Life Sciences Division, Korea Institute of Science and Technology, 39-1 Hawolgok, Seongbuk, Seoul 136-791, Korea.
Biochemistry. 2010 Feb 23;49(7):1435-47. doi: 10.1021/bi901783s.
The death effector domain (DED) of the mammalian apoptosis mediator, Fas-associated death domain protein (FADD), induces Escherichia coli cell death under aerobic culture conditions, yet the mechanisms by which FADD-DED induces cell death are not fully understood. Oxidative stress has been implicated as one of the mechanisms. Using a proteomic approach and validation by coexpression analysis, we illustrate that overexpression of FADD-DED in E. coli invokes protein expression changes that facilitate conversion of pro-oxidant NADH into antioxidant NADPH. Typically, isocitrate dehydrogenase, phosphoenolpyruvate carboxykinase, and pyruvate kinase are downregulated and malate dehydrogenase is upregulated. We reasoned that such a change in E. coli cells is an active response to reduce the size of the NADH pool, thereby decreasing the level of ROS generation. From the coexpression studies, we observed that DNA binding protein Hns, which induces growth arrest when overexpressed heterologously, alleviated the cell killing effect of FADD-DED. FADD-DED was expressed as a noncovalently linked multimeric protein in the membrane of E. coli. Exogenous treatment of E. coli cells with FADD-DED in the presence of a membrane component induced cell death, which was accompanied by a shift of the redox balance and a decrease in the cellular ATP level. Cell death was blocked by prior expression of thioredoxin. Localization of FADD-DED to the membrane may shift the cells into a state that stimulates and fuels ROS generation. The cell death mechanism mediated by ROS may mimic antibiotic-mediated bacterial cell death or Bax-mediated apoptosis in mammalian cells. Our results provide a common mechanistic feature of ROS-involved cell death throughout prokaryotes and eukaryotes.
哺乳动物凋亡介质 Fas 相关死亡结构域蛋白（FADD）的死亡效应结构域（DED）在需氧培养条件下诱导大肠杆菌细胞死亡，但 FADD-DED 诱导细胞死亡的确切机制尚不完全清楚。氧化应激已被认为是其中的一种机制。我们使用蛋白质组学方法并通过共表达分析进行验证，结果表明 FADD-DED 在大肠杆菌中的过表达会引起蛋白表达变化，从而促进将前氧化剂 NADH 转化为抗氧化剂 NADPH。通常，异柠檬酸脱氢酶、磷酸烯醇丙酮酸羧激酶和丙酮酸激酶下调，而苹果酸脱氢酶上调。我们推断，大肠杆菌细胞中的这种变化是一种主动响应，旨在减小 NADH 池的大小，从而降低 ROS 生成水平。从共表达研究中，我们观察到当异源过表达时，DNA 结合蛋白 Hns 会诱导生长停滞，从而减轻 FADD-DED 的细胞杀伤作用。FADD-DED 在大肠杆菌的膜上以非共价连接的多聚体形式表达。在膜成分存在的情况下，外源处理大肠杆菌细胞会诱导细胞死亡，这伴随着氧化还原平衡的转变和细胞内 ATP 水平的降低。硫氧还蛋白的预先表达可阻断细胞死亡。FADD-DED 定位于膜可能会使细胞进入刺激和促进 ROS 生成的状态。ROS 介导的细胞死亡机制可能模拟抗生素介导的细菌细胞死亡或 Bax 介导的哺乳动物细胞凋亡。我们的研究结果为原核生物和真核生物中涉及 ROS 的细胞死亡提供了一个共同的机制特征。
