Collaborative Research:The ferric uptake regulator (Fur) regulates intracellular iron homeostasis via reversible binding of a [2Fe-2S] cluster in Escherichia coli

该NSF奖将支持huang ending博士（路易斯安那州立大学）和Codrina V. Ewbank-Popescu博士（圣托马斯大学）的合作项目，用于研究生和本科生的研究。从分子生物学到光谱学的研究活动都试图了解蛋白质调节细菌内铁含量的分子机制。铁是所有生物必需的元素。然而，在有氧条件下，铁超载会对细胞造成危险的氧化损伤。因此，严格调节铁含量对细胞生长至关重要。在细菌中，铁摄取调节剂（Fur）是一种全局铁调节剂。当铁含量过高时，Fur成为一种转录抑制因子，阻断铁转运体的形成，防止细胞中过量铁的积累。然而，Fur感知细胞内铁含量的机制在很大程度上仍然难以捉摸。这个项目是基于初步研究表明，在大肠杆菌中，当细胞内铁含量高时，Fur可以结合一簇铁与硫结合。该项目的目标是确定Fur如何响应细胞内铁浓度的变化而与[2Fe-2S]-簇结合，进而调节细菌中铁代谢相关基因的表达。这个项目将使我们更好地了解细菌，包括我们组织中的感染细菌，是如何从动物和植物细胞中提取生长所必需的铁，并将其用于细菌生长的。这将使我们了解细菌与生态系统、农业和健康领域的大型生物之间的相互作用。该项目还将为本科生和研究生提供参与充满活力和令人兴奋的跨学科研究活动的机会。铁调节剂（Fur）是细菌中高度保守的全局铁调节剂。目前的假设认为，当细胞内“游离”铁含量升高时，毛皮结合亚铁形成“铁结合”毛皮，并通过控制特定基因的表达来调节细胞内铁的稳态。然而，“铁结合”皮毛从未从任何细菌中分离出来。初步研究表明，在细胞内铁含量升高的大肠杆菌突变细胞中，大肠杆菌皮毛可以通过保守的半胱氨酸残基结合[2Fe-2S]簇。该项目的目标是验证一个假设，即Fur通过[2Fe-2S]簇的可逆结合来调节细胞内铁稳态。目的1：结合生物化学和光谱学（包括紫外-可见吸收光谱、电子顺磁共振（EPR）和Mössbauer光谱）方法，确定Fur中[2Fe-2S]簇的氧化还原性能和稳定性。目的2通过分析[2Fe-2S]簇结合Fur的体外特异性DNA结合活性和[2Fe-2S]簇结合Fur在大肠杆菌细胞中调控的基因表达谱，探讨[2Fe-2S]簇结合Fur的生理功能。目的3是探索其他细菌皮毛蛋白中[2Fe-2S]簇结合的保守性。由于Fur在细菌中高度保守，该项目的成功有望阐明Fur通过铁硫簇调节大肠杆菌和其他细菌细胞内铁稳态的新机制。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This NSF award will support a collaborative project of Dr. Huangen Ding (Louisiana State University) and Dr. Codrina V. Ewbank-Popescu (University of St. Thomas) for research conducted with graduate and undergraduate students. Research activities ranging from molecular biology to spectroscopy seek to understand the molecular mechanism by which the protein Fur regulates iron content inside bacteria. Iron is an essential element for all living organisms. However, iron overload can cause dangerous oxidative damage to cells under aerobic conditions. Thus, tight regulation of the iron content is essential for cells to grow. In bacteria, the Ferric Uptake Regulator (Fur) is a global iron regulator. When iron content is too high, Fur becomes a transcription repressor to block formation of the iron transporters and prevent accumulation of excess iron in cells. However, the mechanism by which Fur senses the intracellular iron content remains largely elusive. This project is based on preliminary studies showing that in the bacterium E coli, Fur can bind a cluster of Iron bound to Sulfur when the intracellular iron content is high. The goal of the project is to determine how Fur may undergo the binding of this [2Fe-2S]-cluster in response to changes of the intracellular iron concentration and in turn regulate the expression of the genes related to iron metabolism in bacteria. This project will provide greater understanding of how bacteria, including infective bacteria in our tissues, can pull iron essential for growth from inside animal and plant cells, to be used instead for bacterial growth. This will inform our knowledge of the interactions of bacteria with larger creatures in the ecosystem, agriculture, and health. The project will also provide opportunities for undergraduate and graduate students to participate in vigorous and exciting interdisciplinary research activities.The ferric iron regulator (Fur) is a highly conserved global iron regulator in bacteria. The current hypothesis states that when the intracellular “free” iron content is elevated, Fur binds ferrous iron to form an “iron-bound” Fur and regulates the intracellular iron homeostasis by controlling the expression of specific genes. However, the “iron-bound” Fur has never been isolated from any bacteria. Preliminary studies revealed that the Escherichia coli Fur can bind a [2Fe-2S] cluster via the conserved cysteine residues in the E. coli mutant cells in which the intracellular iron content is elevated. The goal of this project is to test a hypothesis that Fur regulates the intracellular iron homeostasis via reversible binding of a [2Fe-2S] cluster. Aim 1 is to determine the redox properties and stability of the [2Fe-2S] cluster in Fur by combining biochemical and spectroscopic (including UV-Visible absorption spectroscopy, electron paramagnetic resonance (EPR) and Mössbauer spectroscopy) approaches. Aim 2 is to investigate the physiological function of the [2Fe-2S] cluster-bound Fur by analyzing its specific DNA binding activity in vitro and the gene expression profile regulated by the [2Fe-2S] cluster-bound Fur in E. coli cells. Aim 3 is to explore the conserved nature of the [2Fe-2S] cluster binding in Fur proteins from other bacteria. Because Fur is highly conserved among bacteria, success of the project is expected to illustrate a novel mechanism by which Fur regulates the intracellular iron homeostasis in E. coli and other bacteria via an iron-sulfur cluster.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.