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

 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。在终末分化细胞如红细胞、精子等的情况下，这些变化的规模和速度是显著的。蛋白质组的整体重塑包括细胞中大多数通用成分的程序性消除，同时大量合成小的 一些新的细胞类型特异性蛋白质，如珠蛋白。网织红细胞是蛋白质组快速转换的典型例子，然而，即使在这种情况下，驱动正常稳定蛋白质快速转换的机制在很大程度上仍有待确定。我们发现Ube 2 O/E2- 230 K是一种选择性表达于红细胞系的泛素结合酶，其基因的无效突变具有高度特异性的小细胞性贫血表型。进一步的分析表明，这种小鼠突变体hem 9，Ube 2 O是一种广谱的泛素化酶，在红细胞蛋白质组的全球重塑中起着关键作用的假设。为了评估这个模型，提出了一套综合的方法，将突变分析，质谱和体外生物化学。一个主要的焦点将是以公正和全球的方式确定Ube 2 O的底物。到目前为止，在这一目标上已经取得了重大进展，因为珠蛋白伴侣α-血红蛋白稳定蛋白、珠蛋白本身和核糖体被暂时指定为Ube 2 O底物。因此，hem 9的独特表型是80 S核糖体水平显著升高。相反，低核糖体水平是Diamond Blackfan贫血的标志性特征。发现Ube 2 O足以通过在HEK 293细胞中的dox诱导表达诱导核糖体降解;不需要其他红细胞特异性因子。α-血红蛋白稳定蛋白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。在这些生化调查的同时，Ube 2 O损失的生理效应将通过分析突变型网织红细胞中的蛋白质合成以更高的分辨率进行研究，骨髓移植将用于测试hem 9红细胞表型是否是细胞自主的，正如Ube 2 O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Diamond Blackfan贫血的潜在机制提供新的见解。

 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.