Deciphering Death's Circuitry

Courtesy of Upstate Cell Signaling Solutions CONNECT THE DOTS: Apoptosis is a highly complex cellular process with many discrete and interacting signaling pathways. Apoptosis is about as complex a cellular choreography as one can imagine. Death signals impinge, chromatin cleaves, mitochondria release cell-destroying contents, and membranes undulate and form blebs, eventually shrink-wrapping the shattered cell into neat packages destined for the innards of a phagocyte. Many research groups are deciphering the cascades of proteins that orchestrate the program. Stanley Korsmeyer, a Howard Hughes Medical Institute researcher at the Dana-Farber Cancer Center in Boston, is one of them. In experiments spanning more than a decade, his group has clarified the roles of certain key proteins that regulate the mitochondrial arm of apoptosis. This issues' Hot Papers1,2 "represent milestones in the research on how BCL-2 family members control the process of cell death," says Luca Scorrano, an assistant scientist at the Dulbecco Telethon Institute at Padova University in Italy and a recent member of the Korsmeyer team. Data derived from the Science Watch/Hot Papers database and the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. M.C. Wei et al., "Proapoptotic BAX and BAK: a requisite gateway to mitochon-drial dysfunction and death," Science, 292:727-30, April 27, 2001. (Cited in 205 papers) E. Cheng et al., "BCL-2, BCL-XL sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis," Mol Cell, 8:705-11, September 2001. (Cited in 87 papers) Sten Orrenius, professor of toxicology at the Karolinska Institute in Stockholm, adds, "The role of the mitochondria in the regulation of cell death is one of the hottest topics in current apoptosis research. At least a dozen apoptosis-related proteins are released from these organelles during cell death, and many research groups are elucidating the various events in this pathway. Much interest is focused on the actual release mechanisms of how the outer mitochondrial membrane permeabilizes. The findings by Korsmeyer and associates have provided a solid platform for these studies." Specifically, Korsmeyer and company linked truncated BID (tBID), a BH3-only protein that activates BAK and BAX, to mitochondrial release of prodeath factors. THE BCL-2 PROTEIN FAMILY Built of subsets of four domains, the BCL-2 proteins interact to provoke or block the mitochondrion from releasing cytochrome c and other death-inducing molecules. "The cell is like a big soup filled with pro- and antiapoptotic molecules. If there are more pro, the cell is more likely to die than if there are more anti," explains Anthony Letai, an instructor at Harvard Medical School and a group member. Korsmeyer uses such descriptive language as "evil twins," "rheostat," and "gateway to death" to describe the biochemical balancing act behind apoptosis in the mitochondrion, and the acronyms of apoptosis--many beginning with B--are reminiscent of a Dr. Seuss book. The molecules in the mitochondrial apoptotic pathway interact like a falling row of dominoes. Once a death receptor binds to the doomed cell's surface, BH3-only proteins (so-called because they include only the third of the four domains) stimulate BAX and BAK, which are embedded in the outer mitochondrial membrane, to form mostly self-pairs. They contort in such a way that pores open, through which the molecular messengers of death exit. Countering BAX and BAK are the proteins BCL-2 and the similar BCL-XL. Letai explains their relationships with the characteristic curious mix of initials and descriptors: "The BH3-only proteins BIM, BID, and BAD are triggers; BAX and BAK are the executioners; and BCL-2 and BCL-XL soak up BH3." In 1990, Korsmeyer's team cloned the breakpoint of a translocation between human chromosomes 14 and 18 associated with follicular B-cell lymphoma.3 The translocation places the gene Bcl-2 in a place where it is overexpressed, making it an oncogene. Two years earlier, David Vaux and Jerry Adams at the Walter and Eliza Hall Institute of Medical Research in Melbourne had identified the gene as a homolog of the apoptosis gene ced-3 in Caenorhabditis elegans.4 "Korsmeyer cloned the chromo-somal breakpoint, identified and recognized Bcl-2 is an oncogene that has a novel function, blocking cell death, and then localized the protein to the mitochondria," says Scorrano. Adds Paolo Bernardi, a professor of biomedical sciences at Padova, another major contributor to the field who also was Scorrano's mentor, "The studies of Stan Korsmeyer have greatly contributed to filling the gap between signaling through the BCL-2 protein family and mitochondria." But BCL-2 was just the beginning. "Then we began finding these evil twins such as tBID--closely related members of the BCL-2 family that were prodeath molecules," recalls Korsmeyer. Meanwhile, other investigators were filling in the downstream pieces of the puzzle. Xiaodong Wang, at the University of Texas Southwest Medical Center, discovered the starring role of cytochrome c as the chief executioner5, while Guido Kroemer, at France's National Center for Scientific Research (CNRS) in Villejuif, identified apoptosis-inducing factor and other proteins that rush out of the mitochondria as the cell begins to die.6 Although others had implicated the mitochondrion in the death dance, Korsmeyer's work subtly shifted research there. "Apoptosis originally had a morphological definition, but now it is biochemical as well," says Donald A. Fox, a professor of vision science, biology and biochemistry, and pharmacology at the University of Houston. "The first definition was also nucleocentric. Everyone focused on the nucleus, because chromatin was cleaved. Pathologists called it 'shrinking necrosis.'" Cell biologists now recognize that apoptosis has nuclear, cytoplasmic, and organellar components. Courtesy of Donald A. Fox MITOCHONDRIAL VIEW: Three-dimensional views of an apoptotic rod photoreceptor inner segment mitochondrion from a lead-exposed wild-type mouse (left panel) and Bcl-xL transgenic mouse (right panel). DOUBLE KNOCKOUTS Korsmeyer explains how his team linked truncated BID to the mitochondrial release of prodeath factors. "We knew that BID had to be activated by death receptors on the cell surface. But then we began to ask how this activated tBID and its cousin molecules, [and] triggered the beginning of apoptosis in the mitochondria. We believed it had to cause a central initiating event that released cytochrome c from the mitochondria. We had evidence that tBID just couldn't do it by itself." The experiments reported in the Science paper1 zeroed in on the go-betweens BAK and BAX. The investigators transfected fibroblasts from murine embryos knocked out for either BAK or BAX, or both, with a retrovirus carrying tBID. "We wondered whether both downstream molecules were required, which is why we tested double knockout cells lacking both BAX and BAK. Taking out both proteins was extremely synergistic--these cells were highly resistant to apoptosis," he says. Cells that could produce either BAK or BAX died a normal death. "The Bax/Bak double knockout provides compelling evidence in support of models that envision a default pathway for death that must be constantly suppressed by proteins such as BCL-2 and BCL-XL for cell survival," says John Reed, president and CEO of the Burnham Institute in La Jolla, Calif. The Molecular Cell Hot Paper2 concerns BCL-2 and BCL-XL, which block BH3-only triggers from binding BAX and BAK. And more recent work in the Korsmeyer lab has addressed the ability of tBID to remodel the mitochondrial cristae as it mobilizes cytochrome c stores into apoptotic action.7 Letai summarizes the results: "The mitochondrion is the integrator of apoptotic signals. It needs either BAK or BAX. They oligomerize in response to a signal, and somehow change the permeability of the mitochondrial outer membrane. This releases molecules into the cytoplasm, and then the cell is at the point of no return. That cell is going to die." The tango featuring tBID, BAK, BAX, BCL-2 and their brethren elicits more than academic interest. Dysregulation can spell disaster: too little apoptosis and cancer results; too much causes degenerative conditions. And so experiments are headed in a clinical direction. Recently, Letai, Korsmeyer, and colleagues trimmed BH3-only to 20 amino acids, and found that this piece fits pocket-like receptors on BCL-2, BAK, and BAX. Binding of the 20-mer to BCL-2's pocket shuts off apoptosis; binding BAK or BAX promotes it.8 Developing peptides such as the 20-mer into drugs will target specificities. Says Letai: "BAX and BAK are not specific to cancer, whereas a lot of cancer cells are protected from death by BCL-2. If we can develop a peptide that specifically inhibits BCL-2, it would likely kill cancer cells. And that is the jump from understanding a mechanism to developing a therapeutic. The papers were critical to that jump by giving a clear view of the mechanism of how BCL-2 family members interact." Ricki Lewis (rickilewis@nasw.org) is a contributing editor. References 1. M.C. Wei et al., "Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death," Science, 292:727-30, April 27, 2001. (Cited in 205 papers) 2. E. Cheng et al., "BCL-2, BCL-XL sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis," Mol Cell, 8:705-11, September 2001. (Cited in 87 papers) 3. D.M. Hockenbery et al., "Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death," Nature, 348:334-6, 1990. 4. D. Vaux, J. Adams, "Mammalian homolog of ced-3 in Caenorhabditis elegans," Nature, 335:440-4, 1988. 5. X. Liu et al., "Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c," Cell, 86:147-57, 1996. 6. S.A. Susin et al., "Bcl-2 inhibits the mitochondrial release of an apoptogenic protease," J Exp Med, 184:1331-41, 1996. 7. L. Scorrano et al, "A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis," Dev Cell, 2:55-67, 2002. 8. A. Letai et al., "Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics," Cancer Cell, 2:183-92, September 2002.

Deciphering Death's Circuitry

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