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

 描述(由申请人提供)：细胞分化在基础水平上是对全球蛋白质补体或蛋白质组的规范。这些变化的规模和速度在终末分化细胞，如红细胞、精子和其他细胞中是显著的。蛋白质组的整体重塑包括程序性地消除细胞的大多数通用成分，同时大量合成小分子 新的、特定于细胞类型的蛋白质的数量，如珠蛋白。网织红细胞是蛋白质组快速转变的典型例子，然而，即使在这种情况下，驱动正常稳定蛋白质快速周转的机制在很大程度上仍有待确定。我们发现Ube2O/E2-230K是一种在红系中选择性表达的泛素结合酶，其基因零突变具有高度特异性的小细胞性贫血表型。进一步分析这个小鼠突变体hem9，提出Ube2O是一种广谱泛素化酶的假设，它在红系蛋白质组的全球重塑中发挥关键作用。为了评估这一模型，提出了一套综合的方法，包括突变分析、质谱学和体外生物化学。一个主要的焦点将是以公正和全球的方式确定Ube2O的底物。到目前为止，在实现这一目标方面取得了重大进展，因为珠蛋白伴侣AHSP、珠蛋白本身和核糖体被暂时指定为UBE2O底物。因此，hem9的一个独特表型是80S核糖体水平显著升高。相比之下，低核糖体水平是钻石黑扇贫血的标志性特征。研究发现，Ube2O足以通过DOX诱导表达在HEK293细胞中诱导核糖体降解；不需要其他红系特异性因子。AHSP和核糖体的泛素化和降解将在体外和高纯度系统中重组；这些反应的特异性决定因素将被绘制在底物和酶上。在这些生化研究的同时，Ube2O丢失的生理效应将通过分析突变的网织红细胞中的蛋白质合成以更高的分辨率进行研究，并将使用骨髓移植来测试是否如Ube2O重塑蛋白质组假设所预测的那样，hem9红系表型是细胞自主的。最终，我们的研究有望阐明蛋白质组重构的基本过程，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。

 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.