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：
10354245
负责人：
Maria Elena Zoghbi
金额：
$ 22.59万
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 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10354245
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.

项目概要人类线粒体内膜有 3 个 ABC 转运蛋白：ABCB7 涉及在铁转运（突变导致 X 连锁铁粒幼细胞贫血伴共济失调）中，ABCB8 参与线粒体 K+ 通道的调节和针对阿霉素毒性的保护，以及 ABCB10，它对于红细胞发育、防止氧化应激至关重要，并且它的完全缺失导致小鼠胚胎死亡。尽管其重要性显而易见，但分子研究却很少对这些人类线粒体转运蛋白的影响，主要是因为人类膜蛋白的产生难的。我们相信，促进这些转运蛋白的生产将加速所需的研究更好地从分子角度理解其功能背后的机制。人类 ABC 转运蛋白有只能在昂贵且耗时的真核系统中生产，直到我们小组最近有了在细菌中生产 ABCB10 的想法。我们的初步研究表明细菌产生的 ABCB10 是功能性的。在这里，我们建议使用这种新颖的方法来简单、快速且廉价地生产 ABCB7 和 ABCB8。目标 1 将开发功能性 ABCB7 的表达和纯化方法细菌中的ABCB8。目标 2 将优化将纯化的转运蛋白重构为脂质的方法双层（纳米圆盘和脂质体）用于进行功能测定。我们的建议具有高风险，因为我们将测试在细菌中产生这些人类线粒体转运蛋白的可行性。这个提议是意义重大，因为它将为两种产品的生产和功能测试提供优化的方法几乎没有被研究过的重要人类蛋白质，这将带来很高的回报。我们的经验 ABC 转运蛋白分子研究小组结合这些新方法，将提供更雄心勃勃的 R01 提案的途径，旨在研究基本分子机制这些人类线粒体转运蛋白。