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.

项目总结 ABCB10是一种人线粒体内膜三磷酸腺苷结合盒(ABC)转运体，它利用能量 从三磷酸腺苷的水解中将底物从基质中输出。此传送器对于防止 红细胞生成过程中的氧化应激(abcb10基因敲除小鼠因贫血和氧化损伤在子宫中死亡) 和保护心脏(ABCB10蛋白水平在人类缺血心肌中上调)。尽管它的 对于治疗贫血和保护心脏免受氧化的潜在临床意义， ABCB10‘S底物未知，直到我们小组最近发现胆绿素，一种具有 抗氧化性，作为这种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，像底物一样增加转运体的基础ATPase活性。 对这些底物的鉴定为本研究中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种重要的 传送器工作正常。我们的实验方法包括使用功能性(ATPase分析)、光谱 (发光共振能量转移，LRET)和脂类重组ABCB10的突变分析 纳米盘。该实验系统对ABC转运蛋白的体外研究具有许多优势。 比如“在生理温度下的双层脂类。我们可以在细菌中产生功能性的人类ABCB10， 促进了这项研究所需的大量突变体的生产。目标1将决定 ABCB10在其基本的ATP水解循环中经历的构象变化以及这些分子是如何 在激活过程中，底物对运动进行修改。根据我们的初步数据，这一目标是意料之中的 以证明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将确定底物-转运体相互作用，这是ABCB10‘S刺激的关键。我们会研究 蛋白质的ATPase活性和相关的构象变化对a)化学变化的响应 底物群和b)假定底物结合口袋中的残基的诱变。我们的预赛 结果表明底物的羧基和结合口袋中的两个精氨酸对 ABCB10‘S刺激。这些精氨酸的突变会导致ABCB10的结构性激活(功能增益)。 在这里，我们希望获得关于底物特异性的信息，寻找假定的抑制剂，识别必要的残基 在结合口袋中，并定义伴随蛋白质功能变化的构象变化。在……里面 一般而言，这个项目将提供分子信息，以验证ABC中当前的结构模型 转运蛋白的领域，并提供了想法，以调节ABCB10的S活动的治疗目的。