic should be more prone to traverse the bilayer. Future investigations on the structural intrinsic properties of tilted peptides and the influence of CPP on the bilayer organization using the techniques described in this chapter should help to further understand the molecular determinants of the peptide/lipid inter-relationships.     

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.     

Pore formation by a Bax-derived peptide: effect on the line tension of the membrane probed by AFM

Bax is a critical regulator of physiological cell death that increases the permeability of the outer mitochondrial membrane and facilitates the release of the so-called apoptotic factors during apoptosis. The molecular mechanism of action is unknown, but it probably involves the formation of partially lipidic pores induced by Bax. To investigate the interaction of Bax with lipid membranes and the physical changes underlying the formation of Bax pores, we used an active peptide derived from helix 5 of this protein (Bax-alpha5) that is able to induce Bax-like pores in lipid bilayers. We report the decrease of line tension due to peptide binding both at the domain interface in phase-separated lipid bilayers and at the pore edge in atomic force microscopy film-rupture experiments. Such a decrease in line tension may be a general strategy of pore-forming peptides and proteins, as it affects the energetics of the pore and stabilizes the open state.     

Bid,Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane

Bcl-2 family proteins regulate the release of proteins like cytochrome c from mitochondria during apoptosis. We used cell-free systems and ultimately a vesicular reconstitution from defined molecules 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, activated monomeric Bax to produce membrane openings that allowed the passage of very large (2 megadalton) dextran molecules, explaining the translocation of large mitochondrial proteins during apoptosis. This process required cardiolipin and was inhibited by antiapoptotic Bcl-x(L). We conclude 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-x(L).     

Inner but Not Outer Membrane Leaflets Control the Transition from Glycosylphosphatidylinositol-anchored Influenza Hemagglutinin-induced Hemifusion to Full Fusion

Cells that express wild-type influenza hemagglutinin (HA) fully fuse to RBCs, while cells that express the HA-ectodomain anchored to membranes by glycosylphosphatidylinositol, rather than by a transmembrane domain, only hemifuse to RBCs. Amphipaths were inserted into inner and outer membrane leaflets to determine the contribution of each leaflet in the transition from hemifusion to fusion. When inserted into outer leaflets, amphipaths did not promote the transition, independent of whether the agent induces monolayers to bend outward (conferring positive spontaneous monolayer curvature) or inward (negative curvature). In contrast, when incorporated into inner leaflets, positive curvature agents led to full fusion. This suggests that fusion is completed when a lipidic fusion pore with net positive curvature is formed by the inner leaflets that compose a hemifusion diaphragm. Suboptimal fusion conditions were established for RBCs bound to cells expressing wild-type HA so that lipid but not aqueous dye spread was observed. While this is the same pattern of dye spread as in stable hemifusion, for this “stunted” fusion, lower concentrations of amphipaths in inner leaflets were required to promote transfer of aqueous dyes. Also, these amphipaths induced larger pores for stunted fusion than they generated within a stable hemifusion diaphragm. Therefore, spontaneous curvature of inner leaflets can affect formation and enlargement of fusion pores induced by HA. We propose that after the HA-ectodomain induces hemifusion, the transmembrane domain causes pore formation by conferring positive spontaneous curvature to leaflets of the hemifusion diaphragm.     

Visual and functional demonstration of growing Bax-induced pores in mitochondrial outer membranes

Bax induces mitochondrial outer membrane permeabilization (MOMP), a critical step in apoptosis in which proteins are released into the cytoplasm. To resolve aspects of the mechanism, we used cryo-electron microscopy (cryo-EM) to visualize Bax-induced pores in purified mitochondrial outer membranes (MOMs). We observed solitary pores that exhibited negative curvature at their edges. Over time, the pores grew to ∼100–160 nm in diameter after 60–90 min, with some pores measuring more than 300 nm. We confirmed these results using flow cytometry, which we used to monitor the release of fluorescent dextrans from isolated MOM vesicles. The dextran molecules were released gradually, in a manner constrained by pore size. However, the release rates were consistent over a range of dextran sizes (10–500 kDa). We concluded that the pores were not static but widened dramatically to release molecules of different sizes. Taken together, the data from cryo-EM and flow cytometry argue that Bax promotes MOMP by inducing the formation of large, growing pores through a mechanism involving membrane-curvature stress.     

PB1-F2, an influenza A virus-encoded proapoptotic mitochondrial protein, creates variably sized pores in planar lipid membranes

A frameshifted region of the influenza A virus PB1 gene encodes a novel protein, termed PB1-F2, a mitochondrial protein that can induce cell death. Many proapoptotic proteins are believed to act at the mitochondrial outer membrane to form an apoptotic pore with lipids. We studied the interaction of isolated, synthetic PB1-F2 (sPB1-F2) peptide with planar phospholipid bilayer membranes. The presence of nanomolar concentrations of peptide in the bathing solution induced a transmembrane conductance that increased in a potential-dependent manner. Positive potential on the side of protein addition resulted in a severalfold increase in the rate of change of membrane conductance. sPB1-F2-treated membranes became permeable to monovalent cations, chloride, and to a lesser extent, divalent ions. Despite various experimental conditions, we did not detect the distinctive conductance levels typical of large, stable pores, protein channels, or even pores that are partially proteinaceous. Rather, membrane conductance induced by sPB1-F2 fluctuated and visited almost all conductance values. sPB1-F2 also dramatically decreased bilayer stability in an electric field, consistent with a decrease in the line tension of a lipidic pore. Since similar membrane-destabilizing profiles are seen with proapoptotic proteins (e.g., Bax) and the cytoplasmic helix of human immunodeficiency virus gp41, we suggest that the basis for sPB1-F2-induced cell death may be the permeabilization and destabilization of mitochondrial membranes, leading to macromolecular leakage and apoptosis.     

Effects of Spontaneous Bilayer Curvature on Influenza Virus–mediated Fusion Pores

Cells expressing the hemagglutinin protein of influenza virus were fused to planar bilayer membranes containing the fluorescent lipid probes octadecylrhodamine (R18) or indocarbocyanine (DiI) to investigate whether spontaneous curvature of each monolayer of a target membrane affects the growth of fusion pores. R18 and DiI lowered the transition temperatures for formation of an inverted hexagonal phase, indicating that these probes facilitate the formation of negative curvature structures. The probes are known to translocate from one monolayer of a bilayer membrane to the other in a voltage-dependent manner. The spontaneous curvature of the cis monolayer (facing the cells) or the trans monolayer could therefore be made more negative through control of the polarity of voltage across the planar membrane. Electrical admittance measurements showed that the open times of flickering fusion pores were shorter when probes were in trans monolayers and longer when in cis monolayers compared with times when probe was symmetrically distributed. Open times were the same for probe symmetrically distributed as when probes were not present. Thus, open times were a function of the asymmetry of the spontaneous curvature between the trans and cis monolayers. Enriching the cis monolayer with a negative curvature probe reduced the probability that a small pore would fully enlarge, whereas enriching the trans monolayer promoted enlargement. Lysophosphatidylcholine has positive spontaneous curvature and does not translocate. When lysophosphatidylcholine was placed in trans leaflets of planar membranes, closing of fusion pores was rare. The effects of the negative and positive spontaneous curvature probes do not support the hypothesis that a flickering pore closes from an open state within a hemifusion diaphragm (essentially a “flat” structure). Rather, such effects support the hypothesis that the membrane surrounding the open pore forms a three-dimensional hourglass shape from which the pore flickers shut.     

The Bak core dimer focuses triacylglycerides in the membrane

Apoptosis, the intrinsic programmed cell death process, is mediated by the Bcl-2 family members Bak and Bax. Activation via formation of symmetric core dimers and oligomerization on the mitochondrial outer membrane (MOM) leads to permeabilization and cell death. Although this process is linked to the MOM, the role of the membrane in facilitating such pores is poorly understood. We recently described Bak core domain dimers, revealing lipid binding sites and an initial role of lipids in oligomerization. Here we describe simulations that identified localized clustering and interaction of triacylglycerides (TAGs) with a minimized Bak dimer construct. Coalescence of TAGs occurred beneath this Bak dimer, mitigating dimer-induced local membrane thinning and curvature in representative coarse-grain MOM and model membrane systems. Furthermore, the effects observed as a result of coarse-grain TAG cluster formation was concentration dependent, scaling from low physiological MOM concentrations to those found in other organelles. We find that increasing the TAG concentration in liposomes mimicking the MOM decreased the ability of activated Bak to permeabilize these liposomes. These results suggest that the presence of TAGs within a Bak-lipid membrane preserves membrane integrity and is associated with reduced membrane stress, suggesting a possible role of TAGs in Bak-mediated apoptosis.     

A tale of two mitochondrial channels,MAC and PTP,in apoptosis

The crucial step in the intrinsic, or mitochondrial, apoptotic pathway is permeabilization of the mitochondrial outer membrane. Permeabilization triggers release of apoptogenic factors, such as cytochrome c, from the mitochondrial intermembrane space into the cytosol where these factors ensure propagation of the apoptotic cascade and execution of cell death. However, the mechanism(s) underlying permeabilization of the outer membrane remain controversial. Two mechanisms, involving opening of two different mitochondrial channels, have been proposed to be responsible for the permeabilization; the permeability transition pore (PTP) in the inner membrane and the mitochondrial apoptosis-induced channel (MAC) in the outer membrane. Opening of PTP would lead to matrix swelling, subsequent rupture of the outer membrane, and an unspecific release of intermembrane proteins into the cytosol. However, many believe PTP opening is a consequence of apoptosis and this channel is thought to principally play a role in necrosis, not apoptosis. Activation of MAC is exquisitely regulated by Bcl-2 family proteins, which are the sentinels of apoptosis. MAC provides specific pores in the outer membrane for the passage of intermembrane proteins, in particular cytochrome c, to the cytosol. The electrophysiological characteristics of MAC are very similar to Bax channels and depletion of Bax significantly diminishes MAC activity, suggesting that Bax is an essential constituent of MAC in some systems. The characteristics of various mitochondrial channels and Bax are compared. The involvement of MAC and PTP activities in apoptosis of disease and their pharmacology are discussed.     

Syringomycin E channel: a lipidic pore stabilized by lipopeptide?

Highly reproducible ion channels of the lipopeptide antibiotic syringomycin E demonstrate unprecedented involvement of the host bilayer lipids. We find that in addition to a pronounced influence of lipid species on the open-channel ionic conductance, the membrane lipids play a crucial role in channel gating. The effective gating charge, which characterizes sensitivity of the conformational equilibrium of the syringomycin E channels to the transmembrane voltage, is modified by the lipid charge and lipid dipolar moment. We show that the type of host lipid determines not only the absolute value but also the sign of the gating charge. With negatively charged bilayers, the gating charge sign inverts with increased salt concentration or decreased pH. We also demonstrate that the replacement of lamellar lipid by nonlamellar with the negative spontaneous curvature inhibits channel formation. These observations suggest that the asymmetric channel directly incorporates lipids. The charges and dipoles resulting from the structural inclusion of lipids are important determinants of the overall energetics that underlies channel gating. We conclude that the syringomycin E channel may serve as a biophysical model to link studies of ion channels with those of lipidic pores in membrane fusion.     

Bax assembles into large ring‐like structures remodeling the mitochondrial outer membrane in apoptosis

The Bcl‐2 family proteins Bax and Bak are essential for the execution of many apoptotic programs. During apoptosis, Bax translocates to the mitochondria and mediates the permeabilization of the outer membrane, thereby facilitating the release of pro‐apoptotic proteins. Yet the mechanistic details of the Bax‐induced membrane permeabilization have so far remained elusive. Here, we demonstrate that activated Bax molecules, besides forming large and compact clusters, also assemble, potentially with other proteins including Bak, into ring‐like structures in the mitochondrial outer membrane. STED nanoscopy indicates that the area enclosed by a Bax ring is devoid of mitochondrial outer membrane proteins such as Tom20, Tom22, and Sam50. This strongly supports the view that the Bax rings surround an opening required for mitochondrial outer membrane permeabilization (MOMP). Even though these Bax assemblies may be necessary for MOMP, we demonstrate that at least in Drp1 knockdown cells, these assemblies are not sufficient for full cytochrome c release. Together, our super‐resolution data provide direct evidence in support of large Bax‐delineated pores in the mitochondrial outer membrane as being crucial for Bax‐mediated MOMP in cells.     

Electrophysiological study of a novel large pore formed by Bax and the voltage-dependent anion channel that is permeable to cytochrome c

The Bcl-2 family of proteins, consisting of anti-apoptotic and pro-apoptotic members, regulates cell death by controlling mitochondrial membrane permeability that is crucial for apoptotic signal transduction. We have recently shown that some of these proteins, such as Bcl-x(L), Bax, and Bak, directly modulate the mitochondrial voltage-dependent anion channel (VDAC) and thus regulate apoptogenic cytochrome c release and potential loss. To elucidate the molecular mechanisms of VDAC regulation by Bcl-2 family proteins, an electrophysiological study was carried out. It was found that VDAC and pro-apoptotic Bax created a large pore, with conductance levels 4- and 10-fold greater than those of the VDAC and Bax channels, respectively. Although the VDAC and Bax channels both show ion selectivity and voltage-dependent modulation of their activity, the VDAC-Bax channel had neither of their properties. Anti-apoptotic Bcl-x(L) and its BH4 oligopeptide completely closed the VDAC, in contrast to the Bax. Cytochrome c passed through a single VDAC-Bax channel but not through the VDAC or Bax channel in a planar lipid bilayer. These data provide direct evidence that VDAC forms a novel large pore together with Bax.