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Production of human mitochondrial ABC transporters for structural and biochemical studies

生产用于结构和生化研究的人类线粒体 ABC 转运蛋白

基本信息

批准号：
10482375
负责人：
Maria Elena Zoghbi
金额：
$ 19.03万
依托单位：
UNIVERSITY OF CALIFORNIA, MERCED
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-10 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10482375
关键词：
ABCB1 gene ATP Hydrolysis ATP phosphohydrolase ATP-Binding Cassette Transporters Affect Affinity Chromatography Anemia Ataxia Attention Bacteria Binding Biochemical Biological Assay Caliber Cell membrane Cells Cessation of life Circular Dichroism Consumption Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Detergents Development Doxorubicin Embryo Erythrocytes Escherichia coli Future Generations Genetic Goals Hereditary Sideroblastic Anemia Hour Human Individual Inner mitochondrial membrane Insecta Ion Channel Iron Knockout Mice Laboratories Life Link Lipid Bilayers Lipids Liposomes Mammalian Cell Measurement Membrane Membrane Proteins Membrane Transport Proteins Methodology Methods Micelles Mitochondria Mitochondrial Membrane Protein Modeling Molecular Molecular Sieve Chromatography Multi-Drug Resistance Mus Mutation Nutrient Organelles Orphan Outcome Oxidative Stress Pathway interactions Phospholipids Phylogenetic Analysis Physiological Potassium Channel Production Prokaryotic Cells Property Proteins Regulation Research Sample Size Scaffolding Protein Structure System Techniques Temperature Testing Time Toxic effect Uterus Validation Xenobiotics Yeasts experience feasibility testing heart function high reward high risk lipid transport milligram molecular transporter mutant nanodisk novel novel strategies reconstitution spectroscopic survey success

项目摘要

Project Summary The human mitochondrial inner membrane has three ABC transporters: ABCB7 implicated in iron transport (with mutations causing X-linked sideroblastic anemia with ataxia), ABCB8 involved in regulation of a mitochondrial K+ channel and protection against doxorubicin toxicity, and ABCB10, which is essential for red blood cell development, protection against oxidative stress and whose complete absence leads to death of mice embryos. Despite their clear importance, little molecular research has been done on these human mitochondrial transporters, mainly because production of human membrane proteins is difficult. We believe that facilitating the production of these transporters will accelerate the research needed for a better molecular understanding of the mechanisms underlying their function. Human ABC transporters had only been produced in expensive and time-consuming eukaryotic systems until our group recently had the idea of producing ABCB10 in bacteria. Our preliminary studies show that ABCB10 produced in bacteria is functional. Here, we propose to use this novel approach for the easy, fast, and inexpensive production of ABCB7 and ABCB8. Aim 1 will develop methodologies for expression and purification of functional ABCB7 and ABCB8 in bacteria. Aim 2 will optimize methodologies for reconstitution of the purified transporters into a lipid bilayer (nanodiscs and liposomes) to perform functional assays. Our proposal is of high risk because we will test the feasibility of producing these human mitochondrial transporters in bacteria. This proposal is significant because it will provide optimized methodologies for production and functional testing of two important human proteins that have barely been studied, which will be of high reward. The experience of our group on ABC transporters molecular research, combined with these new methodologies, will provide a pathway for a more ambitious R01 proposal aimed at studying the fundamental molecular mechanisms of these human mitochondrial transporters.

人类线粒体内膜有三种ABC转运蛋白：ABCB 7参与在铁转运中（突变导致X连锁铁粒幼细胞性贫血伴共济失调），ABCB 8参与调节线粒体K+通道和保护免受阿霉素毒性，以及ABCB 10，对红细胞发育、保护免受氧化应激是必需的，导致小鼠胚胎死亡。尽管它们的重要性显而易见，但几乎没有进行过分子研究这些人类线粒体转运蛋白，主要是因为人类膜蛋白的生产，难我们相信，促进这些转运体的生产将加速所需的研究，更好地了解其功能背后的分子机制。人类ABC转运蛋白只有在昂贵和耗时的真核系统中才能产生，直到我们的团队最近才有了在细菌中产生ABCB 10的想法。我们的初步研究表明，细菌中产生的ABCB 10是不降低在这里，我们建议使用这种新的方法，简单，快速，廉价的生产 ABCB7和ABCB8。目的1将开发表达和纯化功能性ABCB 7的方法，细菌中的ABCB8。目的2将优化重组纯化的转运蛋白到脂质中的方法双层（纳米盘和脂质体）以进行功能测定。我们的建议是高风险的，因为我们将测试在细菌中生产这些人类线粒体转运蛋白的可行性。这项建议是重要的是，它将提供优化的生产和功能测试方法，重要的人类蛋白质，几乎没有被研究，这将是高回报。我们的经验 ABC转运蛋白分子研究小组，结合这些新的方法，将提供一个一个更雄心勃勃的R01建议，旨在研究的基本分子机制，这些人类线粒体转运蛋白。