Molecular Physiology of Mitochondrial Calcium Transporters

项目摘要/摘要 线粒体钙转运系统调节线粒体钙水平以控制重要的细胞 这些过程包括ATP的产生、细胞死亡途径和细胞内钙信号的缓冲。 线粒体钙转运功能障碍可导致心肌缺血再灌注损伤和神经变性， 促进癌症转移，并在人类疾病中引发许多其他有害条件。这个系统 包括三个主要的角色，线粒体钙单一转运体复合体，钠/钙交换器(由 NCLX蛋白)和H+/Ca~(2+)交换(可能由Letm1介导)。尽管线粒体钙离子 单一转运蛋白已经得到了广泛的研究，另外两种钙离子的转运和调控机制 交易所大多仍不为人所知。这些交换器对生理学很重要，因为心脏特有的 NCLX基因的缺失会导致心力衰竭，而人类LETM1拷贝的丢失会导致致命的癫痫 遗传病沃尔夫-赫希霍恩综合征。在这里，我们建议研究这些问题的基本机制。 线粒体钙离子交换器及其对线粒体钙稳态的贡献。在目标1中，我们将 使用多种方法确定Letm1的跨膜拓扑结构和转运机制， 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，单一... 分子光漂白和免疫共沉淀。此外，我们将采用新一代CRISPR Prime-EDITOR工具验证Letm1是介导线粒体H+/Ca~(2+)的蛋白质的假设 在生理条件下，它可以将钙离子加载到线粒体中。在目标2中，我们开发了一个 纯化人NCLX并在脂质体中重组蛋白质的新方法。这个强大的工具将是 用来建立Na~+/Ca~(2+)交换的化学计量学、米氏动力学参数和 离子识别的潜在机制。它还将使我们能够确定一个小分子，膜- 预期化合物CGP-37157有效地抑制Nclx，从而为进一步改进提供了有用的信息 这种药物具有潜在的临床应用价值。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钙转运蛋白， 并将为未来努力设计治疗衰弱疾病的新治疗策略铺平道路 由线粒体钙离子转运和动态平衡异常引起。

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.