計畫性細胞凋亡在不同的真核生物中的胚胎發育及組織的發展恆定扮演十分重要角色。在正常細胞受到刺激之後，有兩條不同的途徑會引發細胞凋亡。其一途徑是外在受體受到刺激所引發的細胞凋亡，其次是與細胞中的粒腺體相關的內在途徑。這兩條訊息傳遞路徑都已經有相關的文獻研究。其中細胞色素C在於細胞受到刺激之後會從粒腺體中釋放出來並且與Apaf-1蛋白結合成apoptosome進而引發內在路徑的細胞凋亡。細胞色素C是與Apaf-1的WD40區域產生蛋白交互作用活化procaspase 9並進而產生caspase 3的活化造成細胞凋亡。但是並無相關文獻指出關於細胞色素C是藉由哪些氨基酸與Apaf-1產生交互作用。因此，我們先使用牛的transducin綜合體 (2TRC)與大腸桿菌Tolb蛋白(1CRZ)模擬出可能的Apaf-1的WD40(7)與WD40(6)區域之結構。藉由結構分析，我們假設人類細胞色素C之鹼性氨基酸Lysine可能與Apaf-1的WD40區域之酸性氨基酸Aspartic acid存在有重要的相互作用。因此，我們將人類細胞色素C上的K72, K73, K86, K87, K88突變成Ala，我們想要去驗證是否上述的Lys在與Apaf-1蛋白交互作用中扮演的角色。此外，有文獻研究指出Y67的硝化作用會造成細胞色素C的結構改變並影響其活化caspases的能力。因此，我們也對於Y67做了Y67W、Y67H、Y67A、Y67F之突變。我們將上述的人類細胞色素C之突變株表現在大腸桿菌中並使用K562細胞株細胞液去進行caspase 3的活性分析。我們在實驗中所使用的受質是螢光且具特異性的caspase 3之受質Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin(DEVD-AFC)，進而分析人類細胞色素C造成caspases活化參與的胺基酸。我們在實驗結果中發現K86、K87、K88若圖變成Ala則會使得活化Caspase 3的能力降低。因此，我們猜測這些胺基酸可能是參與和Apaf-1作用的胺基酸。藉由這些分析，我們對於細胞凋亡的機制將有更進一步的瞭解。

　　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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