Iron Storage by Ferritin and Microbial Growth

铁蛋白和微生物生长的铁储存

• 批准号: RGPIN-2015-04802
• 负责人: Murphy, Michael
• 金额: $ 3.79万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=613175
• 关键词: Iron; Storage; Ferritin; Microbial; Growth

Iron is essential for almost all forms of life. It is a cofactor for cell sustaining reactions such as mediating single electron transfer reactions of the electron transport chain or the formation of DNA building blocks by ribonucleotide reductases. The unique reactivity of metals such as iron necessitates control of uptake and intracellular handling to avoid toxic side effects. Understanding the cellular mechanisms of iron homeostasis in microbes is the central direction of my research and the focus of this research program is the role of ferritins. Ferritins are iron storage proteins constructed of 12 or 24 subunits that assemble into a hollow sphere. Ferrous iron is oxidized to ferric ions by oxygen or hydrogen peroxide at ferroxidase sites before storage inside the sphere as an iron oxide mineral. By removing ferrous iron and consuming dioxygen or hydrogen peroxide, ferritins have an antioxidant role. Less is known about the mechanism of iron release from ferritin or how iron storage is regulated. I hypothesize that the mineral core of ferritin regulates the concentration of readily exchangeable cellular iron and that iron release is dependent on the reduction potential of the cell. Ferritin is proposed to be used by bloom-forming pennate diatoms to store excess iron for use in subsequent cell divisions thereby out competing centric diatoms that do not possess ferritin. Our hypothesis is that this ferritin has distinct functional features to supply iron to the photosynthetic machinery of the diatom. Escherichia coli has 3 different types of ferritin with potential distinct roles in iron storage and detoxification and is a model system to investigate ferritin function. Specific aims are: 1. To identify putative amino acid residues directly involved in Fe storage and release by ferritins by site-directed mutagenesis. Crystal structures, kinetics of the ferroxidase reaction, iron mineralization, and iron release of recombinant wild-type and variants will assayed. 2. To show that a ferritin stores iron in cell, metal content analysis by ICP-MS will be done with native ferritin isolated from cells grown under varying iron availability and oxidative stress. 3. Iron release from ferritins will be examined by incubating iron loaded samples in the presence of physiological levels of ferrous iron chelating metabolites (eg. histidine) and reductants such as flavins and NAPH. A screen will be undertaken to search for proteins involved in iron release. The results of this work will contribute to understanding of diatom blooms in the open ocean and the provide a foundation to understanding how pathogens cope with iron restriction imposed by host plants and animals.

铁对几乎所有形式的生命都是必不可少的。它是细胞维持反应的辅因子，如介导电子传输链的单电子转移反应或通过核糖核苷酸还原酶形成DNA构建块。铁等金属的独特反应性需要控制摄取和细胞内处理，以避免毒副作用。了解微生物体内铁稳态的细胞机制是我研究的中心方向，本研究计划的重点是铁蛋白的作用。 铁蛋白是由12或24个亚基组成的储铁蛋白，这些亚基组装成一个中空球。在以氧化铁矿物的形式储存在球体内之前，亚铁在氧化铁酶位置上被氧或过氧化氢氧化成铁离子。通过去除亚铁和消耗氧气或过氧化氢，铁蛋白具有抗氧化作用。人们对铁蛋白释放铁的机制或铁储存是如何调节的知之甚少。我假设铁蛋白的矿物质核心调节细胞内易交换的铁的浓度，铁的释放依赖于细胞的还原潜力。铁蛋白被花朵形成的羽状硅藻用来储存多余的铁，以供随后的细胞分裂使用，从而击败没有铁蛋白的竞争中心硅藻。我们的假设是，这种铁蛋白具有独特的功能特征，可以为硅藻的光合作用机制提供铁。大肠杆菌有3种不同类型的铁蛋白，在铁的储存和解毒方面具有不同的潜在作用，是研究铁蛋白功能的模型系统。 具体目标是： 1.通过定点突变鉴定与铁储存和铁释放直接相关的氨基酸残基。将分析重组野生型和变异体的晶体结构、铁氧合酶反应动力学、铁矿化和铁释放。 2.为了证明铁蛋白在细胞中储存铁，从不同铁可利用性和氧化应激条件下生长的细胞中分离出天然铁蛋白，用电感耦合等离子体质谱(ICP-MS)进行金属含量分析。 3.铁蛋白的铁释放将通过在生理水平的亚铁螯合代谢物(例如。组氨酸)和还原剂，如黄素和NAPH。将进行一项筛选，以寻找与铁释放有关的蛋白质。 这项工作的结果将有助于了解开放海洋中的硅藻水华，并为了解病原体如何应对宿主动植物施加的铁限制提供基础。