Abstract
Protein posttranslational modifications such as neddylation play crucial roles in regulating protein function. Only a few neddylated substrates have been validated to date, and the role of neddylation remains poorly understood. Here, using Trypanosoma brucei as the model organism, we investigated the function and substrates of TbNedd8. TbNedd8 is distributed throughout the cytosol but enriched in the nucleus and the flagellum. Depletion of TbNedd8 by RNAi abolished global protein ubiquitination, caused DNA re-replication in postmitotic cells, impaired spindle assembly, and compromised the flagellum attachment zone filament, leading to flagellum detachment. Through affinity purification and mass spectrometry, we identified 70 TbNedd8-conjugated and -associated proteins, including known Nedd8-conjugated and -associated proteins, putative TbNedd8 conjugation system enzymes, proteins of diverse biological functions, and proteins of unknown function. Finally, we validated six Cullins as bona fide TbNedd8 substrates and identified the TbNedd8 conjugation site in three Cullins. This work lays the foundation for understanding the roles of protein neddylation in this early divergent parasitic protozoan.
Highlights
Protein posttranslational modifications such as neddylation play crucial roles in regulating protein function
The conjugated Nedd8 can be hydrolyzed by deneddylation enzymes, and the most studied deneddylation enzyme is the COP9 signalosome, which is mainly involved in deneddylation of the Cullin subunit in the Cullin-RING ubiquitin ligase (CRL)5 [3]
To validate the candidacy of the six Cullins as TbNedd8 substrates, they were each tagged with a triple HA epitope at the N terminus and ectopically expressed in T. brucei
Summary
Protein posttranslational modifications such as neddylation play crucial roles in regulating protein function. Many putative Nedd8-associated proteins in humans have been identified through proteomic approaches (4 –7), but their candidacy as genuine Nedd substrates remains to be experimentally confirmed. These studies all demonstrated that Cullins are the most abundant Nedd substrates. Other Nedd substrates include a number of oncogenic proteins, such as Von Hippel-Lindau (VHL) tumor suppressor, p53, MDM2, BCA3, EGF receptor, and some ribosomal proteins Neddylation of these proteins regulates transactivation function and protein-protein interaction (p53 and BCA3), increases protein stability (MDM2 and ribosomal proteins), or stimulates ubiquitination (EGF receptor) [1, 8]. Given the unusual biology of T. brucei, investigation of the cellular function of TbNedd and identification of its conjugated substrates may uncover novel regulatory pathways, which could provide hints about the evolution of the neddylation pathway
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