Proteomic analysis of ubiquitinated proteins in normal hepatocyte cell line Chang liver cells.
Post-translational modification by ubiquitin (Ub) and Ub-like modifiers is one of the most important mechanisms regulating a wide range of cellular processes in eukaryotes. Through mediating 26S proteasome-dependent degradation of substrates, the covalent modification of proteins by multiple Ub (ubiquitination) can regulate many different cellular functions such as transcription, antigen processing, signal transduction and cell cycle. To better understand ubiquitination and its functions, proteomic approaches have been developed to purify and identify more protein substrates. The S5a subunit of the 26S proteasome binds to poly-Ub chains containing four or more Ub. In this study, immobilized GST-S5a fusion protein was used to affinity-purify ubiquitinated proteins from Chang liver cells. The purified proteins were then identified with multi-dimensional LC combined with MS/MS. Eighty-three potential ubiquitination substrates were identified. From these proteins, 19 potential ubiquitination sites on 17 potential substrates were determined. These potential ubiquitination substrates are mainly related to important cellular functions including metabolism, translation and transcription. Our results provide helpful information for further understanding of the relationship between ubiquitination machinery and different cell functions.
Tan
Fengwei
F
The National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China.
Lu
Lifang
L
Cai
Yun
Y
Wang
Jinglan
J
Xie
Yunfei
Y
Wang
Lin
L
Gong
Yanhua
Y
Xu
Bing-E
BE
Wu
Jun
J
Luo
Ying
Y
Qiang
Boqin
B
Yuan
Jiangang
J
Sun
Xiaoqing
X
Peng
Xiaozhong
X
D000595:Amino Acid Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D005982:Glutathione Transferase / Q000302:isolation & purification / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D046988:Proteasome Endopeptidase Complex / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D016601:RNA-Binding Proteins; D053719:Tandem Mass Spectrometry; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*
C498506:PSMD4 protein, human; D020543:Proteome; D016601:RNA-Binding Proteins; D025801:Ubiquitin; D005982:Glutathione Transferase; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease