Bridging Apoptotic Signaling Gaps

Above, from left: Xu Luo, Xiaodong Wang, and Imawati Budihardjo. In apoptosis, death signals from outside a cell are conveyed to various organelles inside a cell through an intricate network of molecules acting as messengers. These two Cell papers outline two independent identifications of a critical missing link in this signaling pathway. They show that the cytosolic protein Bid carries a death signal from the cell membrane to the mitochondria. Identifying Bid's signaling role allowed research

Aug 21, 2000
Nadia Halim


Above, from left: Xu Luo, Xiaodong Wang, and Imawati Budihardjo.
In apoptosis, death signals from outside a cell are conveyed to various organelles inside a cell through an intricate network of molecules acting as messengers. These two Cell papers outline two independent identifications of a critical missing link in this signaling pathway. They show that the cytosolic protein Bid carries a death signal from the cell membrane to the mitochondria. Identifying Bid's signaling role allowed researchers to connect two existing stories into one big picture.

Researchers had a good idea of what happens before the death signal reaches the mitochondria. Activation of the Fas and tumor necrosis factor (TNF) death receptors on the cell surface leads to activation of caspase 8 (Casp8). Caspases cleave proteins involved in apoptosis. Researchers also had a handle on cellular events occurring after mitochondria receive the apoptotic signal. The mitochondrial outer membrane becomes permeable to cytochrome c, a protein that transfers electrons within the mitochondrial respiratory chain. Once released into the cytosol, cytochrome c activates downstream caspases, which leads to the rapid degradation of the cell.1 However, how Casp8 induces mitochon-drial damage was a mystery before publication of these Cell papers. The senior authors of the papers agree that this is one reason they are highly cited.

Xiaodong Wang, associate professor of biochemistry and Howard Hughes Medical Institute assistant investigator at the University of Texas Southwestern Medical Center in Dallas, approached the problem from the angle of cytochrome c release, his research concentration. "From previous studies it became clear that the major regulatory event in apoptosis is cytochrome c release, which is potentially regulated by the Bcl-2 family of proteins," explains Wang.2 These proteins share defined regions of homology, but some members act to suppress apoptosis, and others are proapoptotic. His lab combined mitochondria isolated from mouse liver, cytosol from normal HeLa cells, and recombinant Casp8 to create an assay for molecules mediating cytochrome c release through Casp8. Interestingly, the researchers pinpointed the activity to Bid, a protein in the Bcl-2 family.


Junying Yuan
Junying Yuan, associate professor of cell biology at Harvard Medical School, took a completely different approach. "We work on caspases mostly and got into this area [Bid research] by accident," she says. Not much was known about downstream targets of caspases and how caspases induce cell death. So the Yuan lab used small pool expression cloning to screen for Casp8 substrates. This method isolated small pools of 100 cDNAs each from mouse spleen. The cDNAs in each pool were then transcribed and translated in vitro in the presence of a radioactive label. The resulting pools of labeled proteins were incubated with Casp8 to find its substrates. "Many proteins were cleaved by Casp8, but Bid just stood out," says Yuan.

Stan Korsmeyer, professor of pathology at Harvard Medical School, and his lab3 initially cloned Bid. The researchers identified Bid through a screen to find proteins that interact with Bcl-2. Overexpression of Bid caused cells to undergo apoptosis, which led to its name: Bcl-2 interactive death agonist. Bid is unique because it is usually free in the cytosol; most other members of the Bcl-2 family are attached to the cell membrane.

"When we found Bid it clearly made a lot of sense based on what was known at the time," says Wang. Yuan agrees: "We immediately thought it [Bid] may play a role in mediating signaling from the membrane to the mitochondria because the Bcl-2 family of proteins were known to play a role in mitochondrial protection and damage." Once Bid was identified as a key player, each lab worked out the signal transduction mechanism. As a full-length protein, Bid is inactive in the cytosol. Only after Casp8 cleaves Bid does the C terminus of the protein travel to the mitochondria and induce cytochrome c release. "These papers show that activating cell surface receptors will also activate mitochondrial pathways," explains Wang. The papers are unique also because they connect caspases and a proapoptotic member of the Bcl-2 family, indicates Yuan.

For this article, Nadia S. Halim interviewed Xiaodong Wang, associate professor of biochemistry at the University of Texas Southwestern Medical Center in Dallas and Howard Hughes Medical Institute assistant investigator, and Junying Yuan, associate professor of cell biology at Harvard Medical School. Data from 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.

 

X. Lu, I. Budihardjo, H. Zou, C. Slaughter, and X. Wang, "Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors," Cell, 94:481-90, Aug. 21, 1998. (Cited in more than 250 papers since publication)

 

H.L. Li, H. Zhu, C.J. Xu, and J.Y. Yuan, "Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis," Cell, 94:491-501, Aug. 21, 1998. (Cited in more than 250 papers since publication)

Since publication of the two Cell papers, Yuan has collaborated with Gerhard Wagner, professor of biological chemistry and molecular pharmacology at Harvard Medical School, to elucidate the nuclear magnetic resonance structure of Bid. They showed that Bid's overall structure is preserved after Casp8 cleavage.4 At the same time, Korsmeyer and his colleagues also published the Bid structure.5 Last year a Korsmeyer paper validated Bid's role in the apoptotic pathway.6 His lab generated mice deficient in Bid. Upon Fas activation nearly all Bid-deficient mice survived, whereas wild-type mice died of liver injury. This study demonstrated the importance of Bid in vivo: Without Bid, the Fas-activated apoptotic signaling pathway was blocked.

Though Yuan is not pursuing further research on Bid at this time, Wang and his colleagues are exploring why Bid travels specifically to the mitochondria after cleavage. "We are working on a paper that identifies a specific mitochondrial target for Bid. We have also mapped three alpha helices within Bid as the portion that specifies targeting to mitochondria," says Wang.

 

Nadia S. Halim can be contacted at nhalim@the-scientist.com.

 

References

1. X.S. Liu et al., "Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c," Cell, 86:147-57, 1996.

2. J. Yang et al., "Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked," Science, 275:1129-32, 1997.

3. K. Wang et al., "BID: a novel BH3 domain-only death agonist," Genes and Development, 10:2859-69, 1996.

4. J.J. Chou et al., "Solution structure of BID, an intracellular amplifier of apoptotic signaling," Cell, 96:615-24, 1999.

5. J.M. McDonnell et al., "Solution structure of the proapoptotic molecule BID: a structural basis for apoptotic agonists and antagonists," Cell, 96:625-34, 1999.

6. X.M. Yin et al., "Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis," Nature, 400:886-91, 1999.