果蠅的TBP功能缺失所導致的細胞凋亡是透過氧化壓力及p53的活化

Abstract

先前研究發現TATA box binding protein gene (TBP) 失去功能，會導致老鼠和斑馬魚早期發育停止及細胞凋亡。TBP如何調控動物發育及細胞凋亡的機制目前並不很清楚的，本論文的主要目的在於研究TBP導致細胞凋亡的性狀在果蠅是否保守，並了解其調控機制，我們發現TBP突變果蠅僅發育至胚胎晚期，且細胞死亡數大量增加。進一步分析TBP缺失細胞即為死亡細胞，顯見TBP對細胞凋亡的調控為自發性的，遺傳分析結果顯示，表現負顯性P53或DIAP1可抑制TBP缺失引起的細胞凋亡，反之大量表視TBP則可抑制P53引起之細胞死亡，顯示TBP為P53引發細胞凋亡作用路徑的成員；此外相較野生型，TBP突變果蠅的總蛋白質量和總核酸量並沒有顯著的減少，顯示TBP可能僅介由影響少數特定基因的表現造成細胞死亡，利用基因微陣列分析，TBP突變果蠅其Prx2540-2基因表現大量降低，Prx蛋白具水解過氧化氫(H2O2)活性，為細胞抵抗氧化壓力之重要成員。實驗發現TBP突變果蠅其過氧化氫濃度相較野生型果蠅為高，且餵食抗氧化劑可有效增長TBP突變株果蠅的生存壽命。綜言之，我們証實TBP功能缺失可籍由活化P53及氧化壓力造成細胞死亡。Previous studies revealed that loss of TBP function causes developmental arrest and apoptosis in mice and zebrafish. How TBP regulates animal development and apoptosis is currently elusive. The main objectives of this study are to test whether function of Drosophila TBP (dTbp) in regulating apoptosis is conserved, and to dissect the mechanisms by which dTbp regulates apoptotic pathway. We find that homozygous dTbp mutant flies die during late embryogenesis, and ectopic cell death was observed in the dying embryos. Somatic clone analysis reveals that loss of dTbp causes apoptosis in a cell autonomous manner. In genetic analyses, we find that ectopic expression of dominant negative P53 (P53DN) or DIAP1 can suppress deactivated dTbp induced cell death, indicating dTbp is a member of P53 mediated apoptotic pathway. We also find that total protein and ribonucleic acid are not reduced significantly in dTbp mutant flies, suggesting that dTbp might affect only a few genes to regulate apoptosis. Through gene profiling experiments, we find that the expression of Prx2540-2 is greatly reduced in dTbp mutants. Prx normally reduces hydrogen peroxide, and can protect cells from oxidative stress. In our preliminary studies, we find that H2O2 is higher in dTbp mutant flies, and antioxidants can extend the lifespan of dTbp mutants. In sum, our result suggests that activation of P53 and oxidative stress contribute to deactivated dTbp induced apoptosis.