Molecular Physiology of Mitochondrial Calcium Transporters

线粒体钙转运蛋白的分子生理学

• 批准号: 10487518
• 负责人: Ming-Feng Tsai
• 金额: $ 32.26万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10487518
• 关键词: Active Biological Transport; Acute; Address; Apoptosis; Architecture; Attention; Buffers; Calcium; Cardiac; Carrier Proteins; Cell Death; Cell physiology; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Co-Immunoprecipitations; Complex; Coupled; Cysteine; Disease; Engineering; Epilepsy; Functional disorder; Future; Generations; Genetic Diseases; Heart Diseases; Heart failure; Homeostasis; Human; Human Pathology; Investigation; Ion Transport; Ions; Kinetics; Knowledge; Label; Link; Liposomes; Mediating; Medicine; Membrane; Methods; Mitochondria; Mitochondrial Matrix; Molecular; Movement; Myocardial Ischemia; Nature; Neoplasm Metastasis; Nerve Degeneration; Neurodegenerative Disorders; Oxidative Phosphorylation; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Photobleaching; Physiological; Physiology; Play; Positioning Attribute; Potassium Channel; Procedures; Production; Property; Proteins; Regulation; Reperfusion Injury; Research; Role; Scanning; Side; Signal Transduction; Specificity; System; Testing; Wolf-Hirschhorn Syndrome; Work; antiport; antiporter; calcium uniporter; cell growth; design; follow-up; human disease; improved; inhibitor; insight; knock-down; loss of function mutation; mutant; novel; novel therapeutic intervention; predictive test; prime editor; reconstitution; single molecule; small molecule; stoichiometry; tool; virtual

Project Summary/Abstract The mitochondrial Ca2+ transport system modulates mitochondrial Ca2+ levels to control important cellular processes including ATP generation, cell-death pathways, and buffering of intracellular Ca2+ signals. Malfunction of mitochondrial Ca2+ transport induces cardiac ischemia-reperfusion injury and neurodegeneration, facilitates cancer metastasis, and provokes many other detrimental conditions in human disease. This system includes three major players, the mitochondrial Ca2+ uniporter complex, the Na+/Ca2+ exchanger (mediated by the NCLX protein), and the H+/Ca2+ exchanger (possibly mediated by Letm1). Although the mitochondrial Ca2+ uniporter has been studied extensively, the transport and regulatory mechanisms of the other two Ca2+ exchangers remain mostly unknown. These exchangers are important for physiology, because cardiac-specific deletion of NCLX causes heart failure, and loss of a copy of LETM1 in humans induces epilepsy in the deadly genetic disease Wolf-Hirschhorn syndrome. Here, we propose to study the fundamental mechanisms of these mitochondrial Ca2+ exchangers and their contribution to mitochondrial Ca2+ homeostasis. In Aim 1, we will determine the transmembrane topology and transport mechanisms of Letm1 using a wide range of methods, including functional analysis of liposome-reconstituted proteins, substituted cysteine accessibility scan, single- molecule photobleaching, and co-immunoprecipitation. Furthermore, we will employ new-generation CRISPR prime-editor tools to test the hypothesis that Letm1 is the protein that mediates mitochondrial H+/Ca2+ exchange and that it can load Ca2+ into mitochondria under physiological conditions. In Aim 2, we developed a novel procedure to purify human NCLX and reconstitute the protein in liposomes. This powerful tool will be employed to establish the Na+/Ca2+ exchange stoichiometry, Michaelis-Menten kinetic parameters, and the mechanisms underlying ion recognition. It will also allow us to determine how a small-molecule, membrane- permeant compound CGP-37157 potently inhibits NCLX, thus providing useful information to further improve this drug for potential clinical use. Completing the proposed work will fundamentally improve the scientific knowledge of two mitochondrial Ca2+ transport proteins that play important roles in human pathophysiology, and will pave the way for future endeavors to design new therapeutic strategies to treat debilitating diseases caused by abnormal mitochondrial Ca2+ transport and homeostasis.

项目总结/摘要 线粒体Ca 2+转运系统调节线粒体Ca 2+水平，以控制重要的细胞增殖。 包括ATP生成、细胞死亡途径和细胞内Ca 2+信号缓冲的过程。 线粒体Ca ~（2+）转运功能障碍导致心肌缺血再灌注损伤和神经退行性变， 促进癌症转移，并在人类疾病中引起许多其他有害的状况。该系统 包括三个主要参与者，线粒体Ca 2+单向转运体复合物，Na+/Ca 2+交换器（由 NCLX蛋白）和H+/Ca 2+交换剂（可能由Letm 1介导）。虽然线粒体Ca 2 + 单转运体已被广泛研究，其他两个Ca 2+的运输和调节机制 交换器仍然是未知的。这些交换器对生理学很重要，因为心脏特异性 NCLX的缺失会导致心力衰竭，而人类LETM 1拷贝的缺失会导致致命的癫痫。 遗传性疾病沃尔夫-赫希霍恩综合征在这里，我们建议研究这些的基本机制， 线粒体Ca 2+交换器及其对线粒体Ca 2+稳态的贡献。在目标1中，我们 使用多种方法确定Letm 1的跨膜拓扑结构和转运机制， 包括脂质体重构蛋白的功能分析、取代的半胱氨酸可及性扫描、单- 分子光漂白和免疫共沉淀。此外，我们将采用新一代CRISPR技术， prime-editor工具来检验Letm 1是介导线粒体H+/Ca 2+的蛋白质这一假设 在生理条件下，它可以将Ca 2+负载到线粒体中。在目标2中，我们开发了一个 纯化人NCLX并在脂质体中重构蛋白质的新方法。这个强大的工具将是 用于建立Na+/Ca 2+交换化学计量，Michaelis-Menten动力学参数，和 离子识别的基本机制。它也将使我们能够确定一个小分子，膜- 渗透性化合物CGP-37157有效地抑制NCLX，从而提供有用的信息，以进一步改善 这种药物的潜在临床应用。完成拟议的工作将从根本上提高科学性 了解在人类病理生理学中起重要作用的两种线粒体Ca 2+转运蛋白， 并将为未来设计新的治疗策略来治疗衰弱性疾病铺平道路 由线粒体钙离子转运和稳态异常引起。