Mitochondria: pharmacological manipulation
of cell death (original) (raw)
The Bcl-2 family of proteins (Table 1) includes both proapoptotic and antiapoptotic members, each containing 1 or more Bcl-2 homology domains (BH1 through BH4) (40). Antiapoptotic members such as Bcl-2, Bcl-xL, and Mcl-1 contain all 4 BH domains. The proapoptotic members are divided into 2 subgroups. One of these consists of Bax, Bak, and Bok, which possess domains BH1, BH2, and BH3; the other group consists of the more numerous BH3-only proteins, which include Bid, Bad, Puma, and several others (40).
Subdivisions of Bcl-2–family proteins, based on function and sequence homology
While several Bcl-2–family proteins possess ion channel activity in lipid bilayers, only the multidomain proapoptotic proteins Bax and Bak can render membranes permeable to cytochrome c or larger macromolecules (15, 20). These 2 proteins exhibit a degree of functional redundancy in the mouse, as the ablation of both bax and bak genes results in a much more dramatic apoptotic phenotype than single bax or bak knockouts. Indeed, tissues and cells deficient in both genes are resistant to most stimuli that proceed through mitochondrial and endoplasmic reticulum–dependent pathways (41–44). The 2 proteins do not behave identically, however, since in nonapoptotic cells Bax is mostly free in the cytosol while Bak is constitutively localized in membranes of the mitochondria and endoplasmic reticulum (45).
Regardless of localization, Bax and Bak both seem to be in an inactive state in nonapoptotic cells. An activation process, which is not completely understood, is required for Bax to oligomerize and insert stably into the membrane (20, 46, 47). The tertiary structure of the antiapoptotic Bcl-2 family member Bcl-xL shows that its helical domains form a hydrophobic groove where the BH3-only proteins bind. Bax has a similar structural fold; however, in unactivated, soluble Bax, the C-terminal helix α9 occupies the BH3-binding groove (48), and helix α8 is also positioned to interfere with potential binding of BH3 domains (49). Thus, in order to interact with BH3 domains in other proteins, Bax must undergo a conformational alteration. Indeed, a Bax conformational change is commonly detected through the binding of conformation-sensitive antibodies (50), and the interaction of Bax with membranes is sufficient to enable a conformational change but not membrane insertion or pore formation (51). Bax oligomerization does not occur in solution (20), except in the presence of certain detergents (46), consistent with the idea that interaction with a hydrophobic environment is necessary to destabilize the Bax molecule and allow activation and oligomerization.
In what is commonly known as the “rheostat” model, cell survival is determined by the balance between antiapoptotic Bcl-2–family proteins such as Mcl-1, Bcl-xL, or Bcl-2, and proapoptotic BH3-only proteins. Based on recent findings, we can now consider a more complex “switched rheostat” model (Figure 2) with several additional elements: First, Bax and Bak are the positive effector molecules, based on the biochemical and genetic studies mentioned above. Second, the antiapoptotic relatives act as inhibitors of this permeabilization process, not effectors of an independent survival mechanism, at least with regard to direct effects on MOMP. Third, MOMP mediated by Bax and Bak must be triggered by “direct activator” proteins, such as the BH3-only proteins Bim or Bid; indeed, synthetic peptides corresponding to the BH3 domains of these proteins are able to activate Bax (20, 47). The p53 protein can also directly induce Bax-mediated membrane permeabilization in cells and liposomes, although it lacks a clearly identifiable BH3 domain (52, 53). Fourth, the antiapoptotic family members can oppose Bax and Bak as well as the BH3-only proteins (20). Fifth, BH3-only proteins fall into 2 functional classes: the direct activators Bim and Bid mentioned above, and the others, which act as “derepressors” by binding competitively to the antiapoptotic family members and thus freeing up the direct activator proteins to induce Bax/Bak–mediated MOMP (47, 54, 55). Finally, interactions between various BH3-only proteins and the antiapoptotic Bcl-2–family proteins exhibit a pattern of overlapping specificities that was previously unappreciated. Recent studies have shown that some of the BH3-only proteins bind to only a subset of the antiapoptotic proteins, and conversely the antiapoptotic proteins each bind to a subset of the BH3-only proteins (47, 56). For example, the BH3-only protein Noxa binds specifically to Mcl-1 and A1. In contrast, Puma, another BH3-only protein, binds to at least 5 different antiapoptotic relatives (56). Moreover, Bak is differentially inhibited by antiapoptotic Bcl-2–family proteins, further illustrating the intricacy of this protein network (57).
Models of Bcl-2–family function at the mitochondrion during apoptosis. (A) The traditional simple rheostat model assumes that the antiapoptotic Bcl-2–family proteins antagonize the BH3-only proteins, in an equal and opposite manner. (B) Recent results suggest a more inclusive and detailed model, which we term the “switched rheostat.” Bax and Bak are the effectors of MOMP. Certain BH3-only proteins (“direct activators”) and p53 switch on Bax (and possibly Bak) directly and are antagonized by antiapoptotic Bcl-2–family proteins. Other BH3-only proteins (“derepressors”) do not activate Bax directly but act by antagonizing the antiapoptotic family members, thereby freeing the direct activators to trigger Bax/Bak–induced MOMP. The dashed lines indicate that the derepressor BH3-only proteins have differing specificities for antiapoptotic family members.
Adding to this complexity, a number of proteins unrelated to the Bcl-2 family interact with Bax and either inhibit (58–61) or enhance (58, 62) its activation. Bak has been reported to associate with VDAC2 at the mitochondrial membrane, and this inhibits the oligomerization and proapoptotic activity of Bak (63). Although their roles in cell death are not well understood, these proteins that modulate Bax or Bak activation could be important therapeutic targets (64).