Mechanisms of action of Bcl-2 family proteins

Abstract

The Bcl-2 family of proteins controls a critical step in commitment to apoptosis by regulating permeabilization of the mitochondrial outer membrane (MOM). The family is divided into three classes: multiregion proapoptotic proteins that directly permeabilize the MOM; BH3 proteins that directly or indirectly activate the pore-forming class members; and the antiapoptotic proteins that inhibit this process at several steps. Different experimental approaches have led to several models, each proposed to explain the interactions between Bcl-2 family proteins. The discovery that many of these interactions occur at or in membranes as well as in the cytoplasm, and are governed by the concentrations and relative binding affinities of the proteins, provides a new basis for rationalizing these models. Furthermore, these dynamic interactions cause conformational changes in the Bcl-2 proteins that modulate their apoptotic function, providing additional potential modes of regulation.

Figure 1.

Schematic overview of the Bcl-2 family of proteins. The family is divided into two subgroups containing proteins that either inhibit apoptosis or promote apoptosis. The proapoptotic proteins are further subdivided functionally into those that oligomerize and permeabilize the MOM, such as Bax and Bak, or those that promote apoptosis through either activating Bax or Bak or inhibiting the antiapoptotic proteins, such as tBid, Bim, Bad, and Noxa. Proteins are included in the Bcl-2 family based on sequence homology to the founding member, Bcl-2, in one of the four Bcl-2 homology (BH) regions. All the antiapoptotic proteins, as well as Bax, Bak, and Bid, have multiple BH regions, are evolutionarily related, and share a three-dimensional (3D) structural fold. The BH3 proteins contain only the BH3 region, are evolutionarily distant from the multiregion proteins, and are intrinsically unstructured. Most members of the Bcl-2 family proteins contain a membrane-binding region (MBR) on their carboxyl termini in the form of a tail anchor, mitochondrial-targeting sequence, or as a hydrophobic amino acid sequence that facilitates binding and localization of these proteins to the MOM or to the endoplasmic reticulum (ER) membrane.

Figure 2.

Schematics of the core mechanisms proposed by various models for the regulation of MOMP by Bcl-2 proteins. (↑) Activation; (⊥) inhibition; (⊥↑) mutual recruitment/sequestration. Paired forward and reverse symbols indicate the model makes explicit reference to equilibria. (A) The direct activation model divides the different BH3 proteins by qualitative differences in function. The BH3 proteins with high affinity for binding and activating Bax and Bak are termed as “activators,” whereas those that only bind the antiapoptotic proteins are termed “sensitizers.” The activator BH3 proteins directly interact with and activate Bax and Bak to promote MOMP. The antiapoptotic proteins inhibit MOMP by specifically sequestering the BH3 activators. The BH3 sensitizer proteins can compete for binding with the antiapoptotic proteins, thus releasing the BH3 activator proteins to avidly promote MOMP through activation and oligomerization of Bax and Bak. (B) The displacement model categorizes the BH3 proteins solely based on their affinities of binding for the antiapoptotic proteins (hence, does not recognize them as activators). In this model, Bax and Bak are constitutively active and oligomerize and induce MOMP unless held in check by the antiapoptotic proteins. Therefore, for a cell to undergo apoptosis, the correct combination of BH3 proteins must compete for binding for the different antiapoptotic proteins to liberate Bax and Bak and for MOMP to ensue. (C) The embedded together model introduces an active role for the membrane and combines the major aspects of the previous models. The interactions between members of the Bcl-2 family are governed by equlibria and therefore are contingent on the relative protein concentrations as well as their binding affinities. The latter are determined by posttranslational modifications, fraction of protein bound to the membrane, and cellular physiology. At membranes, the activator BH3 proteins directly activate Bax and Bak, which then oligomerize, inducing MOMP. Both activator and sensitizer BH3 proteins can recruit and sequester antiapoptotic proteins in the membrane. The antiapoptotic proteins inhibit apoptosis by sequestering the BH3 proteins and Bax and Bak in the membrane or by preventing their binding to membranes. At different intracellular membranes, the local concentrations of specific subsets of Bcl-2 family members alter the binding of Bcl-2 proteins to the membrane and the binding equilibria between family members. As a result, Bcl-2 family proteins have distinct but overlapping functions at different cellular locations. (D) The unified model builds on the embedded together model by proposing that the antiapoptotic proteins sequester the activator BH3 proteins (mode 1) and sequester Bax and Bak (mode 2). It differs in that in the unified model, inhibition of apoptosis through mode 1 is less efficient (smaller arrow in panel D) and therefore easier to overcome by sensitizer BH3 proteins. In addition, the unified model extends the role of Bcl-2 family proteins and the regulation of MOMP to mitochondria dynamics (not shown).

Figure 3.

Models of Bax and Bak dimer formation. (A) Symmetrical dimers: Active Bax and Bak monomers with helices embedded within the MOM expose their BH3 regions, which, in turn, bind to the “front pocket” composed of the hydrophobic BH1-3 groove of an adjacent monomer. This binding changes the conformation of the “rear pocket” composed of helices 1 and 6, allowing homodimers to form tetramers and further propagate oligomerization. (B) Asymmetrical dimers: Active Bax and Bak expose their BH3 regions, which interact with the rear pocket on an adjacent monomer, forming an oligomer through subsequent rear pocket:BH3 region interactions.

Figure 4.

Schematic overview of the embedded together model. The role of the membrane is highlighted as the “locus of action” where the effects of the interactions between the Bcl-2 family members are manifest. After the cell receives a death signal, an activator BH3 protein migrates to and inserts into the MOM, where it recruits cytoplasmic Bax. Bax undergoes conformational changes at membranes that allow it to respond to chemical changes in the cell such as the generation of reactive oxygen species, ion concentration, and pH. Membrane-bound Bax or Bak changes its conformation such that they oligomerize, leading to MOMP and/or recruit other cytoplasmic Bax. Both the activator and the sensitizer BH3 proteins sequester the antiapoptotic proteins (such as Bcl-XL) by recruiting and strongly binding to them at the MOM, thereby preventing the inhibition of Bax and Bak. Bcl-XL changes its conformation depending on its binding partner. Upon binding to a BH3 protein or Bax/Bak, Bcl-XL changes from form 1 (cytoplasmic or loosely attached to the MOM) to form 2 (helix 9 inserted into MOM) or to form 3 (helices 5, 6, and 9 bound to or inserted into MOM), respectively. It is likely that form 2 binds primarily BH3 proteins but also recruits additional Bcl-XL to the membrane, whereas form 3 binds primarily Bax and Bak. No function has yet been ascribed to Bcl-XL form 1, although one is likely. Thus, by causing the proteins to adopt different conformations, the membrane regulates their function in determining the fate of the cell. Unlike other models that propose unidirectional interactions, in this model, all of the functional interactions are governed by dynamic equilibria of protein–membrane and protein–protein interactions.