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.
Highlights
The eukaryotic translation factor, Eukaryotic initiation factor 5A (eIF5A), is a translation factor essential for protein synthesis, cell growth and animal development
The highly conserved eukaryotic translation factor eIF5A is strictly indispensable for the survival of eukaryotic cells. eIF5A was initially isolated from rabbit reticulocyte lysates[1] as a factor that stimulates methionyl puromycin synthesis, a model assay for the first peptide bond formation
A remarkable reduction in eIF5A was observed with Ad-eIF5A-shRNA transduction while no/little decrease of eIF5A was observed in cells transduced with the control scramble shRNA
Summary
Effects of adenoviral eIF5A shRNA transduction on eIF5A level, cell growth, viability and protein synthesis in HeLa cells. At 96 h, 9 (KHDRBS1, EIF5, EIF3A, FASN, IMPDH1, NPEPPS, RBM14, SEC24C, U2AF2) out of 41 decreased proteins and 9 (ZYX, BAG3, CKAP4, EEF1B2, GOLGA3, HSPA5, KARS, PRKCSH, TP53BP1) out of 39 increased proteins contained polyproline motifs (Table 1) Among these polyproline proteins identified by iTRAQ, the percent of increased proteins was higher than that of decreased proteins upon eIF5A depletion at both 72 and 96 h. All of the proteins except BAG3 displayed high or higher fold changes at 120 h (Table S1), indicating intensification of stress conditions with extended deprivation of eIF5A Increased expression of these proteins and certain other proteins in the ‘Translation’ category may reflect compensatory mechanisms of these cells to cope with eIF5A-deficient conditions. The ‘response to ER stress’ is the most popular node with the highest number of edges interconnected to 8 different nodes (Fig. 4B) and with 11 different chaperone proteins i.e. CCT7, BAG3, Gene
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