TFEB와 TFE3 핵자리옮김에 대한 단백질번역 억제제와 eIF2alpha 인산화 역할 연구

Abstract

Macroautophagy/autophagy is important catabolic process responsible for the degradation of unnecessary or dysfunction cellular components via lysosomal pathway. Autophagy is triggered in diverse stress conditions such as food deprivation, hypoxia, misfolded proteins, damaged organelles, or intracellular pathogens. Therefore, autophagy is essential for balancing energy sources for protein synthesis. Moreover, protein biosynthesis is one of the major metabolic processes, which are crucial for maintaining cellular functions including autophagy. In part 1, using three translation inhibitors with distinct inhibitory mechanisms, I analyzed their effects on the regulation of TFEB/ TFE3 activity and autophagy. Cycloheximide (CHX) is a translation elongation inhibitor, which prevents tRNA translocation by skewing the binding of deacylated tRNA to the E-site. Lactimidomycin (LTM) is another translation elongation inhibitor, which binds to the ribosomal E-site and prevents translocation of the P-site tRNA into the E-site. However, CHX stalls ribosome during ongoing translation, whereas LTM preferentially arrests ribosomes during the very first round of elongation. Lastly, rocaglamide A (RocA) is an eIF4A RNA helicase inhibitor, which inhibits the translation of not only purine-rich 5’ leader containing mRNAs but also normally unresponsive mRNAs via blockade of 43S PI scanning, 43S PIC recruitment block and bystander effect by eIF4F sequestration. In this study, I found that these translation inhibitors enhance TFEB/TFE3 autophagy master regulators nuclear translocation via dephosphorylation and 14-3-3 dissociation and significantly increases autophagy-related genes. Furthermore, I demonstrated that translation inhibition increased autophagosome biogenesis but impaired the degradative autolysosome formation because of lysosomal dysfunction. In addition, these findings suggest a new biological function of translation inhibition in autophagy regulation. In part 2, I reveal the essential role of eIF2α phosphorylation in the nuclear translocation of TFEB and TFE3. Eukaryotic translation of the mRNA molecule consists of 4 stages: initiation, elongation, termination, and recycle. Inhibition at any stages during translation process can lead to either change in the protein structure or their gene expression. However, protein synthesis is mostly and sensitively controlled at the initiation stage rather than elongation or termination stage. Phosphorylation of the α subunit of the translation initiation factor eIF2alpha (eIF2α) at serine 51 mediates translational control and necessary for cell adaptation to cellular stress. Several reports suggest that the eIF2α phosphorylation could play a key role in autophagy regulation. I found that eIF2α phosphorylation-deficient (A/A) cells which have Serine to Alanine mutation at 51st amino acid have defective in autophagy process including autophagosome formation, autophagosome-lysosome fusion and autolysosome formation. These effects mainly caused by defective in TFEB and TFE3 nuclear translocation and reduced their activation in A/A cells during ER stress conditions. Therefore, eIF2α phosphorylation was important for TFEB and TFE3 nuclear translocation under diverse autophagy inducing conditions including ER stress. I also found that TFEB dephosphorylation at both S211 and S142 residues and dissociation from 14-3-3 were not sufficient for its nuclear translocation in A/A cells during ER stress. However, overexpression of the activated ATF6α form was necessary and sufficient to induce both TFEB dephosphorylation and its nuclear translocation in A/A cells during ER stress. Consequently, the activated ATF6α or TFEB forms overexpression could restore impaired autophagic defects in eIF2α phosphorylation-deficient (A/A) cells during ER stress. The data highlight a new mechanism controlling TFEB subcellular localization and activity via an eIF2α phosphorylation-dependent component of UPR signaling pathways under ER stress conditions. Furthermore, the finding revealed that how eIF2α phosphorylation connects the UPR pathways to autophagy. Altogether, this study reveals that translation inhibition by translation inhibitors and eIF2α phosphorylation have an important role in nuclear translocation of TFEB and TFE3. Both translation inhibitors-mediated translation inhibition and eIF2α phosphorylation-mediated translation inhibition may share the TFEB/TFE3 nuclear regulatory mechanism(s) controlling TFEB/TFE3 nuclear translocation and activation.