A novel program of ubiquitination in global remodeling of the erythroid proteome

红系蛋白质组全局重塑中的泛素化新程序

• 批准号: 8886115
• 负责人: MARK D FLEMING
• 金额: $ 48.88万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-15 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8886115
• 关键词: Address; Anemia; Architecture; Biochemical; Biochemistry; Bone Marrow Transplantation; Cells; Complement; Complex; Data; Defect; Development; Diamond-Blackfan anemia; Disease; Enzymes; Erythroblasts; Erythrocytes; Erythroid; Event; Exhibits; Generic Drugs; Genes; Genetic; Genetic Translation; Globin; Goals; Hemoglobin; Immunoblotting; In Vitro; Individual; Inherited; Investigation; Label; Light; Lysine; Maps; Mass Spectrum Analysis; Mediating; Messenger RNA; Modeling; Molecular Chaperones; Mus; Mutant Strains Mice; Oxygen; Pathway interactions; Phase; Phenotype; Physiologic pulse; Physiological; Play; Polyribosomes; Problem Solving; Process; Protein Biosynthesis; Proteins; Proteome; Proteomics; Reaction; Resolution; Reticulocytes; Ribosomal Proteins; Ribosomes; Signal Transduction; Site; Specificity; Speed; Substrate Specificity; System; Testing; Therapeutic; Time; Tissues; Translations; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination; cell type; cofactor; cyclin D3; enzyme substrate; insight; interest; meetings; microcytic anemia; multicatalytic endopeptidase complex; mutant; novel; null mutation; prevent; programs; public health relevance; reconstitution; selective expression; sperm cell; success; yeast two hybrid system

 DESCRIPTION (provided by applicant): Cellular differentiation is at a basic level a specification of the global protein complement, or proteome. The scale and speed of these changes are remarkable in the case of terminally differentiated cells such as erythrocytes, spermatozoa, and others. Global remodeling of the proteome consists of the programmed elimination of most generic constituents of the cell in parallel with abundant synthesis of a small number of new, cell-type-specific proteins such as globin. Reticulocytes are a canonical example of a proteome in rapid transition, yet, even in this case, the mechanisms that drive rapid turnover of normally stable proteins largely remain to be identified. We have found that Ube2O/E2-230K is an ubiquitin-conjugating enzyme expressed selectively in the erythroid lineage and that null mutations in its gene have a highly specific phenotype of microcytic anemia. Further analysis of this murine mutant, hem9, suggests the hypothesis that Ube2O is a broad-spectrum ubiquitinating enzyme that plays a key role in the global remodeling of the erythroid proteome. To assess this model, an integrated set of approaches is proposed, incorporating mutant analysis, mass spectrometry, and in vitro biochemistry. A major point of focus will be to identify substrates of Ube2O in an unbiased and global manner. To date there has been significant progress towards this goal, as the globin chaperone AHSP, globin itself, and ribosomes have been provisionally assigned as Ube2O substrates. Accordingly, a distinctive phenotype of hem9 is markedly elevated levels of 80S ribosomes. In contrast, low ribosome levels are the signature feature of Diamond Blackfan anemia. Ube2O was found to be sufficient to induce ribosome degradation by dox-inducible expression in HEK293 cells; no other erythroid-specific factors are needed. The ubiquitination and degradation of AHSP and ribosomes will be reconstituted in vitro and in highly purified systems; the specificity determinants of these reactions will be mapped for both substrate and enzyme. In parallel with these biochemical investigations, the physiological effects of Ube2O loss will be studied at higher resolution through analysis of protein synthesis in the mutant reticulocytes, and bone marrow transplantation will be used to test whether hem9 erythroid phenotypes are cell-autonomous, as predicted by the hypothesis that Ube2O reshapes the proteome. Ultimately, our studies are expected to shed light on the fundamental process of proteome remodeling and provide new insights into underlying mechanisms of both microcytic anemia and Diamond Blackfan anemia.

 描述（由申请人提供）：细胞分化在基本水平上是整体蛋白质补体或蛋白质组的规范。对于红细胞、精子等终末分化细胞来说，这些变化的规模和速度是显着的。蛋白质组的整体重塑包括细胞中大多数通用成分的程序化消除，同时大量合成少量的蛋白质。 许多新的细胞类型特异性蛋白质，例如珠蛋白。网织红细胞是快速转变的蛋白质组的典型例子，然而，即使在这种情况下，驱动通常稳定的蛋白质快速转变的机制在很大程度上仍有待确定。我们发现 Ube2O/E2-230K 是一种在红系谱系中选择性表达的泛素结合酶，其基因的无效突变具有高度特异性的小细胞性贫血表型。对这种小鼠突变体 hem9 的进一步分析表明，Ube2O 是一种广谱泛素化酶，在红系蛋白质组的整体重塑中发挥关键作用。为了评估该模型，提出了一套综合方法，结合突变分析、质谱和体外生物化学。一个主要关注点是以公正和全局的方式识别 Ube2O 的底物。迄今为止，在实现这一目标方面已取得重大进展，球蛋白伴侣 AHSP、球蛋白本身和核糖体已被暂时指定为 Ube2O 底​​物。因此，hem9 的一个独特表型是 80S 核糖体水平显着升高。相比之下，低核糖体水平是戴蒙德·布莱克凡贫血的标志性特征。发现 Ube2O 足以在 HEK293 细胞中通过 dox 诱导表达来诱导核糖体降解；不需要其他红细胞特异性因子。 AHSP和核糖体的泛素化和降解将在体外和高度纯化的系统中重建；这些反应的特异性决定因素将被映射为底物和酶。与这些生化研究并行，将通过分析突变网织红细胞中的蛋白质合成以更高分辨率研究 Ube2O 损失的生理影响，并将使用骨髓移植来测试 hem9 红系表型是否是细胞自主的，正如 Ube2O 重塑蛋白质组的假设所预测的那样。最终，我们的研究有望揭示蛋白质组重塑的基本过程，并为小细胞性贫血和钻石黑扇贫血的潜在机制提供新的见解。