title: The Protein Neddylation Pathway in Trypanosoma brucei: FUNCTIONAL CHARACTERIZATION AND SUBSTRATE IDENTIFICATION.
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.
authors.affiliation: From the Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China and.|the Department of Microbiology and Molecular Genetics, McGovern Medical School, University of Texas Health Science Center, Houston, Texas 77030.
authors.forename: Shanhui
authors.initials: S
authors.lastname: Liao
authors.affiliation: the Department of Microbiology and Molecular Genetics, McGovern Medical School, University of Texas Health Science Center, Houston, Texas 77030.
authors.forename: Huiqing
authors.initials: H
authors.lastname: Hu
authors.affiliation: the Department of Microbiology and Molecular Genetics, McGovern Medical School, University of Texas Health Science Center, Houston, Texas 77030.
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.affiliation: From the Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China and xmtu@ustc.edu.cn.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Tu
authors.affiliation: the Department of Microbiology and Molecular Genetics, McGovern Medical School, University of Texas Health Science Center, Houston, Texas 77030 ziyin.li@uth.tmc.edu.
authors.forename: Ziyin
authors.initials: Z
authors.lastname: Li
mesh_terms: D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism*; D005407:Flagella / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
chemical_list: D044842:Cullin Proteins; D015800:Protozoan Proteins; D014452:Ubiquitins
keywords: Cullin; DNA replication; Nedd8; Trypanosoma brucei; flagellum; mitosis; mitotic spindle; neddylation; parasitology