Production of human mitochondrial ABC transporters for structural and biochemical studies

人类线粒体内膜有三种ABC转运蛋白：ABCB7 在铁转运中(突变导致X连锁铁粒母细胞贫血并共济失调)，ABCB8参与了 线粒体K通道的调节和对阿霉素毒性的保护，以及ABCB10，它 对红细胞发育、防止氧化应激是必不可少的，它完全不存在 会导致小鼠胚胎死亡。尽管它们显然很重要，但几乎没有人做过分子研究 在这些人类线粒体转运体上，主要是因为人膜蛋白的产生 很难。我们相信，促进这些转运体的生产将加速 更好地从分子上理解它们的作用机制。人类ABC转运蛋白 只在昂贵和耗时的真核系统中生产，直到我们小组最近获得了 在细菌中生产ABCB10的想法。我们的初步研究表明，在细菌中产生的ABCB10 功能齐全。在这里，我们建议使用这种新的方法来轻松、快速和廉价地生产 ABCB7和ABCB8。AIM 1将开发功能性ABCB7和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组为脂质的方法 双分子膜(纳米盘和脂质体)进行功能分析。我们的提议风险很高，因为我们将 测试在细菌中生产这些人线粒体转运蛋白的可行性。这项建议是 意义重大，因为它将为生产和功能测试提供优化的方法 几乎没有人研究过的重要的人类蛋白质，这将是很高的回报。我们的经历 ABC转运蛋白分子研究小组，结合这些新方法，将提供一种 更雄心勃勃的R01提案的途径，旨在研究 这些人类线粒体转运体。

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.