Proteomic Analysis of eIF5B Silencing-Modulated Proteostasis

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.

Eukaryotic initiation factor 5B (eIF5B) provides a critical cell survival switch to glioblastoma cells via regulation of apoptosis

“…8). Our findings are consistent with a body of work suggesting that eIF5B is involved in regulating hepatocellular carcinoma proliferation and metastasis 40 , pro-growth pathways 41 , central carbon metabolism, and hypoxia adaptation of glioblastoma 19 .

Fig. 8Model for regulation of apoptosis by eIF5B in U343 glioblastoma cells.eIF5B (gray oval) promotes translation of XIAP, Bcl-xL, cIAP1, c-FLIP S , and Nrf2 (green ovals).

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Eukaryotic translation initiation factors as promising targets in cancer therapy

“…The inhibition of eIF4E has been shown to have therapeutic potential for the treatment of several types of cancer [ 154 – 158 ]. Additionally, the overexpression of eIF4E has been found in breast [ 117 ], lung [ 120 ], prostate [ 121 ], colorectal [ 122 ], skin [ 123 ], head and neck [ 124 ], and cervical cancers (Table 1 ) [ 125 ], and elevated expression levels of eIF4E are strongly related to poor prognosis and decreased survival.…”

Therapeutic targeting nudix hydrolase 1 creates a MYC-driven metabolic vulnerability

“…Collected supernatant was quantified by BCA kit, and 100 µg of protein from each sample were used for digestion and TMT labeling. Quantitative proteomics were performed using Deng’s method 39 . Equal amounts of protein were reduced with 5 mM dithiothreitol and alkylated with 13 mM iodoacetamide.…”