受體相關凋亡路徑 (death receptor-mediated apoptosis pathway) 及粒線體凋亡路徑 (mitochondrial-mediated apoptotic pathway) 為兩條較清楚的路徑調控著細胞凋亡；在內部凋亡傳遞路徑中，前胸腺素 (Prothymosin α, ProTα) 在細胞體外試驗中具有抑制細胞凋亡體形成及caspase-3活化的現象。更進一步證據顯示在哺乳動物細胞中前胸腺素α與P8會形成複合體，以及此複合體會於staurosporine引起細胞凋亡中更強烈抑制抗細胞凋亡的作用。ProTα為一個酸性 (pI=3.55) 且缺少二級結構之蛋白質，P8為一80胺基酸所組成且無特殊三級結構之鹼性蛋白質，此二蛋白質皆為核蛋白且被視為共轉錄因子。在此研究中，我們的興趣在於這些核蛋白如何調控細胞質所發生之事件，如細胞凋亡。
為了研究ProTα調控粒線體凋亡路徑是透過直接抑制細胞質中細胞凋亡體的形成或於細胞核內表現抗細胞凋亡蛋白基因，我們於此研究構築一將ProTα與綠螢光蛋白 (Enhance Green Fluorescence Protein, EGFP) 融合之轉殖基因，以及建立於HeLa細胞過度表現ProTα-EGFP融合蛋白之穩定轉殖株。螢光顯微鏡分析顯示ProTα-EGFP融合蛋白持續表現及聚集於轉殖株的細胞核內。利用西方墨點法分析轉殖株的ProTα-EGFP融合蛋白及上游凋亡蛋白表現；比較野生型HeLa細胞及轉殖株，Bcl-2及Bax於過度表現ProTα-EGFP轉殖株中分別呈現調升及調降現象。這些結果加強ProTα可能作用於改變粒線體凋亡路徑上游細胞凋亡相關蛋白表現。 Death receptor-mediated pathway and mitochondrial-mediated pathway are two well-defined apoptotic pathways regulating the progression of apoptosis. In the regulation of mitochondrial-mediated apoptotic pathway, Prothymosin α (ProTα) has an inhibitory effect on the formation of apoptosome and on the activation of capase-3 in vitro. Furthermore, accumulating evidences demonstrated that ProTα could form a complex with P8 in mammalian cells and that this complex could significantly enhance the antiapoptoic effect induced by staurosporine. Both ProTα, a highly acidic (pI=3.55) protein lacking apparent secondary structure, and P8, a highly basic 80-aa protein lacking non-specific tertiary structure, are nuclear proteins, and it bas been postulated that these two proteins function as co-transcription factor. In this study, we intend to address how these nuclear proteins regulate the cytosolic events, such as apoptosis.
In order to investigate whether ProTα regulates mitochondrial-mediated apoptosis pathway through direct inhibition of apoptosome formation in cytosol or through gene expression of antiapoptotic proteins in nucleus, we have constructed a transgene encoding ProTα fusion with EGFP and established HeLa stable clones overexpression of ProTα-EGFP fusion protein. Fluorescence microscope analysis has shown that ProTα-EGFP fusion protein is consitutively expressed and localized in the nuclei of transfectants. The expression levels of ProTα-EGFP fusion protein and upstream apoptotic proteins in transfectants also have been measured by western blotting analysis. Compared with wild type of HeLa cell, Bcl-2 and Bax showed up-regulation and down-regulation, respectively in ProTα expressing transfectants. These results raises the possibility that ProTα affects the progression of mitochondrial-mediated apoptosis through altering the expression levels of apoptosis-associated proteins.
