Investigation Of The Regulation Mechanism Of EIF2α Kinases In Mitochondrial Unfolded Protein Response

Mitochondrial proteins are mainly encoded by the nucleus genome,and a small number of proteins are encoded by mitochondrial DNA,these proteins will transmit signals to the nucleus and the cytoplasm when misfolded proteins accumulating in the mitochondria,leading to activation of a series of chaperones,proteases and other genes to restore protein homeostasis and function,this response is called mitochondrial unfolded protein response(UPR^mt),which plays an important role in the innate immunity against pathogens in metazoan.Until now there are only relatively few studies on UPR^mt in mammals,it remains unclear how accumulation of the misfolded proteins in the mitochondria transmits signals to the outside of mitochondria and induces the activation of nuclear transcriptional programs.The transcription factors ATF4,ATF5 and CHOP are thought to be involved in transcriptional regulation of UPR^mt;meanwhile,the evidence showed that activation of UPR^mt can cause eIF2αphosphorylation and translational blockade.How eIF2αphosphorylation regulates the activation of ATF4,ATF5 and CHOP during UPR^mt?how the accumulation of the misfolded protein in the mitochondria activates eIF2αkinases and phosphorylates eIF2α?Does mammalian UPR^mt have a role in innate immunity similar that UPR^mt in C.elegans?To address these questions,the following research was therefore carried out:Firstly,a rapid and specific induction system of UPR^mt was established in this study.Gamitrinib TPP（G-TPP）is a specific inhibitor of mitochondrial chaperone HSP90,mitochondrial protein was misfolded and UPR^mt was activated with G-TPP treatment,in order to facilitate this study,we firstly screened a series of cell lines and finally found that the human liver cancer cell lines SMMC7721cell line and the male chronic myeloid leukemia HAP1 cell line were responded well to G-TPP treatment,eIF2αphosphorylation and ATF4 protein level were significantly up-regulated,and CHOP was up-regulated expression at both transcription level（P<0.01）and protein level.In this study,we carefully analyzed the response time and intensity in two types of cells with G-TPP treatment.The results showed that eIF2αphosphorylation was induced at 30 min and ATF4 protein level was increased significantly at 1h with G-TPP treatment.This phenomenon still existed when extending treatment time to 9 h and 12 h.It was also found that up-regulation expression of CHOP protein,but there was no difference in the expression of ATF5 at both transcriptional and protein level.In addition,BIP gene,a marker of endoplasmic reticulum stress response was not induced with G-TPP treatment at both transcriptional and protein levels.ATF4 protein level was inhibited with ISRIB treatment（ISR inhibitor）,we constructed eIF2αS51A^+/-heterozygous mice,and prepared MEFs cells treated with G-TPP,the increase of ATF4 was significantly lower than in wild-type MEFs cells at protein level,it is proved that eIF2αphosphorylation is essential for inducing increase of ATF4 protein level.ATF4 knockdown cells were constructed,and found that CHOP was inhibited at transcriptional level,which indicating that the transcriptional expression of CHOP gene is regulated by ATF4.eIF2αphosphorylation cause decrease of cellular overall protein translational level,but the translation of some chaperones and proteases are increase.In order to study change of cellular active translation genes under G-TPP induced UPR^mt,we conducted Ribo-Seq profiling and found that HSPA1B,HSPA1A,HSPA6,HSPA8,DNAJB1 and other heat shock proteins,cytochrome P450enzymes CYP1A1 and CYP1B1,and transcription factor DDIT4 were significantly bond to polyribosomes,indicating that active translation of these genes occurred;meanwhile,ATF4 was up-regulated at translational level and CHOP was up-regulated at both transcriptional and translational level.It was worth noting that expression of ATF5,HSP60,HSPE1,CLPP and LONP1 were not increased at transcriptional and translational level,which is consistent with our previous data.The differentially expressed genes（DEGs）（q value<0.05）related to mitochondrial function were screened for GO enrichment and KEGG pathway enrichment analysis,we found that DEGs were mainly enriched in protein folding,apoptosis,cellular stress response,and cellular homeostasis,lifespan regulation,endoplasmic reticulum protein processing,Alzheimer’s disease,PI3K-Akt and MAPK signaling pathways.We constructed knockdown and knockout cell lines of four eIF2αkinases（PERK,PKR,GCN2 and HRI）through RNA interference and CRISPR-Cas9 gene editing technique,respectively,and confirmed that eIF2αwas phosphorylated in the process of HRI-mediated UPR^mt.In HRI^-/-cell lines,the decline of ROS was alleviated,depolarization of mitochondrial membrane potential was significantly reduced,and apoptosis was aggravated compared to the control group,which indicating that HRI also regulates mitochondria function under the context of UPR^mt.We explored the mechanism of mitochondrial misfolded protein activating HRI and eIF2αphosphorylation.Firstly,we examined mitochondria morphology by electron microscopy,mitochondria morphology was normal with G-TPP treatment for 4 h,however,mitochondria morphology were rounding,swelling,and ridge was disappeared after G-TPP treatment 6 h,endoplasmic reticulum structure is normal,which indicate that G-TPP treatment only specifically effected on mitochondria,Since G-TPP activated UPR^mt occurring within 1 to 2 h,mitochondrial morphology was not significantly abnormality during this period.ROS was not increased with G-TPP treatment.Mitochondrial membrane potential was depolarized with G-TPP treatment,oxidative phosphorylation process and glycolysis process were severely damaged with G-TPP treatment,which suggesting the metabolic rate of mitochondrial oxidized respiratory chain protein is very fast,and short-term inhibition of mitochondrial protein folding will cause a decrease of oxidative phosphorylation.We used MAVS-Turbo ID labeling the outer mitochondrial membrane and confirmed that HRI in the cytoplasm migrated to the vicinity of the outer mitochondrial membrane.HRI-Turbo ID was re-expressed in HAP1 cells of HRI knock outing and showed that sixty-six mitochondrial proteins interacted with HRI.G-TPP induced-UPR^mt can inhibit the translation of mitochondrial protein,such as COX,CS,and TIM23,however,there was no significant difference in the translation of cytoplasmic proteins,such as GOT and LAMP1.The above results indicate that under UPR^mt condition,phosphorylation eIF2αinhibits mainly the translation of mitochondrial proteins,reduces the import of mitochondria proteins to reduce the burden on mitochondria and give enough space and time to restore normal function and protein homeostasis.Base on the mechanism of UPR^mt elucidated in mammal it this study,we preliminarily explored the influence of UPR^mt on virus replication,the results that G-TPP induced UPR^mt inhibited protein synthesis of vesicular stomatitis virus（VSV）,furtherly,we analyzed regulating mechanism of eIF2αkinase on virus replication in SMMC7721 cells with eIF2αkinase genes knock outing or knock downing,respectively.The results of western blot showed that PERK inhibited VSV replication,PKR and GCN2 promoted virus replication,and HRI had no significant effect on virus replication.The preliminary results indicated that UPR^mt involve in the regulation of virus replication.In summary,this study clarified that G-TPP can quickly induce UPR^mt through HRI-eIF2α-ATF4-CHOP signaling pathway.HRI is essential for UPR^mt through phosphorylating eIF2α;HRI migrated to the vicinity of the outer mitochondrial membrane to interact with a variety of proteins during UPR^mt.eIF2αphosphorylation mainly inhibits mitochondrial function.In addition,G-TPP-induced UPR^mt participated in the regulation of VSV replication,which lay the foundation for further elucidating the role of UPR^mt in viral infection,innate immunity and disease treatment.