A ubiquitin-conjugating enzyme critical for terminal erythroid differentiation

一种对终末红细胞分化至关重要的泛素结合酶

• 批准号: 8779851
• 负责人: Anthony Tuan Nguyen
• 金额: $ 3.5万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8779851
• 关键词: Affect; Alleles; Amino Acids; Anemia; Animals; Back; Biology; Blood; Cell model; Cells; Centrifugation; Congenital Anemia; Data; Defect; Development; Diamond-Blackfan anemia; Disease; Employee Strikes; Erythrocytes; Erythroid; Erythropoiesis; Ethylnitrosourea; Exhibits; Failure; Flow Cytometry; Functional disorder; Genes; Globin; Goals; Hematological Disease; Hemoglobin; Hereditary Disease; Human; In Vitro; Inherited; Lead; Ligase; Literature; Mass Spectrum Analysis; Measures; Mentors; Messenger RNA; Mitochondria; Modeling; Molecular; Molecular Weight; Mus; Mutate; Mutation; Output; Pathogenesis; Pathway interactions; Peptides; Phenotype; Physiological; Polyribosomes; Process; Protein Biosynthesis; Proteins; Proteomics; Radiolabeled; Recombinants; Regulation; Reticulocytes; Ribosomal Proteins; Ribosomes; Role; Sickle Cell; Site; Staging; System; Testing; Thalassemia; Therapeutic; Time; Translations; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination; Western Blotting; Work; disease phenotype; erythroid differentiation; family genetics; human disease; insight; interest; microcytic/hypochromic anemia; multicatalytic endopeptidase complex; mutant; novel therapeutic intervention; null mutation; protein degradation; public health relevance; radiotracer; reconstitution; research study; synthetic protein; theories

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.

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的发病机制与血红蛋白调节有关，但越来越多的人认识到， 以贫血为重要后遗症的许多遗传性疾病都是由核糖体缺陷引起的。这些疾病的红细胞特异性表型是蛋白质合成机制在调节红细胞生成中的重要性的基础。该提案探讨了红细胞生成中的蛋白质降解。泛素-蛋白酶体系统在网织红细胞中被发现，在那里它是高度活跃的。然而，终末分化中细胞重塑的途径 仍然在很大程度上是未知的，没有具体的泛素化因子已经牵连。E2- 230 K是一种泛素结合酶，在网织红细胞阶段高度选择性上调。最近，在共同导师Mark Fleming的实验室中发现了小鼠E2- 230 K基因中的无效突变，称为hem 9。纯合子hem 9小鼠表现出低色素性、小细胞性贫血，并且似乎在红系谱系中特别受到影响。我们发现，所有主要的泛素蛋白结合带的网织红细胞，这是低分子量的，大大降低了水平的提取物hem 9突变体，我们认为是前所未有的表型在泛素文献。E2- 230 K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，hem 9也是th 3-地中海贫血等位基因的强抑制因子，这表明E2- 230 K可能具有除珠蛋白之外的关键底物。当来自突变体的网织红细胞裂解物用重组E2- 230 K重建时，核糖体蛋白（RP）被证明是E2- 230 K靶的主要类别。因此，hem 9网织红细胞具有升高的核糖体水平和异常的多核糖体谱。此外，在网织红细胞离体成熟为红细胞的过程中，似乎存在核糖体消除的主要缺陷。因此，本项目的目标是表征E2- 230 K在终末红细胞分化中的作用，特别是在核糖体周转中，以及在贫血的病理生理学中。我们建议进一步验证RP作为E2- 230 K的底物，并进行无偏的蛋白质组学方法，以广泛确定生理相关的目标。我们还将描述E2- 230 K在核糖体泛素化和终末红细胞分化中的降解中的作用。最后，我们将描述hem 9 对网织红细胞翻译输出的影响，以了解泛素化的失败如何导致贫血。我们的工作将阐明E2- 230 K和泛素化在红细胞生成中的作用。这些见解的机制红细胞重塑可能有有趣的治疗意义的血液系统疾病，如地中海贫血和钻石黑帆贫血。