Ubiquitination in erythropoiesis and the pathophysiology of anemia

描述（由申请人提供）：血红蛋白生成障碍导致贫血是人类最常见的遗传性疾病之一。这些疾病的发病机制不仅与血红蛋白a2 ss 2四聚体的缺乏有关，还与过量、不稳定、未配对的α-珠蛋白链的积累以及异常基因产物本身的积累有关。在网织红细胞中发现了泛素-蛋白酶体系统，它在网织红细胞中高度活跃，通过基本上未知的途径降解特别是过量的球蛋白。十多年前，我们发现一种异常大的泛素结合酶E2- 230 K在红细胞系中被特异性诱导。事实上，E2- 230 K是富含网织红细胞的小鼠红细胞（RBC）中第二丰富的mRNA。我们的特点是小鼠hem 9突变系统乙基亚硝基脲筛选血液突变。hem 9纯合子具有低色素小细胞性贫血。我们发现hem 9是E2- 230 K基因的无义突变，明显无效。我们建议，以确定如何在发展中的红细胞泛素化失败导致贫血。由于低色素性小细胞性贫血是珠蛋白或卟啉合成或铁获取/利用异常的唯一结果，我们预计E2- 230 K缺乏将影响这三种途径之一。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。我们已经通过几个生化步骤纯化了这些物种，并对它们进行了质谱分析。值得注意的是，由此鉴定的一种缀合物是α-珠蛋白-泛素缀合物。因此，我们的数据表明，E2- 230 K可能是网织红细胞中α-珠蛋白的主要泛素连接酶。我们建议确定E2- 230 K是否对过量的α-珠蛋白具有高度选择性，或者为网织红细胞中的蛋白质质量控制提供一般途径。我们还发现hem 9是th-3地中海贫血等位基因的抑制基因。这一结果表明，E2- 230 K除了α-珠蛋白外，还可能具有关键的接合靶标。因此，我们建议进一步验证α-珠蛋白作为底物的这种分配，以及进行无偏见的蛋白质组学方法，以广泛确定E2- 230 K的生理相关目标。真正的底物将被指定为需要E2- 230 K在网织红细胞中进行泛素化并被纯化的E2- 230 K泛素化的蛋白质。为此，我们成功地在大肠杆菌中表达了可溶性的、具有酶活性的E2- 230 K。杆菌因此，我们的广泛目标是解释hem 9贫血，我们将确定E2- 230 K是否作为一种通用的蛋白质质量控制机制，或作为一种特定的质量控制机制，以消除潜在的毒性过量球蛋白，以及E2- 230 K是否也可能在终末红细胞分化中具有更广泛的影响。显然，了解网织红细胞如何通过泛素依赖性机制控制球蛋白和其他关键蛋白质的降解，可能对我们治疗红细胞遗传性疾病的方法产生重大影响。 公共卫生相关性：该项目旨在了解蛋白质在红细胞中如何正常降解，以及该途径中的缺陷如何导致贫血的发展。

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.