Mapping the blood cell protein complexosome

摘要 红细胞及其祖细胞表达不同的蛋白质，这些蛋白质构成了它们独特的 生物学，这为许多血液疾病提供了分子基础，包括各种贫血，如 在低红细胞产量和寿命所导致的肾功能衰竭期间出现的这些症状。重要的是 哺乳动物红细胞缺乏细胞核和其他主要细胞器，因此既不能通过RNA- 测序或重组转染法--其他细胞类型的强大技术--都可以用来揭示RBC 基因的功能和途径。相比之下，蛋白质组学方法允许对红细胞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%)，但在 由1500到2000种不同的蛋白质组成。这些蛋白质中的许多在红细胞功能中起着关键作用，包括 关键的代谢和生物能量作用，以及控制红细胞细胞形状的细胞骨架作用。超过500人 红细胞中的蛋白质具有完全未知的功能。因此，红细胞生物学中的一个基本问题是，所有的 这些蛋白质共同支持红细胞的正常功能和发育。建筑深部机械化 对红细胞生物学的理解需要准确地描绘蛋白质复合体的精确成员 这是红细胞特有的，因为它们执行这些细胞独有的关键功能。我们建议执行第一个 使用一种强大的新技术，系统地、全球地探索红细胞中天然蛋白质-蛋白质相互作用(PPI) 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蛋白质生物化学、定量质谱学和蛋白质组学 以及集成的计算机建模以可靠地定义扩展的PPI网络和多蛋白复合体 原生红细胞，有助于为解释红细胞生物学奠定丰富的新机制基础。到这个结束的时候 格兰特，我们将在天然蛋白质复合体上进行近2000次质谱学实验 从原始红细胞及其祖细胞中分离出来，定义了红细胞相互作用组，包括共享和 新的蛋白质复合体，达到了前所未有的程度。作为这项工作的结果，红细胞将成为第一个初选 人类细胞类型，稳定蛋白质复合体的近乎完整的图谱，为研究红细胞提供了新的见解 生物学和发育，并为未来尝试进行化学或遗传干预奠定基础， 在影响这些关键细胞的疾病中。

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.