| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 92 === Apoptosis is an important form of programmed cell death required for the embryonic development and tissue homeostasis of multicellular organisms. The extrinsic death receptors- and intrinsic mitochrondria-dependent pathways following the apoptotic stimulus leading to caspases activation have been well characterized. Cytochrome c released from mitochondria functions as a trigger for the formation of apoptosome in the intrinsic apoptotic pathway. The apoptosome is a heptameric complex comprised of apoptotic protease activating factor-1 (Apaf-1) and cytochrome c. The apoptosome binds and activates procaspase-9, resulting in activation of further caspases, such as caspase-3, which orchestrate the final packaging of the apoptotic cell. Apaf-1 is a 130-kDa protein consisting of a caspase recruitment domain (CARD), an arm domain, and two WD-40 repeats. It was shown that the WD-40 repeats act as a recognition domain for mitochondrial damage through binding to cytochrome c, allowing Apaf-1 to oligomerize and interact with procaspase-9 through the CARD-CARD interaction. However, little is known about how human cytochrome c interacts with Apaf-1. Using 3D structure of bovine transducin complex (2TRC) and E. coli Tolb protein (1CRZ) as structural templates, 3D model structures of two WD-40 repeats (WD-40 (7) and WD-40 (6)) was generated using homology modeling. Based on the structural analyses of human cytochrome c docking into two WD-40 repeats of Apaf-1 model, we hypothesized that the basic residues K72, K73, K86, K87 and K88 of cytochrome c maybe involved in the interaction with the acidic D residues of two WD-40 domains of Apaf-1. It was reported that the nitration of Y67 residue promoted a conformational change, resulting in affecting activation of caspases by cytochrome c. To identify the residues of cytochrome c involved in activation of caspases, we used site-directed mutagenesis on human cytochrome c and cell-free caspase activation assay to carry out the study. In this study we have expressed seven cytochrome c mutants (K72A/K73A, K72A/K86A, K86A/K87A/K88A, Y67A, Y67F, Y67H, and Y67W) and purified them to homogeneity with the yields of 5-15 mg/L. To identify the interaction between human cytochrome c and Apaf-1, we determined the biological activity of recombinant cytochrome c by using a cell-free caspase activation assay. This method indirectly measures their ability to bind Apaf-1, and the fluorogenic Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (DEVD-AFC) was used as the caspase-3 substrate. The mutations of K72, K86, K87, and K88 to alanines caused the decrease in activating caspase 3, suggesting that these residues maybe involved in Apaf-1 binding. These results may be important not only for identifying the binding residues of cytochrome c to apaf-1, but also for understanding cell apoptosis.
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