Mapping the blood cell protein complexosome

摘要 红细胞（红细胞; RBC）及其祖细胞表达不同的蛋白质，这是其独特的基础。 生物学，并提供了许多血液疾病的分子基础，包括各种贫血，如 在肾衰竭期间由于红细胞生成和寿命低而引起的那些。重要的是， 哺乳动物红细胞缺乏细胞核和其他主要细胞器，因此， 测序或重组转染-其他细胞类型的强大技术-可用于揭示红细胞 基因功能和途径。相比之下，蛋白质组学方法允许对RBC蛋白质进行详细分析， 开创性的研究表明，红细胞虽然以血红蛋白为主（98%），但表达的顺序是 1,500到2,000种不同的蛋白质。其中许多蛋白质在红细胞功能中发挥关键作用，包括 关键的代谢和生物能量作用，以及控制RBC细胞形状的细胞骨架作用。500多 RBC中的蛋白质具有完全未知的功能。因此，RBC生物学的一个基本问题是， 这些蛋白质一起工作以支持适当的RBC功能和发育。建立深层机制 理解红细胞生物学需要准确描述蛋白质复合物的精确成员 这是RBC特有的，因为它们执行这些细胞特有的关键功能。我们建议执行第一个 红细胞中天然蛋白质-蛋白质相互作用（PPI）的系统性，全球性探索，使用强大的新 直接检测人类和其他哺乳动物内源性蛋白质之间相互作用的技术 红细胞。我们提出的实验结合了联合收割机蛋白质生物化学、定量质谱和蛋白质组学 和综合计算机建模，以可靠地定义扩展的PPI网络和多蛋白质复合物 原生于RBC，有助于为解释RBC生物学奠定丰富的新机制基础。在年底前 格兰特，我们将在天然蛋白质复合物上进行近2,000次质谱实验， 分离自原代RBC及其祖细胞，定义RBC相互作用组，包括共享和 新的蛋白质复合物，达到前所未有的程度。由于这项工作，红细胞将是第一个主要的 人类细胞类型与稳定蛋白质复合物的几乎完整的地图，使新的见解红细胞 生物学和发展，并为未来试图进行化学或遗传干预奠定基础， 影响这些关键细胞的疾病

ABSTRACT Erythrocytes (red blood cells; RBCs) and their progenitors express distinct proteins, which underlie their unique biology, and which provide a molecular basis for many blood diseases, including diverse anemias, such as those arising during renal failures as a result of low red blood cell production and lifespan. Importantly, mammalian RBCs lack nuclei and other major organelles, and hence neither transcriptional profiling by RNA- sequencing nor recombinant transfection—powerful techniques in other cell types—can be used to reveal RBC gene functions and pathways. Proteomics methods, in contrast, allow for a detailed analysis of RBC proteins, and pioneering studies have revealed that RBCs, while dominated by hemoglobin (98%), express on the order of 1,500 to 2,000 distinct proteins. Many of these proteins play critical roles in erythrocyte function, including key metabolic and bioenergy roles, and cytoskeletal roles in controlling RBC cell shape. More than 500 proteins in RBCs are of entirely unknown function. A fundamental question in RBC biology is thus how all of these proteins work together to support proper RBC function and development. Building deep mechanistic understanding of RBC biology requires accurately delineating the precise membership of protein complexes specific to RBCs, as these carry out key functions unique to these cells. We propose to perform the first systematic, global exploration of native protein-protein interactions (PPIs) in RBCs, using a powerful new technology to examine those interactions directly among endogenous proteins in human and other mammalian RBCs. Our proposed experiments combine protein biochemistry, quantitative mass spectrometry proteomics and integrative computer modeling to reliably define the extended PPI networks and multiprotein complexes native to RBCs, helping to lay rich new mechanistic foundations for interpreting RBC biology. By the end of this grant, we will have performed nearly 2,000 mass spectrometry experiments on native protein complexes isolated from primary RBCs and their progenitors, defining the RBC interactome, including both shared and novel protein complexes, to an unprecedented degree. As a result of this work, RBCs will be the first primary human cell type with a near complete map of stable protein complexes, giving new insights into erythrocyte biology and development, and laying the foundation for future attempts to intervene, chemically or genetically, in diseases affecting these critical cells.