Mechanisms of proteome curation during red blood cell differentiation - PROJECT SUMMARY Red blood cells are responsible for circulating oxygen around the human body. To achieve this, differentiating red blood cells must drastically restructure their proteome to enrich for hemoglobin, a tetramer of four globin proteins that coordinate heme to bind oxygen. This occurs at a staggering rate: approximately two million blood cells, each containing over 95% hemoglobin, must be produced every second to maintain health. A critical factor for red blood cell production is UBE2O, a unique ubiquitylation enzyme within the ubiquitin-proteasome system that degrades proteins. UBE2O has a quality control function of eliminating `orphan' proteins that fail to assemble into the appropriate protein complex. These substrates include unassembled globin and ribosomal proteins. UBE2O is also upregulated during late stages of red blood cell differentiation to mediate the clearance of entire ribosome, the molecular machines that synthesize proteins. While ribosomes are needed during initial stages of red blood cell differentiation to synthesize globin proteins, they must be eliminated for terminal red blood cell differentiation to concentrate hemoglobin. How UBE2O accomplishes these tasks essential for red blood cell function is not known. We recently established experimental and conceptual foundations to dissect the mechanisms of protein ubiquitylation by UBE2O. In Aim 1, we will build on these foundations to reconstitute the proteasomal degradation of UBE2O substrates with purified factors. This will establish the minimal requirements for UBE2O-mediated protein quality control that occurs through a unique multi-mono-ubiquitylation activity not typically associated with proteasomal degradation. In Aim 2, we will use unbiased proteomics and structural studies to explore how different substrate adaptors tune the identities of the proteins UBE2O targets for degradation. In Aim 3, we will identify new factors that work with UBE2O to degrade entire ribosomes, which are large ~4 Megadalton complexes of approximately 80 proteins scaffolded on 4 rRNA molecules, through proteasomes, which are restricted to degrading one protein at a time. Successful completion of these aims will elucidate unique mechanisms that operate in the ubiquitin- proteasome system to mediate protein quality control, ribosome homeostasis, and red blood cell differentiation. Such molecular-level insights will generate precise hypotheses to test in complex physiological models and facilitate our understanding of how these processes contribute to blood disorders and the identification of new therapeutic strategies.
