The assembly of iron-sulphur proteins in germinating seeds

发芽种子中铁硫蛋白的组装

• 批准号: BB/K008838/1
• 负责人: Janneke Balk
• 金额: $ 43.17万
• 依托单位: University of East Anglia
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK008838%2F1
• 关键词: assembly; iron; sulphur; proteins; germinating

Seeds contain large amount of carbon and minerals to help the germinating seedling put down a root and fold out its first leaves, before it can obtain its own water, nutrition and energy. This fascinating process of seedling establishment is underpinned by dramatic biochemical changes invisible to the naked eye. Storage lipids are broken down and converted to sugars and protein, while the photosynthetic machinery and green pigments are rapidly assembled. In this proposal we will investigate how one important nutrient, iron (Fe), is mobilized from its stores in seed and is incorporated into the catalytic sites of Fe enzymes. Iron is an essential mineral that functions predominantly in electron transfer and catalytic reactions. Its chemical versatility is enhanced by how the iron interacts with the enzyme, such as in a ring structure in haem, or together with inorganic sulphide in Fe-S clusters. The latter form of Fe is most dominant in plants, and therefore an important source of human iron nutrition. Because free Fe and sulphide are toxic, the assembly of Fe-S clusters needs to be tightly controlled, involving proteins that carefully handle the Fe and S, and deliver it safely to the newly produced enzymes without inadvertent release of the intermediates. At least 30 proteins have so far been identified that are involved in Fe-S cluster assembly in plants. The proteins have been assigned to three different pathways, located in different parts of the cell: the mitochondria, chloroplasts and cytosol. While the former two are fairly well studied because the pathways are similar in bacteria, our knowledge on the cytosolic pathway is very limited. This pathway is essential for a range of critical Fe-S enzymes, however, such as a key enzyme in seedling lipid mobilization (aconitase), and more than 20 enzymes involved in DNA replication, repair and regulation.In the past years, we have started to characterize a protein, NBP35, that is thought to play a key role in the Fe-S assembly pathway in the cytosol. In addition, we have evidence that the sulphide is provided by the mitochondria. In the next few years we would like to find the source of iron and how it is delivered to NBP35, and identify other proteins that function together with NBP35.To investigate the source of iron, we will extend our initial studies on iron transporter mutants, and analyse how they are affected in the activity of Fe-S enzymes in different cell compartments. Since little is known about the mobilization of Fe in germinating seedlings, we will also compile a data set with changes in Fe distribution over day 1 - 10, and compare this to changes in protein and transcript levels of genes that play a role in handling or regulating Fe. The systems approach will be combined with a molecular-biochemical study of S and Fe delivery to NBP35. To find direct interaction partners of NBP35, we will precipitate the protein from cell extract with specific antibodies. Proteins that co-precipitate will be identified using mass spectrometry, and their interaction with NBP35 will be further characterized in so called yeast-2-hybrid assays. Last but not least, any genes of interest that come out of the systems- or protein-interaction studies will be further investigated using mutant analyses. This will reveal whether the proteins indeed function in cytosolic Fe-S cluster assembly. Knowledge on the mobilization of Fe in seeds and how it enters cofactor biosynthesis pathways is important for improving germination success rates and crop yields. This knowledge will also be important for expressing Fe-S proteins like nitrogenase in plants. This project links in with research to improve seeds as a source for human Fe nutrition.

种子含有大量的碳和矿物质，帮助发芽的幼苗在获得自己的水分，营养和能量之前放下根并展开第一片叶子。这个迷人的幼苗建立过程是由肉眼看不到的戏剧性生化变化支撑的。储存脂质被分解并转化为糖和蛋白质，而光合作用机制和绿色色素迅速组装。在这个建议中，我们将调查一个重要的营养，铁（Fe），是如何从其在种子中的商店动员，并纳入铁酶的催化位点。铁是一种重要的矿物质，主要在电子转移和催化反应中发挥作用。它的化学多功能性通过铁如何与酶相互作用而增强，例如在血红素中的环状结构中，或与Fe-S簇中的无机硫化物一起。后一种形式的铁在植物中占主导地位，因此是人体铁营养的重要来源。由于游离的Fe和硫化物是有毒的，因此需要严格控制Fe-S簇的组装，包括小心处理Fe和S的蛋白质，并将其安全地传递给新产生的酶，而不会无意中释放中间体。到目前为止，至少有30种蛋白质已被确定参与植物中的Fe-S簇组装。这些蛋白质被分配到三个不同的途径，位于细胞的不同部分：线粒体，叶绿体和细胞质。虽然前两个是相当好的研究，因为在细菌中的途径是相似的，我们对胞质途径的知识是非常有限的。这条途径是必不可少的一系列关键的Fe-S酶，然而，如在幼苗脂质动员的关键酶（乌头酸酶），和超过20个酶参与DNA复制，修复和regulation.In过去几年中，我们已经开始表征蛋白质，NBP35，这被认为是在细胞质中的Fe-S组装途径中发挥关键作用。此外，我们有证据表明硫化物是由线粒体提供的。在接下来的几年里，我们希望找到铁的来源，以及它是如何传递到NBP35的，并确定其他蛋白质与NBP35一起发挥作用。为了研究铁的来源，我们将扩展我们对铁转运蛋白突变体的初步研究，并分析它们如何影响不同细胞室中Fe-S酶的活性。由于对发芽幼苗中Fe的动员知之甚少，我们还将编制一个数据集，其中包含第1 - 10天Fe分布的变化，并将其与在处理或调节Fe中发挥作用的基因的蛋白质和转录水平的变化进行比较。该系统的方法将结合分子生物化学研究的S和Fe交付NBP35。为了找到NBP35的直接相互作用伙伴，我们将用特异性抗体从细胞提取物中沉淀蛋白质。共沉淀的蛋白质将使用质谱法鉴定，并且它们与NBP35的相互作用将在所谓的酵母-2-杂交测定中进一步表征。最后但并非最不重要的是，任何感兴趣的基因，来自系统或蛋白质相互作用的研究将进一步研究使用突变分析。这将揭示蛋白质是否确实在胞质Fe-S簇组装中起作用。种子中铁的动员以及它如何进入辅因子生物合成途径的知识对于提高发芽成功率和作物产量是重要的。这些知识对于在植物中表达Fe-S蛋白如固氮酶也很重要。该项目与研究相联系，以改善种子作为人类铁营养的来源。

项目成果

NBP35 interacts with DRE2 in the maturation of cytosolic iron-sulphur proteins in Arabidopsis thaliana.

• DOI: 10.1111/tpj.13409
• 发表时间: 2017-02
• 期刊: The Plant journal : for cell and molecular biology
• 作者: Bastow EL;Bych K;Crack JC;Le Brun NE;Balk J
• 通讯作者: Balk J

A conserved mitochondrial ATP-binding cassette transporter exports glutathione polysulfide for cytosolic metal cofactor assembly.

• DOI: 10.1074/jbc.m114.553438
• 发表时间: 2014-08-22
• 期刊: The Journal of biological chemistry
• 作者: Schaedler TA;Thornton JD;Kruse I;Schwarzländer M;Meyer AJ;van Veen HW;Balk J
• 通讯作者: Balk J