Ubiquitination in erythropoiesis and the pathophysiology of anemia

描述（由申请人提供）：导致贫血的血红蛋白生成障碍是人类最普遍的遗传性疾病之一。这些疾病的发病机制不仅与血红蛋白a2ss2四聚体缺乏有关，还与过量、不稳定、不成对的α -珠蛋白链的积累以及异常基因产物本身的积累有关。泛素-蛋白酶体系统是在网状细胞中发现的，在那里它是高度活跃的，通过一种基本上未知的途径降解过量的珠蛋白。十多年前，我们发现一个异常大的泛素结合酶E2-230K在红系中被特异性诱导。事实上，E2-230K是富网织红细胞小鼠红细胞（rbc）中第二丰富的mRNA。我们通过系统乙基亚硝基脲筛选血液学突变体，对小鼠hem9突变进行了表征。Hem9纯合子有低色性小细胞贫血。我们发现它是E2-230K基因的无义突变，显然是无效的。我们建议确定红细胞泛素化的失败是如何导致贫血的。由于低色素小细胞贫血是珠蛋白或卟啉合成或铁获取/利用异常的独特结果，我们预计E2-230K缺乏将影响这三条途径中的一条。我们发现，在hem9突变体的提取物中，所有低分子量的网状红细胞的主要泛素蛋白结合带的水平都大大降低，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纯化了这些物种，并对它们进行了质谱分析。值得注意的是，这样鉴定的一个缀合物是α -球蛋白-泛素缀合物。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是否对过量的α -珠蛋白具有高度选择性，或者提供了网状红细胞中蛋白质质量控制的一般途径。我们还发现hem9是th3地中海贫血等位基因的抑制因子。这一结果表明E2-230K除了α -珠蛋白外可能还有关键的共轭靶点。因此，我们建议进一步验证α -球蛋白作为底物的这种分配，并开展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E2-230K才能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，我们成功地在大肠杆菌中表达了E2-230K的可溶性酶活性形式。因此，我们的大目标是解释hem9贫血，我们将确定E2-230K是否作为一般的蛋白质质量控制机制，还是作为清除潜在毒性过量球蛋白的特定质量控制机制，以及E2-230K是否也可能在终末期红细胞分化中具有更广泛的影响。显然，了解网状红细胞如何通过泛素依赖机制控制珠蛋白和其他关键蛋白的受控降解，可能对我们治疗遗传性红细胞疾病的方法产生重大影响。

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.