IRON REGULATION OF GENE EXPRESSTION

铁是大多数生物生存所必需的，并在许多生物过程中发挥作用，包括氧气和电子传递、固氮和DNA合成。游离铁对细胞是有毒的，因为它能够形成活性羟基，导致脂膜过氧化和DNA损伤。细胞通过严格调节细胞吸收的铁量和储存在细胞中的铁量来维持铁的动态平衡。铁稳态是由铁调节蛋白(IRPS)调节的。铁调节蛋白1和2(Irp1和2)是参与细胞铁稳态的关键蛋白。IRP1和IRP2是胞质RNA结合蛋白，调节mRNAs的翻译或稳定性，编码与铁的吸收、隔离和利用有关的蛋白。IRP1和IRP2与特定的茎环结构结合，称为铁反应元件(IRES)，位于特定mRNA5‘-或3’-非翻译区。IRPS与5‘IRES结合导致翻译抑制，而IRPS与3’IRES结合导致mRNA稳定。虽然IRP1和IRP2调节一些相同的ire-mRNAs，但它们对特定的ire-mRNAs有偏好。根据细胞铁水平的不同，Irp1表现出两种相互排斥的活性：当铁缺乏时，Irp1与IRES结合，调节mRNA的翻译或稳定性；当铁丰富时，Irp1表现出乌头酸酶活性，催化柠檬酸和异柠檬酸的相互转化。与IRP1不同的是，IRP2缺乏乌头酸酶活性，它受铁的蛋白分解调节。除了铁以外，IRPS还受到低氧的调节，这表明它们在低氧条件下调节铁的动态平衡方面发挥着重要作用。我们计划讨论这两个IRPS在调节细胞铁代谢中的作用。这项建议的具体目的是)利用功能基因组学筛选方法分离新的ire mRNAs，2)确定改变乌头酸酶转基因小鼠和培养细胞铁稳态的机制，以及3)确定在低氧条件下调节irp1和irp2的机制。

Iron is required by most organisms for survival and plays a role in many biological processes including oxygen and electron transport, nitrogen fixation, and DNA synthesis. Free iron is toxic to cells due to its ability to form reactive hydroxyls that cause peroxidation of lipid membranes and damage DNA. Cells maintain iron homeostasis by tightly regulating the amount of iron taken up by cells and the amount of iron stored in cells. Iron homeostasis is regulated by the iron-regulatory proteins (IRPs). Iron regulatory proteins 1 and 2 (IRP1 and 2) are key proteins involved in cellular iron homeostasis. IRP1 and IRP2 are cytosolic RNA binding proteins that regulate the translation or the stability of mRNAs encoding proteins involved in uptake, sequestration and utilization of iron. IRP1 and IRP2 bind to specific stem-loop structures, termed the Iron-responsive elements (IREs), located in either the 5'- or 3'-untranslated regions of specific mRNAs. The binding of IRPs to 5' IREs results in translational repression, while the binding of IRPs to 3' IREs results in mRNA stabilization. Although IRP1 and IRP2 regulate some of the same IRE- mRNAs, they have preferences for specific IRE-mRNAs. IRP1 exhibits two mutually exclusive activities depending on cellular iron levels: When iron is scarce, IRP1 binds to IREs, regulating mRNA translation or stability; when iron is abundant, IRP 1 exhibits aconitase activity, catalyzing the interconversion of citrate and isocitrate. Unlike IRP 1, IRP2 lacks aconitase activity and it is regulated by iron by proteolysis. In addition to iron, IRPs are regulated by hypoxia, indicating that they have important roles in the regulation of iron homeostasis during low oxygen conditions. We plan to address the roles of the two IRPs in regulating cellular iron metabolism. The specific aims of this proposal l) to isolate novel IRE mRNAs using a functional genomics screening approach, 2) to determine the mechanisms altering iron homeostasis in aconitase transgenic mice and in cultured cells, and 3) to determine the mechanisms regulating IRP1 and IRP2 during hypoxia.