Abstract
BackgroundUBE2O is proposed as a ubiquitin-conjugating enzyme, but its function was largely unknown.MethodsMass spectrometry was applied to identify c-Maf ubiquitination-associated proteins. Immunoprecipitation was applied for c-Maf and UBE2O interaction. Immunoblotting was used for Maf protein stability. Luciferase assay was used for c-Maf transcriptional activity. Lentiviral infections were applied for UBE2O function in multiple myeloma (MM) cells. Flow cytometry and nude mice xenografts were applied for MM cell apoptosis and tumor growth assay, respectively.ResultsUBE2O was found to interact with c-Maf, a critical transcription factor in MM, by the affinity purification/tandem mass spectrometry assay and co-immunoprecipitation assays. Subsequent studies showed that UBE2O mediated c-Maf polyubiquitination and degradation. Moreover, UBE2O downregulated the transcriptional activity of c-Maf and the expression of cyclin D2, a typical gene modulated by c-Maf. DNA microarray revealed that UBE2O was expressed in normal bone marrow cells but downregulated in MGUS, smoldering MM and MM cells, which was confirmed by RT-PCR in primary MM cells, suggesting its potential role in myeloma pathophysiology. When UBE2O was restored, c-Maf protein in MM cells was significantly decreased and MM cells underwent apoptosis. Furthermore, the human MM xenograft in nude mice showed that re-expression of UBE2O delayed the growth of myeloma xenografts in nude mice in association with c-Maf downregulation and activation of the apoptotic pathway.ConclusionsUBE2O mediates c-Maf polyubiquitination and degradation, induces MM cell apoptosis, and suppresses myeloma tumor growth, which provides a novel insight in understanding myelomagenesis and UBE2O biology.
Highlights
Ubiquitin-conjugating enzyme E2 O (UBE2O) is proposed as a ubiquitin-conjugating enzyme, but its function was largely unknown
UBE2O interacts with c-Maf To find out specific enzymes responsible for c-Maf ubiquitination, a LC/MS/MS assay was performed after affinity purification with a c-Maf specific antibody [12]
To verify the interaction between UBE2O and c-Maf, a UBE2O and a c-Maf plasmid were co-transfected into HEK293T cells, from which the lysates were coimmunoprecipitated with a specific anti-HA antibody followed by immunoblotting
Summary
UBE2O is proposed as a ubiquitin-conjugating enzyme, but its function was largely unknown. The ubiquitin-conjugating enzymes (E2s) transfer activated ubiquitin molecules to a ubiquitin ligase (E3) or directly mediate substrate ubiquitination. The detailed mechanisms are not fully understood, the Maf family transcription factors raise high attention [8]. This family is comprised of seven members of which c-Maf, MafA, and MafB belong to the large Maf subfamily because these transcription factors contain the complete structure and functional domains including the DNAbinding domain and the transcription activation domain [8]. All c-Maf, MafA, and MafB are found to be highly overexpressed in MM cells in association with chromosomal translocations and other unknown mechanisms [9].
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