Investigating Calcium Homeostasis During a Key Mitochondrial Stress Pathway

项目摘要 线粒体是细胞代谢、信号传导、蛋白质稳态、免疫和凋亡的中心。一 线粒体有效应对线粒体和细胞需求变化的方式之一是， 钙信号Ca2+离子通过称为线粒体的蛋白质通道进入线粒体基质。 钙单向转运体（MCU），其位于线粒体的内膜上。MCU介导的改变 线粒体钙信号传导参与多种人类神经退行性疾病， 帕金森氏病，阿尔茨海默氏病，遗传性痉挛性截瘫。此外，这些疾病 已经显示出表现出线粒体蛋白质应激的特征，包括升高的蛋白质水平， 线粒体伴侣蛋白线粒体蛋白应激可由错误折叠或未折叠的蛋白引起 在线粒体中积累。Sancak实验室的初步研究表明，MCU介导的 钙信号在促进线粒体蛋白应激反应中起关键作用。数据显示 在线粒体蛋白应激期间MCU mRNA和蛋白质的增加表明MCU是上调的， 线粒体蛋白应激反应的一部分更有趣的是，相对于WT和MCU拯救细胞系， MCU敲除细胞具有较低的线粒体伴侣和转录因子ATF 4的基线水平 是细胞应激反应的核心此外，当诱导线粒体蛋白应激时， ATF4和这些分子伴侣的增加远低于WT或救援细胞。这个问题的核心假设是 建议是改变的MCU调节和钙信号传导包括迄今为止未表征的途径 调节线粒体蛋白应激反应的转录。Aim 1将提供有关 调控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激如何改变钙信号传导和转录。成功 完成这一建议将通过确定新的球员在鲜为人知的领域推进该领域 细胞核信号通路。这些发现还将揭示线粒体的一种新功能， 钙信号在线粒体蛋白应激转录反应调节中的作用 反应，并将揭示用于治疗神经退行性疾病的潜在治疗靶点。 目标1的完成将为基因编辑与药理学工具配对提供有价值的培训， MCU是一种与疾病相关性很大的蛋白质，其功能和调控。目标2的完成将提供 培训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压力。 华盛顿大学资金充足，设备齐全。它的顶级教职员工和技术人员， 各种先进的设施将确保这项拟议工作的成功。

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.