Congenital anemias are a prevalent and multifaceted subset of inherited human diseases. While the pathogenesis of certain inherited anemias such as thalassemias and sickle cell is related to hemoglobin regulation, it is increasingly appreciated that a number of inherited diseases with anemia as an important sequela are caused by ribosomal defects. The erythroid-specific phenotype of these diseases underlies the importance of the protein synthetic machinery in regulating erythropoiesis. This proposal explores protein degradation in erythropoiesis. The ubiquitin-proteasome system was discovered in reticulocytes, where it is highly active. However, the pathways of cellular remodeling in terminal differentiation remain largely unknown, and no specific ubiquitinating factor has been implicated. E2-230K is a ubiquitin-conjugating enzyme that is highly and selectively upregulated in the reticulocyte stage. Recently, a null mutation in the murine E2-230K gene, known as hem9, was identified in the lab of co-mentor Mark Fleming. Homozygous hem9 mice exhibit a hypochromic, microcytic anemia, and appear to be affected specifically in the erythroid lineage. 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. Preliminary identification of E2-230K substrates suggested that it is possibl the first known globin ligase in reticulocytes. However, hem9 is also a strong suppressor of the th3 -thalassemic allele, which suggests that E2-230K may have key substrates in addition to globin. When reticulocyte lysates from the mutants were reconstituted with recombinant E2-230K, ribosomal proteins (RPs) proved to be a major class of E2-230K targets. Accordingly, hem9 reticulocytes have elevated ribosome levels and aberrant polysome profiles. Moreover, during the ex vivo maturation of reticulocytes into erythrocytes, there appears to be a major defect in the elimination of ribosomes. Thus, the goal of this project is to characterize the role f E2-230K in terminal erythroid differentiation, specifically in ribosome turnover, and in the pathophysiology of anemia. We propose to further verify RPs as substrates of E2-230K as well as to carry out unbiased proteomic approaches to broadly determine physiologically relevant targets. We will also characterize the role of E2-230K in ribosome ubiquitination and degradation in terminal erythroid differentiation. Finally, we will characterize the effect of hem9 on reticulocyte translational output to understand how a failure of ubiquitination can lead to anemia. Our proposed work should clarify the role of E2-230K and ubiquitination in erythropoiesis. These insights into the mechanism of erythroid cellular remodeling may have interesting therapeutic implications for hematologic diseases such as -thalassemia and Diamond Blackfan Anemia.
The proposed work will examine the gene encoding E2-230K, a ubiquitin-conjugating enzyme that is highly upregulated during the reticulocyte stage of erythroid differentiation, and that, when mutated, causes hypochromic microcytic anemia while also ameliorating -thalassemia in a murine model. Unexpectedly, we identified a major perturbation of the protein synthesis machinery in this mutant, a phenotype related to a family of genetic disorders collectively known as Diamond Blackfan Anemia. The overarching goal is to understand the molecular mechanisms that regulate protein turnover in late erythrocyte differentiation, which could have interesting therapeutic implications for many diseases of the blood.