title: Global quantitative proteomics reveal up-regulation of endoplasmic reticulum stress response proteins upon depletion of eIF5A in HeLa cells.
abstract: The eukaryotic translation factor, eIF5A, is a translation factor essential for protein synthesis, cell growth and animal development. By use of a adenoviral eIF5A shRNA, we have achieved an effective depletion of eIF5A in HeLa cells and undertook in vivo comprehensive proteomic analyses to examine the effects of eIF5A depletion on the total proteome and to identify cellular pathways influenced by eIF5A. The proteome of HeLa cells transduced with eIF5A shRNA was compared with that of scramble shRNA-transduced counterpart by the iTRAQ method. We identified 972 proteins consistently detected in three iTRAQ experiments and 104 proteins with significantly altered levels (protein ratio ≥1.5 or ≤0.66, p-value ≤0.05) at 72 h and/or 96 h of Ad-eIF5A-shRNA transduction. The altered expression levels of key pathway proteins were validated by western blotting. Integration of functional ontology with expression data of the 104 proteins revealed specific biological processes that are prominently up- or down-regulated. Heatmap analysis and Cytoscape visualization of biological networks identified protein folding as the major cellular process affected by depletion of eIF5A. Our unbiased, quantitative, proteomic data demonstrate that the depletion of eIF5A leads to endoplasmic reticulum stress, an unfolded protein response and up-regulation of chaperone expression in HeLa cells.
authors.affiliation: Molecular and Cellular Biochemistry Section, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bldg.30 Rm. 3A300, Bethesda, MD 20892, USA.
authors.forename: Ajeet
authors.initials: A
authors.lastname: Mandal
authors.affiliation: Molecular and Cellular Biochemistry Section, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bldg.30 Rm. 3A300, Bethesda, MD 20892, USA.
authors.forename: Swati
authors.initials: S
authors.lastname: Mandal
authors.affiliation: Molecular and Cellular Biochemistry Section, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bldg.30 Rm. 3A300, Bethesda, MD 20892, USA.
authors.forename: Myung Hee
authors.initials: MH
authors.lastname: Park
mesh_terms: D000256:Adenoviridae / Q000378:metabolism; D049109:Cell Proliferation; D002470:Cell Survival; D019295:Computational Biology; D059865:Endoplasmic Reticulum Stress; D063990:Gene Ontology; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D018832:Molecular Chaperones / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D014176:Protein Biosynthesis; D040901:Proteomics / Q000379:methods*; D034741:RNA, Small Interfering / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014161:Transduction, Genetic; D056811:Unfolded Protein Response; D015854:Up-Regulation; D000097574:Eukaryotic Translation Initiation Factor 5A
chemical_list: D018832:Molecular Chaperones; D010448:Peptide Initiation Factors; D010455:Peptides; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A; C011083:polyproline