Proteomics and model organism humanization to decode human genetics

摘要/摘要 虽然人类基因组提供了20,000种蛋白质的部分清单，但在很大程度上仍不清楚这些蛋白质是如何 蛋白质组装成“分子机器”来执行它们的生物角色。这一点对于基本的 人类基因的特征和理解大多数人类遗传特征的机制 以及疾病，这些疾病通常是由共同工作的蛋白质系统中的缺陷引起的。我们专注于&gt；5000 人类蛋白质在真核生物中共享，并可追溯到真核生物的最后一个共同祖先。这些古老的 蛋白质执行关键的细胞过程，包括DNA复制、修复、转录、剪接、 线粒体和纤毛突起，以及贩运，等等。他们是不成比例的驱动因素 人类疾病，与一系列疾病有关，包括癌症、出生缺陷、代谢紊乱、 帕金森病、亨廷顿病、肌萎缩侧索硬化症、精神发育迟缓等等。更多 在这些高度保守的人类蛋白质中，有750多种仍然完全没有特征。一个根本问题 所有这些蛋白质是如何协同工作来支持细胞功能的。然而，一个关键的限制仍然是缺乏 直接询问这些蛋白质的表达、相互作用和激活状态的大规模数据。当前 量化蛋白质组的方法才刚刚开始研究在哺乳动物中表达的蛋白质。 电池对任何重要深度都不敏感，并且始终存在灵敏度低和吞吐量低的问题。这些限制 减缓了医疗应用，例如生物标记物的发现，包括质谱学在内的技术 而且抗体阵列往往缺乏足够的灵敏度和量化准确性来发挥作用。我们建议 主要有三个方面的研究：第一，我们建议从生物化学的角度来定义人类的主要蛋白质。 复合体，为解释不同的人类遗传和疾病提供了机制基础。我们将专注于 主要基于进化上保守的人类蛋白质，利用其他物种的研究，由于这些 蛋白质对细胞功能的关键重要性。第二，我们正在开发替代功能分析，用于 通过系统地人源化酵母细胞，取代每一种基本酵母，深度保守人类蛋白质 基因反过来又是人类的版本。由此产生的菌株成为研究人类的新的物理试剂。 基因在简化的生物环境中，开启了对人类基因功能的简单高通量分析， 人类基因变异对基因功能的影响，药物的筛选和再利用，以及快速 耐药机制的确定。最后，我们的目标是推进一种新的蛋白质组学技术， 单分子蛋白质测序，这可能潜在地解决目前限制该领域的问题，通过 敏感度和吞吐量提高了数量级。这些目标的成功将带来新的见解 进入基本的人体细胞生物学和生物化学，为未来的干预尝试奠定了基础， 在化学或遗传方面，这些大分子对细胞的功能最关键。

Summary/Abstract While the human genome provides a parts list of >20,000 proteins, it is still largely unknown how these proteins assemble into `molecular machines' to carry out their biological roles. This is important both for basic characterization of human genes and for understanding the mechanisms underlying most human genetic traits and diseases, which often arise from defects in systems of proteins working together. We focus on the >5,000 human proteins shared across eukaryotes and dating to the last eukaryotic common ancestor. These ancient proteins carry out critical cellular processes, including DNA replication, repair, transcription, splicing, mitochondrial and ciliary processes, and trafficking, among others. They are disproportionately drivers of human disease, linked to a wide array of disorders, spanning cancers, birth defects, metabolic disorders, Parkinson disease, Huntington disease, amyotrophic lateral sclerosis, mental retardations, and more. More than 750 of these deeply conserved human proteins are still entirely uncharacterized. A fundamental question is how all of these proteins work together to support cell function. However, a key limitation remains the lack of large-scale data directly interrogating these proteins' expression, interactions, and activation states. Current approaches for quantifying the proteome are only beginning to survey the proteins expressed in mammalian cells to any significant depth, and consistently suffer from low sensitivity and throughput. These limitations have slowed medical applications, e.g. biomarker discovery, where techniques including mass spectrometry and antibody arrays often lack sufficient sensitivity and quantification accuracy to be effective. We propose research in three broad areas: First, we propose a major effort to biochemically define the main human protein complexes, providing a mechanistic basis for interpreting diverse human genetics and diseases. We will focus primarily on evolutionarily conserved human proteins, leveraging studies in other species, due to these proteins' critical importance to cellular function. Second, we are developing surrogate functional assays for deeply conserved human proteins by systematically humanizing yeast cells, replacing each essential yeast gene in turn by its human version. The resulting strains serve as new physical reagents for studying human genes in a simplified organismal context, opening up simple high-throughput assays of human gene function, the impact of human genetic variation on gene function, the screening and repurposing of drugs, and the rapid determination of mechanisms of drug resistance. Finally, we aim to advance a new proteomics technology, single-molecule protein sequencing, which could potentially solve problems currently limiting the field, by orders-of-magnitude improvements in sensitivity and throughput. Success of these aims will give new insights into basic human cell biology and biochemistry, laying the foundation for future attempts to intervene, chemically or genetically, with those macromolecules most critical to the functioning of cells.