Molecular Physiology of Mitochondrial Calcium Transporters

线粒体钙转运蛋白的分子生理学

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要线粒体 Ca2+ 转运系统调节线粒体 Ca2+ 水平以控制重要的细胞过程包括 ATP 生成、细胞死亡途径和细胞内 Ca2+ 信号缓冲。线粒体 Ca2+ 转运功能障碍导致心脏缺血再灌注损伤和神经退行性变，促进癌症转移，并引发人类疾病中的许多其他有害状况。这个系统包括三个主要参与者：线粒体 Ca2+ 单向转运蛋白复合物、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+ uniporter 已被广泛研究，另外两种 Ca2+ 的运输和调节机制交换者大多仍不为人所知。这些交换器对于生理学很重要，因为心脏特异性 NCLX 缺失会导致心力衰竭，而人类 LETM1 副本的缺失会导致致命性癫痫。遗传病沃尔夫-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 Ca2+ 交换器及其对线粒体 Ca2+ 稳态的贡献。在目标 1 中，我们将使用多种方法确定 Letm1 的跨膜拓扑和运输机制，包括脂质体重组蛋白的功能分析、取代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 用于检验 Letm1 是介导线粒体 H+/Ca2+ 的蛋白质这一假设的 prime-editor 工具交换，并且在生理条件下它可以将 Ca2+ 加载到线粒体中。在目标 2 中，我们开发了纯化人类 NCLX 并在脂质体中重建蛋白质的新程序。这个强大的工具将用于建立 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膜如何渗透化合物 CGP-37157 有效抑制 NCLX，从而为进一步改善提供有用的信息该药物具有潜在的临床用途。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发挥重要作用的两种线粒体 Ca2+ 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 Ca2+ 转运和稳态异常引起。