Modular UBE2H-CTLH E2-E3 complexes regulate erythroid maturation

Abstract

The development of haematopoietic stem cells into mature erythrocytes – erythropoiesis – is a controlled process characterized by cellular reorganization and drastic reshaping of the proteome landscape. Failure of ordered erythropoiesis is associated with anaemias and haematological malignancies. Although the ubiquitin system is a known crucial post-translational regulator in erythropoiesis, how the erythrocyte is reshaped by the ubiquitin system is poorly understood. By measuring the proteomic landscape of in vitro human erythropoiesis models, we found dynamic differential expression of subunits of the CTLH E3 ubiquitin ligase complex that formed maturation stage-dependent assemblies of topologically homologous RANBP9- and RANBP10-CTLH complexes. Moreover, protein abundance of CTLH’s cognate E2 ubiquitin conjugating enzyme UBE2H increased during terminal differentiation, and UBE2H expression depended on catalytically active CTLH E3 complexes. CRISPR-Cas9-mediated inactivation of CTLH E3 assemblies or UBE2H in erythroid progenitors revealed defects, including spontaneous and accelerated erythroid maturation as well as inefficient enucleation. Thus, we propose that dynamic maturation stage-specific changes of UBE2H-CTLH E2-E3 modules control the orderly progression of human erythropoiesis.

RanBP9 controls the oligomeric state of CTLH complex assemblies

“…It will be interesting to investigate the role of RanBP10, which, as a close paralog, may replace the organizing role of RanBP9, for example, as already demonstrated in stage-dependent assemblies important for erythropoiesis ( 32 ) and thereby dictate the ability of the CTLH complex to bind to distinct substrates. Whether this modulation of function is based on its scaffolding function or occurs via direct substrate interactions remains to be determined.…”

“…CTLH activities have been further implicated in cell proliferation ( 20 , 21 ), aging ( 22 ), metabolism ( 23 , 24 ), and developmental processes. The complex is also responsible for an organized maternal-to-zygotic transition ( 25 , 26 ) and is important for neurodevelopment ( 27 , 28 , 29 , 30 ) as well as hematopoiesis ( 31 , 32 , 33 , 34 , 35 , 36 ). While the name CTLH complex suggests that there is a single complex with a defined subunit architecture, there are in fact multiple alternative subunits, which may lead to differential CTLH complexes.…”

“…In summary, regulation of substrate recognition of the GID–CTLH complex relies both on suitable SRs specifically targeting N-terminal degron sequences paired with an oligomeric assembly, which assists in the positioning and selective targeting of substrates ( 1 ). Compared with the yeast complex, the CTLH complex displays a greater variety of interchangeable subunits, thus possibly modulating its oligomeric assembly: Rmnd5b and RanBP10 can replace their paralogs Rmnd5a and RanBP9 ( 20 , 32 ), Armc8β its longer isoform Armc8α ( 21 ), and muskelin, presumably, the more distantly related Wdr26 ( Fig. 1 B ).…”

Identifying E3 Ligase Substrates With Quantitative Degradation Proteomics

“…In contrast to the substrates with observed CTLH‐dependent degradation, the degradation of the cognate E2 enzyme for CTLH E3 ligase, UBE2H, was 4.2‐fold accelerated in the Y394A mutant (from 57.8 h to 13.8 h, Figure 6E). This is due to the inefficient transfer of ubiquitin to the substrates, resulting in the autoubiquitylation of UBE2H and its subsequent degradation, as reported previously [36] . These examples highlight another advantage of the differential degradomics approach: the differences in both steady‐state protein levels and the degradation kinetics can be projected simultaneously, thus providing in‐depth information about the homeostasis of the protein of interest.…”

“… [12,15,16] Two of the seven components, MAEA and RMND5 (a or b), contain the RING domain for E3 ligase activity, which form a heterodimeric complex (Figure 1B). While the role of the CTLH ligase has been implicated in various cellular processes such as erythropoiesis, microtubule dynamics, and chromosome segregation, its direct ubiquitylation substrates in the affected pathways are poorly characterized [17–20] . To determine degradation substrates of the CTLH complex in mammalian cells, we engineered HEK293T cells to delete the MAEA gene (Figure 1C).…”