Systems-to-structure approaches for defining mitochondrial protein function

项目概要 线粒体是新陈代谢和信号传导的中心，其功能对除少数真核细胞外的所有细胞都至关重要 类型。尽管线粒体是细胞生物学的标志性动力源，但线粒体的许多方面仍然存在 非常模糊——这一事实导致我们几乎完全无法解决线粒体功能障碍 治疗上。这种功能障碍与一系列罕见的先天性代谢错误和 常见疾病的数量不断增加，包括帕金森氏症、阿尔茨海默氏症、各种癌症和 2 型癌症 糖尿病——通常通过不同的方式。例如，异常的线粒体生物合成可能无法正确设置 细胞线粒体含量；信号传导过程失调可能无法校准线粒体活性 改变细胞需求；蛋白质故障会使核心生物能量过程失效。一个专业 理解并最终解决这些过程的瓶颈是驱动它们的蛋白质 常常未被识别。同时，数百种已知线粒体蛋白的功能可能实现 这些角色是未定义的，或者充其量是人们对其知之甚少。 2008年，我领导了一项综合努力，以创建一个 哺乳动物线粒体蛋白质组综合纲要（称为 MitoCarta）使 已知哺乳动物线粒体蛋白的数量，并暴露了这一知识上的重大差距：惊人的约 300 约 1100 种蛋白质中的约 50 种没有注释功能，其中约 50 种蛋白质现在与人类直接相关 疾病。因此，我的研究计划的高级目标是实现更全面的 通过系统地建立孤儿的功能来了解线粒体生物学 线粒体蛋白及其在疾病相关过程中的作用。我们首先设计小说来做到这一点， 多维分析旨在在这些蛋白质之间建立新的联系并建立 途径和过程。这些包括定制的、高通量的蛋白质-蛋白质相互作用筛选、大 基于规模质谱分析的酵母和人类细胞基因敲除，以及计算 接近。然后，我们采用机械和结构方法来定义所选蛋白质的功能 生化深度，包括古老的非典型激酶和脂质结合蛋白，使线粒体 辅酶 Q 生物合成途径和其他重要的代谢过程。最后，我们研究了如何事后 转录和翻译后调节因子的作用是建立定制的线粒体基础设施 能够满足不断变化的细胞需求。总体而言，通过有目的地阐明未探索的领域 随着线粒体生物学的发展，我们正在迅速对这些细胞器的作用有了更全面的了解， 它们的蛋白质成分如何实现其多种功能。这些努力有望帮助建立深入的、 对线粒体生物化学的机制理解将激发新的治疗策略 大量人类疾病源于线粒体功能障碍。 ！

PROJECT SUMMARY Mitochondria are centers of metabolism and signaling whose function is essential to all but a few eukaryotic cell types. Despite their position as the iconic powerhouses of cellular biology, many aspects of mitochondria remain remarkably obscure—a fact that contributes to our near complete inability to address mitochondrial dysfunction therapeutically. Such dysfunction is associated with a spectrum of rare inborn errors of metabolism and an increasing number of common diseases—including Parkinson’s, Alzheimer’s, various cancers, and type 2 diabetes—often through distinct means. For instance, aberrant mitochondrial biogenesis can fail to properly set cellular mitochondrial content; dysregulated signaling processes can fail to calibrate mitochondrial activity to changing cellular needs; and malfunctioning proteins can render core bioenergetic processes ineffectual. A major bottleneck to understanding—and ultimately addressing—these processes is that the proteins driving them have often not been identified. Concurrently, the functions of hundreds of known mitochondrial proteins that may fulfill these roles are undefined, or at best are poorly understood. In 2008, I led an integrative effort to generate a comprehensive compendium of the mammalian mitochondrial proteome—termed MitoCarta—that doubled the number of known mammalian mitochondrial proteins and exposed this major gap in knowledge: A striking ~300 of the ~1100 proteins had no annotated function, including ~50 that are now directly associated with human disease. Thus, the high-level goal of my research program is to achieve a more comprehensive understanding of mitochondrial biology by systematically establishing the functions of orphan mitochondrial proteins and their roles within disease-related processes. We do so by first devising novel, multi-dimensional analyses designed to make new connections between these proteins and established pathways and processes. These include customized, high-throughput protein-protein interaction screens, large- scale mass spectrometry-based profiling of yeast and human cell gene knockouts, and computational approaches. We then employ mechanistic and structural approaches to define the functions of select proteins at biochemical depth, including ancient and atypical kinases and lipid binding proteins that enable the mitochondrial coenzyme Q biosynthesis pathway and other essential metabolic processes. Finally, we investigate how post- transcriptional and post-translational regulators operate to establish a customized mitochondrial infrastructure capable of meeting changing cellular needs. Overall, by purposefully elucidating the unexplored areas of mitochondrial biology, we are rapidly arriving at a more complete understanding of what these organelles do and how their protein componentry enables their myriad functions. These efforts promise to help establish a deep, mechanistic understanding of mitochondrial biochemistry that will motivate novel therapeutic strategies for the vast array of human disorders rooted in mitochondrial dysfunction. !