Mechanism of Apoptosis Inhibition By Bcl-XL

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DESCRIPTION(provided by applicant): Neuronal cell death by apoptosis is often associated with cerebral ischemia. Currently, a number of factors that influence neuronal cell death have been identified. Among them, members of the Bcl-2 family represent a group of proteins that serve as key regulators of apoptosis by promoting either ceR survival as in the case of Bcl-2 and Bcl-XL, or cell death as in the case of Bax. Upregulation of the pro-apoptotic factor Bax has been reported in the affected area of the brain, implicating the participation of this protein in promoting neuronal cell death. In healthy living cells, Bax is predominantly a cytosolic protein and its ability to modulate cell death is associated with its translocation from the cytosol to mitochondria during apoptosis. This pro-apoptotic activity of Bax however, can be blocked by coexpression with Bcl-XL, a pro-survival member of the Bcl-2 family that is essential for neuronal development and is localized mainly to mitochondria. The long-range goal of this project is to define the underlying mechanisms by which Bax and Bcl-XL regulate apoptosis to enable the development of neuroprotective compounds. The objective of this application is to define the molecular basis by which Bcl-XL inhibits Bax activity as a first step towards accomplishing the long-range goal. The central hypothesis for this proposal is that Bcl-XL blocks the pro-apoptotic function of Bax by preventing its redistribution from the cytosol to mitochondria during apoptosis. We have developed a simple method of tracking the movement of Bax by tagging it to the green fluorescent protein. This enables us to determine how the presence of various Bcl-XL mutants affects the intracellular distribution of Bax by confocal microscopy. In addition, we have begun to study how Bcl-XL exerts its effect. With a unique epitopespecific rnonoclonal antibody that we have generated against Bcl-XL, we have purified a major Bcl-X1 associated protein by immunoaffinity chromatography and identified it as ATP synthase subunit. To accomplish the objective of this application, the following specific aims will be pursued: 1) determine the functional domains of BCI-XL involved in inhibiting Bax translocation to mitochondria, 2> determine the specificity and universality of BCI-XL interaction with ATP synthase subunit, 3) determine the sites of interaction between Bcl-XL and ATP synthase subunit, 4) determine the role of ATP synthase subunit in regulating Bax redistribution to mitochondria and cell death, and 5) determine whether ATP synthase subunit can regulate the channel forming properties of BcIXL Upon completion of this proposal, we expect to have gained further understanding of the structural/functional properties of BCI-XL and its binding protein and the underlying mechanism of Bax inhibition by Bcl-X1 during apoptosis.