Proteomic Analysis of eIF5B Silencing-Modulated Proteostasis

PLoS One. 2016 Dec 13;11(12):e0168387. doi: 10.1371/journal.pone.0168387. eCollection 2016.

Abstract

Protein translational machinery is an important component of the proteostasis network that maintains cellular proteostasis and regulates aging and other cellular processes. Ample evidence indicates that inhibition of translation initiation factor activities enhances stress resistance in model organisms. Eukaryotic translation initiation factor 5B (eIF5B) acts by joining the pre-40S subunit with the 60S ribosomal unit to form an 80S-like complex during protein translational initiation. Reduced eIF5B expression may disrupt proteostasis and trigger cellular processes associated with stress responses. In this study, the physiological effects of altered eIF5B expression were examined in 293T and HepG2 cells. Cells with eIF5B-knockdown (eIF5B-KN) grew more slowly than control cells, and had a lower level of intracellular reactive oxygen species (ROS), increased resistance to oxidative stress and enhanced autophagy. Proteomic analysis showed that eIF5B knockdown resulted in upregulation of 88 proteins and downregulation of 130 proteins compared with control cells. The differentially expressed proteins were associated with diverse cellular processes including amino acid metabolism, RNA processing and protein metabolism, and DNA synthesis. Autonomous downregulation of the mitogen-activated protein kinase (MAPK) signaling pathway was identified as confirmed by western blotting and qPCR. We proposed that deactivation of MAPK pathway modulated proteostasis and induced prolonged S-phase of the cell-cycle, contributing to the slow growth of eIF5B-KN cells. eIF5B silencing also inactivated the mTOR pathway, downregulated glutamine transporters, enhanced autophagy, and decreased 28S rRNA and 5.8S rRNA expression levels which were reversed by restoration of eIF5B expression. Taken together, these results suggest that eIF5B silencing provides a negative feedback to deactivate MAPK signaling, leading to reduced cell growth. These findings provide a useful resource to further biological exploration of the functions of protein synthesis in regulation of proteostasis and stress responses.

MeSH terms

  • Autophagy
  • CRISPR-Cas Systems
  • Cell Cycle
  • Cell Proliferation
  • Eukaryotic Initiation Factors / genetics*
  • Gene Silencing*
  • HEK293 Cells
  • Hep G2 Cells
  • Humans
  • MAP Kinase Signaling System
  • Metabolomics
  • Oxidative Stress
  • Polymerase Chain Reaction
  • Proteomics / methods*
  • Reactive Oxygen Species / metabolism
  • Up-Regulation

Substances

  • Eukaryotic Initiation Factors
  • Reactive Oxygen Species
  • eukaryotic initiation factor-5B

Grant support

This work was supported by the National Natural Science Foundation of China 31270871 (HTD), the Ministry of Education of China 2012Z02293 (HTD) and the Chinese Ministry of Science and Technology (2014CBA02005 and 2014AA020907) and the Global Science Alliance Program of Thermo-Fisher Scientific. The funders had not played a role in study design, data collection and analysis.