Molecular Mechanisms of The Human Mitochondrial ABC Transporter ABCB10

人类线粒体 ABC 转运蛋白 ABCB10 的分子机制

• 批准号: 10596638
• 负责人: Maria Elena Zoghbi
• 金额: $ 30.44万
• 依托单位: UNIVERSITY OF CALIFORNIA, MERCED
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10596638
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Adopted; Affect; Amino Acids; Anemia; Antioxidants; Arginine; Bacteria; Biliverdine; Binding; Biochemical; Biological Assay; Biological Transport; Cell membrane; Cessation of life; Coupling; Cryoelectron Microscopy; Data; Detergents; Development; Dimerization; Embryo; Environment; Erythropoiesis; Goals; Heart; Heme; Human; Hydrolysis; In Vitro; Inner mitochondrial membrane; Knockout Mice; Lead; Lipid Bilayers; Lipids; Measurement; Measures; Mechanics; Membrane; Methodology; Mitochondria; Modeling; Molecular; Molecular Conformation; Movement; Mus; Mutagenesis; Mutation; Mutation Analysis; Myocardial Ischemia; Nucleotides; Organelles; Oxidative Stress; Physiological; Population; Power stroke; Production; Property; Protein Conformation; Proteins; Publications; Pump; Reporting; Research; Role; Site; Structural Models; Structure; Substrate Domain; Substrate Specificity; System; Techniques; Temperature; Testing; Therapeutic; Time; Translational Research; Transmembrane Domain; Uterus; Variant; Work; analog; base; chemical group; clinically relevant; dimer; functional group; gain of function; heme a; in utero; inhibitor; luminescence resonance energy transfer; mutant; nanodisk; oxidation; oxidative damage; reconstitution; reconstruction; response; translational medicine; zinc mesoporphyrin

PROJECT SUMMARY ABCB10 is a human mitochondrial inner membrane ATP binding cassette (ABC) transporter that uses energy from ATP hydrolysis to export a substrate out of the matrix. This transporter is essential for protection against oxidative stress during erythropoiesis (abcb10 knock-out mice die in uterus due to anemia and oxidative damage) and protecting the heart (ABCB10 protein level is upregulated in human ischemic myocardium). Despite its potential clinical relevance for treating anemia and protecting the heart against oxidation, the identity of ABCB10’s substrate was unknown until our group recently identified biliverdin, a heme degradation product with antioxidant properties, as the physiological substrate for this transporter. We have also found that zinc mesoporphyrin, a heme analog, increases the basal ATPase activity of the transporter like substrates do. Identification of these substrates has opened the door to the biochemical and structural studies proposed in this project, which will contribute to a better understanding of the molecular mechanisms by which this important transporter works. Our experimental approach involves the use of functional (ATPase assays), spectroscopic (Luminescence Resonance Energy Transfer, LRET), and mutational analysis of ABCB10 reconstituted in lipid nanodiscs. This experimental system has many advantages for the in vitro study of ABC transporters in a “native- like” lipid bilayer and at physiological temperature. We can produce functional human ABCB10 in bacteria, facilitating the production of the numerous mutants needed for this research. Aim 1 will determine the conformational changes that ABCB10 undergoes during its basal ATP hydrolysis cycle and how those molecular movements are modified during activation by substrate. According to our preliminary data, this aim is expected to prove that ABCB10 functions through small conformational changes. If our hypothesis is correct, our findings will challenge the generally accepted idea that all related ABC exporters follow a similar molecular mechanism. Aim 2 will determine substrate-transporter interactions that are critical for ABCB10’s stimulation. We will study the protein’s ATPase activity and associated conformational changes in response to a) variations in the chemical groups of the substrates and b) mutagenesis of residues in a putative substrate binding pocket. Our preliminary results suggest that the substrate’s carboxyl groups and two arginines in the binding pocket are critical for ABCB10’s stimulation. Mutagenesis of these arginines cause constitutive ABCB10 activation (gain-of-function). Here, we expect to gain information about substrate specificity, find putative inhibitors, identify essential residues in the binding pocket, and define conformational changes that accompany alterations in protein’s function. In general, this project will provide molecular information that can validate current structural models in the ABC transporters field and provide ideas to modulate ABCB10’s activity for therapeutic purposes.

项目概要 ABCB10 是一种利用能量的人线粒体内膜 ATP 结合盒 (ABC) 转运蛋白 ATP 水解将底物从基质中导出。该转运蛋白对于防止 红细胞生成过程中的氧化应激（abcb10 敲除小鼠因贫血和氧化损伤在子宫内死亡） 保护心脏（人缺血心肌中ABCB10蛋白水平上调）。尽管其 治疗贫血和保护心脏免受氧化的潜在临床意义， ABCB10 的底物直到我们小组最近鉴定出胆绿素（胆绿素）为止都是未知的，胆绿素是一种血红素降解产物，具有 抗氧化特性，作为该转运蛋白的生理底物。我们还发现，锌 中卟啉是一种血红素类似物，可像底物一样增加转运蛋白的基础 ATP 酶活性。 这些底物的鉴定为本文提出的生化和结构研究打开了大门 项目，这将有助于更好地理解这一重要的分子机制 运输机工作。我们的实验方法涉及使用功能性（ATP 酶测定）、光谱 （发光共振能量转移，LRET）以及在脂质中重组的 ABCB10 的突变分析 纳米圆盘。该实验系统对于 ABC 转运蛋白的“天然”体外研究具有许多优势。 就像“脂质双层和在生理温度下。我们可以在细菌中生产功能性人类ABCB10， 促进本研究所需的大量突变体的产生。目标 1 将确定 ABCB10 在其基础 ATP 水解循环期间经历的构象变化以及这些分子如何 运动在底物激活过程中发生改变。根据我们的初步数据，这个目标是预期的 证明 ABCB10 通过微小的构象变化发挥作用。如果我们的假设是正确的，我们的发现 将挑战所有相关 ABC 出口商都遵循类似分子机制的普遍接受的观点。 目标 2 将确定对于 ABCB10 的刺激至关重要的底物-转运蛋白相互作用。我们将学习 蛋白质的 ATP 酶活性和相关的构象变化，以响应 a) 化学物质的变化 b) 假定的底物结合袋中残基的诱变。我们的初步 结果表明，底物的羧基和结合口袋中的两个精氨酸对于 ABCB10的刺激。这些精氨酸的诱变导致 ABCB10 组成型激活（功能获得）。 在这里，我们期望获得有关底物特异性的信息，找到假定的抑制剂，识别必需的残基 在结合口袋中，并定义伴随蛋白质功能改变的构象变化。在 一般来说，该项目将提供可以验证 ABC 中当前结构模型的分子信息 转运蛋白场并提供调节 ABCB10 活性以达到治疗目的的想法。