Loss of UBE2O Mitigates Beta Thalassemia Through Broad Proteomic Changes

Abstract

UBE2O is a ubiquitin-conjugating enzyme that is upregulated during terminal erythroid differentiation. We previously showed that UBE2O selectively ubiquitinates ribosomal proteins (RPs) and drives the elimination of ribosomes in reticulocytes and non-erythroid cells. Here, we showed that Ube2o-/- reticulocytes have a severe defect in the elimination of RPs by immunoblot analysis. We then used quantitative mass spectrometry to analyze the reticulocyte proteome in a global and unbiased manner. Of the 1235 proteins quantified, we observed significant elevations of 183 proteins, many of which were RPs. Similarly, we quantified the proteomic changes in HEK293-derived cells upon expression of UBE2O. Nearly 10% of all proteins, including RPs, were perturbed by UBE2O expression, which was consistent with the role of UBE2O in broadly remodeling the proteome. Since UBE2O preferentially targets basic proteins, we quantified the intracellular amino acid pools in mutant reticulocytes and found a depletion of free amino acids. As such, deletion of GCN2, an amino acid sensor, partially ameliorated the Ube2o-/- anemic phenotype. Using ribosome profiling, we showed that there is decreased ribosome occupancy of the globin genes in Ube2o-/- reticulocytes, which is consistent with an activation of the integrated stress response through GCN2. The attenuated translation of globin may likely underlie the anemic phenotype of the mutant. We next showed that alpha globin is a direct substrate of UBE2O. Finally, loss of UBE2O was shown to mitigate phenotypes of globin excess in a mouse model of beta thalassemia. In addition to RPs, Ube2o-/- reticulocytes have elevated levels of ferritin as a result of the depression of ferritin translation. Moreover, these mutant cells have elevated levels of three additional non-RP substrates, RIOK1, PTRF, and NOP16. Using a pull-down assay, we showed that UBE2O has multiple conserved substrate recognition domains and is capable of multiple substrate recognition modalities. In summary, UBE2O is a hybrid ubiquitinating enzyme that broadly remodels the proteome during terminal erythroid differentiation. Defects in protein degradation during erythroid differentiation result in altered translation of globins and ferritins. Attenuated translation of globin appears to ameliorate beta thalassemia; thus, UBE2O is a novel and potent therapeutic target for this prevalent genetic disease.