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(16) THE ROLE OF BID IN APOPTOSIS A. Gross, Y. Zaltsman, R. Sarig,
K. Arnold and M. Greenberg
The proteins from the BCL-2 family are among the more prominent regulators of apoptosis. The cell death regulatory activity of these molecules mostly depends on their ability to modulate mitochondrial function. ‘BH3-domain only’ members of the BCL-2 family including the pro-apoptotic molecule BID connect signal transduction pathways with the death effector mechanism based at mitochondria. Activation of the TNF-R1/Fas cell surface ‘death’ receptors leads to early caspase-mediated cleavage of full-length p22 BID (FL-BID). The truncated product of BID (p15 tBID) translocates to the mitochondria and induces the release of cytochrome c (cyt c), which, upon release, propagates a downstream caspase cascade. Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis, indicating that BID is a critical substrate in-vivo for signaling by death-receptor agonists. Currently, it is unknown how tBID induces cyt c release, whether this is tBID’s major role and which mitochondrial components are involved in this process. It is also unclear whether FL-BID plays an active role in the apoptotic process. To identify proteins that associate with tBID at the mitochondrial membrane, mitochondria prepared from cells pretreated with TNF were treated with protein crosslinkers and tBID was purified by anti-BID antibodies. Using this approach we have identified a new ~46kD BID-immunoreactive band. An identical ~46kD band was visualized when purified mouse liver mitochondria were incubated in-vitro with recombinant tBID and treated with crosslinkers. Reprobing these blots with a variety of antibodies indicated that BAX, BAK, BCL-2, BCL-XL, VDAC and ANT were not part of the BID complex. We are now in the process of identifying this associating protein. A second approach we have taken to identify mitochondrial proteins that are involved in BID’s death pathway was to use yeast. Inducible expression of FL-BID or tBID in yeast had no detectable effect on growth or survival. Subcellular fractionations indicated that FL-BID was mostly localized in the cytosol, whereas tBID was localized to mitochondria. Despite the fact that tBID localized to mitochondria, cyt c was not released and Dym was not altered. Thus, yeast seem to be missing a protein(s), present in mammalian cells, that is essential for BID’s mitochondrial/death function. In order to study the role of the uncleaved FL-BID in the apoptotic process, we have expressed a non-cleavable FL-BID mutant (D59A) in Bid-/- mouse embryonic fibroblasts. Expression of this mutant led to mitochondrial alterations and death rates that were similar to the ones observed with wild type BID. Thus, cleavage of BID in this paradigm does not seem to be essential for inducing apoptosis. In addition, immunolocalization studies of BID and of cyt c indicated that the majority of BID (both wild type and mutant) was localized to the cytosol whereas cyt c remained in the mitochondria. Thus, FL-BID and tBID seem to differ in their ways to induce apoptosis.
For further information contact...Carmen Mannella: carmen@wadsworth.org |
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