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

摘要 线粒体是多方面的细胞器，在包括能量在内的多种细胞功能中发挥着重要作用。 产生、新陈代谢、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，并代表着 几种神经性疾病的融合。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。适当的线粒体功能和健康依赖于一个健康的蛋白质组。 因此，线粒体蛋白质组受到精心而完整的蛋白质质量控制的监控。 网络。最近对小鼠的研究发现，大脑中线粒体蛋白的平均半衰期 从几分钟到几天不等。值得注意的是，我们自己最近基于发现的蛋白质组学分析显示，一个小的 线粒体蛋白质组的亚组在小鼠和大鼠的脑、心脏和眼球中持续存在数月。考虑到至关重要的 线粒体在细胞健康和生存中的作用，以及线粒体的高度动态性质，我们发现 线粒体蛋白质可以在健康组织中持续数月是意想不到的，而且具有潜在的重要性。 这个项目的首要目标是在此背景下表征线粒体长寿命蛋白(mt-llps)。 线粒体的动态平衡。我们目前对mt-LLP的理解是基于 组织匀浆，我们缺乏对哪些特定类型的细胞含有这些特殊蛋白质的了解。 有几条证据表明，具有超长寿命的蛋白质不可避免地存在二分法。一对一 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 确保一个紧凑的能源产生化学反应堆。与此同时，他们的长期坚持性使有限责任公司 与生俱来地增加了与年龄相关的恶化的风险。因此，我们的总体目标是使用整体- 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 含有mt-LLP的类型(目标1)，同时调查线粒体DNA寿命(目标2)。最后，我们的目标是确定 如果山脊结构完整性对于mt-LLP持久性(目标3.1)是必需的，并且与mt-LLP的存在相关联 线粒体膜电位(目标3.2)。了解含有mt-LLP的细胞和结构， 以及对线粒体适合性的影响，可能会开辟新的研究途径，并为 在老年性神经变性过程中对线粒体网络动力学的调控。

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.