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.

项目摘要 ABCB 10是一种人类线粒体内膜ATP结合盒（ABC）转运蛋白， 从ATP水解中将底物输出到基质外。这个传送器对于保护 红细胞生成过程中的氧化应激（abcb 10基因敲除小鼠由于贫血和氧化损伤而在子宫内死亡） 和保护心脏（ABCB 10蛋白水平在人缺血心肌中上调）。尽管 治疗贫血和保护心脏免受氧化的潜在临床相关性， ABCB 10的底物是未知的，直到我们的小组最近发现了胆绿素，一种血红素降解产物， 抗氧化特性，作为这种转运蛋白的生理底物。我们还发现锌 中卟啉，一种血红素类似物，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物化学和结构研究打开了大门。 项目，这将有助于更好地了解分子机制，这一重要的 传送器工作。我们的实验方法包括使用功能（ATP酶测定），光谱分析， （发光共振能量转移，LRET）和在脂质中重构的ABCB 10的突变分析 纳米盘该实验系统对于在体外研究“天然的”细胞中的ABC转运蛋白具有许多优点。 就像“脂双层和生理温度下。我们可以在细菌中产生功能性的人类ABCB 10， 促进了这项研究所需的大量突变体的产生。目标1将决定 ABCB 10在其基础ATP水解周期中经历的构象变化以及这些分子如何在其基础ATP水解周期中发生构象变化。 在激活期间通过衬底改变运动。根据我们的初步数据， 来证明ABCB 10通过微小的构象变化发挥作用。如果我们的假设是正确的 将挑战普遍接受的观点，即所有相关的ABC出口商都遵循类似的分子机制。 目标2将确定对ABCB 10的刺激至关重要的底物-转运蛋白相互作用。我们将研究 蛋白质的ATP酶活性和响应于a）化学物质的变化的相关构象变化 和B）诱变推定的底物结合口袋中的残基。我们的初步 结果表明，底物的羧基和结合口袋中的两个甘氨酸对于 ABCB 10的刺激。这些氨基酸的突变导致组成型ABCB 10激活（功能获得）。 在此，我们期望获得关于底物特异性的信息，发现推定的抑制剂，鉴定必需残基， 在结合口袋中，并定义伴随蛋白质功能改变的构象变化。在 总的来说，该项目将提供分子信息，以验证ABC中现有的结构模型 转运蛋白场和提供的想法，以调节ABCB 10的活性用于治疗目的。