Molecular Physiology of Mitochondrial Calcium Transporters

线粒体钙转运蛋白的分子生理学

• 批准号: 10676910
• 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 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10676910
• 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; Cytoplasm; 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; Photobleaching; Physiological; Physiology; Play; Positioning Attribute; Potassium Channel; Procedures; Production; Property; Proteins; Regulation; Reperfusion Injury; Research; Role; Scanning; Signal Transduction; Specificity; Structure; 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; pharmacologic; prime editor; protein reconstitution; 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.

项目摘要/摘要 线粒体Ca2+传输系统调节线粒体Ca2+水平以控制重要的细胞 包括ATP生成，细胞死亡途径以及细胞内CA2+信号缓冲的过程。 线粒体Ca2+转运的故障会诱导心脏缺血 - 再灌注损伤和神经退行性， 促进癌症转移，并激发人类疾病中许多其他有害状况。这个系统 包括三个主要参与者，即线粒体Ca2+ Uniporter综合体，Na+/Ca2+交换器（由 NCLX蛋白）和H+/Ca2+交换器（可能由LETM1介导）。虽然线粒体Ca2+ 已经对Uniporter进行了广泛的研究，即其他两个CA2+的运输和调节机制 交换机仍然大多未知。这些交换器对生理很重要，因为心脏特异性 NCLX的删除会导致心力衰竭，而在人类中的LETM1副本的丢失会引起致命的癫痫 遗传疾病沃尔夫·希尔施霍恩综合征。在这里，我们建议研究这些基本机制 线粒体Ca2+交换器及其对线粒体Ca2+稳态的贡献。在AIM 1中，我们将 使用多种方法确定LETM1的跨膜拓扑和传输机制， 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，单个 分子光漂白和共免疫沉淀。此外，我们将采用新产生CRISPR 测试LETM1是介导线粒体H+/Ca2+的蛋白质的假设的主要编辑工具 交换，并且可以在生理条件下将Ca2+加载到线粒体中。在AIM 2中，我们开发了一个 纯化人NCLX并重建脂质体中蛋白质的新方法。这个强大的工具将是 用于建立Na+/Ca2+交换化学计量，Michaelis-Menten动力学参数和 离子识别的机制。它还将使我们能够确定小分子如何，膜 - Pereant复合CGP-37157有效抑制NCLX，从而提供有用的信息以进一步改善 该药物用于潜在的临床用途。完成拟议的工作将从根本上改善科学 了解两个线粒体Ca2+转运蛋白在人类病理生理学中起重要作用的知识， 并将为未来的努力铺平道路，以设计新的治疗策略来治疗衰弱的疾病 由线粒体Ca2+运输和稳态引起的。