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 ABCB10是一种使用能量的人线粒体内膜ATP结合盒（ABC）转运蛋白 从ATP水解到从基质中输出基质。该运输蛋白对于防止 红细胞生成期间的氧化应激（ABCB10敲除小鼠因贫血和氧化损伤而在子宫中死亡） 并保护心脏（ABCB10蛋白水平在人缺血性心肌中进行了更新）。尽管有它 治疗贫血和保护心脏免受氧化的潜在临床相关性， ABCB10的底物是未知的 抗氧化特性，作为该转运蛋白的物理底物。我们还发现锌 中磷脂是一种血红素类似物，像底物一样增加了转运蛋白的基本ATPase活性。 这些底物的识别已为此开辟了生化和结构研究的大门 项目，这将有助于更好地理解这种重要的分子机制 运输蛋白作品。我们的实验方法涉及使用功能（ATPase分析），光谱法 （发光共振能量传递，LRET）和ABCB10的突变分析在脂质中重构 纳米盘。该实验系统在“天然 - 喜欢”脂质双层和在物理温度下。我们可以在细菌中产生功能性的人ABCB10， 促进这项研究所需的众多突变体的产生。 AIM 1将确定 ABCB10在其基本ATP水解周期中经历的构象变化以及这些分子如何 在底物激活过程中修改运动。根据我们的初步数据，预计这一目标 为了证明ABCB10通过微小的咨询更改发挥作用。如果我们的假设正确，我们的发现 将挑战所有相关的ABC出口商遵循类似的分子机制的观念。 AIM 2将确定对ABCB10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ATPase活性和相关的会议变化响应于a）化学物质的变化 底物和b）在推定的底物结合袋中残留物的诱变。我们的初步 结果表明，结合口袋中的基板的羧基和两个精氨酸对于 ABCB10的刺激。这些精氨酸的诱变会导致宪法ABCB10激活（功能获得）。 在这里，我们希望获得有关底物特异性的信息，找到假定的抑制剂，确定必要的保留 在结合口袋中，并定义涉及蛋白质功能改变的构象变化。 一般，该项目将提供可以验证ABC中当前结构模型的分子信息 运输者领域并提供想法，以调节ABCB10的活动以进行治疗。