Ubiquitination in erythropoiesis and the pathophysiology of anemia

描述(申请人提供)：导致贫血的血红蛋白产生障碍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a2ss2四聚体缺乏有关，而且还与过量的、不稳定的、不成对的α-珠蛋白链的积累和异常基因产物本身的积累有关。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，特别是通过一种基本上未知的途径降解多余的珠蛋白。十多年前，我们发现一个异常大的泛素结合酶，E2-230K，在红系中被特异性地诱导。事实上，在富含网织红细胞的小鼠红细胞中，E2-230K是第二丰富的mRNA。我们已经从系统的乙基亚硝脲血液学突变筛查中鉴定了小鼠的hem9突变。Hm9纯合子有低色素性小细胞性贫血。我们发现hem9是E2-230K基因的无义突变，明显为零。我们建议确定发育中的红细胞泛素化失败如何导致贫血。由于低色素性小细胞性贫血是珠蛋白或卟啉合成异常或铁的获取/利用异常的唯一结果，我们预计E2-230K缺乏将影响这三条途径中的一条。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。我们通过几个生化步骤提纯了这些物种，并对它们进行了质谱分析。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可能是网织红细胞中α-珠蛋白的主要泛素连接酶。我们建议确定E2-230K对过量的α-珠蛋白是高度选择性的，还是为网织红细胞中的蛋白质质量控制提供了一条通用的途径。我们还发现hem9是TH-3地中海贫血等位基因的抑制因子。这一结果表明，除了α-珠蛋白外，E2-230K可能还有关键的结合靶点。因此，我们建议进一步验证α-珠蛋白作为底物的分配，并进行无偏倚的蛋白质组学方法来广泛确定E2-230K的生理相关靶点。真正的底物将被指定为需要在网织红细胞中泛素化的E2-230K的蛋白质，并被纯化的E2-230K泛素化。为此，我们成功地在大肠杆菌中以可溶性的、具有酶活性的形式表达了E2-230K。因此，我们的主要目标是解释H9贫血，我们将确定E2-230K是作为一般的蛋白质质量控制机制，还是作为清除潜在有毒过量珠蛋白的特殊质量控制机制，以及E2-230K是否也可能在终末红系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的机制控制珠蛋白和其他关键蛋白质的降解，可能会对我们治疗遗传性红细胞疾病的方法产生重大影响。 与公共卫生相关：这个项目旨在了解蛋白质在红细胞中的正常降解方式，以及该途径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.