**6.1. Mechanisms of MOMP**

*Bax/Bak pore formation*. The first proposed mechanism of MOMP is mediated by the Bcl-2 family of proteins that directly act on the outer mitochondrial membrane. This family consists of about 17 members, some of which are proapoptotic and others antiapoptotic. The antiapoptotic members (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1, Bcl-B, etc.) contain 3-4 BH domains (Bcl-2 homology regions: BH-1, BH-2, BH-3 and BH-4) and are named "Bcl-2 like" proteins.

Among the proapoptotic members, some contain 2-3 BH domains (Bax, Bak, Bok, etc.) and the "BH3-only" proteins (Bid, Bad, Bim, Bik, Bmf, Hrk, Bnip3, Noxa, Puma, Spike, etc.) have the biggest proapoptotic potential. BH-3 only proteins exert their effects either by inhibiting antiapoptotic Bcl-2 like members or by directly activating Bak and Bax. In the first mechanism, "facilitators" such as Bad interact with antiapoptotic Bcl-2 like proteins, dissociating them and creating MMP. In the second mechanism, however, "activators" such as truncated Bid (tBid) activate proapoptotic proteins by stimulating translocation of Bax to the mitochondrial membrane or by activating Bak [36].

Cell Death and Cancer, Novel Therapeutic Strategies 75

channel which allows the influx of water and small solutes. PTP is a multiprotein complex located in the mitochondrial membrane that spans contact sites between the inner and the outer mitochondrial membranes. It is composed of three proteins: the voltage-dependent anion channel (VDAC), which is the most abundant protein of the outer membrane, the soluble matrix protein cyclophilin D (CypD), and the adenine nuclease translocase (ANT) located in the inner membrane. Other proposed PTP components are: hexokinase, creatine kinase and peripheral benzodiazepine receptor. The requirement of ANT is controversial. Distinct VDAC isoforms may interact with Bcl-2 members in a different manner. PTP opening leads to ∆Ψm dissipation, uncoupling of oxidative phosphorylation, water and ions influx, matrix swelling, outer membrane rupture and release of intermembrane space proteins, such as cytochrome c. Ca2+ favours PTP opening and permeability transitions are regulated by ∆Ψm, pH of mitochondrial matrix, redox potential, adenine nucleotides and bivalent metallic ions. PTP regulation by Bcl-2 proteins is still a matter of debate. It has been proposed that proapototic members promote pore opening, whereas antiapoptotic proteins favour pore closure [41]. Bcl-2 members can indirectly regulate PTP opening through Ca2+ efflux from the ER. In healthy cells, Bcl-2 or Bcl-XL can be located not only inserted in the mitochondrial outer membrane but also in the ER. Calcium ions exit the ER through inositol phosphate 3 receptors (IP3R), which can be blocked upon Bcl-2 binding. The proapoptotic members Bax and Bak induce Ca2+ movement from the ER to the mitochondrion [42]. However, Ca2+ interchange through PTP can be regulated by proapoptotic tBid [43,44].

**Figure 5.** Proposed model of mitochondrial membrane permeabilization. **Permeability transition pore complex.** OM: outer membrane. IMS: intermembrane space. IM: inner membrane. VDAC: voltagedependent anion channel. HK: hexokinase. PBR: peripheral benzodiazepine receptor. CK: creatine kinase. ANT: adenine nuclease translocase. CypD: cyclophilin D (modified from Kroemer et al. 2007).

The rise of mitochondrial matrix Ca2+ induces oxidative metabolism. However, upon apoptotic stimulus, Ca2+ can cause PTP opening, which can be transient and provide a fast calcium release mechanism, or persistent, giving rise to outer membrane rupture and release of apoptotic factors. Additionally, a different channel has been identified in the mitochondrion, composed of ceramide, a lipid that can form hydrogen bonds giving rise to ceramide structures that form channels which allow the efflux of proteins up to 60 kDa [42].

The interplay between antiapoptotic and proapoptotic proteins decides the final fate of the damaged cell. Antiapoptotic proteins can be found in the outer mitochondrial membrane, in the cytoplasm or in the Endoplasmic Reticulum (ER). Some propapoptotic members such as Bax or Bid reside in the cytosol, but translocate to the outer membrane upon triggering of a death stimulus, where they oligomerize to form a channel, either with themselves or with membrane anchored-Bak or tBid. The importance of the interactions between proteins and lipids is becoming more evident. In fact, it is now thought that Bak or Bax destabilize lipid bilayers instead of forming pores. BH3-only proteins activity is transcriptionally and posttranslationally regulated. Bid, for instance, is regulated by caspase-8, cathepsins, granzyme B or calpain [37-39]. The proapototic protein Bad in inhibited through phosphorylation by Akt and activated through dephosphorylation by calcineurin [40].

**Figure 4.** Proposed model of mitochondria membrane permeabilization. **Bax/Bak pore formation.** Bcl-2 family members regulation.

*Permeability transition pore complex.* The second proposed mechanism of MOMP is based on the formation of the permeability transition pore (PTP) complex, a high conductance channel which allows the influx of water and small solutes. PTP is a multiprotein complex located in the mitochondrial membrane that spans contact sites between the inner and the outer mitochondrial membranes. It is composed of three proteins: the voltage-dependent anion channel (VDAC), which is the most abundant protein of the outer membrane, the soluble matrix protein cyclophilin D (CypD), and the adenine nuclease translocase (ANT) located in the inner membrane. Other proposed PTP components are: hexokinase, creatine kinase and peripheral benzodiazepine receptor. The requirement of ANT is controversial. Distinct VDAC isoforms may interact with Bcl-2 members in a different manner. PTP opening leads to ∆Ψm dissipation, uncoupling of oxidative phosphorylation, water and ions influx, matrix swelling, outer membrane rupture and release of intermembrane space proteins, such as cytochrome c. Ca2+ favours PTP opening and permeability transitions are regulated by ∆Ψm, pH of mitochondrial matrix, redox potential, adenine nucleotides and bivalent metallic ions. PTP regulation by Bcl-2 proteins is still a matter of debate. It has been proposed that proapototic members promote pore opening, whereas antiapoptotic proteins favour pore closure [41]. Bcl-2 members can indirectly regulate PTP opening through Ca2+ efflux from the ER. In healthy cells, Bcl-2 or Bcl-XL can be located not only inserted in the mitochondrial outer membrane but also in the ER. Calcium ions exit the ER through inositol phosphate 3 receptors (IP3R), which can be blocked upon Bcl-2 binding. The proapoptotic members Bax and Bak induce Ca2+ movement from the ER to the mitochondrion [42]. However, Ca2+ interchange through PTP can be regulated by proapoptotic tBid [43,44].

74 Apoptosis and Medicine

family members regulation.

Among the proapoptotic members, some contain 2-3 BH domains (Bax, Bak, Bok, etc.) and the "BH3-only" proteins (Bid, Bad, Bim, Bik, Bmf, Hrk, Bnip3, Noxa, Puma, Spike, etc.) have the biggest proapoptotic potential. BH-3 only proteins exert their effects either by inhibiting antiapoptotic Bcl-2 like members or by directly activating Bak and Bax. In the first mechanism, "facilitators" such as Bad interact with antiapoptotic Bcl-2 like proteins, dissociating them and creating MMP. In the second mechanism, however, "activators" such as truncated Bid (tBid) activate proapoptotic proteins by stimulating translocation of Bax to

The interplay between antiapoptotic and proapoptotic proteins decides the final fate of the damaged cell. Antiapoptotic proteins can be found in the outer mitochondrial membrane, in the cytoplasm or in the Endoplasmic Reticulum (ER). Some propapoptotic members such as Bax or Bid reside in the cytosol, but translocate to the outer membrane upon triggering of a death stimulus, where they oligomerize to form a channel, either with themselves or with membrane anchored-Bak or tBid. The importance of the interactions between proteins and lipids is becoming more evident. In fact, it is now thought that Bak or Bax destabilize lipid bilayers instead of forming pores. BH3-only proteins activity is transcriptionally and posttranslationally regulated. Bid, for instance, is regulated by caspase-8, cathepsins, granzyme B or calpain [37-39]. The proapototic protein Bad in inhibited through phosphorylation by Akt and activated through dephosphorylation by calcineurin [40].

**Figure 4.** Proposed model of mitochondria membrane permeabilization. **Bax/Bak pore formation.** Bcl-2

*Permeability transition pore complex.* The second proposed mechanism of MOMP is based on the formation of the permeability transition pore (PTP) complex, a high conductance

the mitochondrial membrane or by activating Bak [36].

**Figure 5.** Proposed model of mitochondrial membrane permeabilization. **Permeability transition pore complex.** OM: outer membrane. IMS: intermembrane space. IM: inner membrane. VDAC: voltagedependent anion channel. HK: hexokinase. PBR: peripheral benzodiazepine receptor. CK: creatine kinase. ANT: adenine nuclease translocase. CypD: cyclophilin D (modified from Kroemer et al. 2007).

The rise of mitochondrial matrix Ca2+ induces oxidative metabolism. However, upon apoptotic stimulus, Ca2+ can cause PTP opening, which can be transient and provide a fast calcium release mechanism, or persistent, giving rise to outer membrane rupture and release of apoptotic factors. Additionally, a different channel has been identified in the mitochondrion, composed of ceramide, a lipid that can form hydrogen bonds giving rise to ceramide structures that form channels which allow the efflux of proteins up to 60 kDa [42].
