Elucidation of the mammalian mitochondrial unfolded protein response network

线粒体包含一个专用的蛋白质质量控制系统，以确保线粒体蛋白质组的适当折叠。线粒体蛋白的酶活性是细胞功能的核心，线粒体蛋白质组的扰动与衰老和许多疾病（如神经退行性疾病）有关。当线粒体蛋白错误折叠时，线粒体激活线粒体未折叠蛋白反应（UPRmt）以恢复线粒体蛋白平衡和功能。尽管我们对秀丽隐杆线虫的upmt有广泛的了解，但对哺乳动物的upmt的蛋白质和机制知之甚少。哺乳动物的UPRmt有两个功能分支：一个是增加线粒体伴侣水平的转录反应，另一个是我最近发现的导致线粒体翻译减少的翻译反应。考虑到UPRmt网络在理解线粒体对应激、衰老和疾病的反应方面的重要作用，哺乳动物的UPRmt研究还远远不够，主要是由于缺乏在明确的急性条件下研究UPRmt的实验范例。我之前的工作建立了高度特异性的线粒体蛋白错误折叠的化学诱导来研究急性哺乳动物UPRmt，并提供了可能在调节UPRmt中发挥作用的蛋白质和机制。在这些数据的指导下，我提出了四个目标作为本提案的一部分，主要结合生化、基于定量质谱的、细胞生物学和基因编辑技术来阐明和发现在哺乳动物UPRmt中起关键作用的蛋白质，并破译哺乳动物UPRmt的传感和信号传导机制：1)阐明线粒体内UPRmt的传感和信号传导，特别是在钙信号传导和研究LETM1的作用的背景下，我发现它调节UPRmt；2)确定通过UPRmt控制进程的时间概况，以及在亲渴望UPRmt激活后向亲死亡反应的过渡；3)扩展我的初步和建立进一步的遗传筛选，以确定UPRmt必需的蛋白质，并阐明其作用机制；4)破译UPRmt传递至细胞核并引发转录反应的信号通路。总之，这些目标将在很大程度上扩展我们对蛋白质形成和指导哺乳动物UPRmt机制的认识，并将为理解线粒体对衰老或疾病时线粒体蛋白质稳态扰动的反应过程奠定必要的基础。

Mitochondria contain a dedicated protein quality control system to ensure proper folding of the mitochondrial proteome. The enzymatic activity of mitochondrial proteins is central to cellular function and perturbations of the mitochondrial proteome have been implicated in ageing and numerous diseases, such as neurodegenerative diseases. Upon mitochondrial protein misfolding, mitochondria activate the mitochondrial unfolded protein response (UPRmt) to restore mitochondrial proteostasis and function. Despite our extensive knowledge of the C. elegans UPRmt, little is known about the proteins and mechanisms underlying the mammalian UPRmt. Two functional branches form the mammalian UPRmt: a transcriptional response to increase mitochondrial chaperones levels and a translational response, recently discovered by me, that leads to decreased mitochondrial translation. Considering the important role of the UPRmt network for understanding the mitochondrial responses to stresses, ageing, and disease, the mammalian UPRmt has been vastly understudied, mainly due to the lack of experimental paradigms to study the UPRmt under defined, acute conditions.My previous work established highly specific chemical induction of mitochondrial protein misfolding to study the acute mammalian UPRmt and provided with proteins and mechanisms likely playing a role in regulating the UPRmt. Guided by this data, I propose four objectives as part of this proposal, combining mainly biochemical, quantitative mass spectrometry-based, cell biological, and gene editing techniques to elucidate and discover proteins playing a crucial role in the mammalian UPRmt and to decipher the mechanisms underlying sensing and signaling of the mammalian UPRmt: 1) The elucidation of sensing and signaling of UPRmt inside mitochondria, especially in the context of calcium signaling and studying the role of LETM1, which I identified to regulate the UPRmt; 2) Defining the temporal profile controlling progression through the UPRmt and the transition towards a pro-death response upon pro-longed UPRmt activation; 3) The extension of my preliminary and establishment of further genetic screens to identify the proteins essential for the UPRmt and to elucidate the mechanisms of their action; 4) To decipher the signaling pathways transmitting the UPRmt to the nucleus and eliciting the transcriptional response. Together, these objectives will largely extent our knowledge of the proteins forming and the mechanisms guiding the mammalian UPRmt and will form the essential foundation for understanding the processes underlying the mitochondrial response to perturbations in mitochondrial proteostasis upon ageing or disease.