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.
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.