Investigating Calcium Homeostasis During a Key Mitochondrial Stress Pathway

项目总结 线粒体是细胞新陈代谢、信号传递、蛋白质动态平衡、免疫和细胞凋亡的中心。一 线粒体对不断变化的线粒体和细胞需求做出有效反应的方式之一是 钙信号。钙离子通过一种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单转运体(MCU)。更改的MCU中介 线粒体钙信号与多种人类神经退行性疾病有关，包括 帕金森氏症、阿尔茨海默病和遗传性痉挛截瘫。此外，这些同样的疾病 已显示出线粒体蛋白质应激的特征，包括高水平的蛋白质水平 线粒体伴侣。线粒体蛋白质应激可由错误折叠或未折叠的蛋白质引起 在线粒体内堆积。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着关键作用。数据显示 线粒体蛋白应激过程中MCU mRNA和蛋白的增加提示MCU作为一部分上调 线粒体蛋白应激反应。更耐人寻味的是，相对于WT和MCU救援细胞系， MCU基因敲除细胞线粒体伴侣和转录因子ATF4的基线水平较低 中枢到细胞的应激反应。此外，当诱导线粒体蛋白应激时，mRNA水平 ATF4和这些伴侣蛋白的增加要比WT或救援细胞少得多。这一点的中心假设是 有人提出，MCU调节和钙信号的改变构成了一条迄今尚不明确的途径 它调节转录，以应对线粒体蛋白压力。Aim 1将提供关于 调控MCU转录、翻译和蛋白质稳定性的分子机制。功能界别 对AIM 2的研究将揭示线粒体蛋白应激如何改变钙信号和转录。成功 这项提议的完成将通过在鲜为人知的领域中发现新的参与者来推动这一领域的发展 线粒体到核的信号通路。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中心转录反应调控中的作用 反应，并将揭示治疗神经退行性疾病的潜在靶点。 目标1的完成将在将基因编辑与解剖的药理工具配对方面提供宝贵的培训 MCU是一种与疾病密切相关的蛋白质，其功能和调节。目标2的竣工将提供 接受使用激光扫描共聚焦显微镜和实时成像技术研究线粒体应激的培训。 华盛顿大学资金雄厚，装备精良。它的顶尖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。

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.