Ubiquitination in erythropoiesis and the pathophysiology of anemia

红细胞生成中的泛素化和贫血的病理生理学

基本信息

批准号：
8301161
负责人：
MARK D FLEMING
金额：
$ 27.33万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-15 至 2014-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8301161
关键词：
Accounting Affect Alleles Anemia Animals Back Binding Biochemical Cells Data Defect Development Disease Employee Strikes Enzymes Equilibrium Erythrocytes Erythroid Erythropoiesis Escherichia coli Ethylnitrosourea Failure Functional disorder Genes Globin Goals Health Heme Hemoglobin Hemoglobinopathies Homozygote Human In Vitro Inherited Iron Label Lead Ligase Literature Mass Spectrum Analysis Messenger RNA Modeling Molecular Weight Mus Mutant Strains Mice Mutation Nature Nonsense Mutation Pathogenesis Pathway interactions Phase Phenotype Phosphotransferases Physiologic pulse Physiology Porphyrins Production Proteins Proteomics Quality Control Recombinants Reticulocytes Sickle Cell Site Specificity Streptavidin System Testing Thalassemia Thalassemia intermedia Therapeutic Time Tissues Toxic effect Translations Ubiquitin Ubiquitin-Conjugating Enzymes Ubiquitination Work alpha Globin base disease phenotype enzyme activity erythroid differentiation heme a insight microcytic/hypochromic anemia mouse model multicatalytic endopeptidase complex mutant novel therapeutic intervention null mutation ubiquitin ligase

项目摘要

DESCRIPTION (provided by applicant): Disorders of hemoglobin production leading to anemia are among the most prevalent inherited diseases in humans. The pathogenesis of these diseases is not only related to the deficiency of hemoglobin a2ss2 tetramers, but also the accumulation of excess, unstable, unpaired alpha-globin chains and accumulation of the abnormal gene product itself. The ubiquitin-proteasome system was discovered in reticulocytes, where it is highly active, degrading in particular excess globin through a pathway that remains essentially unknown. Over ten years ago, we found that an unusually large ubiquitin-conjugating enzyme, E2-230K, was specifically induced in the erythroid lineage. Indeed, E2-230K is the second most abundant mRNA in reticulocyte-rich mouse red blood cells (RBCs). We have characterized the murine hem9 mutation from a systematic ethylnitrosourea screen for hematological mutants. hem9 homozygotes have hypochromic microcytic anemia. We found hem9 to be a nonsense mutation in the E2-230K gene, an apparent null. We propose to determine how a failure of ubiquitination in the developing RBC leads to anemia. Since hypochromic microcytic anemias are uniquely the result of abnormalities of globin or porphyrin synthesis or iron acquisition/utilization, we expect that E2-230K deficiency will affect one of these three pathways. 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. We have purified these species through several biochemical steps and subjected them to mass spectrometry. Remarkably, the one conjugate thus identified was an alpha-globin-ubiquitin conjugate. Thus, our data suggest that E2-230K may be the major ubiquitin ligase for alpha-globin in reticulocytes. We propose to determine whether E2-230K is highly selective for excess alpha-globin, or provides a general pathway for protein quality control in reticulocytes. We have also found that hem9 is a suppressor of the th-3 thalassemic allele. This result suggests that E2-230K may have key conjugative targets in addition to alpha-globin. Thus, we propose to further verify this assignment of alpha-globin as a substrate as well as to carry out unbiased proteomic approaches to broadly determine the physiologically relevant targets of E2-230K. True substrates will be assigned as proteins that require E2-230K for ubiquitination in reticulocytes and are ubiquitinated by purified E2-230K. To this end, we have successfully expressed E2-230K in a soluble, enzymatically active form in E. coli. Thus, with our broad goal being to explain the hem9 anemia, we will determine whether E2-230K serves as a general protein quality control mechanism, or as a specific quality control mechanism to scavenge potentially toxic excess globins, and also whether E2- 230K may also have a more global influence in terminal erythroid differentiation. Clearly, understanding how reticulocytes manage the controlled degradation of globin and other key proteins through ubiquitin-dependent mechanisms could have a major impact on our therapeutic approach to inherited diseases of the red cell. PUBLIC HEALTH RELEVANCE: This project aims to understand how proteins are normally degraded in red blood cells and how defects in that pathway can contribute to the development of anemia.

描述（由申请人提供）：导致贫血的血红蛋白产生疾病是人类最普遍的遗传疾病之一。这些疾病的发病机理不仅与血红蛋白A2SS2四聚体的缺乏有关，而且还与过量，不稳定，未配对的α-珠链链的积累以及异常基因产物本身的积累相关。在网状细胞中发现了泛素 - 蛋白酶体系统，在网状细胞中，它具有高度活跃，特别是通过基本未知的途径降解过多的球蛋白。十多年前，我们发现一种异常大的泛素偶联酶E2-230K是在红系谱系中专门诱导的。实际上，E2-230K是富含网状细胞的小鼠红细胞（RBC）中第二大丰富的mRNA。我们已经表征了来自系统的乙基硝基筛膜筛查的鼠HEM9突变，用于血液学突变体。 HEM9纯合子具有低色素微细胞性贫血。我们发现HEM9在E2-230k基因（明显的无效）中是一个胡说八道的突变。我们建议确定发展中RBC中泛素化的失败是如何导致贫血的。由于低色素微细胞性贫血是球蛋白或卟啉合成或铁的采集/利用异常的独特结果，因此我们希望E2-230K缺陷会影响这三种途径之一。我们发现，所有主要的泛素蛋白 - 蛋白偶联物带的网状细胞均具有低分子量，在HEM9突变体的提取物中的水平大大降低了，我们认为，在泛素文献中，我们认为这种表型是前所未有的。我们已经通过几个生化步骤纯化了这些物种，并将它们予以质谱法。值得注意的是，这样鉴定的一个结合物是α-珠蛋白 - 泛素结合物。因此，我们的数据表明E2-230K可能是网状细胞中α-珠蛋白的主要泛素连接酶。我们建议确定E2-230K是否对过量的α-珠蛋白具有很高的选择性，或者为网状细胞中蛋白质质量控制提供了一般途径。我们还发现，HEM9是Th-3 Thalassemic等位基因的抑制剂。该结果表明，除α-珠蛋白外，E2-230K除了α-珠蛋白外可能还具有关键的共轭靶标。因此，我们建议进一步验证α-珠蛋白作为底物的分配，并采用公正的蛋白质组学方法，以广泛确定E2-230K的生理相关靶标。真正的底物将被分配为需要E2-230K在网状细胞中泛素化的蛋白质，并被纯化的E2-230K泛素化。为此，我们以大肠杆菌的可溶性，酶活性形式成功地表达了E2-230K。因此，我们的广泛目标是解释HEM9贫血，我们将确定E2-230K是否充当通用蛋白质质量控制机制，还是作为清除潜在有毒过量全球蛋白的特定质量控制机制，以及E2-230K是否也可能在末端红细胞区分中具有更全球的影响。显然，了解网状细胞如何通过泛素依赖性机制管理球蛋白和其他关键蛋白的受控降解可能对我们对红细胞遗传疾病的治疗方法产生重大影响。公共卫生相关性：该项目旨在了解通常如何在红细胞中降解蛋白质以及该途径中的缺陷如何有助于贫血的发展。