Person: Nguyen, Anthony Tuan
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Nguyen
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Anthony Tuan
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Nguyen, Anthony Tuan
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Publication A Novel Program of Ubiquitination Remodels the Erythroid Proteome During Terminal Differentiation(2016-05-17) Nguyen, Anthony Tuan; Liberles, Stephen; Goldberg, Alfred; Lodish, HarveyThe ubiquitin-proteasome system was initially discovered in reticulocytes, which undergo massive and rapid proteome remodeling. During terminal differentiation, hundreds of generic constituents of the cell undergo programmed elimination. However, the mechanisms that drive the turnover of normally stable proteins remain largely unknown. Two decades ago, an unusually large ubiquitin-conjugating enzyme, Ube2O, was found to be strongly and specifically upregulated in terminally differentiating reticulocytes, contemporaneously with the induction of globin. A null mutation in the murine Ube2O gene, known as hem9, resulted in a hypochromic, microcytic anemia, suggesting that Ube2O may be a major ubiquitinating factor in erythropoiesis. To understand the role of Ube2O in terminal differentiation, we first found that all major low molecular weight ubiquitin-protein conjugate bands are greatly reduced in levels in hem9 reticulocytes. When null reticulocyte lysates were treated with recombinant Ube2O, ribosomal proteins were overwhelmingly the major class of targets. Accordingly, hem9 reticulocytes have elevated ribosomal protein levels and 80S ribosomes. This phenotype of elevated ribosome abundance was accounted for by a defect in the elimination of ribosomes. Furthermore, overexpression of Ube2O was sufficient to drive ribosomal degradation in non-erythroid 293 cells. Quantitative mass spectrometry on these cells confirmed the destabilization of ribosomal proteins and indicated that Ube2O has a specific, yet broad ubiquitination program. Interestingly, the hem9 defect was phenocopied by treating wild-type reticulocytes with proteasome inhibitors. To confirm this finding, we reconstituted the degradation of ribosomal proteins in a cell-free reticulocyte lysate system. We also reconstituted the ubiquitination of purified ribosomes by recombinant Ube2O in vitro, and the degradation of several specific ribosomal proteins with purified proteasomes. Next, we found that the initiation factor eIF2α is hyperphosphorylated in hem9 reticulocytes, suggesting a global inhibition of protein synthesis. This was independent of HRI, a dominant regulator of translation in reticulocytes; instead, another eIF2α kinase, GCN2 was activated in the null mutant. Consistent with these findings, null reticulocytes were deficient in free amino acids, a phenotype that was recapitulated by proteasome inhibition. In summary, Ube2O selectively ubiquitinates ribosomal proteins and targets them to the proteasome for degradation, thus playing a central role during terminal erythroid differentiation.Publication Loss of UBE2O Mitigates Beta Thalassemia Through Broad Proteomic Changes(2018-05-15) Nguyen, Anthony TuanUBE2O 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.