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基因的机制 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；9000人 真核生物共有的蛋白质，可以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 纤毛突起和贩运等。它们是人类疾病的不成比例的驱动因素，与 各种各样的疾病，包括癌症、出生缺陷、代谢障碍、帕金森病、亨廷顿 疾病、肌萎缩侧索硬化症等等。在这些高度保守的人类蛋白质中，有近1300种 尽管几乎可以肯定具有重要的细胞角色，但大多数情况下仍然没有特征性。一个根本的问题是如何 所有这些蛋白质共同作用以支持细胞功能。然而，一个关键的限制仍然是缺乏大型- 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。当前 量化蛋白质组的方法才刚刚开始对哺乳动物细胞中的蛋白质进行研究 显著的深度，并且始终存在灵敏度低和吞吐量低的问题。这些限制已经放慢了 医疗应用，例如发现生物标记物，其中包括质谱学和抗体等技术 阵列往往缺乏足够的灵敏度和量化精度来发挥作用。我们建议在三个方面进行研究 广泛的领域：首先，我们提出了一项重大努力，以生化方式定义主要的人类蛋白质复合体， 为解释不同的人类基因和疾病提供了机制基础。我们将重点关注 进化上保守的人类蛋白质，因为这些蛋白质对细胞功能至关重要，利用 在其他物种中使用比较蛋白质组学方法进行研究。第二，我们正在开发代孕产品 通过系统地人源化酵母细胞，取代每个酵母细胞，对高度保守的人类蛋白质进行功能分析 关键的酵母基因依次由其人类版本表达。由此产生的菌株可作为新的物理试剂用于 在简化的生物体环境中研究人类基因，开启简单的人类高通量分析 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， 以及耐药机制的快速确定。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，这两项技术都使新的 与许多相关的蛋白质表达和物理组织的高度敏感的特征类型 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人类细胞有新的认识 生物学和生物化学，为未来尝试进行化学或遗传干预奠定了基础 那些对细胞功能最关键的大分子。

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.