antagonists Reaper, Hid, and Grim (RHG) localize to mitochondria upon their expression through a Grim homology (GH)-3 domain that binds to lipids. The primary function of RHG proteins is to antagonize the action of the Drosophila IAP, DIAP-1. Their action releases active caspases from DIAP-1, which then cleave important proapoptotic substrates. How or why the OMM may be involved in this process remains unclear. In contrast to C. elegans and the vertebrates, BCL-2 family proteins in Drosophila do not affect cell survival or OMM integrity.

9. CONCLUSIONS

Mitochondria play a dual role in the fate of a cell: mitochondria provide the majority of energy in the form of ATP, yet mitochondria translate cell death signals initiated by other cells but also from within themselves. In the mitochondrial pathways of cell death, the bioenergetic functions of mitochondria are compromised in specific ways. During apoptosis, fragmentation, loss of OMM integrity, dilution of IMS proteins, activation of caspases, and loss of mitochondrial membrane potential all contribute to the demise of mitochondrial function. On the other hand, during necrosis the IMM loses selectivity and mitochondria lose membrane potential and swell, causing OMM rupture. In addition, the deathpromoting signals emanating from the mitochondria such as cytochrome c and SMAC/Diablo release coincide with or happen slightly before mitochondrial dysfunction. By specifically disrupting mitochondrial function, the cell’s fate to die is favored due to the demise of cellular ATP levels and thereby loss of metabolic control coupled with positive death signals at the same time. These two phenomena make the mitochondrial steps of cell death pathways a point of no return from which it is difficult for a cell to recover. In long-lived cells, such as neurons, or cells attempting to avoid death, such as

tumor cells, mechanisms may be in place to delay cell death processes after the mitochondrial steps and allow cellular survival. By studying the role of mitochondria in cell death pathways, we have gleaned important information regarding cell death, but also insights into mitochondrial physiology.

SUGGESTED READINGS

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1. INTRODUCTION

A fundamental step in the commitment to apoptosis via the intrinsic pathway is mitochondrial outer membrane permeabilization (MOMP). This step allows the release of proapoptotic proteins from the mitochondrial intermembrane space into the cytoplasm. Once in the cytoplasm, they induce caspase activation, oligonucleosomal DNA cleavage, and other hallmarks of apoptosis. The details of these important events that occur downstream of MOMP are described in Chapter 4. Here we restrict our attention to the molecular mechanisms by which the cell controls this critical event, molecular mechanisms that primarily involve interactions among the family of proteins known as the BCL-2 family.

The BCL-2 family gets its name from the B-cell leukemia/lymphoma-2 gene that was cloned from the breakpoint of the t(14;18) present in nearly all cases of follicular lymphoma, as well as a minority of diffuse large B-cell lymphomas. Although previously discovered oncogenes had been found to increase the rate of proliferation of malignant cells, BCL-2 was found instead to foster accumulation of malignant cells by inhibiting cell death. It was found that BCL-2 had homologs in virtually all metazoans studied, and it was functionally interchangeable with the Ced9 gene of the roundworm Caenorhabditis elegans. These results suggested that control of cell death by homologs of BCL-2 is a phylogenetically ancient property of metazoan cells.

In the years since the cloning of BCL-2 in 1985, an entire family of proteins has been discovered that is related to BCL-2 by sequence homology, as well as by involvement in control of apoptosis (Figure 5-1). The BCL-2 family contains proteins that induce as well as those that prevent MOMP and apoptosis. Next we describe how BCL-2 family proteins interact to adjudi-

cate whether the cell takes the critical step of commitment to apoptosis.

2. ACTIVATING APOPTOSIS: BAX AND BAK AND

THE ACTIVATOR BH3-ONLY PROTEINS

BAX and BAK are essential effectors of apoptotic signaling at the mitochondrion. Models of combined deletion of BAX and BAK show that in the absence of BAX and BAK, MOMP cannot take place. In response to upstream death signaling, carried at least in part by select proapoptotic BH3-only proteins (see below, Sections 4 and 5), BAX and BAK are activated, homooligomerize, and form pores in the mitochondrial outer membrane (MOM). Although BAX and BAK are necessary components of the apoptotic pore in the MOM, it remains possible that they cooperate with other proteins to form this pore in vivo. Nonetheless, BAX alone can form pores in liposomal vesicles of sufficient size to permit efflux of cytochrome c, an intermembrane protein that is released after MOMP to assist in caspase activation by the apoptosome.

It is obvious that the mitochondrial membrane permeabilizing function of BAX and BAK must be tightly controlled. This control is not exerted primarily at the level of protein expression, as apoptosis can be observed over very short time scales, on the order of minutes, a time insufficient for alterations in BAX or BAK levels. Furthermore, BAX and BAK protein levels typically do not change significantly during execution of an apoptotic signal. How then are BAX and BAK converted from quiescent proteins compatible with survival to rapid and essential effectors of cell death?

The main steps for BAX activation are translocation to the mitochondrion, conformation change, insertion into the mitochondrial membrane, oligomerization,

and, finally, pore formation. These events can take place over mere minutes in a cell enduring death signaling. In the case of BAX, subcellular localization is an important mode of control. Before the induction of apoptosis, BAX exists as a monomer either in the cytosol or loosely attached to the mitochondrial outer membrane in an alkali-labile fashion. Control over translocation from the cytosol to the mitochondria is poorly understood, but enforced dimerization of BAX has been observed to cause translocation, as does cytosol from cells undergoing apoptosis and the protein Peg3/Pw1. BCL-2 and Ku70 have been found to inhibit translocation of BAX to mitochondria. Because the distribution of BAX between a purely cytosolic state and one in which it is loosely attached to the mitochondria varies considerably among cell types, it seems likely that control over this localization may vary similarly.

Once localized to the mitochondrion, BAX must undergo a conformational change and insert into the mitochondrion to execute its mitochondrial permeabilizing function. Because BAK is already inserted into the mitochondrion, it requires only conformational change, oligomerization, and pore formation for complete activation. In both cases, the conformational change exposes an N-terminal epitope that can be specifically recognized by conformation-specific antibodies. This insertion and conformational change can be induced by a subset of BH3only proteins called activators, which includes BID and BIM, and possibly PUMA (see also discussion that follows). The interaction between activators and BAX and BAK appears to be a catalytic “hit and run” type interaction, and complexes between activators and BAX and BAK are difficult, though not impossible, to observe. The importance of activators may perhaps best be seen in defined liposomal systems. In these systems, unilamellar liposomal vesicles are made with phospholipids that mimic mitochondrial composition. Recombinant BCL-2 family proteins can be added to the vesicles, and the ability to permeabilize can be measured. BAX or BID by themselves are unable to efficiently permeabilize the liposomes, but if BID is added to BAX,

permeabilization becomes drastically more efficient. Furthermore, BAX is recruited and inserted into the membrane, and a conformation change exposing its N-terminus is induced. Stoichiometric studies show that BID can activate fully a great molar excess of BAX, supporting the model of a transient “hit and run” interaction. Thus all the important properties of activation of BAX can be recapitulated by the addition of BID protein. Other proteins that have been implicated as activators of BAX and BAK include the BH3-only protein PUMA and p53, and it is possible that other molecules, perhaps even non-proteins, can participate as activators. In addition, increasing temperature to 43◦ C has also been found to activate BAX and BAK, likely either by alteration of the proteins themselves or perhaps alteration of the lipid environment.

Once activated, BAX and BAK oligomerize. Predominantly homo-oligomers have been observed, although it may be that oligomerization of one facilitates the oligomerization of the other. After oligomerization, BAX and BAK participate in the formation of a pore that permits the efflux of macromolecules from the intermembrane space. It is not known whether additional proteins are required for this permeabilization. Supporting an independent role for BAX and BAK is the finding that BAX can, by itself, create pores that permit the efflux of cytochrome c in liposomes. Furthermore, conductance studies suggest that the properties of channels isolated from cells and those made from recombinant BAX are similar. However, it cannot be ruled out that other proteins participate. It appears that this permeabilization is independent of the mitochondrial permeability transition pore (PTP) on the basis of the generally observed insensitivity of MOMP to cyclosporine A treatment and to deletion of cyclophilin D, a key component of the PTP. Another important channel in mitochondria is the voltage-dependent anion channel (VDAC). MOMP appears independent of this channel as well, as deletion of VDAC proteins again has little effect on MOMP.

BOK is a proapoptotic BCL-2 family protein that by sequence homology and function resembles BAX and BAK. Little is known about this protein, although its expression seems to be largely limited to reproductive tissues. Because loss of BAX and BAK alone appears sufficient to prevent MOMP in several cell types, it appears that BOK does not play an important role in many tissues. However, study of this protein is quite limited, at least in part by the paucity of good antibodies that selectively recognize BOK. It remains possible that BOK is an important effector of apoptotic signaling in response to select stresses in certain tissues.

3. INHIBITING APOPTOSIS

As might be expected with such an important cell fate decision, there are additional factors that negatively regulate commitment to apoptosis (Figure 5-2). Most prominent among these are the antiapoptotic BCL-2 family proteins, the best studied of which include BCL- 2, BCL-XL, MCL-1, BCL-w, and BFL-1. As mentioned previously, BCL-2 was the first of these proteins to be discovered and thus lends its name to the entire protein family. Forced expression of any of these antiapoptotic proteins allows a cell to survive a wide variety of insults that might otherwise induce apoptosis, including growth factor withdrawal, DNA damage, microtubule disruption, and kinase inhibition. These proteins inhibit

Damage/Derangement Signals

-Checkpoint violation

-DNA damage

-Oncogene activation

Figure 5-2. Model of BCL-2 family control of programmed cell death. Death signals cause induction or post-translational activation of BH3-only proteins. Activator BH3-only proteins, including BID and BIM, induce oligomerization of BAX and/or BAK, causing MOMP, cytochrome c release, and caspase activation, resulting in cell death. Antiapoptotic proteins prevent apoptosis by sequestering activator BH3-only proteins and activated BAX/BAK, upstream of BAX/BAK oligomerization. Sensitizer BH3-only proteins promote cell death by binding the antiapoptotic proteins, displacing activator BH3-only proteins to trigger BAX/BAK oligomerization. Reprinted from Certo M, Del Gaizo Moore V, Nishino M, et al. Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members. Cancer Cell. 2006;9:351–65, Copyright C 2006 with permission from Elsevier.

cell death by binding proapoptotic proteins to inhibit the processes described previously. Perhaps their most important function is to bind and sequester activator BH3-only proteins to prevent their interaction with and activation of BAX and BAK. In fact, all of the antiapoptotic proteins identified can bind BID and BIM, as well as PUMA. Antiapoptotic proteins contain a hydrophobic pocket formed by the hydrophobic faces of the BH1, BH2, and BH3 domains. The BH3 domain of the activator proteins is itself an amphipathic helix, the hydrophobic face of which binds into the hydrophobic pocket of the antiapoptotic proteins.

In addition, antiapoptotic proteins can bind BAX and BAK, particularly after BAX and BAK undergo the conformational change that accompanies activation.