Roles of eIF2alpha phosphorylation in anti-oxidative stress response and autophagy

Abstract

Endoplasmic reticulum (ER) stress caused by an accumulation of improperly folded proteins can trigger the unfolded protein response (UPR) to restore ER homeostasis. UPR-mediated restoration of ER homeostasis can be achieved by diverse signaling pathways to increase ER protein-folding capacity and reduce misfolded protein accumulation in the ER. The UPR can activate degradation systems such as the autophagy lysosome system and the ubiquitin proteasome system to remove misfolded ER proteins. In response to ER stress, the eukaryotic translation initiation factor 2 alpha subunit (eIF2α) suppresses the translation of general mRNA, to reduce the accumulation of misfolded proteins in the ER. To investigate the role of eIF2α phosphorylation in autophagy induced by ER stress, I used a hepatocyte-specific eIF2α phosphorylation-deficient mouse model. Upon treatment with tunicamycin, an ER stress inducer, deficiency in eIF2α phosphorylation produced an abnormal ER structure and defective mitochondrial dynamics. Interestingly, the deficiency in eIF2α phosphorylation downregulated the expression of autophagy genes but caused a marked accumulation of LC3, an autophagosome marker, and P62/sequestosome-1, a selective autophagy substrate, in ER-stressed hepatocytes. In addition, during ER stress, eIF2α phosphorylation-deficient hepatocytes displayed an accumulation of LC3/P62-positive autophagic vesicles but decreased co-localization between LC3 and LAMP1, which suggests that eIF2α phosphorylation is not necessary for autophagosome formation but is required for autophagosome maturation, including the fusion of the autophagosome with a late endosome or lysosome. eIF2α phosphorylation deficiency disrupts lysosome positioning as well as lysosome functionality which can affect autophagic flux upon ER stress. Importantly, QaSNARE syntaxin 17 (STX17), which is required for autophagosomal fusion with lysosomes, was not co-localized with LC3-positive vesicles in eIF2α phosphorylation-deficient hepatocytes during ER stress. Accordingly, at the late stage of ER stress, the mis-localization of STX17 could be responsible for blocking autophagic flux in eIF2α phosphorylation-deficient hepatocytes. Lastly, I observed that eIF2α phosphorylation was responsible for maintaining the expression of lysosomal genes and autophagosome-lysosome fusion related genes, which are the target genes of the master autophagy regulators TFEB and TFE3, during ER stress. Therefore, TFEB overexpression could restore lysosome functionality and mitochondrial dynamics in eIF2α phosphorylation-deficient primary hepatocytes upon ER stress. In other words, TFEB overexpression promotes autophagosome-lysosome fusion in eIF2α phosphorylation-deficient primary hepatocytes upon ER stress. Collectively, these observations suggest that eIF2α phosphorylation is an important event in modulating autophagy to ameliorate ER stress. Therefore, it is possible that ER stress-mediated liver damage in the eIF2α phosphorylation-deficient mouse model could be caused by the impairment of lysosomal degradation. Alternatively, modulating TFEB activity which stimulates autophagy, lysosomal and mitochondrial biogenesis, lysosomal motility, and SNARE-mediated fusion, is an attractive therapeutic avenue for diseases related to defects in the autophagy-lysosomal pathway. |Reactive oxygen species (ROS) play a significant role in intracellular signaling and regulation, particularly when they are maintained at physiologic levels. However, excess of ROS can cause cell damage and induce cell death. I recently reported that eIF2α phosphorylation protects hepatocytes from oxidative stress and liver fibrosis induced by fructose metabolism. Here, I found that hepatocyte-specific eIF2α phosphorylation-deficient mice have significantly reduced expression of the EGF receptor (EGFR) and altered EGFR-mediated signaling pathways. EGFR-mediated signaling pathways are important for cell proliferation, differentiation, and survival in many tissues and cell types. Therefore, I studied whether the reduced amount of EGFR is responsible for the eIF2α phosphorylation-deficient hepatocytes’ vulnerability to oxidative stress. Reactive oxygen species (ROS) such as hydrogen peroxide and superoxides induce both EGF receptor tyrosine phosphorylation and eIF2α phosphorylation. eIF2α phosphorylation-deficient primary hepatocytes, or EGFR knockdown cells, have decreased ROS scavenging ability compared to normal cells. Therefore, these cells are particularly susceptible to oxidative stress. However, overexpression of EGFR in these eIF2α phosphorylation-deficient primary hepatocytes increased ROS scavenging ability and alleviated ROS-mediated cell death. Therefore, I hypothesize that the reduced EGFR level in eIF2α phosphorylation–deficient hepatocytes is one of critical factors responsible for their susceptibility to oxidative stress.