| Apoptosis is induced by chemicals and death receptor activation. Although the stimuli are different, common proteins are activated downstream. The Bcl-2 family of proteins plays an important role in mediating apoptotic signaling at the mitochondria resulting in cytochrome c release and apoptosis. These proteins can be divided into three categories: anti-apoptotic (Bcl-X L, Bcl-2), pro-apoptotic (Bax, Bak), and BH3-only (Bid, Bim). The BH3-only proteins are hypothesized to assist in the activation of pro-apoptotic proteins. This thesis focused on the interplay between these proteins and their impact on apoptois. It was concluded that BH3-only proteins are likely activated specifically depending on apoptotic insult and that these proteins are the key mediators in the activation of pro-apoptotic Bcl-2 proteins. Chemical inhibitors and over-expression systems were used to investigate the temporal activation of BH3-only proteins and subsequently the pro-apoptotic Bcl-2 family members.; In both chemical and receptor apoptosis, the protein phosphatase inhibitor calyculin A prevented Bax translocation and the subsequent markers of apoptosis. Caspase inhibition by z-VAD-fmk showed caspase 8 and Bid cleavage are required for Bax translocation in the receptor pathway, but not in the chemical pathway. Therefore, Bax activation is not dependent on Bid in the chemical pathway. Over-expression of Bcl-XL prevented Bax translocation presumably because there is insufficient BH3-only protein to inhibit over-expressed Bcl-X L. Zinc inhibited apoptosis at the level of Bax and Bak oligomerization in the chemical pathway, but not in the receptor pathway, indicating that Bid activation is not sensitive to inhibition by zinc. Finally, Bax translocation was inhibited by taxol in the chemical but not the receptor pathway, suggesting that activation of the BH3-only protein required for Bax translocation in the chemical pathway is inhibited by taxol.; The above conclusions establish the importance of BH3-only proteins in both pathways of apoptosis. They confirm that in the receptor pathway of apoptosis Bid is the primary BH3-only protein mediating the activation of Bax and Bak, while Bid plays no role in Bax and Bak activation in the chemical pathway. Using chemical modulators of signaling, zinc was established to be important in inhibiting the chemical pathway, but not the receptor pathway, while calyculin A inhibited both pathways effectively. This establishes that zinc may be mediating its effects on a specific BH3-only protein, while calyculin A is targeting a protein phosphorylation that presumably impacts different phosphoproteins in each pathway. In summary, these results establish a temporal relationship for several apoptotic proteins including members of the Bcl-2 family and mechanisms for their regulation that may be suitable pharmacologic targets to combat diseases in which apoptosis plays a key role. |
