Development of a fluorescent reporter to assess iron regulatory protein activity in living cells.
Development of a fluorescent reporter to assess iron regulatory protein activity in living cells.
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开发荧光报告基因以评估活细胞中铁调节蛋白的活性。
DOI：
10.1016/j.bbamcr.2004.09.032
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Connor,JamesR
中科院分区：
文献类型：
--
作者：
Henderson,RebeccaJ;Patton,StephanieM;Connor,JamesR
通过将铁反应元件 (IRE) 插入 pd2ECFP 载体，我们展示了一种非侵入性方法，用于确定铁调节蛋白 (IRP) 活性的变化，从而导致活细胞中蛋白质翻译的变化。该构建体利用 IRP 之间特异性铁依赖性相互作用，将 mRNA 上的 IRE 结合起来，以类似于铁蛋白产生的方式转录后调节蛋白质表达。在本报告中，我们使用 HEK-293 细胞证明，IRE 驱动的荧光报告基因可用于观察足以改变蛋白质合成的细胞铁状态的变化。当铁利用率降低时，青色荧光蛋白 (CFP) 表达量减少，表明 IRP 与 IRE 结合并阻断蛋白质翻译。相反，将细胞暴露于铁会增加 CFP 荧光。该构建体比传统使用的染料和现有的 IRE 驱动构建体具有优势，因为它可用于在较长时间内重复研究铁影响的蛋白质生产。该构建体的未来应用包括研究细胞突变如何影响细胞铁代谢，以及各种类型的外源应用营养剂、应激剂和治疗剂如何影响细胞铁代谢。
Through the insertion of an iron responsive element (IRE) into a pd2ECFP vector, we demonstrate a noninvasive method for determining alterations in iron regulatory protein (IRP) activity that results in changes in protein translation in living cells. This construct takes advantage of the specifically iron-dependent interaction between IRPs that bind IREs on mRNAs to posttranscriptionally regulate protein expression in a manner similar to ferritin production. In this report, we demonstrate, using HEK-293 cells, that an IRE-driven fluorescent reporter can be used to observe changes in cellular iron status that are sufficient to alter protein synthesis. When iron availability was decreased, there was less cyan fluorescent protein (CFP) expression, suggesting that IRPs bind to the IRE and block protein translation. Conversely, exposing the cells to iron increased CFP fluorescence. This construct has advantages over traditionally used dyes and existing IRE driven constructs because it can be used to repeatedly study iron-influenced protein production over extended periods of time. The future applications of this construct include investigation of how mutations in cells may impact cellular iron metabolism and how various types of exogenously applied trophic, stress, and therapeutic agents may impact cellular iron metabolism.