APOPTOSIS

R.M. Kluck, E. Bossy-Wetzel, D.R. Green, D.D. Newmeyer, "The release of cytochrome c from mitochondria: A primary site for Bcl-2 regulation of apoptosis," Science, 275:1132-6, 1997. (Cited in more than 410 papers since publication) Donald D. Newmeyer Xiaodong Wang Comments by Donald D. Newmeyer , an assistant member of the division of cellular immunology at the La Jolla Institute for Allergy and Immunology in La Jolla, Calif. J. Yang, X. Liu, K. Bhalla, C.N. Kim, A.M. Ibrado, J. Cai, T.I. Peng, D.P. Jones, X. Wang, "Prevention of apoptosis by Bcl-2: Release of cytochrome c from mitochondria blocked," Science, 275:1129-32, 1997. (Cited in more than 420 papers since publication) Comments by Xiaodong Wang , an assistant Howard Hughes Medical Institute investigator and an assistant professor of biochemistry at the University of Texas Southwest Medical Center in Dallas Two years ago, these two papers inexorably linked mitochondria--the eukaryotic organelles that serve as the site of cellular respiration--with the proteins Bcl-2 and cytochrome c in a sequence of biochemical events that leads to apoptosis. Cytochrome c, a mitochondrial protein and critical link in the respiratory chain, had for some time been known to reside in the inner mitochondrial membrane; the Bcl-2 protein, found on the mitochondrial outer membrane, had long been recognized as a critical antiapoptotic agent. But only in the last few years have scientists shown that Bcl-2 somehow regulates cytochrome c release on mitochondria to help direct apoptosis. These two companion papers simultaneously reached the same conclusion in different models (one a human cell line, the other Xenopus eggs): Bcl-2 blocks the release of cytochrome c and, as a result, often the apoptotic process as well. One of the first indications that mitochondria are critical players in the apoptotic process came in 1994 when Donald Newmeyer and colleague John C. Reed of the Burnham Institute in La Jolla, Calif., using a cell-free system based on extracts from Xenopus eggs, demonstrated that mitochondria are required for a form of apoptosis that's inhibitable by Bcl-2,1 a protein previously shown to be an important antiapoptotic molecule associated with mitochondrial membranes.2 As part of follow-up work aimed at better understanding mitochondrial function, Newmeyer's team found that the organelles release a soluble factor that, when put into a fresh extract, causes the rapid activation of caspases and subsequent apoptosis. While they were attempting to purify and identify that factor, Newmeyer's group received a bit of surprise news. "We were stunned to find out from the work of Xiaodong Wang that this factor was cytochrome c," Newmeyer says of Wang's 1996 Cell paper,3 which itself has been cited more than 350 times. While studying the biochemical mechanism of caspase activation, Wang's group had discovered that cell extracts could be used to study the processing of caspase-3, a key protease in the apoptotic pathway. Later they found that these extracts contained three protein factors that worked together to activate caspase-3 and induce apoptosis. Purification of those factors revealed one of them to be cytochrome c, which was linked to apoptosis for the first time. Both Newmeyer and Wang suggest that their 1997 Science papers were the natural extension of their prior work: cytochrome c, known to be located on the outer surface of the inner mitochondrial membrane, had now been implicated in apoptosis near mitochondria; the Bcl-2 protein, a known antiapoptotic agent, had previously been shown to reside on the outer membrane of mitochondria. The next logical step, therefore, was to see whether Bcl-2 regulated cytochrome c release. According to Wang, however, the real novelty of this series of findings was the initial isolation of cytochrome c reported in the 1996 Cell paper. "It was met with suspicion because it was such a novel finding," says Wang, adding that he's somewhat surprised that the 1997 paper has received a greater number of citations. "This Bcl-2 [1997 Science] paper seems to connect cytochrome c to a well-studied apoptotic phenomenon." "When his Cell paper came out there was a lot of skepticism," remarks Newmeyer. "It took some confirmation for people to start to believe it." Newmeyer also attributes the 1997 Science papers' high impact to their addressing the fundamental question of how Bcl-2 might work to block apoptosis, a causal process that many investigators are struggling with. "This is one clear way in which it does so," suggests Newmeyer. "[But it's] probably not the only way." Although Newmeyer and Wang reached the same conclusions, Newmeyer notes that his team also found that Bcl-2 acts directly on the mitochondria to which it's bound rather than in a more global manner. Both teams investigated whether mitochondria had to undergo something called the permeability transition prior to the release of cytochrome c--a process signified by the loss of the mitochondrial membrane potential. Results suggested that the membrane potential remained pretty much constant, indicating no permeability transition. Other labs have reported contradictory results, however. Says Newmeyer, "It's quite a confusing area of apoptosis research at the moment." In studies since the 1997 Science papers, the cytochrome c-mediated pathway has been shown to be essential in neurons, but not in other cells--cells can use cytochrome c for apoptosis, but many may not rely on it. There may be, says Newmeyer, multiple parallel pathways, or other factors in mitochondria that are released. "We know that several factors are released along with cytochrome c from the intermembrane space," he explains. "Some of these may have proapoptotic function or cause cell death independent of cytochrome c .... The story has become much more complicated since our [1997 Science ] paper was published." Newmeyer's group is currently trying to understand the mechanism of cytochrome c release from mitochondria. Wang's group has continued to focus on caspases, and has uncovered the pathway from cytochrome c to caspase-3, which includes two intermediary molecules: caspase 9, and Apaf-1, the human homologue of CED-4 in C. elegans and the last missing piece in the conserved evolutionary apoptotic pathway between the nematode worm and humans.4 "Right now we are working to reconstitute this entire system using recombinant protein so we can study the detailed biochemistry of it, break down all the intermediate steps," says Wang. D.D. Newmeyer et al., "Cell-free apoptosis in Xenopus egg extracts--inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria," Cell, 79:353-64, 1994. D. Hockenbery et al., "Bcl-2 is an inner mitochondrial membrane protein that blocks cell death," Nature, 348:334-6, 1990. X.S. Liu et al., "Induction of apoptotic program in cell-free extracts: Requirement for dATP and cytochrome c," Cell, 86:147-57, 1996. H. Zou et al., "Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3." Cell, 90:405-13, 1997.

APOPTOSIS

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