Proteomics and model organism humanization to decode human genetics

总结/摘要 虽然人类基因组提供了> 20，000种蛋白质的部分列表，但这些蛋白质如何在很大程度上仍然是未知的。 蛋白质组装成“分子机器”来执行它们的生物学角色。这对于基础 人类基因的表征和了解大多数人类遗传学的机制 疾病，这往往是由于蛋白质系统的缺陷共同作用。我们专注于9,000多人 真核生物共有的蛋白质，可追溯到最后一个真核生物共同祖先。这些古老的蛋白质 进行关键的细胞过程，包括DNA复制，修复，转录，剪接，线粒体和 睫状突起和贩运等。它们是人类疾病的不成比例的驱动因素， 一系列的疾病，包括癌症、出生缺陷、代谢紊乱、帕金森病、亨廷顿舞蹈症、 肌萎缩性侧索硬化症等等。这些高度保守的人类蛋白质中有近1,300种是 尽管几乎可以肯定具有重要的细胞作用，但大多数仍未被表征。一个根本问题是 所有这些蛋白质一起工作以支持细胞功能。然而，一个关键的限制仍然是缺乏大的- 规模数据直接询问这些蛋白质的表达，相互作用和激活状态。电流 定量蛋白质组的方法才刚刚开始调查哺乳动物细胞中的蛋白质， 显著深度，并且始终遭受低灵敏度和吞吐量。这些限制已经减缓 医学应用，例如生物标志物发现，其中包括质谱和抗体的技术 阵列通常缺乏足够的灵敏度和定量准确性而不能有效。我们建议在三个研究 广泛的领域：首先，我们提出了一个主要的努力，以生物化学定义的主要人类蛋白质复合物， 为解释不同的人类遗传学和疾病提供了机制基础。我们将专注于 进化上保守的人类蛋白质，由于这些蛋白质对细胞功能至关重要， 使用比较蛋白质组学方法对其他物种进行研究。第二，我们正在开发代理 通过系统地人源化酵母细胞，替换每个 基本的酵母基因反过来由它的人类版本。由此产生的菌株作为新的物理试剂， 在简化的有机体环境中研究人类基因，开辟了人类基因的简单高通量测定方法。 基因功能，人类遗传变异对基因功能的影响，药物的筛选和再利用， 以及快速确定耐药机制。最后，我们的目标是推进新的蛋白质组学 技术，单分子蛋白质测序和鸟枪电子显微镜，这两者都使新的 蛋白质表达和物理组织的高灵敏度表征类型与许多 人类细胞生物学和疾病的各个方面。这些目标的成功将使人们对基本的人类细胞有新的认识。 生物学和生物化学，为未来试图干预奠定基础，化学或遗传， 这些大分子对细胞的功能至关重要。

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 diseases, which often arise from defects in systems of proteins working together. We focus on the >9,000 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, and more. Nearly 1,300 of these deeply conserved human proteins are still mostly uncharacterized, despite almost certainly having important cell roles. 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 to quantify the proteome are only beginning to survey the proteins 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 on evolutionarily conserved human proteins due to these proteins’ critical importance to cellular function, leveraging studies in other species using a comparative proteomics approach. 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 new proteomics technologies, single-molecule protein sequencing and shotgun electron microscopy, both of which enable new types of highly sensitive characterization of protein expression and physical organization relevant to many aspects of human cell biology and disease. 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.