Investigating Calcium Homeostasis During a Key Mitochondrial Stress Pathway

研究关键线粒体应激途径中的钙稳态

• 批准号: 10553100
• 负责人: Melissa Jane Macewen
• 金额: $ 4.51万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-16 至 2024-01-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10553100
• 关键词: Alzheimer' s Disease; Apoptosis; Biological Assay; CRISPR screen; Calcium; Calcium Signaling; Calcium ion; Cell Line; Cell Nucleus; Cells; Cellular Stress; Cellular biology; Chimeric Proteins; Clustered Regularly Interspaced Short Palindromic Repeats; Computer software; Cytosol; Data; Disease; Educational process of instructing; Ensure; Event; Exhibits; Faculty; Financial cost; Funding; Genes; Genetic; Genetic Transcription; Goals; Health; Heat shock proteins; Hereditary Spastic Paraplegia; Homeostasis; Human; Image Analysis; Imaging Techniques; Immunity; Inner mitochondrial membrane; Ions; Knock-out; Knowledge; Laser Scanning Confocal Microscopy; Lead; Link; Mediating; Messenger RNA; Metabolism; Mitochondria; Mitochondrial Matrix; Mitochondrial Proteins; Molecular; Molecular Chaperones; Mutation; Neurodegenerative Disorders; Nuclear; Parkinson Disease; Pathway interactions; Phosphotransferases; Play; Prevention; Proteins; Regulation; Reporter; Research; Role; Signal Pathway; Signal Transduction; Stress; Testing; Training; Transcriptional Regulation; Translations; Universities; Washington; Western Blotting; Work; biological adaptation to stress; calcium uniporter; candidate identification; career; computerized data processing; experimental study; genome-wide; insight; knock-down; live cell imaging; live cell microscopy; microscopic imaging; misfolded protein; mitochondrial metabolism; new therapeutic target; novel; optical imaging; pharmacologic; programs; protein degradation; proteostasis; response; skills; success; therapeutic target; tool; transcription factor; treatment strategy; uptake

PROJECT SUMMARY Mitochondria are central to cellular metabolism, signaling, protein homeostasis, immunity, and apoptosis. One of the ways by which mitochondria respond effectively to changing mitochondrial and cellular needs is with calcium signaling. Ca2+ ions enter the mitochondrial matrix through a protein channel called the mitochondrial calcium uniporter (MCU) that resides on the inner membrane of the mitochondria. Altered MCU-mediated mitochondrial calcium signaling is involved in a variety of human neurodegenerative diseases including Parkinson's disease, Alzheimer's disease, and hereditary spastic paraplegia. Furthermore, these same diseases have been shown to exhibit the hallmarks of mitochondrial protein stress, including elevated protein levels of mitochondrial chaperones. Mitochondrial protein stress can be caused by misfolded or unfolded proteins accumulating within the mitochondria. Preliminary studies in the Sancak Lab suggest that MCU-mediated calcium signaling plays a critical role in facilitating the mitochondrial protein stress response. Data showing increased MCU mRNA and protein during mitochondrial protein stress suggests that MCU is upregulated as part of a mitochondrial protein stress response. Even more intriguingly, relative to WT and MCU rescue cell lines, MCU knockout cells have lower baseline levels of mitochondrial chaperones and ATF4, a transcription factor central to cellular stress responses. Furthermore, when mitochondrial protein stress is induced, the mRNA levels of ATF4 and these chaperones increase much less than in WT or rescue cells. The central hypothesis of this proposal is that altered MCU regulation and calcium signaling comprise a heretofore uncharacterized pathway that regulates transcription in response to mitochondrial protein stress. Aim 1 will offer new insight about the molecular mechanisms regulating the transcription, translation and protein stability of MCU. The functional studies of Aim 2 will reveal how mitochondrial protein stress alters calcium signaling and transcription. Successful completion of this proposal will advance the field by identifying novel players in the poorly understood mitochondria-to-nuclear signaling pathway. These findings will also uncover a new function for mitochondrial calcium signaling in the regulation of a transcriptional response central to the mitochondrial protein stress response, and will reveal potential therapeutic targets for the treatment of neurodegenerative disease. Completion of Aim 1 will provide valuable training in pairing gene editing with pharmacological tools to dissect the function and regulation of MCU, a protein with great disease relevance. Completion of Aim 2 will provide training in using laser-scanning confocal microscopy and live imaging techniques to study mitochondrial stress. The University of Washington is very well funded and well equipped. Its top tier faculty, staff, and technicians, and variety of state-of-the-art facilities will ensure the success of this proposed work.

项目摘要 线粒体对于细胞代谢，信号传导，蛋白质稳态，免疫和凋亡是核心。一 线粒体对线粒体和细胞需求的有效反应的方式与 钙信号传导。 Ca2+离子通过称为线粒体的蛋白质通道进入线粒体基质 位于线粒体内膜上的钙Uniporter（MCU）。 MCU介导的改变 线粒体钙信号传导与多种人类神经退行性疾病有关 帕金森氏病，阿尔茨海默氏病和遗传性痉挛性截瘫。此外，这些相同的疾病 已显示出表现出线粒体蛋白质应激的标志，包括蛋白质水平升高 线粒体伴侣。线粒体蛋白质应激可能是由错误折叠或展开的蛋白质引起的 在线粒体内积聚。 Sancak实验室的初步研究表明MCU介导 钙信号传导在促进线粒体蛋白质应激反应中起关键作用。显示的数据 线粒体蛋白质应激期间MCU mRNA和蛋白质的增加表明MCU被上调为一部分 线粒体蛋白质应激反应。更有趣的是，相对于WT和MCU营救细胞系， MCU基因敲除细胞的线粒体伴侣和ATF4的基线水平较低，ATF4是转录因子 细胞应激反应的中心。此外，当诱导线粒体蛋白质应激时，mRNA水平 ATF4和这些伴侣的增加远低于WT或救援细胞。中心假设 提案是改变MCU调节和钙信号传导包括迄今未经特色的途径 这对线粒体蛋白应激的响应调节转录。 AIM 1将提供有关有关的新见解 调节MCU转录，翻译和蛋白质稳定性的分子机制。功能 AIM 2的研究将揭示线粒体蛋白应力如何改变钙信号传导和转录。成功的 该提案的完成将通过识别不太了解的新颖球员来推进该领域 线粒体至核信号通路。这些发现还将发现线粒体的新功能 在调节线粒体应激的转录响应中的钙信号传导 反应，并将揭示用于治疗神经退行性疾病的潜在治疗靶标。 AIM 1的完成将提供有价值的培训，以配对基因编辑与药理学工具进行剖析 MCU的功能和调节，MCU，一种具有很大疾病相关的蛋白质。 AIM 2的完成将提供 使用激光扫描共聚焦显微镜和实时成像技术来研究线粒体应激的培训。 华盛顿大学的资金丰富且设备齐全。它的顶级教职员工和技术人员， 各种最先进的设施将确保这项拟议工作的成功。