| 摘要: | 前胸腺素 (Prothymosin α, PA) 為一個由111個胺基酸所組成的核蛋白。根據以往的研究指出，PA 發揮重要作用在細胞增殖、轉錄調控、染色質重塑、氧化應激反應、免疫反應以及細胞凋亡。PA 過度表現刺激細胞增生，而縮短細胞週期的 G1 期來干擾分化。而轉錄因子E2F 及一些原致癌基因如 c-myc等會促進 ProT 的表現。在一些腫瘤細胞中發現 ProT 的含量會增加。
PA 分子機制尚未清楚，除了前胸腺素抗凋亡活性是經由抑制凋亡體 (apopsome) 形成。先前研究指出，PA 藉由負調控 caspase-9 活性而阻斷凋亡體形成而阻止 caspase 效應的活化。更進一步研究證明，p8 和 PA 形成一個複合體參與 staurosporine 誘導細胞凋亡 (apoptosis) 的調控，且可採取一致行動調控細胞凋亡級聯。p8 為一個 80 胺基酸所組成且無特殊三級結構的鹼性 (pI=9.98) 蛋白質，PA 為一個酸性 (pI=3.55) 且缺少二級結構的蛋白質，此兩蛋白質皆為核蛋白且被視為共轉錄因子。在這項研究中，由於 PA 及 p8 同為核蛋白，所以我們研究 PA-p8 複合體調節抗凋亡基因以達抑制細胞凋亡。我們於此構築 p8 與紅螢光蛋白融合之轉殖基因，以及建立過度表現 p8-RFP 於 HeLa 細胞株、 p8 與 PA 融合蛋白及 p8 與刪減型 PA 融合蛋白之穩定轉殖株。螢光顯微鏡分析顯示 p8-RFP 融合蛋白II持續表現及聚集於轉殖株的細胞質及細胞核內。利用西方墨點法分析短暫性轉染 p8-RFP 於 PA 融合蛋白轉殖株及上游凋亡蛋白表現；比較野生型 HeLa 細胞及短暫性轉染之轉殖株，caspase-3 及 Bax 於過度表現刪減型 PA 轉殖株中呈現調降現象。
為了研究 PA 調控粒線體凋亡路徑是透過抑制細胞質中細胞凋亡小體的形成，我們於此研究中構築一刪減型 PA 與綠螢光蛋白融合之轉殖基因，以及建立過度表現刪減型 PA-EGFP 融合蛋白之穩定轉殖株。螢光顯微鏡分析顯示刪減型 PA-EGFP 融合蛋白持續表現及聚集於轉殖株的細胞質。利用西方墨點法分析轉殖株的刪減型 PA-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，caspase-3、cytochrome c 及 Bax 於過度表現刪減型 PA 轉殖株中呈現調降現象。這些結果顯示 PA 與 p8 複合體及刪減型 PA 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。
Prothymosin α (PA), a nuclear protein of 111 amino acids plays essential roles in cell proliferation, transcriptional regulation, chromatin remodeling, oxidative stress- response, immune response, and apoptosis. Overexpression of PA stimulates cell proliferation, shortening the G1-phase of the cell cycle, and interfering with differentiation. The transcription factor E2F and the proto-oncogene c-myc directly control the expression of PA.
The molecular mechanisms of PA activities are largely unknown, except for its antiapoptotic activity, which is mediated through blocking of apoptosome formation by negatively regulating caspase-9 activation. Further studies demonstrated that p8 and PA form a complex involved in the regulation of staurosporine-induced apoptosis, and they could act in concert to regulate the apoptotic cascade. The structures of both p8 and PA are still unresolved. Both p8, a highly basic (pI=9.98) 80-aa protein lacking non-specific tertiary structure, and PA, a highly acidic (pI=3.55) protein lacking apparent secondary structure, is a nuclear protein, and it is postulated that these two proteins function as co-transcription factors. In this study, we investigate how the PA-p8 complex regulates the anti-apoptotic genes to achieve their function in the inhibition of apoptosis. We have constructed a transgene encoding p8 fusion with RFP and established HeLa stable clones overexpression of p8-RFP fusion protein, PA stable clones overexpression of p8-RFP fusion protein, and truncated PA stableclones overexpression of p8-RFP fusion protein. Fluorescence microscope analysis has shown that p8-RFP fusion protein is constiutively expressed and localized in the nuclei and the cytosol of transfectants. The transient transfection expression levels of p8-RFP fusion protein in PA stable clones and upstream apoptotic proteins in transfectants also have been measured by western blotting analysis. Compared with wild type of HeLa cell, caspase-3 and Bax showed down-regulation, respectively in PA-p8 complex expressing transfectants.
In order to investigate whether PA regulates mitochondrial-mediated apoptosis pathway through inhibition of apoptosome formation in cytosol, We have constructed a transgene encoding truncated PA fusion with EGFP and established HeLa stable clones overexpression of truncated PA-EGFP fusion protein. Fluorescence microscope analysis has shown that truncated PA-EGFP fusion protein is constiutively expressed and localized in the cytosol of transfectants. The expression levels of truncated PA-EGFP fusion protein and upstream apoptotic proteins in transfectants also have been measured by western blotting analysis. Compared with wild type of HeLa cell, caspase-3 and Bax showed down-regulation, respectively in truncated PA expressing transfectants.
The results that PA-p8 complex and truncated PA affects the progression of mitochondrial-mediated apoptosis through altering the expression levels of apoptosis-associated proteins. |
