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

该NSF奖项将支持Huangen Ding博士（路易斯安那州立大学）和Codrina V. Ewbank-Popescu博士（圣托马斯托马斯大学）与研究生和本科生进行的研究合作项目。 从分子生物学到光谱学的研究活动旨在了解蛋白质Fur调节细菌内铁含量的分子机制。 铁是所有生物体的必需元素。 然而，在有氧条件下，铁过载会对细胞造成危险的氧化损伤。 因此，铁含量的严格调节对细胞生长至关重要。 在细菌中，铁吸收调节剂（Fur）是一种全局铁调节剂。 当铁含量过高时，Fur成为转录抑制因子，以阻止铁转运蛋白的形成并防止过量铁在细胞中的积累。 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。 该项目基于初步研究，表明在大肠杆菌中，当细胞内铁含量高时，毛皮可以结合一簇与硫结合的铁。 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，从而调节细菌中与铁代谢相关的基因的表达。该项目将提供更好的了解细菌，包括我们组织中的感染性细菌，如何从动物和植物细胞内提取生长所必需的铁，用于细菌生长。 这将为我们了解细菌与生态系统，农业和健康中较大生物的相互作用提供信息。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。三价铁调节剂（Fur）是细菌中高度保守的全球铁调节剂。目前的假说认为，当细胞内“游离”铁含量升高时，Fur结合亚铁形成“铁结合”Fur，并通过控制特定基因的表达来调节细胞内铁稳态。 然而，“铁结合”的毛皮从未从任何细菌中分离出来。 初步研究表明，大肠杆菌Fur可以通过大肠杆菌Fur蛋白中保守的半胱氨酸残基与[2Fe-2S]簇结合。大肠杆菌突变体细胞，其中细胞内铁含量升高。 本项目的目标是验证一个假设，即毛皮调节细胞内铁稳态通过可逆结合的[2Fe-2S]簇。 目的1是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）和穆斯堡尔谱）方法确定毛皮中[2Fe-2S]团簇的氧化还原性质和稳定性。目的二是通过分析[2Fe-2S]簇结合的Fur的体外特异性DNA结合活性和对大肠杆菌基因表达谱的调控，探讨其生理功能。coli细胞。 目的3：探讨其他细菌Fur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调节E.该奖项反映了NSF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.