Proteomics and model organism humanization to decode human genetics

蛋白质组学和模型生物人性化以解码人类遗传学

• 批准号: 10330772
• 负责人: EDWARD M MARCOTTE
• 金额: $ 57.31万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10330772
• 关键词: Amyotrophic Lateral Sclerosis; Animal Model; Antibodies; Area; Biochemical; Biochemistry; Biological; Biological Assay; Biology; Cardiovascular Diseases; Cell physiology; Cells; Cellular biology; Chemicals; Congenital Abnormality; DNA biosynthesis; Defect; Disease; Drug Interactions; Drug resistance; Electron Microscopy; Eukaryota; Foundations; Future; Genes; Genetic Diseases; Genetic Transcription; Genetic Variation; Health; Human; Human Genetics; Human Genome; Huntington Disease; Link; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Measures; Medical; Medicine; Metabolic Diseases; Mitochondria; Molecular Machines; Nerve Degeneration; Parkinson Disease; Peptide Sequence Determination; Proteins; Proteome; Proteomics; RNA Splicing; Reagent; Research; Role; Shotguns; Structure of ciliary processes; Supporting Cell; Surveys; System; Techniques; Technology; Work; Yeasts; biomarker discovery; comparative; drug repurposing; gene function; high throughput screening; human disease; insight; large scale data; macromolecule; protein complex; protein expression; repaired; screening; single molecule; success; trafficking

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.

摘要/摘要 虽然人类基因组提供了> 20,000蛋白的零件清单，但仍然未知这些 蛋白质组装成“分子机”，以扮演其生物学作用。这对基本很重要 人类基因的表征和理解大多数人基因的机制 疾病通常是由于蛋白质系统的缺陷引起的。我们专注于> 9,000人 跨真核生物共享的蛋白质并与最后一个真核生物共同的祖先约会。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 睫状工艺和贩运等。它们是人类疾病的驱动因素不成比例的，与 各种各样的疾病，跨癌症，先天缺陷，代谢疾病，帕金森病，亨廷顿 疾病，肌萎缩性侧索硬化症等等。这些深厚保守的人蛋白中有近1,300个是 仍然大部分没有特色，dospite几乎肯定具有重要的细胞角色。一个基本问题是如何 所有这些蛋白质共同作用以支持细胞功能。但是，关键限制仍然缺乏大的 扩展数据直接询问这些蛋白质的表达，相互作用和激活状态。当前的 量化蛋白质的方法才开始对哺乳动物细胞中的蛋白质进行调查 敏感性和吞吐量始终遭受巨大的深度。这些限制已减慢 医疗应用，例如生物标志物发现，其中技术包括质谱和抗体 阵列通常缺乏足够的灵敏度和数量准确性，无法有效。我们提出了三个 广泛领域：首先，我们提出了一项主要的努力，以生化定义主要的人类蛋白质复合物，即 提供了解释潜水人类遗传学和疾病的机械基础。我们将重点关注 由于这些蛋白质对细胞功能至关重要，因此在进化上构成了人类蛋白质 使用比较蛋白质组学方法在其他物种中进行研究。第二，我们正在发展代理人 通过系统地人性化的酵母细胞，更换每个人，对深处保守的人蛋白的功能测定 基本的酵母基因依次通过其人类版本。由此产生的菌株作为新的物理试剂 在简单的有机环境中研究人类基因，打开了人类的简单高通量测定 基因功能，人类遗传变异对基因功能的影响，药物的筛查和重新利用， 以及快速确定耐药性机制。最后，我们旨在推进新的蛋白质组学 技术，单分子蛋白测序和shot弹枪电子显微镜，这两者都启用了新的 与许多人相关的蛋白质表达和物理组织的高度敏感表征的类型 人类细胞生物学和疾病的各个方面。这些目标的成功将为基本人类细胞提供新的见解 生物学和生物化学，为未来的化学或遗传学干预奠定基础 这些大分子对细胞功能最重要。