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

基本信息

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
项目类别：
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

项目摘要

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。对于终末分化细胞（例如红细胞、精子等），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（例如球蛋白）是快速转变的蛋白质组的典型例子，但即使在这种情况下，驱动通常稳定的蛋白质快速更新的机制在很大程度上仍有待确定。进一步分析发现，Ube2O/E2-230K是一种在红细胞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。这种小鼠突变体 hem9 提出了这样的假设：Ube2O 是一种广谱泛素化酶，在红细胞蛋白质组的整体重塑中发挥着关键作用。为了评估该模型，提出了一套综合方法，包括突变分析、质量分析。光谱测定法和体外生物化学的一个主要焦点是以公正和全球性的方式鉴定 Ube2O 的底物，迄今为止，在这一目标方面已经取得了重大进展。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 的独特表型是 80S 核糖体水平显着升高，相反，低核糖体水平是 Diamond Blackfan 贫血的标志性特征。足以在 HEK293 细胞中通过 dox 诱导表达来诱导核糖体降解；没有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；与这些生化研究同时，将绘制这些反应的特异性决定因素。通过分析突变网织红细胞中的蛋白质合成以更高分辨率进行研究，并将使用骨髓移植来测试 hem9 红系表型是否如预测的那样具有细胞自主性最终，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 Diamond Blackfan 贫血的潜在机制提供新的见解。