King, Randall WSeguinot Zapata, Bryan O.2026-07-0720262026-06-052026Seguinot Zapata, Bryan O.. 2026. Proteome Remodeling in Erythropoiesis Mediated by TBCEL and YPEL5. Doctoral Dissertation, Harvard University Graduate School of Arts and Sciences.32668862https://dash.harvard.edu/handle/1/42744071Erythropoiesis requires extensive proteome remodeling, resulting in approximately 98% of soluble protein being hemoglobin following the differentiation of erythroid precursors into red blood cells (Roux-Dalvai et al., 2008). Major protein complements, including ribosomes, RNA-binding proteins, and the microtubule cytoskeleton, are selectively dismantled (Zhu et al., 2023). Rather than carrying out nonspecific bulk turnover, erythroid cells deploy specialized ubiquitin-proteasome system factors to orchestrate targeted protein clearance. This dissertation identifies and mechanistically characterizes TBCEL and YPEL5 as key regulators of cytoskeletal and RNA-binding-protein remodeling during erythropoiesis. A defining event in erythroid differentiation is the transition from a radial microtubule network to a cortical actin-spectrin cytoskeleton, requiring extensive degradation of α/β-tubulin heterodimers (Liu et al., 2010). The mechanisms governing tubulin turnover in cells and during this specific differentiation process have remained elusive. Transcriptomic analysis identified TBCEL (Tubulin Binding Cofactor E-like), a tubulin-associated factor, as a highly induced factor during late erythropoiesis (Nguyen et al., 2017). Proteomic analysis of TBCEL knockout reticulocytes revealed a specific accumulation of tubulins, establishing TBCEL as a major and selective tubulin degradation factor in vivo. Biochemical and structural analyses demonstrate that TBCEL directly binds α/β-tubulin heterodimers through conserved interfaces within its leucine-rich repeat and ubiquitin-like domains. Additionally, structural, bioinformatic, and biochemical analyses suggest that TBCEL binding induces a distorted tubulin conformation with a remarkable 30° rotation of tubulin subunits with respect to each other, consistent with destabilization and disruption of the heterodimer. These data support a model in which TBCEL acts not as a tubulin chaperone, but on the contrary as a tubulin-disrupting factor that converts the highly stable heterodimers into structurally altered species competent for downstream degradation. Functional studies revealed that TBCEL overexpression induces ubiquitin-dependent tubulin degradation via the proteasome. Interactome analyses suggest mechanistic divergence between tubulin subunits: α-tubulin associates with proteasome shuttling factors consistent with direct targeting, whereas β-tubulin associates with ubiquitin ligases, consistent with ubiquitin-dependent breakdown. These findings uncover a previously unrecognized tubulin clearance mechanism in which dimer disruption precedes proteasomal targeting and reveal subunit-specific degradation pathways. Beyond erythropoiesis, TBCEL regulates tubulin homeostasis in the central nervous system. Loss of TBCEL results in differential accumulation of specific tubulin isotypes correlating with severe age-dependent motor deficits, and developmental brain enlargement in mice. This highlights that alterations in tubulin turnover that are not so dramatic as to result in wholesale elimination of microtubules, as in reticulocytes, can have profound physiological consequences. These phenotypes are likely to reflect isotype imbalances, implying that TBCEL may be a key "writer" of the tubulin code in neural tissues. Phylogenetic analyses indicate that TBCEL arose just prior to the emergence of metazoans, supporting a conserved and ancient role in cytoskeletal regulation. A second erythropoietic pathway identified in this work is the CTLH ubiquitin ligase complex, with the YPEL5 protein acting as a novel substrate receptor. The YPEL5-CTLH complex promotes the turnover of over 100 RNA-granule components, possibly acting on the granules themselves, during erythropoiesis. The conserved CULT domain of YPEL5 is required for substrate ubiquitination, as demonstrated by mutational analysis and in vitro ubiquitination assays. YPEL5 also defines a new family of substrate receptors for the ubiquitin system. Interactome studies and structural modeling identify FBXO42, an RNA granule-associated ubiquitin ligase receptor, as a candidate interactor linking YPEL5 to RNA-granule substrates. In vitro biochemical assays validate this interaction and show CTLH-YPEL5-dependent ubiquitination of purified FBXO42. These findings support a model where YPEL5 redirects CTLH ligase activity toward RNA granule proteins to facilitate their clearance during erythroid maturation. Collectively, these studies define TBCEL and YPEL5 as novel ubiquitin-proteasome system components that specify and drive selective proteome remodeling during erythropoiesis. More broadly, this work establishes new principles for cytoskeletal and RNA-protein homeostasis to reveal how lineage-specific degradation programs shape cellular differentiation and organismal physiology.application/pdfenerythropoiesisprotein degradationsubstrate receptortubulinubiquitin ligaseubiquitin proteasome systemBiochemistryCellular biologyMolecular biologyThesis or Dissertation2026-07-070000-0002-2651-8145