Long-lived proteins as pillars of mitochondrial architecture in rodent brains

摘要 线粒体是多方面的细胞器，在包括能量在内的无数细胞功能中发挥重要作用 生产、代谢、钙稳态和细胞死亡。人们普遍认为， 线粒体质量是线粒体功能障碍、衰老的关键因素，并且代表了 几种神经系统疾病的融合。然而，功能失调的线粒体究竟是如何导致 这些条件仍然难以捉摸。正常的线粒体功能和健康取决于健康的蛋白质组。 因此，线粒体蛋白质组是由一个精心设计的和综合的蛋白质质量控制监测 网络最近对小鼠的研究发现，平均而言， 从几分钟到几天不等。值得注意的是，我们自己最近发现的蛋白质组学分析显示，一个小的 在小鼠和大鼠的脑、心脏和眼球中，线粒体蛋白质组的一个子集持续数月。鉴于至关重要的 线粒体在细胞健康和存活中的作用，以及线粒体的高度动态特性，我们的发现， 线粒体蛋白质可以在健康组织中持续数月是出乎意料的并且具有潜在的重要性。 该项目的总体目标是在特定的背景下表征线粒体长寿命蛋白（mt-LLP）。 线粒体内环境稳定我们目前对mt-LLP的理解是基于以下综合指标： 组织匀浆，我们缺乏对哪些特定细胞类型含有这些特殊蛋白质的了解。 几条证据表明，具有超长寿命的蛋白质不可避免地会出现二分法。一 另一方面，由于其持久性，mt-LLP作为线粒体结构的支柱，提供结构稳定性 确保紧凑的能量产生化学反应器。同时，他们的长期坚持使有限责任合伙人 与年龄相关的恶化风险增加。因此，我们的总体目标是利用整个- 动物稳定同位素脉冲标记结合生化和蛋白质组学分析鉴定脑细胞 携带mt-LLP的类型（目的1）和平行研究mtDNA寿命（目的2）。最后，我们的目标是确定 如果嵴结构完整性是mt-LLP持久性所必需的（目标3.1），并与mt-LLP的存在相关 线粒体膜电位（Aim 3.2）。了解携带mt-LLP的细胞和结构， 以及对线粒体适应性的影响，可以开辟新的研究途径，并提供分子靶点， 在年龄相关的神经变性过程中调节线粒体网络动力学。

ABSTRACT Mitochondria are multifaceted organelles that play vital roles in a myriad of cellular functions, including energy production, metabolism, calcium homeostasis, and cell death. It is generally accepted that a decline in mitochondria quality is a key contributor to mitochondrial dysfunction, aging, and represents a key point of convergence for several neurological disorders. Yet, precisely how dysfunctional mitochondria contribute to these conditions remains elusive. Proper mitochondrial function and fitness depends on a healthy proteome. Therefore, the mitochondrial proteome is monitored by an elaborate and integrated protein quality control network. Recent studies in mice have found that, on average, half-lives of mitochondrial proteins in the brain vary from minutes to days. Notably, our own recent discovery-based proteomic analysis revealed that a small subset of mitochondrial proteome persists for months in brain, heart, and eyeball in mice and rats. Given the vital role of mitochondria in cell health and survival, and the highly dynamic nature of mitochondria our discovery that mitochondrial proteins can persist for months in healthy tissues is unexpected and of potential importance. The overarching goal of this project is to characterize mitochondrial long-lived proteins (mt-LLPs) in the context of mitochondrial homeostasis. Our current understanding of mt-LLPs are based on composite measures from tissue homogenates and we lack an understanding of which specific cell types harbor these exceptional proteins. Several lines of evidence point to an inevitable dichotomy of proteins with exceptionally long lifespans. On one hand, due to their persistence, mt-LLPs serve as pillars of mitochondrial architecture, providing structural stability ensure a compact energy generating chemical reactor. At the same time, their long-term persistence puts LLPs at an inherently increased risk for age-related deterioration. Thus, our overall objectives are to use whole- animal stable isotope pulse labelling combined with biochemical and proteomic analyses to identify the brain cell types harboring mt-LLPs (Aim 1) and in parallel investigate mtDNA lifetime (Aim 2). Finally, we aim to determine if cristae structural integrity is required for mt-LLP persistence (Aim 3.1) and correlate the presence of mt-LLPs with mitochondrial membrane potential (Aim 3.2). Understanding the cells and structures harboring mt-LLPs, and the effect on mitochondrial fitness, could open new avenues of research and provide molecular targets for modulating mitochondrial network dynamics in the process of age-related neurodegeneration.