1.A.21 The Bcl-2 (Bcl-2) Family

The Bcl-2 family consists of the apoptosis regulator Bcl-X and its homologues (Adams and Corey 1998). Bcl-X is a dominant regulator of programmed cell death in mammalian cells (Boise et al. 1993; Tsujimoto and Shimizu 2000). The long form (Bcl-X(L), displays cell death repressor activity but the short isoform (Bcl-X(S)) and the β-isoform (Bcl-Xβ) promote cell death. Bcl-X(L), Bcl-X(S) and Bcl-Xβ are three isoforms derived by alternative RNA splicing. Bcl-X(S) forms heterodimers with Bcl-2. Interaction of Bcl-2 with the outer mitochondrial membrane protein, voltage-dependent anion channel 1 (VDAC1) or with peptides derived from VDAC3 protects against cell death by inhibiting cytochrome c release. A direct interaction of Bcl-2 with bilayer-reconstituted purified VDAC was demonstrated, with Bcl-2 decreasing channel conductance (Arbel and Shoshan-Barmatz, 2010). Bcl-2 family members are integrators of survival and death signals (Schinzel et al. 2004). 

A novel type of peptide-induced pore is not necessarily framed by a peptide structure (Qian et al., 2008). Peptide-induced pores in multiple bilayers form a periodically ordered lattice as analyzed by X-ray diffraction. The pores induced by Bax-derived helical peptides were at least partially framed by a lipid monolayer. Evidence suggested that the formation of such lipidic pores is a major mechanism for alpha-pore-forming proteins, including apoptosis-regulator Bax (Bcl2-associated X protein) (Qian et al., 2008).  The ER-mitochondrion interface is a critical cell-signaling junction whereby Bcl-xL dynamically interacts with type 3 inositol 1,4,5-trisphosphate receptors (IP3R3) to coordinate mitochondrial Ca²⁺ transfer and alters cellular metabolism in order to increase the cells' bioenergetic capacity, particularly during periods of stress (Williams et al. 2016). Allosteric cross-talk between the hydrophobic cleft and the BH4 domain of Bcl-2 controls inositol 1,4,5-trisphosphate receptor activity (Shapovalov et al. 2022).

The high resolution structure of the monomeric soluble form of human Bcl-X(L) has been determined by both x-ray crystallography and NMR (Muchmore et al. 1996). The protein contains four domains Bcl-2 homology (BH) domains 1-4. The structure consists of two central primarily hydrophobic α-helices surrounded by amphipathic helices. The three functionally important Bcl-2 homology regions (BH1 BH2 and BH3) are in close spatial proximity. They form an elongated cleft that may provide the binding site for other Bcl-2 family members. The arrangement of the α-helices in Bcl-X(L) resembles that for diphtheria toxin (TC #1.C.7) and the colicins (TC #1.C.1). Diphtheria toxin forms a transmembrane pore and translocates the toxic catalytic domain into the animal cell cytoplasm. The colicins similarly form pores in lipid bilayers. Structural homology therefore suggests that Bcl-2 family members that contain the BH1 and BH2 domains (Bcl-X(L) Bcl-2 and Bax) function similarly.

These proteins are localized to the outer mitochondrial membrane of the animal cell where they are thought to form a complex with the voltage-dependent anion channel porin (VDAC; TC #1.B.8). Bcl-2 has been shown to regulate release of apoptogenic cytochrome c by promoting open channel formation by VDAC in response to cell death signals. Proteins of the Bcl-2 family are also present in the perinuclear envelope and are widely distributed in many body tissues. Their ability to form oligomeric pores in artificial lipid bilayers has been documented but the physiological significance of pore formation is not clear. Each of these proteins has distinctiveproperties including some degree of ion selectivity (Antonsson et al. 2000).

Bcl-X(L) is 233 amino acyl residues long and exhibits a single very hydrophobic putative transmembrane α-helical segment (residues 210-226) when in the membrane. A large conformational change analogous to that observed when the soluble protein encounters a lipid bilayer occurs when the protein is exposed to certain detergents. These may cause the internally buried putative TMS in the soluble form to become exposed initiating membrane insertion under normal physiological conditions. Homologues of Bcl-X include the Bax (rat; 192 aas; spQ63690) and Bak (mouse; 208 aas; spO08734) proteins which also influence apoptosis. Bcl-2 and Bcl-X(L) inhibit the NLRP1 inflammasome by loop domain-dependent suppression of ATP binding and oligomerization. Anti-apoptotic proteins Bcl-2 and Bcl-X(L) bind to and inhibit NLRP1 in cells. Bcl-2 and Bcl-X(L) inhibited caspase-1 activation induced by NLRP1 with a K(i) of approximately 10 nM. Bcl-2 and Bcl-X(L) also inhibit ATP binding to NLRP1, which is required for oligomerization of NLRP1, and Bcl-X(L) interferes with NLRP1 oligomerization. Deletion of the flexible loop regions of Bcl-2 and Bcl-X(L), which are located between the first and second alpha-helices of these anti-apoptotic proteins and which were previously shown to be required for binding NLRP1, abrogated the ability to inhibit caspase-1 activation, ATP binding and oligomerization of NLRP1 (Faustin et al., 2009).

Using isolated mitochondria, recombinant Bax and Bak have been shown to induce dye loss, swelling and cytochrome c release (Narita et al., 1998). All of these changes are dependent on Ca²⁺ and are prevented by cyclosporin A and bongkrekic acid, both of which are known to close permeability transition pores (megachannels). Coimmunoprecipitation studies revealed that Bax and Bak interact with VDAC to form permeability transition pores. Thus, even though they can form channels in artificial membranes at acidic pH, proapoptotic Bcl-2 family proteins (including Bax and Bak) probably induce the mitochondrial permeability transition and cytochrome c release by interacting with permeability transition pores, the most important component for pore fomation of which is VDAC (Shimizu et al., 1999). Willis et al. (2007) showed that Bax and Bak can mediate apoptosis without discernable association with the putative BH3-only activators (Bim, Bid, and Puma), even in cells with no Bim or Bid and reduced Puma. BH3-only proteins induce apoptosis primarily by engaging the multiple pro-survival relatives guarding Bax and Bak.

Kuwana et al. (2002) used cell-free systems and vesicular reconstitution from defined molecules to show that pore formation requires only Bid or its BH3-domain peptide, activated monomeric Bax, to produce large pores that allow passsage of 2 megadalton dextran molecules. The process required cardiolipin and was inhibited by the anti-apoptotic Bcl-X_L (Kuwana et al., 2002). Thus, this report suggests that VDAC as well as other matrix and membrane mitochondrial proteins are not required for induction of apoptotic channel formation.

Early in mitochondria-mediated apoptosis, the mitochondrial outer membrane becomes permeable to proteins that, when released into the cytosol, initiate the execution phase of apoptosis. Proteins in the Bcl-2 family regulate this permeabilization, but the molecular composition of the mitochondrial outer membrane pore is under debate. Ceramides form stable channels (1.D.10) in mitochondrial outer membranes capable of passing the largest proteins known to exit mitochondria during apoptosis (Siskind et al., 2006). Bcl-2 proteins are not required for ceramide to form protein-permeable channels, but both recombinant human Bcl-x(L) and CED-9, the Caenorhabditis elegans Bcl-2 homologue, disassemble ceramide channels in the mitochondrial outer membranes. Bcl-x(L) and CED-9 disassemble ceramide channels in solvent-free planar phospholipid membranes. Thus, ceramide channel disassembly may result from direct interaction with these anti-apoptotic proteins (Siskind et al., 2008). Thus, ceramide channels may be one mechanism for releasing proteins from mitochondria during the induction phase of apoptosis.

BID, a proapoptotic BCL-2 family member, plays an essential role in the tumor necrosis factor alpha (TNF-alpha)/Fas death receptor pathway in vivo. Activation of the TNF-R1 receptor results in the cleavage of BID into truncated BID (tBID), which translocates to the mitochondria and induces the activation of BAX or BAK. In TNF-alpha-activated FL5.12 cells, tBID becomes part of a 45-kDa cross-linkable mitochondrial complex. Grinberg et al. (2005) described the biochemical purification of this complex and the identification of mitochondrial carrier homolog 2 (Mtch2; TC# 2.A.29.25.2) as part of this complex. Mtch2 is similar to members of the mitochondrial carrier family. Mtch2 is an integral outer membrane protein exposed on the surface of mitochondria. Mtch2 resides in a protein complex of ca. 185 kDa, and the addition of TNF-alpha to these cells leads to the recruitment of tBID and BAX to this complex. Thus, Mtch2 is a mitochondrial target of tBID. The Mtch2-resident complex probably participates in the mitochondrial apoptotic program (Grinberg et al., 2005; Gross, 2005).

Bax/Bak-dependent mitochondrial outer membrane permeabilization (MOMP) represents a central apoptotic event primarily controlled by Bcl-2 family proteins.  Expression of active Bax/Bak in bacteria, the putative origin of mitochondria, has revealed  functional similarities to the λ bacteriophage (λ) holin (Pang et al. 2011). As critical effectors for bacterial lysis, holin oligomers form membrane lesions, through which endolysins, muralytic enzymes, escape the cytoplasm to attack the cell wall at the end of the infection cycle. Active Bax/Bak, but not other Bcl-2 family proteins tested, displays holin behavior, causing bacterial lysis by releasing endolysin in an oligomerization-dependent manner. Replacing the holin gene with active alleles of Bax/Bak results in plaque-forming phages. Active Bax produces large membrane holes, the size of which are controlled by structural elements in the protein. Notably, lysis by active Bax is inhibited by Bcl-xL, and the lysis activity of the wild-type Bax is stimulated by a BH3-only protein. Together, these results mechanistically link MOMP to holin-mediated hole formation in the bacterial plasma membrane.

In healthy cells, Bax is largely cytosolic, but it translocates to the mitochondrial outer membrane (MOM) when cells receive an apoptotic stimulus. Like the prosurvival Bcl-2 proteins, cytosolic Bax comprises a globular bundle of nine α helices. The last hydrophobic helix (α9) may regulate Bax activity, as it either anchors Bax in the MOM or resides in a hydrophobic groove on the surface of cytosolic Bax. The homologous groove on the prosurvival proteins, comprising mainly α2 through α5, is the canonical binding site for BH3 domains from both BH3-only proteins and Bax or Bak (Czabotar et al. 2011).

In stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the MOM. The resulting release of cytochrome c and other proapoptotic proteins initiates a proteolytic cascade that ensures the cell's demise. Certain BH3-only relatives directly activate them to mediate this step. Czabotar et al. (2013) determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove, but, unlike their complexes with prosurvival relatives, they dissociated Bax into two domains. The structures defined the sequence signature of activator BH3 domains and revealed how they activate Bax via its groove by favoring release of its BH3 domain. Bax helices α2-α5 alone adopted a symmetric homodimeric structure, supporting the proposal that two Bax molecules insert their BH3 domains into each other's surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. These observations define critical Bax transitions toward apoptosis.

The Bcl-2 (B-cell lymphoma 2) protein Bax (Bcl-2 associated X, apoptosis regulator) can commit cells to apoptosis via outer mitochondrial membrane permeabilization. Bax activity is controlled in healthy cells by prosurvival Bcl-2 proteins. C-terminal Bax transmembrane domain interactions have been implicated in Bax pore formation. Andreu-Fernández et al. 2017 showed that the isolated transmembrane domains of Bax, Bcl-xL (B-cell lymphoma-extra large), and Bcl-2 mediate interactions between Bax and prosurvival proteins inside the membrane in the absence of apoptotic stimuli. Bcl-2 protein transmembrane domains specifically homooligomerize and heterooligomerize in bacterial and mitochondrial membranes. Their interactions participate in the regulation of Bcl-2 proteins, thus modulating apoptotic activity.

Studies indicate that symmetric homodimers are the basic unit of pore formation in Bak and Bax. Each dimer contains an extended hydrophobic surface that lies on the outer membrane, and is anchored at either end by a transmembrane domain. Membrane-remodelling events such as positive membrane curvature have been reported to accompany apoptotic pore formation, suggesting that Bak and Bax form lipidic pores rather than proteinaceous pores. Uren et al. 2017 reviewed how clusters of dimers and their lipid-mediated interactions provide a molecular explanation for the heterogeneous assemblies of Bak and Bax observed during apoptosis.

The interaction between two core Bcl-2 family members, the executor pore-forming protein Bax and the truncated form of the activator protein Bid (tBid) have been determined. They have been imaged at the single particle level in a mitochondria-like planar supported lipid bilayer. The conformations of the proteins were inferred from their mobilities, and their transient interactions were detected using a single particle cross-correlation analysis. Both tBid and Bax have at least two different conformations in the membrane, and their affinities for one another increase by an order of magnitude when they pass from their loosely membrane-associated to their transmembrane form (Rose et al. 2021).

Melatonin attenuates liver ischemia-reperfusion (IR) injury via inhibiting the PGAM5-mPTP pathway (Shi et al. 2024).  Phosphoglycerate mutase/protein phosphatase (PGAM5)-mediated cell death plays an important role in multiple liver diseases.  This established a new paradigm in melatonin-mediated hepatocyte protection under the burden of I/R attack (Shi et al. 2024).

The generalized transport reactions for membrane-embedded, oligomeric Bcl-2 family members are:

cytochrome c (mitochondrial intermembrane space) cytochrome c (cytoplasm).

ions and small molecules (in) ⇌ ions and small molecules (out)