The Characterization of a Mitochondrial System for the Study of Apoptosis and its Inhibitors

Abstract

<P> The study of apoptosis is a rapidly growing field due to its relevance not only to development, but also its relationship to several diseases such as cancer. The Bcl-2 family of proteins function at the mitochondrial membrane, where many apoptotic stimuli converge. There are three main theories on the regulation of the Bcl-2 family proteins, supported by several methods of in vitro and cell-based studies. </p> <p> Mitochondria isolated from wild type and Bak-/-C57BL6 mouse liver are free of Bax, Bci-XL and Bid as determined by immunoblotting. A comparison of the two membranes and the use of recombinant proteins demonstrated that tBid activated Bak or Bax to permeabilize the membrane in this system, and lower concentrations of tBid were required for membrane permeabilization in the presence of both Bak and Bax. Recombinant Bci-XL inhibited this process, indicated by a decrease in cytochrome c release. Mutant recombinant proteins demonstrated that Bci-XL inhibits cytochrome c release through interactions with Bax/Bak and tBid, and by a third protein-independent mechanism. Together, this supports the Embedded Together Model. </p> <p> The mitochondrial system also functions as an intermediate in the study of inhibitors of the Bcl-2 family of proteins. Potential inhibitors 3e and 3e-D2 previously demonstrated to bind Bci-XL through fluorescence polarization were shown to have a Bcl-2 protein-independent method of membrane permeabilization that has not yet been determined. 3e also functioned as an activator of Bax and Bak. Similar to fluorescence polarization experiments, the dimeric compound 3e-D2 was more potent than monomeric 3e. </p> <p> A comparison of membranes in the presence and absence of Bak provides a robust system in which to study multiple facets of apoptosis, including but not limited to regulation of Bcl-2 proteins and the development of proteinspecific inhibitors. </p>