A ubiquitin-conjugating enzyme critical for terminal erythroid differentiation

描述(由申请人提供)：先天性贫血是人类遗传性疾病的一个普遍和多方面的子集。虽然某些遗传性贫血，如地中海贫血和镰状细胞贫血的发病机制与血红蛋白调节有关，但人们越来越认识到这一点。 一些以贫血为重要后遗症的遗传性疾病是由核糖体缺陷引起的。这些疾病的红系特异性表型强调了蛋白质合成机制在调节红细胞生成方面的重要性。这项建议探索了红细胞生成过程中的蛋白质降解。泛素-蛋白酶体系统是在网织红细胞中发现的，它在网织红细胞中高度活跃。然而，终末分化过程中细胞重塑的途径 在很大程度上仍不清楚，也没有具体的泛素化因素牵涉其中。E2-230K是一种泛素结合酶，在网织红细胞阶段高度和选择性上调。最近，在共同导师马克·弗莱明的实验室中，发现了小鼠E2-230K基因的一个零突变，称为hem9。纯合子的hem9小鼠表现出一种低色素性、小细胞性贫血，并且似乎特别在红系中受到影响。我们发现，网织红细胞的所有主要泛素-蛋白质结合带都是低分子的，在hem9突变体的提取物中水平大大降低，我们认为这种表型在泛素文献中是前所未有的。对E2-230K底物的初步鉴定表明，它可能是网织红细胞中第一个已知的珠蛋白连接酶。然而，hem9也是th3地中海贫血等位基因的强抑制因子，这表明除了珠蛋白外，E2-230K可能还有关键的底物。当突变体的网织红细胞裂解物与重组的E2-230K重组时，核糖体蛋白(RPS)被证明是一类主要的E2-230K靶标。相应地，hem9网织红细胞核糖体水平升高，并有异常的多聚体。此外，在网织红细胞体外成熟为红细胞的过程中，核糖体的清除似乎存在一个主要缺陷。因此，本项目的目标是表征E2-230K在红系终末分化中的作用，特别是在核糖体周转和贫血的病理生理学中的作用。我们建议进一步验证RPS作为E2-230K的底物，并进行无偏倚的蛋白质组学方法来广泛确定生理相关的靶点。我们还将研究E2-230K在核糖体泛素化和降解终末红系分化中的作用。最后，我们将描述hem9的作用。 关于网织红细胞的翻译输出，以了解泛素化失败如何导致贫血。我们提出的工作应该阐明E2-230K和泛素化在红细胞生成中的作用。这些对红系细胞重塑机制的洞察可能对地中海贫血和钻石黑粉贫血等血液病具有有趣的治疗意义。

DESCRIPTION (provided by applicant): Congenital anemias are a prevalent and multifaceted subset of inherited human diseases. While the pathogenesis of certain inherited anemias such as thalassemias and sickle cell is related to hemoglobin regulation, it is increasingly appreciated that a number of inherited diseases with anemia as an important sequela are caused by ribosomal defects. The erythroid-specific phenotype of these diseases underlies the importance of the protein synthetic machinery in regulating erythropoiesis. This proposal explores protein degradation in erythropoiesis. The ubiquitin-proteasome system was discovered in reticulocytes, where it is highly active. However, the pathways of cellular remodeling in terminal differentiation remain largely unknown, and no specific ubiquitinating factor has been implicated. E2-230K is a ubiquitin-conjugating enzyme that is highly and selectively upregulated in the reticulocyte stage. Recently, a null mutation in the murine E2-230K gene, known as hem9, was identified in the lab of co-mentor Mark Fleming. Homozygous hem9 mice exhibit a hypochromic, microcytic anemia, and appear to be affected specifically in the erythroid lineage. We find that all major ubiquitin-protein conjugate bands of reticulocytes, which are of low molecular weight, are greatly reduced in levels in extracts from hem9 mutants, a phenotype that we believe to be unprecedented in the ubiquitin literature. Preliminary identification of E2-230K substrates suggested that it is possibl the first known globin ligase in reticulocytes. However, hem9 is also a strong suppressor of the th3 -thalassemic allele, which suggests that E2-230K may have key substrates in addition to globin. When reticulocyte lysates from the mutants were reconstituted with recombinant E2-230K, ribosomal proteins (RPs) proved to be a major class of E2-230K targets. Accordingly, hem9 reticulocytes have elevated ribosome levels and aberrant polysome profiles. Moreover, during the ex vivo maturation of reticulocytes into erythrocytes, there appears to be a major defect in the elimination of ribosomes. Thus, the goal of this project is to characterize the role f E2-230K in terminal erythroid differentiation, specifically in ribosome turnover, and in the pathophysiology of anemia. We propose to further verify RPs as substrates of E2-230K as well as to carry out unbiased proteomic approaches to broadly determine physiologically relevant targets. We will also characterize the role of E2-230K in ribosome ubiquitination and degradation in terminal erythroid differentiation. Finally, we will characterize the effect of hem9 on reticulocyte translational output to understand how a failure of ubiquitination can lead to anemia. Our proposed work should clarify the role of E2-230K and ubiquitination in erythropoiesis. These insights into the mechanism of erythroid cellular remodeling may have interesting therapeutic implications for hematologic diseases such as -thalassemia and Diamond Blackfan Anemia.