Unfolded proteins in the mitochondria activate HRI and shut-down of mitochondrial protein translation

Unfolded proteins in the mitochondria activate HRI and shut-down of mitochondrial protein translation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Unfolded proteins in the mitochondria activate HRI and shut-down of mitochondrial protein translation Yongshu Wu, Yang Yang, Xiaodong Qin, Zhixiong Zhang, Munib Ullah, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4121132/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Mitochondrial unfolded protein response (UPR mt ) is triggered through eIF2α phosphorylation in mammal. However, the mechanisms of UPR mt activation and the influence on mitochondrial protein translation through eIF2α phosphorylation remain unclear. In this study, we confirmed that UPR mt was a rapid and specific stress response through eIF2α phosphorylation with pharmacological induction, along with the protein expression of eIF2α phosphorylation, ATF4, and CHOP. Meanwhile, with the up-regulation of some chaperones, cytochrome P450 enzymes, and DDIT4 determined by RNA-Seq and ribosome profiling, eIF2α phosphorylation is essential for expressing ATF4 and CHOP, then ATF4 traffics into the nucleus and initiates CHOP expression. In addition, the generation of ROS and mitochondrial morphology was unchanged under GTPP induced UPR mt . Furthermore, we unraveled the mechanism that HRI kinase mediates UPR mt induced with mitochondrial unfolded proteins by CRISPR-Cas9 technology and mitochondrial recruitment of HRI and interaction with other proteins. Meanwhile, we confirmed that mitochondrial protein translation and the number of mitochondrial protein imports were inhibited through eIF2α phosphorylation with the accumulation of mitochondrial unfolded protein. These findings provide the molecular mechanism of UPR mt and the impact on cellular protein translation, which will offer a novel insights into the functional research of UPR mt , including its implications for human diseases and pathobiology. mitochondrial unfolded protein response eIF2α phosphorylation heme-regulated inhibitor mitochondrial proteostasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction The mitochondria are the network organelles for energy production and metabolism of amino acids and nucleotides [ 1 – 3 ] and serve as the hubs for many signaling processes, such as innate immune response during viral infection [ 4 , 5 ] , apoptosis [ 6 ] , neurodegenerative disease, and ages [ 7 – 9 ] . The mitochondrial protein folding environment is regulated by nuclear-encoded mitochondrial chaperones that enhance protein folding and proteases to degrade proteins that fail to fold or oligomerize correctly. Mitochondrial unfolded protein response is induced when the accumulation of misfolded or unfolded proteins exceeds the capacity of mitochondrial protein folding in the mitochondria, term as UPR mt . UPR mt was discovered first in cultured mammalian cells. However, many genes about UPR mt were well studied and identified in C. elegans [ 10 , 11 ] . UPR mt was regulated by the basic leucine zipper (bZIP) activating transcriptional factor associated with stress-1 (ATFS-1) in C. elegans , which includes both a mitochondrial targeting sequence (MTS) and a nuclear localization sequence (NLS) [ 12 ] . This function is important in mediating the communication between the mitochondria and the nucleus [ 11 ] . Under the physiologic condition, ATFS-1 is imported into the mitochondria and degraded by LON protease. However, the decrease of mitochondria import of ATFS-1 results in the accumulation of ATFS-1 in the cytosol and traffics into the nucleus, along with protein ubiquitin-like5 (UBL5) and DVE-1 under mitochondrial stress conditions, which activate the transcriptional expression of a set of genes encoding proteases, chaperones, and metabolic enzymes to restore mitochondrial function and cellular homeostasis [ 11 , 13 , 14 ] . Hence, we speculate that cells may utilize mitochondrial import efficiency as a mitochondrial normal function signal through ATFS-1, a sensor that detects mitochondria-to-nucleus signaling in C. elegans . Nucleus accumulation of ATFS-1 can activate the transcription of over 500 genes that regulate many cellular activities [ 11 , 14 , 15 ] . However, the mechanism studying of UPR mt is less in mammals. Research has shown that ATF5, as a bZIP transcriptional factor, has the homologous function to ATFS-1, which is regulated by mitochondria import efficiency to restore mitochondria function [ 16 ] . Apart from ATF5, at least ATF4 and C/EBP homologous protein (CHOP) involving in UPR mt [ 16 – 19 ] . However, the role of these transcriptional factors in UPR mt in the mammal is still unclear. The integrated stress response (ISR) is an intricate signaling pathway existing in eukaryotic cells, which is activated through the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) in response to different physiological changes and pathological conditions, the activation of eIF2α phosphorylation results in decreasing of global protein synthesis and induction of selected genes, such as ATF4. ISR is activated by four known eIF2α kinases, which sense different stresses, PERK is activated by the accumulation of misfolded or unfolded protein in the endoplasmic reticulum (ER), PKR senses double-stranded RNA in the cytosol, and heme-regulated inhibitor (HRI) is induced by heme depletion [ 20 ] , GCN2 responds to amino acid starvation, as well as mitochondria stress [ 21 ] . Activated kinases phosphorylate eIF2α, which reduces overall protein synthesis and preferential expression of mRNAs that include small upstream open reading frames (uORFs) in the 5' untranslated region (UTR) to restore cellular homeostasis. Previous studies showed that UPR mt is triggered through the expression of eIF2α phosphorylation and ATF4 in mammalian cells [ 17 , 22 ] . In addition, the study has demonstrated that ATF4, ATF5, and CHOP are required for UPR mt[ 19 , 23 ] . However, the role of eIF2α phosphorylation in UPR mt and its influence on mitochondria protein translation need further illumination. Whether four eIF2α kinases are involved in the activation of UPR mt remains unclear. To shed light on the underlying functional mechanism of UPR mt , Gamitrinib-triphenylphosphonium (G-TPP) was used in this study, which is a mitochondrial-targeted TNF receptor-associated protein-1 (TRAP1) of HSP90 family inhibitors and chemically interfere with mitochondrial protein folding [ 24 , 25 ] .We elucidated the role of eIF2α kinases in UPR mt through CRISPR-Cas9 technology. In this study, we confirmed that UPR mt was a rapid and specific stress response with pharmacological induction, along with the up-expression of eIF2α phosphorylation, ATF4, and CHOP. Meanwhile, some chaperones, cytochrome P450 enzymes, and DDIT4 were up-regulated and confirmed by RNA-Seq and ribosome profiling. The activation of eIF2α phosphorylation is essential for the expression of ATF4 and CHOP, and ATF4 is trafficked into the nucleus and initiates the expression of CHOP. Furtherly, we unraveled the mechanism that HRI-mediated G-TPP induced UPR mt through the HRI-eIF2α-p-ATF4-CHOP signaling pathway, and HRI interacted with many mitochondrial proteins in the vicinity of mitochondria. G-TPP-induced UPR mt blocked mitochondrial protein translation and the number of mitochondrial proteins imported while not influencing ROS generation and mitochondria morphology change through eIF2α phosphorylation. Materials and methods Cell culture and experimental animals. SMMC7721 cells, HAP1 cells, and HEK-293 T cells were preserved in our laboratory. SMMC7721 cells and HEK-293 T cells were cultured in DMEM basic (1×) (Gibco, C14190500BT) supplemented with 10% fetal bovine serum (Gibco, 10099-141) and 1% penicillin and streptomycin (Gibco, 15140-122) in an incubator (Binder, CB160) containing 5% CO 2 at 37°C. HAP1 cells were maintained in IMDM modified medium (HyClone, SH30228-01). Cells were digested using 0.25% Trypsin-EDTA (Gibco, 25200056). eIF2α S51A heterozygous mice were purchased from Jackson Laboratory, USA, strain: B6; 129-Eif2s1tm1Rjk/J, 017601. The tail tissues of WT mice and offspring of eIF2α S51A heterozygous mice were collected and DNA were extracted, and then were screened and identified by PCR, the primer sequences: Forward: 5'-ACACCCATTCCATGATAGTAAAATG-3', Reverse: GTTGTAGACCCTGACAATGAAGG-3'); MEFs cells were cultured in DMEM basic (1×). MEFs preparation. Fetal rats of 13 ~ 15 days were anesthetized by intraperitoneally injecting 0.5 mL of 2.5% avertin, then euthanized by cervical dislocation. The fetal rat was placed on a paper towel and sprayed enough 75% ethanol to soak the entire mouse body. Transferred fetal rat into biological safety cabinet, cut skin and tissues to expose uterus, removed embryos with a scalpel in 10 cm dish containing PBS, discarded all other tissues, such as the head and visceral tissues. The remaining part of the embryos was washed with PBS 4 times and placed in a clean 10 cm dish and cut embryos into 2 mm pieces with a new scalpel. Added 0.25% trypsin and incubated in 37°C incubators for 10 min. DMEM containing 10% FBS was added to resuspend cells, inoculated into T75 cell flasks, cultured in an incubator containing 5% CO 2 , discarded non-adherent cells, and replaced culture medium to continue culturing. Generation of ATF4-KD and eIF2α kinases-KO cell lines construction. ATF4 knockdown cells (ATF4-KD) were constructed by shRNA, and targeting sequences of ATF4 was available from the website ( https://www.sigmaaldrich.cn/CN/zh ). Firstly, primers were synthesized by Sangon Biotech (Shanghai, China) and cloned into pLKO.1 vector (Addgene, 10878) by digestion and ligation, shRNA sequences for ATF4 were shown in the following: ATF4 : 5'-GCCTAGGTCTCTTAGATGATT-3'. The eIF2α kinases (PERK, PKR, GCN2, and HRI) knockout cell lines (eIF2α kinases-KO) were manufactured using the CRISPR-Cas9 technique. sgRNA were designed using sgRNA Designer from Zhang's lab. The sgRNAs were synthesized and cloned into the Lenti-CRISPR v2 vector (Addgene, 52961) by ligation. sgRNA sequences of eIF2α kinases as follows: PERK : 5'-GTAATTATCAGCACTTTAGA-3'; PKR-1 : 5'-GTACTACTCCCTGCTTCTGA-3', PKR-2 : 5'-GATTATGAACAGTGTGCATCG-3'; GCN2-1 : 5'-CCTCCGGAGAGCTACCCGCAACG-3', GCN2-2 : 5'-TATATGTAAAAGTGGATTTG-3'; HRI-1 : 5'-CCGAGGGCCCGGACCCCGAA-3', HRI-2 : 5'-AAGGGAAGGTTGGCTGTTGT-3'. For lentivirus packing and infection, 4 µg recombinant lentiviral plasmid was co-transfected with 2 µg packaging plasmid psPAX2 (Addgene, 12260) and 1 µg envelope plasmid pMD2.G (Addgene, 12259) into Lenti-™ 293 T cells using Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher Scientific, L3000015). Infectious particles were harvested from 48 h to 96 h after transfection, centrifugated at 4000 rpm/min for 30 min, and concentrated with a final concentration of 20% PEG-8000, then concentrated lentiviruses were added into SMMC7721 or HAP1 cell lines, respectively, cells were selected by adding 250 µg/mL hygromycin (Invitrogen, 10687010) or 5 µg/mL puromycin (Invitrogen, A1113803). For KO cell lines, positive clones were isolated through limiting dilution assay, and knockdown and knockout efficiency were determined by Western blot. Construction of HAP1-sgHRI-HRI-TurboID cell line. The pEGFP-N1-HRI-TurboID plasmid was constructed by inserting the CDs sequence of HRI into the 5' edge of the TurboID cDNA sequence. To select positive clones against hygromycin, a homologous arms assay was constructed with a pSMPUW-Hygro vector, and lentiviral packaging was performed. Packaged lentiviruses were added into HAP1-HRI −/− cells and selected by 250 µg/mL hygromycin (Invitrogen, 10687010). The positive clones were determined by Western blot. Western blot. Cells were washed with cold PBS twice, and ultrasonic protein was extracted on ice in protease and phosphatase inhibitor (Thermo Fisher Scientific, A32959) in RIPA buffer (Beyotime, P0013B) with 1% PMSF (Thermo Fisher, 36978). Lysates were centrifuged at 12000 rpm/min for 10 min. Supernatants were collected and denatured at 95°C for 10 min in a 4 × loading buffer. Equal amounts of protein were resolved on 10% electrophoresis acrylamide gel (Bio-Rad, 1610173), transferred to PVDF membranes (Millipore, ISEQ00010), and blocked in 5% nonfat milk (BD, 232100) in TBS-T (50 mM Tris (pH 7.4), 150 mM NaCl, 0.1% Tween20 (Sigma, P7949-500ML) for 2 h at room temperature (RT). In TBS-T, membranes were incubated with primary antibodies overnight at 4°C in 5% bovine serum albumin (BSA)(Millipore, 9048-46-8). Primary antibodies were used in this study as follows: β-tubulin antibody (66240-1-Ig; 1:5000); EIF2A antibody (11170-1-AP; 1:2000); ATF4 antibody (10835-1-AP; 1:1000); LONP1 antibody (15698-1-AP; 1:1000); CLPP antibody (15440-1-AP; 1:1000); HRI antibody (20499-1-AP; 1:1000); TIM23 antibody (11123-1-AP; 1:1000); GOT1 antibody (14886-1-AP; 1:1000); GOT2 antibody (14800-1-AP; 1:1000) were purchased from Proteintech; PERK antibody (3192s; 1:1000); CHOP antibody (D46F1; 1:1000); HSP60 antibody (D6F1; 1:1000); GAPDH antibody (5174; 1:1000); Histone H3 antibody (4499; 1:5000); BIP antibody (3183S; 1:1000); GCN2 antibody (3302S; 1:1000); COX IV antibody (3E11; 1:1000); COX IV antibody (4844; 1:1000); CS antibody (14309; 1:1000); LAMP1 antibody (9091; 1:1000); Streptavidin-HRP antibody (3999s; 1:1000) were purchased from CST; ATF5 antibody (Ab184923; 1:2000); PKR antibody (Ab32506; 1:1000); Phospho-EIF2A antibody (Ser51) (Ab32157; 1:2000) were purchased from Abcam; HSPE1 antibody (Thermo Fisher, PA5-79415; 1:2000); Flag antibody (Sigma-Aldrich, F1804; 1:1000). Membranes were washed with TBS-T three times, 10 min each time. Membranes were incubated in appropriate HRP-conjugated secondary antibodies (Bio-Rad, 170–6515; 1:5000 and 170–6516; 1:5000) at 1:10,000 dilution in 5% milk in TBS-T for 2 h at RT. Membranes were washed three times with TBS-T, 10 min each time. Membranes were detected using standard chemiluminescence with ECL (Affinity, KF003) and imaged by GE Healthcare Amersham™ Imager 600 in an automatic exposure model to ensure that bands are not saturated. Band intensity was determined using ImageJ software (NIH) and adobe photoshop cc software (Adobe). At least 3 independent experiments were performed. Quantitative Real-Time PCR (RT-qPCR). Total RNA was extracted using RNeasy Plus Universal Mini Kit (50) (QIAGEN, 73404). According to the manufacturer's directions, RNA was converted to cDNA using the ReverTra Ace qPCR RT Master Mix (TOYOBO, FSQ-301). Each reaction includes 2 µL 4×DN Master Mix ( with gDNA Remover), RNA template 0.2 µg, and nuclease-free water 5 µL, 37°C incubated 5 min, and then added 5×RT Master Mix II 2 µL, 37°C 15 min, 50°C 5 min, 98°C 5 min. RT-qPCR was performed using Blaze TaqTM SYBR Green qPCR Mix2.0 kit (Gene Copoeia, QP043). Each reaction of qPCR contains 2×SYBR Green PCR Master Mix 10 µL, forward primer 0.7 µM, reverse primer 0.7 µM, RNase-free water 5.2 µL, template cDNA 2 µL. PCR initial heat activation 95°C 2 min, 95°C, denaturation 5 s, 60°C, extension 10 s, 40 cycles. The primer used in qPCR as following: ACTIN (Human) Forward: 5'-GGACCTGACTGACTACCTCAT-3', Reverse: CGTAGCACAGCTTCTCCTTAAT-3'); LONP1 (Human) (Forward: 5'-TCAATGTCACCCGCAACTAC-3', Reverse: 5'-GAACTCCAGGATGCGTTTCT-3'); CLPP (Human) (Forward: 5'-CTCTTCCTGCAATCCGAGAG-3', Reverse: 5'-GGATGTACTGCATCGTGTCG-3'); HSP60 (Human) (Forward: 5'-TCCAGGGTTTGGTGACAATAG-3', Reverse: 5'-GATTCAGGGTCAATCCCTCTTC-3'); HSPE1 (Human) (Forward: 5'-GCTGCTGAAACTGTAACCAAAG-3', Reverse: 5'-CTCCACCCTTTCCTTTAGAACC-3'); CHOP (Human) (Forward: 5'-GTCTAAGGCACTGAGCGTATC-3', Reverse: 5'-CAGGTGTGGTGATGTATGAAGA-3'); ATF5 (Human) (Forward: 5'-CCCACCTGACCTGGAAGC-3', Reverse: 5'-CCTCGTTGCGGCAGTAGAT-3'); ATF4 (Human) (Forward: 5'-GGAGATAGGAAGCCAGACTACA-3', Reverse: 5'-GGCTCATACAGATGCCACTATC-3'); BIP (Human) (Forward: 5'-AACCATCCCGTGGCATAAA-3', Reverse: 5' -GGACATACATCAAGCAGTACCA-3'’). The sample was analyzed in three biological replicates, and the relative expression values were obtained using the 2 −ΔΔCt method . ROS detection. ROS was detected using DCFDA cellular ROS detection assay kit (Abcam, ab113851). The protocol was as follows: HAP1 cells were harvested and seeded in a dark 96-well assay plate (black with clear flat bottom), allowing cells to be attached overnight. Cells were washed once with 1×Buffer. Cells were stained with 25 µM DCFDA in 1×Buffer for 45 min at 37°C, washed once in 1 × Buffer, and treated with GTPP (10 µM), ISRIB (200 nM), and GTPP + ISRIB, DMSO as a negative control, 55 µM TBHP (Tert-Butyl Hydrogen peroxide) as the positive control. Read signals at Ex/Em: 485/535 nm by PerkinElmer VICTOR Nivo multimode plate reader. Transmission electron microscopy. HAP1 cells were seeded into a 60 mm dish and treated with GTPP (10 µM) for 2 and 4 h when cells were confluence about 80 ~ 90%, with DMSO as the negative control. Method for ultrastructure analysis of mitochondria was performed according to the protocol: fixation: cells were collected and fixed with 2.5% glutaraldehyde at 4°C for 2 h, rinsed with 0.1 mol/l phosphoric acid rinsing solution three times, 15 min each time, fixed with 1% osmium acid for 2 h, rinsed with 0.1mol/L phosphoric acid rinsing solution 3 times, 15 min. Dehydration: dehydrated with 50% of ethanol dehydration for 15 min, dehydrated with different concentrations of ethanol dehydration for 15 min, 70, 90, and 95%, respectively, the above septs were conducted in a fume cupboard, dehydrated with 100% acetone 3 times at RT, 15 min each time. Embedding: cells were embedded with pure acetone along with an embedding solution (2:1) at 37°C for 3 h, embedded with pure acetone along with an embedding solution (1:2) at 37°C for 3 h, and placed in pure embedding solution at 45°C for more than 2 h, and then converted to 60°C. Solidify: cells were placed in a drying oven at 45°C for more than 3 h and at 60°C for 24 h. Sliced by Lycra UC7 ultra-thin microtome to 50–60 nm. Double stained with 3% uranyl acetate-lead citrate. Cellular ultrastructure was observed and filmed with a JEOL JEM-1400 transmission electron microscope. RNA-Seq. Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample were quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA) with a RIN number > 7.0. The final cDNA libraries were constructed using TruSeq® Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA). The 2×150 bp paired-end sequencing (PE150) was performed on an Illumina Novaseq™ 6000 (LC-Biotechnology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol. Analysis of RNA data. Fastp software ( https://github.com/OpenGene/fastp ) was used to remove the reads that contained adaptor contamination, low-quality bases (Q ≤ 20, more than 50%), and undetermined bases (more than 10%). Then, sequence quality was also verified using fastp. HISAT2 ( https://ccb.jhu.edu/software/hisat2 ) was used to map reads to the reference genome of Homo sapiens Ensembl_release101. The mapped reads of each sample were assembled using StringTie ( https://ccb.jhu.edu/software/stringtie ) with default parameters. Then, all transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using gffcompare ( https://github.com/gpertea/gffcompare/ ). RSEM was used to perform gene expression levels by calculating fragments per kilobase million (FPKM). The differentially expressed genes were selected with |log2 Fold Change| > 1 and FDR < 0.05 by R package DESeq2 ( http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html ). Ribo-Seq. Cycloheximide was added to the cell medium to a final 100 µg/ml concentration to block translation. The resuspended extracts in lysis buffer were transferred into a clean microtube, pipetted several times, and incubated on ice for 10 min. Then cells were triturated 10 times through a 26-G needle. The lysate was centrifuged at 20000 g/min for 10 min at 4°C, and the supernatant was collected. To prepare ribosome footprints (RFs), 7.5 µL of RNase I and 5 µL of DNase I was added to 300 µL of lysate to incubate for 45 min at RT with gentle mixing on a nutator mixer. Nuclease digestion was stopped by adding 10 µL of SUPERase·In RNase inhibitor. Size exclusion columns (Illustra MicroSpin S-400 HR Columns; GE Healthcare; catalog no. 27-5140-01) were equilibrated with 3 ml of polysome buffer by gravity flow and centrifuged at 600 g/min for 4 min at RT. 100 µL of digested RFs were added to the column and centrifuged at 600 g/min for 2 min. Next, 10 µL 10% (wt/vol) SDS was added to the elution, and RFs with a size greater than 17nt were isolated according to the RNA Clean and Concentrator-25 kit (Zymo Research; R1017). rRNA was removed using DNA probes complementary to rRNA sequences. Then RNase H and DNase I were used to digest the probes. RFs were purified using magnet beads (Vazyme). Ribo-seq libraries were constructed using NEBNext® Multiple Small RNA Library Prep Set for Illumina® (catalog no. E7300S, E7300L) after obtaining the ribosome footprints above. PCR products were sequenced using Illumina HiSeq™ 2500 (LC-Biotechnology CO., Ltd., Hangzhou, China). Analysis of Ribo-seq data. Raw reads containing over 50% of low-quality bases or over 10% of N bases were removed. Adapter sequences were trimmed. Reads with lengths between 10 ~ 50 bp were retained for subsequent analysis. Bowtie2 was used for mapping reads to the ribosome RNA (rRNA) database. The rRNA removed reads of each sample were mapped to the reference genome by Bowtie2, allowing no mismatches. Reads number in the open reading frame of coding genes was calculated by the software RiboTaper, and the gene expression level was normalized using the FPKM method, the same as the RNA-Seq. To identify differentially translated genes across sample groups, the edgeR package ( http://www.rproject.org/ ) was used. Genes with |log2 Fold Change| > 1 and FDR < 0.05 in comparison were considered as significant DTGs. DTGs were then subjected to enrichment analysis of GO functions and KEGG pathways. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Cells were treated with G-TPP (10 µM) for 2 and 4 h, respectively, equal volume DMSO was added as the control, and 500 mmol/L Biotin (Sigma, V900418-1G) was added for 30 min before finishing treatment time. The samples were performed by Immunoprecipitation. IP samples were separated by SDS-PAGE, and a gel lane was cut for LC/MS/MS. Briefly, the samples were digested, desalted, and analyzed by Q Exactive high-resolution mass spectrometer (Thermo Scientific) accompanied by Easy-nLC 1200 (Thermo Scientific). Proteome Discover 2.5 software was used to map reads to the reference genome of UniProt-Human and analyze the data. Immunoprecipitation. To investigate the relationship between HRI and MAVS, the pEGFP-N1-MAVS-TurboID plasmid was constructed by inserting the CD sequence of MAVS into the 5' edge of the TurboID cDNA sequence. The pEGFP-N1-MAVS-TurboID plasmid was transfected into HEK 293T cells using Lipofectamine 3000 reagent for 36 h, and cells were treated with G-TPP for 4 h, equal volume DMSO as the control, and 500 mmol/L Biotin (Sigma, V900418-1G) was added for 30 min before finishing treatment time. Rinsed with pre-cooled PBS once, added 400 µL RAPA lysis solution, placed on ice for 30 min to absolutely dissolve, sonicated with cell sonicator for 1 min, 12000 rpm/min, centrifuged for 10 min, and 80 µL of lysate were transferred into a clean 1.5 mL centrifuge tube, and 5×loading buffer was added, heated at 98°C, 10 min, as Input samples. 50 µL of Hydrophilic Streptavidin Magnetic beads (NEB, S1421S) were washed with 500 µL lysate buffer, discarded supernatant, and beads were added into remaining cell lysate, 18 rpm/min, 4°C overnight. The cell lysate was washed 3 times, 500 µL each time and 80 µL of lysate buffer were added into 5×loading buffer, 98°C heated for 10 min. Input samples and IP samples were performed with Western blot. Cytoplasmic and nuclear extraction test. HAP1 cells were seeded into a 60 mm dish, and cells were treated with G-TPP (10 µM) for 2 h when cellular confluence was up to 80% and an equal volume of DMSO as a control. Cells were washed twice with pre-cooled PBS after treatment and discarded PBS. The extraction assay was performed according to MinuteTM Cytoplasmic and Nuclear Extraction Kit (Invent Biotechnologies, Inc., SC-003). 500 µL of cytoplasmic extraction buffer were added, placed on ice for 5 min, then transferred into a pre-cooled 1.5 mL microtube, vortexed vigorously for 15 s, and centrifuged for 5 min at top speed in a microcentrifuge at 4°C. The supernatant (cytosol fraction) was transferred into a fresh pre-chilled 1.5 mL microtube. Appropriate amounts of nuclear extraction buffer were added to the pellets, vortexed vigorously for 15 s, incubated microtube on ice for 1 min, and repeated this step 4 times. Immediately transferred the nuclear extract into a pre-chilled filter cartridge with a collection microtube and centrifuged at top speed (14,000 ~ 16,000 rpm/min) in a microcentrifuge for 30 s at 4°C. Discarded filter cartridge, Added 5×loading buffer in the cytoplasm and the nucleoplasm components, heated at 95°C, 10 min to denature protein, and then performed by Western blot. Drugs treatment. G-TPP (synthesized from Chengdu Ruizhi Chemical Research Co. LTD) is dissolved in DMSO, and the final concentration is 10 µM. ISRIB (Med Chem Express) dissolves in DMSO, and the final concentration is 200 nM. Statistical analysis. Data were expressed as mean ± standard deviation (SD). The significance of the variability between different treatment groups was analyzed by a two-way analysis of variance (ANOVA) test via GraphPad Prism software (version 8.0.2.). P < 0.05 was considered statistically significant. The pictures of Western blot were imaged by both Image J software and photoshop cc software. Results G-TPP induces UPR mt via rapid and specific activation of ISR. To understand the mechanism of UPR mt with G-TPP treatment, the expression of eIF2α phosphorylation, ATF4, ATF5, and CHOP were detected in different cell lines and primary cells with G-TPP treatment at a concentration of 10 µM. The results showed that the human liver cancer cell lines (SMMC7721 cell lines) and the male chronic myeloid leukemia (HAP1 cell lines) responded well to G-TPP treatment, eIF2α phosphorylation, and the protein expression of ATF4 was significantly up-regulated. Hence, both SMMC7721 and HAP1 cell lines were used for subsequent experiments. SMMC7721 and HAP1 cells were treated with G-TPP for 2 and 4 h, respectively. The results showed that eIF2α phosphorylation and ATF4 protein were significantly up-regulated but ATF5 expression was not changed (Fig. 1 A and Extended Data Fig. 1 A). The same results were obtained in MEF cells with G-TPP treatment for 2 and 4 h (Extended Data Fig. 1 B). Furthermore, HAP1 cells were treated with G-TPP for 5, 10, 30 min, and 1 h. The Western blot showed that eIF2α phosphorylation and up-expression of ATF4 occurred at about 30 min and 1 h, respectively. However, the expression of ATF5 was still unchanged (Fig. 1 B). This phenomenon also occurred in SMMC7721 cell lines (Extended Data Fig. 1 C). It suggests that UPR mt is an acute activation process through eIF2α phosphorylation with G-TPP treatment. However, the transcriptional expression of ATF4 was not up-regulated in both cell lines (Fig. 1 C and Extended Data Fig. 1 D), indicating that the ATF4 protein expression-dependent model is activated upon G-TPP-induced UPR mt . The transcriptional expression of ATF5 was also not influenced in the SMMC7721 cell line (Extended Data Fig. 1 E). We furtherly wonder what the role of CHOP in G-TPP-induced UPR mt is. The results showed that CHOP was significantly activated with G-TPP treatment at the transcriptional level ( P < 0.001) and protein level in HAP1 cell lines (Fig. 1 .D-E), suggesting that CHOP is also a general activation transcriptional factor in response to G-TPP-induced UPR mt . We investigated the expression of mitochondrial stress proteins encoded by nuclear genes with G-TPP treatment. However, the expression of HSP60, HSPE1, LONP1, and CLPP was not changed at protein and transcriptional levels with G-TPP treatment (Extended Data Fig. 1 .F-G). These results demonstrate that GTPP can induce UPR mt rapidly through the expression of eIF2α phosphorylation, ATF4, and CHOP. Meanwhile, the expression of BIP, a UPR ER marker protein was not activated at transcriptional and protein levels (Extended Data Fig. 1 .H-I), indicating that a mitochondrial special UPR mt is activated through eIF2α phosphorylation. Furtherly, HAP1 cells were treated with G-TPP for 4 h and sequenced by the RNA-seq and Ribosome profiling, with DMSO as a control. The data showed that the overall trend of differentially expressed genes (DEGs) differed significantly (|log2FC|>1 and FDR < 0.05) with G-TPP treatment compared to the control group at transcriptional and translational levels (Fig. 1 .F). Furthermore, the genes in our study and some genes with DEGs were analyzed, and CHOP expression was up-regulated significantly with G-TPP treatment at both levels compared with DMSO treatment. The expression of ATF4 was nearly twice as higher as that of the control group by Ribo-Seq, although there was no significant difference at the transcriptional level, which is consistent with the results verified in our study. However, at both levels, there was no significant difference in the expression of ATF5, HSP60, HSPE1, CLPP, and LONP1. Interestingly, we found that some DEPs were significantly up-regulated, such as HSPs: HSPA1B, HSPA1A, HSPA6, HSPA8 and DNAJB1, cytochrome P450 enzymes (CYP1A1 and CYP1B1), transcriptional factor DDIT4 (Fig. 1 .G). UPR mt is a rapid and specific stress response expressing eIF2α phosphorylation, ATF4, and CHOP. Extended Data Fig. 1 . G-TPP induce ISR rapidly and specifically . A-C , western blot analysis of eIF2α-P, eIF2α, ATF4, ATF5 and CHOP level treated with DMSO and G-TPP for indicated timepoint in SMMC7721 cells and MEFs cells, respectively. D-E , qPCR of ATF4 and ATF5 mRNA in SMMC7721 cells with or without G-TPP treatment (mean of levels relative to untreated ± s.d.; n = 3 biological replicates). F , western blot analysis of LONP1, CLPP, HSPE1 and HSP60 level upon treatment of HAP1 cells with DMSO and G-TPP for 2 h and 4 h. G , qPCR of LONP1, CLPP, HSPE1 and HSP60 mRNA in HAP1 cells with or without G-TPP treatment (mean of levels relative to untreated ± s.d. ; n = 3 biological replicates). H , western blot analysis of eIF2α-P, eIF2α, ATF4 and BIP level treatment of HAP1 cells with DMSO and G-TPP for indicated timepoint. I , qPCR of BIP mRNA in HAP1 cells with or without G-TPP treatment (mean of levels relative to untreated ± s.d.; n = 3 biological replicates). UPR mt -induced eIF2α phosphorylation is necessary for the expression of ATF4 and CHOP. eIF2α phosphorylation, ATF4, and CHOP was induced with G-TPP-induced UPR mt in our study. Hence, we investigated the relationship between each other. HAP1 cells were treated with ISRIB (ISR inhibitor) for 2 h. The results showed that eIF2α phosphorylation was not inhibited while the protein expression of ATF4 and CHOP were completely decreased (Fig. 2 A). Meanwhile, the transcriptional expression of CHOP was distinctly downregulated with G-TPP treatment compared to the DMSO group ( P < 0.0001) (Fig. 2 B). These results indicate that ISRIB blocks the effects downstream of eIF2α phosphorylation. Furtherly, eIF2α S51A +/− MEFs cells and wild-type (WT) cells were treated with G-TPP for 4 h and DMSO as a control. The results showed that eIF2α phosphorylation was induced in both MEFs WT cells and eIF2α S51A +/− cells, while the protein expression of ATF4 and CHOP in eIF2α S51A +/− cells was significantly lower than that of WT cells (Fig. 2 C). Overall, eIF2α phosphorylation is essential for ATF4 and CHOP expression induction. To identify whether the ATFS-1 homologous gene exists in mammalian cells, we divided cells into the cytosolic fraction and the nuclear fraction after the G-TPP treatment for 2 h. The Western blot results showed that it was ATF4 trafficked into the nucleus under UPR mt (Fig. 2 D), suggesting that ATF4 may play a key role in mito-nuclear communication upon UPR mt . To identify whether the expression of CHOP is controlled by ATF4, ATF4 knockdown cells were performed in HAP1 cells, and knockdown efficiency was determined at transcriptional and protein levels, respectively (Fig. 2 .E-F). eIF2α phosphorylation was increased in both ATF4 knockdown cells and control cells, and ATF4 protein expression was inhibited in ATF4 knockdown cells compared to control cells (Fig. 2 G). Protein expression of CHOP was not obtained in ATF4 knockdown cells. Meanwhile, CHOP's transcriptional expression was abolished in ATF4 knockdown cells compared to control cells (Fig. 2 H). These results indicate that eIF2α is upstream of ATF4 and CHOP, activates ATF4 traffics into the nucleus, and initiates CHOP expression. ROS generation and mitochondrial morphology was unchanged by G-TPP induced UPR mt . We identified that UPR mt was activated through eIF2α phosphorylation in our study. Hence, we investigated the influence of mitochondrial function and morphology upon UPR mt through eIF2α phosphorylation. HAP1 cell lines were treated with G-TPP at indicated timepoint, DMSO as a negative control, and TBHP as a positive control. The results showed that the generation of reactive oxygen species (ROS) was not increased with G-TPP treatment compared to the control cells (Fig. 3 A). In addition, mitochondrial morphology through a transmission electron microscope (TEM) with G-TPP treatment for 2 and 4 h in HAP1 cells. The results showed that mitochondria morphology was normal, and elliptical, with a clear bilayer membrane structure and clear ridge structure with G-TPP treatment compared to the DMSO group (Fig. 3 B-C). Meanwhile, the morphology of ER was not changed with G-TPP treatment (Fig. 3 D). Altogether, activated UPR mt does not affect mitochondrial morphology and ROS generation. HRI mediates UPR mt induced by mitochondrial unfolded proteins. We demonstrated that UPR mt was induced through eIF2α phosphorylation. To identify the key kinase for activation of UPR mt , four known eIF2α kinases were knocked out individually in both HAP1 cell lines and SMMC7721 cell lines using a CRISPR-Cas9 gene-editing system with single-guided RNAs. Knockout efficiency was confirmed at the protein level (Fig. 4 A-D and Extended Data Fig. 4 A-D). In both SMMC7721 and HAP1 cell lines deficient in PERK, PKR, and GCN2, respectively, the protein expression of ATF4 and eIF2α phosphorylation was moderately decreased compared to WT cells, especially the downregulation of ATF4. However, ATF4 protein and eIF2α phosphorylation were inhibited in HRI −/− cells (Fig. 4 E-H and Extended Data Fig. 4 E-H). The transcriptional expression of CHOP was significantly increased in HAP1 cell lines deficient in PERK, PKR, and GCN2 ( P < 0.01, P < 0.001, P < 0.001), respectively. However, the transcriptional expression of CHOP was completely inhibited in HAP1 cell lines deficient in HRI (Fig. 4 I-L). The same results were confirmed in SMMC7721 cell lines. The transcriptional expression of CHOP was significantly increased in knockout cells of PERK and PKR ( P < 0.05, P < 0.001), respectively, excepting GCN2 −/− and HRI −/− cells (Extended Data Fig. 4 I-L). These results demonstrate that HRI mediates UPR mt with the accumulation of mitochondrial unfolded proteins. Extended data Fig. 4 . HRI mediate ISR induced by mitochondrial unfolded proteins . A-D , western blot analysis of knockout efficiency of PERK, PKR, GCN2 and HRI in SMMC7721 cells, respectively. E-F , western blot analysis of eIF2α-P, eIF2α, ATF4 and CHOP level treated with DMSO and G-TPP for 2 h in SMMC7721 wild-type or KO cells of PERK, GCN2, PKR and HRI, respectively. I-L , qPCR of CHOP mRNA with or without G-TPP treatment in SMMC7721 wild-type or KO cells of PERK, GCN2, PKR and HRI, respectively (mean of levels relative to untreated ± s.d.; n = 3 biological replicates). Reduced mitochondrial protein translation is specifically induced by mitochondrial unfolded protein. eIF2α phosphorylation inhibits global protein translation, and we suppose that mitochondrial protein translation rather than cytoplasmic protein translation is mainly suppressed to alleviate the mitochondrial load. Cells were divided into the cytoplasm fraction and the nuclear fraction after G-TPP treatment for 4 h in HAP1 cells. The Western blot results showed that the expression of cytochrome-c oxidase subunit IV (COX IV), a subunit of the electron transport chain (ETC) located within the inner membrane, citrate synthase (CS), translocase of the inner membrane channel subunit 23 (TIM23), the major channel of the TIM complex proteins was decreased obviously, and the number of mitochondria proteins input were also decreased significantly with GTPP treatment compared to DMSO treatment. Lysosome-associated membrane protein1 (LAMP1), glutamic-oxaloacetate transaminase1 (GOT1), and GOT2 belong to cytoplasmic proteins. The quantity of LAMP1, GOT1, and GOT2 in the cytosol was not decreased significantly with G-TPP, G-TPP + ISRIB, respectively, compared to DMSO treatment (Fig. 5 ). These results confirmed our speculation that G-TPP-induced UPR mt causes the translation decrease of mitochondrial proteins and the number of mitochondria proteins imported, while there is no significant effect on the translation of cytoplasmic protein. A pro-survival stress response will be induced by inhibiting mitochondrial protein translation and the number of mitochondria proteins imported to relieve mitochondrial load and promote mitochondrial proteostasis recovery upon UPR mt . Mitochondrial recruitment of HRI and interaction with many mitochondrial proteins upon G-TPP inducedUPR mt . In this study, we confirmed that HRI was involved in G-TPP-induced UPR mt . Hence, we want to know how HRI initiates this process, and we suppose that HRI in the cytosol may migrate to the vicinity of mitochondria and interact with relative proteins. Hence, an EGFP-MAVS-TurboID plasmid was constructed, which contains TurboID proximity labeling. The immunoprecipitant results showed that only HRI was captured nearby of MAVS compared to the other eIF2α kinases, PERK, PKR, and GCN2 (Fig. 6 A). These results suggest that HRI in the cytosol migrates to mitochondrial out member and initiates downstream signal pathway during G-TPP induced UPR mt . Furtherly, we speculate that HRI may play with many proteins to initiate UPR mt . Hence, recovering the expression of HRI in HAP1 cell lines deficient in HRI was constructed, containing TurboID proximity labeling (HAP1-sgHRI-HRI-TurboID), and the HRI protein expression was confirmed by Western blot (Fig. 6 B). HAP1-sgHRI-HRI-TurboID cell lines were treated with G-TPP for 2 and 4 h, respectively, and IP and LC-MS/MS were performed. The results showed that 30 proteins with DMSO treatment, 58 proteins with G-TPP treatment for 2 h, and 86 proteins with G-TPP treatment were obtained for 4 h. 28 proteins and 56 proteins were increased with G-TPP treatment for 2 and 4 h, respectively, compared with the control group (Fig. 6 C). Furthermore, the GO function analysis was screened through a hypergeometric distribution algorithm to show enrichment score ( P ≤ 0.05), enrichment significance for BP (biological process), CC (cellular component), and MF (molecular function) were analyzed by Fisher's test, and the top 10 terms were listed (Fig. 6 D), the number of terms differed significantly between G-TPP treatment group and DMSO treatment group. In addition, KEGG signaling pathways analysis also differed significantly with G-TPP treatment compared to the control group. The top 30 terms were shown (Fig. 6 E). These results suggest that HRI interacts with many proteins during G-TPP-induced UPR mt and initiate the different cellular function and signaling pathways to combat this cellular response. Discussion The mitochondrial-targeted chaperone inhibitor (G-TPP) was used in our study, which induce specially UPR mt [ 25 ] . The study has shown that acute treatment of G-TPP (6 h) was performed for induction of UPR mt , which induces the up-expression of HSPD1 (1.7 fold), ATF4 (2.1 fold), and CHOP (11.3 fold), particularly the expression of CHOP in Hela cells [ 24 ] . Rapid and specific stress response was also induced with the up-expression of eIF2α phosphorylation at 30 min and ATF4 protein at 1 h, respectively, after G-TPP treatment in our study. Meanwhile, the expression of CHOP was also activated (Fig. 1 B, E and Extended data Fig. 1 C). In addition, UPR ER was not activated at transcriptional and protein levels (Extended Data Fig. 1 H-I). It suggests that UPR mt is a more rapid and special stress response with pharmacological treatment to restore cellular homeostasis, which is consistent with the previous study that UPR ER results in UPR mt and mitochondrial dysfunction in an ATF4-dependent manner, but mitochondrial stressors-induced UPR mt did not activate UPR ER in alveolar epithelial cells, such as oligomycin, doxycycline, and antimycin A treatment [ 26 ] . Meanwhile, mitochondrial morphology and the generation of ROS were not changed upon UPR mt (Fig. 3 ). It is speculated that the activation of UPR mt aims to promote a cellular survival response and restore mitochondrial proteostasis quickly. UPR mt is proposed to be triggered by the accumulation of unfolded or misfolded proteins, and it has been shown that a misfolded mitochondrial matrix protein ∆OTC can trigger UPR mt in mammalian cells. The evidence-based on activating CHOP and its target genes in Hela cells with the overexpression of ∆OTC, such as Cpn60, Cpn10, mtDnaJ, and ClpP, represents a classical UPR mt [ 10 ] . However, we found that eIF2α phosphorylation, the expression of ATF4 and CHOP are the main regulators with G-TPP treatment (Fig. 1 A, Extended data Fig. 1 A-B), while chaperone and protease were not up-regulated in our study (Extended data Fig. 1 F-G), which consistent with some recent studies that ATF4 and CHOP are main regulators [ 17 , 26 ] . UPR mt is a protein quality control mechanism that strives to achieve mitochondrial proteostasis in the face of misfolded proteins because of the reliance on both the nuclear and mitochondrial genomes, a perturbation of the coordination of these genomes results in a mito-nuclear imbalance [ 27 , 28 ] . Mitochondrial protein translation and mitochondrial protein import efficiency rather than cytoplasmic proteins were reduced through eIF2α phosphorylation in our study (Fig. 5 ). The study has shown that compromised mitochondrial protein import acts as a signal for the activation of UPR mt , and MTS of ATFS-1 as a sensor for this signal in C. elegans. ATFS-1 accumulates in the cytosol and traffics into the nucleus, which activates mitochondrial-specific chaperon and protease that promotes fold under stress conditions in C. elegans [ 29 ] . The mito-nuclear retrograde response was regulated by ATF4 in our study (Fig. 2 D), which may play a homologous role with ATFS-1 in the mammal. However, a study has demonstrated that ATF5, homologous to ATFS-1, regulated UPR mt during mitochondrial dysfunction in mammalian cells [ 16 ] . We could not detect it, and this discrepancy is unknown. The previous finding has confirmed that the transcriptional and protein expression of ATF4 was induced by other mitochondrial stressors, such as Doxycycline, Actinonin, and ionophore carbonyl cyanide-4 (trifluoromethoxy), phenylhydrazone (FCCP), MitoBloCK-6 (MB) and CDDO, these drugs can alter mitochondrial proteostasis [ 24 ] . In addition, eIF2α phosphorylation and ATF4 pathway were induced by G-TPP treatment in different cells, such as MEFs and Hela cells, and in SMMC7721 and HAP1 cell lines confirmed in our study, it demonstrates that UPR mt can be induced in different cell lines. In combination, eIF2α phosphorylation, the protein expression of ATF4, and the transcriptional expression of CHOP are induced upon UPR mt , but it is different from prototypical UPR mt in worms [ 13 , 17 ] and mammals [ 10 , 24 ] . Meanwhile, some DEPs were determined, such as HSPs: HSPA1B, HSPA1A, HSPA6, HSPA8, and DNAJB1, cytochrome P450 enzymes (CYP1A1 and CYP1B1), transcription factor DDIT4 (Fig. 1 G), which consistent with the previous study that heat shock proteins (HSPA1A, HSPA1B, and DNAJB1) were up-regulated steeply in DELE1- or HRI- knockout cells, and cells treated with ISRIB and CCCP [ 30 ] . It suggests that an alternative stress response program may be induced, and the mechanism needs further elaboration. CHOP, another key transcriptional factor of ISR, was activated by ISR through ATF4. CHOP was also induced during mitochondrial stress [ 20 , 30 ] . The study has shown that the translational expression of CHOP was activated in the context of mammalian UPR mt , such as the knockdown of surf1 and TIM23 , respectively [ 31 , 32 ] . Our study found that CHOP was up-regulated at the transcriptional and translational levels (Fig. 1 D-E). Both HSPE1 and CLPP are deemed as targets of CHOP. However, we did not detect any change in these genes at the transcriptional and translational levels (Extended data Fig. 1 F-G). In addition, CHOP can tune cellular response to different types of stress by initiating apoptotic and non-apoptotic programs [ 30 ] . Hence, the next works should be addressed the role of CHOP in the maintenance of mitochondrial proteostasis. eIF2α phosphorylation is the main regulator upon UPR mt . Our study found that the HRI branch of eIF2α kinases is involved in G-TPP-induced UPR mt . These results are consistent with the previous studies that OMA1 cleaved DELE1 into DELE1-S in the mitochondria and then redistributed to the cytosol, where it interacted with HRI to induce the expression of ATF4 or CHOP in response to mitochondrial stress [ 30 , 33 ] . We expounded the downstream mechanism furtherly that HRI in the cytosol activates eIF2α phosphorylation and the translational expression of ATF4, which traffics into the nucleus and initiates CHOP expression in response to UPR mt . The activation of eIF2α phosphorylation mainly blocks the decrease of mitochondrial protein translation and the number of mitochondrial proteins imported. Meanwhile, HRI interacts with many proteins in the vicinity of mitochondria screened by MS and initiate downstream molecule through different signaling pathways. However, which protein interacts with HRI needs to be furtherly confirmed. Conclusion In conclusion, we unraveled the mechanism of UPR mt through eIF2α phosphorylation in the mammal. The schematic diagram was summarized (Fig. 7 ), which will provide the theoretical foundation or therapeutic target for disease resulting from UPR mt . Declarations Ethics approval and consent to participate Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding This study was supported by Southwest Minzu University Double World-Class Project（XM2023012); Southwest Minzu University Research Startup Funds (16011211013/RQD202100), Natural Science Foundation of Sichuan Province (2022NSFSC0073), CAAS innovation programme fund. Not applicable. Author Contribution X.Q., Z.Z., and Y.L. contributed to the conceptualization of the study. X.Q., Y.Y., and Y.W. were responsible for the methodology design. Y.W., and X.Q. conducted to verify the experimental results. Y.W. and X.Q. wrote the original draft. Z.Z. and Y.L. were involved in writing, reviewing, editing, providing supervision, and funding acquisition. All authors read and approved the final manuscript. Acknowledgments Not applicable. References Wang L (2016) Mitochondrial purine and pyrimidine metabolism and beyond. Nucleosides Nucleotides Nucleic Acids 35(10–12):578–594. 10.1080/15257770.2015.1125001 Wellen KE, Thompson CB (2012) A two-way street: reciprocal regulation of metabolism and signalling. Nat Rev Mol Cell Biol 13(4):270–276. 10.1038/nrm3305 Smyrnias I (2021) The mitochondrial unfolded protein response and its diverse roles in cellular stress. Int J Biochem Cell Biol 133:105934. 10.1016/j.biocel.2021.105934 Bohovych I, Khalimonchuk O (2016) Sending Out an SOS: Mitochondria as a Signaling Hub. Front Cell Dev Biology 4:109. 10.3389/fcell.2016.00109 Weinberg SE, Sena LA, Chandel NS (2015) Mitochondria in the regulation of innate and adaptive immunity. Immunity 42(3):406–417. 10.1016/j.immuni.2015.02.002 Bhola PD, Letai A (2016) Mitochondria-Judges and Executioners of Cell Death Sentences. Mol Cell 61(5):695–704. 10.1016/j.molcel.2016.02.019 Ganguly G, Chakrabarti S, Chatterjee U, Saso L (2017) Proteinopathy, oxidative stress and mitochondrial dysfunction: cross talk in Alzheimer's disease and Parkinson's disease. Drug Des Devel Ther 11:797–810. 10.2147/DDDT.S130514 Sun N, Youle RJ, Finkel T (2016) The Mitochondrial Basis of Aging. Mol Cell 61(5):654–666. 10.1016/j.molcel.2016.01.028 Suomalainen A, Battersby BJ (2018) Mitochondrial diseases: the contribution of organelle stress responses to pathology. Nat Rev Mol Cell Biol 19(2):77–92. 10.1038/nrm.2017.66 Zhao Q, Wang JH, Ilya VL, Stan S, Michael TR, Nicholas JH (2002) A mitochondrial specific stress response in mammalian cells. EMBO J 21(17):4411–4419. 10.1093/emboj/cdf445 Nargund AM, Pellegrino MW, Fiorese CJ, Baker BM, Haynes CM (2012) Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation. Science 337(6094):587–590. 10.1126/science.1223560 Haynes CM, Yang Y, Blais SP, Neubert TA, Ron D (2010) The matrix peptide exporter HAF-1 signals a mitochondrial UPR by activating the transcription factor ZC376.7 in C. elegans. Mol Cell 37(4):529–540. 10.1016/j.molcel.2010.01.015 Houtkooper RH, Mouchiroud L, Ryu D, Moullan N, Katsyuba E, Knott G et al (2013) Mitonuclear protein imbalance as a conserved longevity mechanism. Nature 497(7450):451–457. 10.1038/nature12188 Amrita M, Nargund CJF, Mark W, Pellegrino P, Deng, Cole, Haynes M (2015) Mitochondrial and nuclear accumulation of the transcription factor ATFS-1 promotes OXPHOS recovery during the UPR(mt). Mol Cell 58(1):123–133. 10.1016/j.molcel.2015.02.008 Lin Y-F, Schulz AM, Pellegrino MW, Lu Y, Shaham S, Haynes CM (2016) Maintenance and propagation of a deleterious mitochondrial genome by the mitochondrial unfolded protein response. Nature 533(7603):416–419. 10.1038/nature17989 Fiorese CJ, Schulz AM, Lin YF, Rosin N, Pellegrino MW, Haynes CM (2016) The Transcription Factor ATF5 Mediates a Mammalian Mitochondrial UPR. Curr Biol 26(15):2037–2043. 10.1016/j.cub.2016.06.002 Quiros PM, Prado MA, Zamboni N, D'Amico D, Williams RW, Finley D et al (2017) Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals. J Cell Biol 216(7):2027–2045. 10.1083/jcb.201702058 Martinez-Reyes I, Sanchez-Arago M, Cuezva JM (2012) AMPK and GCN2-ATF4 signal the repression of mitochondria in colon cancer cells. Biochem J 444(2):249–259. 10.1042/BJ20111829 Michel S, Canonne M, Arnould T, Renard P (2015) Inhibition of mitochondrial genome expression triggers the activation of CHOP-10 by a cell signaling dependent on the integrated stress response but not the mitochondrial unfolded protein response. Mitochondrion 21:58–68. 10.1016/j.mito.2015.01.005 Pakos-Zebrucka K, Koryga I, Mnich K, Ljujic M, Samali A, Gorman AM (2016) The integrated stress response. EMBO Rep 17(10):1374–1395. 10.15252/embr.201642195 Baker BM, Nargund AM, Sun T, Haynes CM (2012) Protective coupling of mitochondrial function and protein synthesis via the eIF2α kinase GCN-2. PLoS Genet 8(6):e1002760. 10.1371/journal.pgen.1002760 Bao XR, Ong SE, Goldberger O, Peng J, Sharma R, Thompson DA et al (2016) Mitochondrial dysfunction remodels one-carbon metabolism in human cells. Elife 16(5):e10575. 10.7554/eLife.10575 Teske BF, Fusakio ME, Zhou D, Shan J, McClintick JN, Kilberg MS et al (2013) CHOP induces activating transcription factor 5 (ATF5) to trigger apoptosis in response to perturbations in protein homeostasis. Mol Biol Cell 24(15):2477–2490. 10.1091/mbc.E13-01-0067 Munch C, Harper JW (2016) Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation. Nature 534(7609):710–713. 10.1038/nature18302 Fabienne C, Fiesel EDJ, Hudec R (2017) Wolfdieter Springer. Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy. Oncotarget. 8(63):106233–106248. 10.18632/oncotarget.22287 Dingyuan Jiang HC, Xie N, Banerjee S, Liu R, Dai H, Victor J, Thannickal GL (2020) )ATF4 Mediates Mitochondrial Unfolded Protein Response in Alveolar Epithelial Cells. Am J Respir Cell Mol Biol 63(4):478–489. 10.1165/rcmb.2020-0107OC Sutandy FXR, Gößner I, Tascher G, Münch C (2023) A cytosolic surveillance mechanism activates the mitochondrial UPR. Nature 618(7966):849–854. 10.1038/s41586-023-06142-0 Lee-Glover LP, Shutt TE (2023) Mitochondrial quality control pathways sense mitochondrial protein import. Trends Endocrinol Metabolism 2300243–00246. 10.1016/j.tem.2023.11.004 Rolland SG, Schneid S, Schwarz M, Rackles E, Fischer C, Haeussler S et al (2019)Compromised Mitochondrial Protein Import Acts as a Signal for UPR(mt). Cell Rep 28(7):1659–1669e1655. 10.1016/j.celrep.2019.07.049 Fessler E, Eckl EM, Schmitt S, Mancilla IA, Meyer-Bender MF, Hanf M et al (2020)A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol. Nature 579(7799): 433–437. 10.1038/s41586-020-2076-4 Jovaisaite V, Auwerx J (2015) The mitochondrial unfolded protein response-synchronizing genomes. Curr Opin Cell Biol. 2015 33:74–81. 10.1016/j.ceb.2014.12.003 Oliveira AN, Hood DA (2018) Effect of Tim23 knockdown in vivo on mitochondrial protein import and retrograde signaling to the UPR(mt) in muscle. Am J Physiol Cell Physiol 315(4):C516–C526. 10.1152/ajpcell.00275.2017 Guo X, Aviles G, Liu Y, Tian R, Unger BA, Lin YT et al (2020) Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway. Nature 579(7799):427–432. 10.1038/s41586-020-2078-2 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4121132","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":281168182,"identity":"ddaccdb1-7e9e-4587-9c7b-a68720da9e0d","order_by":0,"name":"Yongshu Wu","email":"","orcid":"","institution":"Zhejiang A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Yongshu","middleName":"","lastName":"Wu","suffix":""},{"id":281168183,"identity":"c61bcf6a-ec1d-4b1c-ab1e-62911879f4b4","order_by":1,"name":"Yang Yang","email":"","orcid":"","institution":"Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Yang","suffix":""},{"id":281168184,"identity":"ee5d7b10-bbaa-4c11-878d-adfff1f3d4c3","order_by":2,"name":"Xiaodong Qin","email":"","orcid":"","institution":"Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiaodong","middleName":"","lastName":"Qin","suffix":""},{"id":281168185,"identity":"f1fcf6f1-ccaa-4547-a520-2f7d7c670703","order_by":3,"name":"Zhixiong Zhang","email":"","orcid":"","institution":"Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zhixiong","middleName":"","lastName":"Zhang","suffix":""},{"id":281168186,"identity":"86b070c8-ee71-4664-8d18-7723c2106bed","order_by":4,"name":"Munib Ullah","email":"","orcid":"","institution":"Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Munib","middleName":"","lastName":"Ullah","suffix":""},{"id":281168187,"identity":"0727c7ee-1acb-42d7-8807-fcbae6f91bee","order_by":5,"name":"Yanmin Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYBACPmYwJcHADxVgbCCkhQ2mRbKBaC0whsEBorWw8xh+LsyxyDM+f8ZMuoDBRnbDAeZnD/A7jMdYeuY2iWKzGzlm0jMY0ow3HGAzNyCgxUCad5tE4rYbvNukeRgOJ244wMMmQciW3yAtm/vPgrT8J0qLGdiWDQy5IC0HiNHCVmYN0jLjRv5nax6DZOOZh9nM8Grh5z+8+TbvtrrE/v5jibd5Kuxk+443P8OrhYGBAzl4QGxm/OqBgP0BQSWjYBSMglEwwgEAbtM8tEGYKeIAAAAASUVORK5CYII=","orcid":"","institution":"Southwest Minzu University","correspondingAuthor":true,"prefix":"","firstName":"Yanmin","middleName":"","lastName":"Li","suffix":""},{"id":281168188,"identity":"259d7581-e8af-45dc-be08-a9659dd98a24","order_by":6,"name":"Zhidong Zhang","email":"","orcid":"","institution":"Southwest Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Zhidong","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-03-18 07:59:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4121132/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4121132/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53152515,"identity":"0fab6a0b-c505-4a8b-824e-3421e05ccbd6","added_by":"auto","created_at":"2024-03-21 08:59:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":41666,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eG-TPP induces ISR rapidly and specifically\u003c/strong\u003e. \u003cstrong\u003eA-B\u003c/strong\u003e, Western blot analysis of eIF2α-P, eIF2α, ATF4, and ATF5 level upon treatment of HAP1 cells with DMSO and G-TPP for indicated timepoint. \u003cstrong\u003eC\u003c/strong\u003e, qPCR of ATF4 mRNA in HAP1 cells with or without G-TPP treatment (mean of levels relative to untreated ±s.d.; n=3 biological replicates). \u003cstrong\u003eD\u003c/strong\u003e, Western blot analysis of eIF2α-P, eIF2α, ATF4, CHOP level upon treatment of HAP1 cells with DMSO and G-TPP for 2 h. \u003cstrong\u003eE\u003c/strong\u003e, qPCR of CHOP mRNA in HAP1 cells with or without G-TPP treatment (mean of levels relative to untreated ±s.d. ; n=3 biological replicates). \u003cstrong\u003eF\u003c/strong\u003e, The hot plot of DEGs for RNA-Seq and Ribo-Seq(|log2FC|\u0026gt;1 and FDR\u0026lt;0.05) in HAP1 cells with or without G-TPP treatment. \u003cstrong\u003eG\u003c/strong\u003e, The volcano plot of DEPs in HP1 cells with or without G-TPP treatment.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4121132/v1/add1e2abbbc8b6094b3073dd.png"},{"id":53153428,"identity":"94347094-bdef-4b76-9859-cd77560219b7","added_by":"auto","created_at":"2024-03-21 09:07:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":482514,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended Data Fig1. G-TPP induce ISR rapidly and specifically\u003c/strong\u003e. \u003cstrong\u003eA-C\u003c/strong\u003e, western blot analysis of eIF2α-P, eIF2α, ATF4, ATF5 and CHOP level treated with DMSO and G-TPP for indicated timepoint in SMMC7721 cells and MEFs cells, respectively. \u003cstrong\u003eD-E\u003c/strong\u003e, qPCR of ATF4 and ATF5 mRNA in SMMC7721 cells with or without G-TPP treatment (mean of levels relative to untreated ±s.d.; n=3 biological replicates). \u003cstrong\u003eF\u003c/strong\u003e, western blot analysis of LONP1, CLPP, HSPE1 and HSP60 level upon treatment of HAP1 cells with DMSO and G-TPP for 2 h and 4 h. \u003cstrong\u003eG\u003c/strong\u003e, qPCR of LONP1, CLPP, HSPE1 and HSP60 mRNA in HAP1 cells with or without G-TPP treatment (mean of levels relative to untreated ±s.d. ; n=3 biological replicates). \u003cstrong\u003eH\u003c/strong\u003e, western blot analysis of eIF2α-P, eIF2α, ATF4 and BIP level treatment of HAP1 cells with DMSO and G-TPP for indicated timepoint. \u003cstrong\u003eI\u003c/strong\u003e, qPCR of BIP mRNA in HAP1 cells with or without G-TPP treatment (mean of levels relative to untreated ±s.d.; n=3 biological replicates).\u003c/p\u003e","description":"","filename":"ExtendedFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4121132/v1/f5bc61554fef7ac37da0ee7a.png"},{"id":53152513,"identity":"67dee50d-3c3b-4038-ab45-828c9fbf067b","added_by":"auto","created_at":"2024-03-21 08:59:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":49006,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 2. eIF2α phosphorylation is necessary for the expression of ATF4 and CHOP\u003c/strong\u003e. \u003cstrong\u003eA,\u003c/strong\u003e Western blot analysis of eIF2α-P, eIF2α, ATF4, and CHOP levels treated with DMSO, G-TPP, ISRIB, and G-TPP+ISRIB for 2 h in HAP1 cells. \u003cstrong\u003eB\u003c/strong\u003e, qPCR of CHOP mRNA in HAP1 cells treated with DMSO, G-TPP, ISRIB, and G-TPP+ISRIB for 2 h in HAP1 cells (mean of levels relative to untreated ±s.d.; n=3 biological replicates). \u003cstrong\u003eC\u003c/strong\u003e, Western blot analysis of eIF2α-P, eIF2α, ATF4, and CHOP level treated with DMSO and G-TPP for 4 h in MEFs wild-type or eIF2α S51A\u003csup\u003e-/+ \u003c/sup\u003ecells. \u003cstrong\u003eD\u003c/strong\u003e, the cytoplasmic and the nuclear fraction extraction assay was performed by MinuteTM Cytoplasmic and Nuclear Extraction Kit, and Western blot analysis of ATF4, ATF5, and CHOP level treated with DMSO and G-TPP for 2 h in HAP1 cells. \u003cstrong\u003eE-F\u003c/strong\u003e, knockdown efficiency of ATF4 was identified at transcriptional and protein levels. \u003cstrong\u003eG\u003c/strong\u003e, Western blot analysis of eIF2α-P, eIF2α, ATF4 level treated with DMSO and G-TPP for 2 h in HAP1-scrambled and shATF4 cells. \u003cstrong\u003eH\u003c/strong\u003e, qPCR of CHOP mRNA with or without G-TPP treatment in HAP1 HAP1-scrambled and shATF4 cells (mean of levels relative to untreated ±s.d.; n=3 biological replicates).\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4121132/v1/7e90ede4772f781dc2006cd4.png"},{"id":53152522,"identity":"d0f077af-d16f-4c51-b5f3-51412837f256","added_by":"auto","created_at":"2024-03-21 08:59:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":162634,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 3. G-TPP induces ISR independent of ROS generation and mitochondrial morphology change.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e, measurement of ROS upon G-TPP treatment using DCFDA cellular ROS detection assay kit. Shown are means of levels relative to untreated (n=3 biological replicates). \u003cstrong\u003eB-D\u003c/strong\u003e, TEM images of mitochondrial morphology (B-C) and endoplasmic reticulum morphology (D).\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4121132/v1/2ea7b23ff6482fe98ce4de44.png"},{"id":53152516,"identity":"940304ce-a102-41f8-bc35-128dcc448fa3","added_by":"auto","created_at":"2024-03-21 08:59:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":52760,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 4. HRI mediates ISR induced by mitochondrial unfolded proteins\u003c/strong\u003e. \u003cstrong\u003eA-D\u003c/strong\u003e, Western blot analysis of the knockout efficiency of PERK, PKR, GCN2, and HRI in HAP1 cells. \u003cstrong\u003eE-F\u003c/strong\u003e, Western blot analysis of eIF2α-P, eIF2α, ATF4, and CHOP level treated with DMSO and G-TPP for 2 h in HAP1 wild-type or KO cells of PERK, GCN2, PKR, and HRI, respectively. \u003cstrong\u003eI-L\u003c/strong\u003e, qPCR of CHOP mRNA with or without G-TPP treatment in HAP1 wild-type or KO cells of PERK, GCN2, PKR, and HRI, respectively (mean of levels relative to untreated ±s.d.; n=3 biological replicates).\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4121132/v1/1fc5c9eb7cc5aca3d7636154.png"},{"id":53152517,"identity":"709cc4ae-2b33-4ce6-93fb-f24e4db63c26","added_by":"auto","created_at":"2024-03-21 08:59:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":465618,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended data Fig4. HRI mediate ISR induced by mitochondrial unfolded proteins\u003c/strong\u003e. \u003cstrong\u003eA-D\u003c/strong\u003e, western blot analysis of knockout efficiency of PERK, PKR, GCN2 and HRI in SMMC7721 cells, respectively. \u003cstrong\u003eE-F\u003c/strong\u003e, western blot analysis of eIF2α-P, eIF2α, ATF4 and CHOP level treated with DMSO and G-TPP for 2 h in SMMC7721 wild-type or KO cells of PERK, GCN2, PKR and HRI, respectively. \u003cstrong\u003eI-L\u003c/strong\u003e, qPCR of CHOP mRNA with or without G-TPP treatment in SMMC7721 wild-type or KO cells of PERK, GCN2, PKR and HRI, respectively (mean of levels relative to untreated ±s.d.; n=3 biological replicates).\u003c/p\u003e","description":"","filename":"ExtendedFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4121132/v1/769b3afd0972ef8e8057d9a3.png"},{"id":53152518,"identity":"915944d1-3e89-4f7d-afff-34c76fe8ef6d","added_by":"auto","created_at":"2024-03-21 08:59:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":632465,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 5. The decrease of mitochondrial protein translation specifically induced by mitochondrial unfolded proteins\u003c/strong\u003e. The cytoplasmic and nuclear fraction extraction assay was performed by MinuteTM Cytoplasmic and Nuclear Extraction Kit and Western blot analysis of COX IV, CS, LAMP1, TIM23, GOT2, GOT1 level treated with DMSO, G-TPP, ISRIB and G-TPP+ISRIB for 4 h in HAP1 cells.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4121132/v1/d63d566844720b18f9d2cc57.png"},{"id":53152520,"identity":"42684820-ba32-4fba-a3da-204225c9ef82","added_by":"auto","created_at":"2024-03-21 08:59:07","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":42307,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 6. Mitochondrial recruitment of HRI and HRI interaction with different proteins upon G-TPP induced UPR\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003emt\u003c/strong\u003e\u003c/sup\u003e. \u003cstrong\u003eA\u003c/strong\u003e, immunoprecipitation analysis of EGFP-MAVS-Turboid with PERK, GCN2, PKR, and HRI, respectively, in HEK293 T cells. \u003cstrong\u003eB\u003c/strong\u003e, Western blot identification of HRI recovery expression in HAP1-HRI\u003csup\u003e-/- \u003c/sup\u003ecells. \u003cstrong\u003eC\u003c/strong\u003e, The Venn diagram of the DEPs in HAP1-sgHRI-HRI-TurboID cells treated with G-TPP for 2 and 4 h. \u003cstrong\u003eD\u003c/strong\u003e, GO enrichment analysis of the DEPs in HAP1-sgHRI-HRI-TurboiD cells treated with G-TPP for 4 h. \u003cstrong\u003eE\u003c/strong\u003e, KEGG pathway enrichment analysis of DEPs in HAP1-sgHRI-HRI-TurboiD cells treated with G-TPP for 4 h.\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4121132/v1/7764725f83f41072e0751205.png"},{"id":53152519,"identity":"d761a538-8dd2-4d58-8a67-128b07743bd2","added_by":"auto","created_at":"2024-03-21 08:59:07","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":33504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 7. Model of HRI mediates UPR\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003emt\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e through ISR in mammalian cells.\u003c/strong\u003e UPR\u003csup\u003emt\u003c/sup\u003e is induced with the accumulation of unfolded proteins treated by G-TPP independent of the generation of ROS and change of mitochondria morphology. Different proteins in the mitochondria interact with HRI in the vicinity of mitochondria, which activate eIF2α phosphorylation. The latter induces protein expression of ATF4, which traffics to the nucleus and initiates the expression of CHOP and other chaperones (HSPA1B, HSPA1A, HSPA6, HSPA8, DNAJB1), cytochrome p450 enzymes, and DDIT4.\u003c/p\u003e","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4121132/v1/59fba7dc8c9d1af5e762b492.png"},{"id":53199138,"identity":"06b1db40-614a-449c-ae89-fa8a8a424208","added_by":"auto","created_at":"2024-03-21 18:53:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3097242,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4121132/v1/af2ac248-d9c6-454f-b308-c838a432fd57.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Unfolded proteins in the mitochondria activate HRI and shut-down of mitochondrial protein translation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe mitochondria are the network organelles for energy production and metabolism of amino acids and nucleotides\u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e and serve as the hubs for many signaling processes, such as innate immune response during viral infection\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, apoptosis\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, neurodegenerative disease, and ages\u003csup\u003e[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. The mitochondrial protein folding environment is regulated by nuclear-encoded mitochondrial chaperones that enhance protein folding and proteases to degrade proteins that fail to fold or oligomerize correctly. Mitochondrial unfolded protein response is induced when the accumulation of misfolded or unfolded proteins exceeds the capacity of mitochondrial protein folding in the mitochondria, term as UPR\u003csup\u003emt\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUPR\u003csup\u003emt\u003c/sup\u003e was discovered first in cultured mammalian cells. However, many genes about UPR\u003csup\u003emt\u003c/sup\u003e were well studied and identified in \u003cem\u003eC. elegans\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. UPR\u003csup\u003emt\u003c/sup\u003e was regulated by the basic leucine zipper (bZIP) activating transcriptional factor associated with stress-1 (ATFS-1) in \u003cem\u003eC. elegans\u003c/em\u003e, which includes both a mitochondrial targeting sequence (MTS) and a nuclear localization sequence (NLS) \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. This function is important in mediating the communication between the mitochondria and the nucleus\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Under the physiologic condition, ATFS-1 is imported into the mitochondria and degraded by LON protease. However, the decrease of mitochondria import of ATFS-1 results in the accumulation of ATFS-1 in the cytosol and traffics into the nucleus, along with protein ubiquitin-like5 (UBL5) and DVE-1 under mitochondrial stress conditions, which activate the transcriptional expression of a set of genes encoding proteases, chaperones, and metabolic enzymes to restore mitochondrial function and cellular homeostasis \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Hence, we speculate that cells may utilize mitochondrial import efficiency as a mitochondrial normal function signal through ATFS-1, a sensor that detects mitochondria-to-nucleus signaling in \u003cem\u003eC. elegans\u003c/em\u003e. Nucleus accumulation of ATFS-1 can activate the transcription of over 500 genes that regulate many cellular activities \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHowever, the mechanism studying of UPR\u003csup\u003emt\u003c/sup\u003e is less in mammals. Research has shown that ATF5, as a bZIP transcriptional factor, has the homologous function to ATFS-1, which is regulated by mitochondria import efficiency to restore mitochondria function \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Apart from ATF5, at least ATF4 and C/EBP homologous protein (CHOP) involving in UPR\u003csup\u003emt [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. However, the role of these transcriptional factors in UPR\u003csup\u003emt\u003c/sup\u003e in the mammal is still unclear.\u003c/p\u003e \u003cp\u003eThe integrated stress response (ISR) is an intricate signaling pathway existing in eukaryotic cells, which is activated through the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) in response to different physiological changes and pathological conditions, the activation of eIF2α phosphorylation results in decreasing of global protein synthesis and induction of selected genes, such as ATF4. ISR is activated by four known eIF2α kinases, which sense different stresses, PERK is activated by the accumulation of misfolded or unfolded protein in the endoplasmic reticulum (ER), PKR senses double-stranded RNA in the cytosol, and heme-regulated inhibitor (HRI) is induced by heme depletion \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e, GCN2 responds to amino acid starvation, as well as mitochondria stress\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Activated kinases phosphorylate eIF2α, which reduces overall protein synthesis and preferential expression of mRNAs that include small upstream open reading frames (uORFs) in the 5' untranslated region (UTR) to restore cellular homeostasis.\u003c/p\u003e \u003cp\u003ePrevious studies showed that UPR\u003csup\u003emt\u003c/sup\u003e is triggered through the expression of eIF2α phosphorylation and ATF4 in mammalian cells \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. In addition, the study has demonstrated that ATF4, ATF5, and CHOP are required for UPR\u003csup\u003emt[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. However, the role of eIF2α phosphorylation in UPR\u003csup\u003emt\u003c/sup\u003e and its influence on mitochondria protein translation need further illumination. Whether four eIF2α kinases are involved in the activation of UPR\u003csup\u003emt\u003c/sup\u003e remains unclear. To shed light on the underlying functional mechanism of UPR\u003csup\u003emt\u003c/sup\u003e, Gamitrinib-triphenylphosphonium (G-TPP) was used in this study, which is a mitochondrial-targeted TNF receptor-associated protein-1 (TRAP1) of HSP90 family inhibitors and chemically interfere with mitochondrial protein folding \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.We elucidated the role of eIF2α kinases in UPR\u003csup\u003emt\u003c/sup\u003e through CRISPR-Cas9 technology. In this study, we confirmed that UPR\u003csup\u003emt\u003c/sup\u003e was a rapid and specific stress response with pharmacological induction, along with the up-expression of eIF2α phosphorylation, ATF4, and CHOP. Meanwhile, some chaperones, cytochrome P450 enzymes, and DDIT4 were up-regulated and confirmed by RNA-Seq and ribosome profiling. The activation of eIF2α phosphorylation is essential for the expression of ATF4 and CHOP, and ATF4 is trafficked into the nucleus and initiates the expression of CHOP. Furtherly, we unraveled the mechanism that HRI-mediated G-TPP induced UPR\u003csup\u003emt\u003c/sup\u003e through the HRI-eIF2α-p-ATF4-CHOP signaling pathway, and HRI interacted with many mitochondrial proteins in the vicinity of mitochondria. G-TPP-induced UPR\u003csup\u003emt\u003c/sup\u003e blocked mitochondrial protein translation and the number of mitochondrial proteins imported while not influencing ROS generation and mitochondria morphology change through eIF2α phosphorylation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003eCell culture and experimental animals.\u003c/b\u003e SMMC7721 cells, HAP1 cells, and HEK-293 T cells were preserved in our laboratory. SMMC7721 cells and HEK-293 T cells were cultured in DMEM basic (1\u0026times;) (Gibco, C14190500BT) supplemented with 10% fetal bovine serum (Gibco, 10099-141) and 1% penicillin and streptomycin (Gibco, 15140-122) in an incubator (Binder, CB160) containing 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. HAP1 cells were maintained in IMDM modified medium (HyClone, SH30228-01). Cells were digested using 0.25% Trypsin-EDTA (Gibco, 25200056).\u003c/p\u003e \u003cp\u003eeIF2α S51A heterozygous mice were purchased from Jackson Laboratory, USA, strain: B6; 129-Eif2s1tm1Rjk/J, 017601. The tail tissues of WT mice and offspring of eIF2α S51A heterozygous mice were collected and DNA were extracted, and then were screened and identified by PCR, the primer sequences: Forward: 5'-ACACCCATTCCATGATAGTAAAATG-3', Reverse: GTTGTAGACCCTGACAATGAAGG-3'); MEFs cells were cultured in DMEM basic (1\u0026times;).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMEFs preparation.\u003c/b\u003e Fetal rats of 13\u0026thinsp;~\u0026thinsp;15 days were anesthetized by intraperitoneally injecting 0.5 mL of 2.5% avertin, then euthanized by cervical dislocation. The fetal rat was placed on a paper towel and sprayed enough 75% ethanol to soak the entire mouse body. Transferred fetal rat into biological safety cabinet, cut skin and tissues to expose uterus, removed embryos with a scalpel in 10 cm dish containing PBS, discarded all other tissues, such as the head and visceral tissues. The remaining part of the embryos was washed with PBS 4 times and placed in a clean 10 cm dish and cut embryos into 2 mm pieces with a new scalpel. Added 0.25% trypsin and incubated in 37\u0026deg;C incubators for 10 min. DMEM containing 10% FBS was added to resuspend cells, inoculated into T75 cell flasks, cultured in an incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e, discarded non-adherent cells, and replaced culture medium to continue culturing.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneration of ATF4-KD and eIF2α kinases-KO cell lines construction.\u003c/b\u003e ATF4 knockdown cells (ATF4-KD) were constructed by shRNA, and targeting sequences of ATF4 was available from the website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.sigmaaldrich.cn/CN/zh\u003c/span\u003e\u003cspan address=\"https://www.sigmaaldrich.cn/CN/zh\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Firstly, primers were synthesized by Sangon Biotech (Shanghai, China) and cloned into pLKO.1 vector (Addgene, 10878) by digestion and ligation, shRNA sequences for ATF4 were shown in the following: \u003cem\u003eATF4\u003c/em\u003e: 5'-GCCTAGGTCTCTTAGATGATT-3'.\u003c/p\u003e \u003cp\u003eThe eIF2α kinases (PERK, PKR, GCN2, and HRI) knockout cell lines (eIF2α kinases-KO) were manufactured using the CRISPR-Cas9 technique. sgRNA were designed using sgRNA Designer from Zhang's lab. The sgRNAs were synthesized and cloned into the Lenti-CRISPR v2 vector (Addgene, 52961) by ligation. sgRNA sequences of eIF2α kinases as follows: \u003cem\u003ePERK\u003c/em\u003e: 5'-GTAATTATCAGCACTTTAGA-3'; \u003cem\u003ePKR-1\u003c/em\u003e: 5'-GTACTACTCCCTGCTTCTGA-3', \u003cem\u003ePKR-2\u003c/em\u003e: 5'-GATTATGAACAGTGTGCATCG-3'; \u003cem\u003eGCN2-1\u003c/em\u003e: 5'-CCTCCGGAGAGCTACCCGCAACG-3', \u003cem\u003eGCN2-2\u003c/em\u003e: 5'-TATATGTAAAAGTGGATTTG-3'; \u003cem\u003eHRI-1\u003c/em\u003e: 5'-CCGAGGGCCCGGACCCCGAA-3', \u003cem\u003eHRI-2\u003c/em\u003e: 5'-AAGGGAAGGTTGGCTGTTGT-3'.\u003c/p\u003e \u003cp\u003eFor lentivirus packing and infection, 4 \u0026micro;g recombinant lentiviral plasmid was co-transfected with 2 \u0026micro;g packaging plasmid psPAX2 (Addgene, 12260) and 1 \u0026micro;g envelope plasmid pMD2.G (Addgene, 12259) into Lenti-\u0026trade; 293 T cells using Lipofectamine\u0026trade; 3000 Transfection Reagent (Thermo Fisher Scientific, L3000015). Infectious particles were harvested from 48 h to 96 h after transfection, centrifugated at 4000 rpm/min for 30 min, and concentrated with a final concentration of 20% PEG-8000, then concentrated lentiviruses were added into SMMC7721 or HAP1 cell lines, respectively, cells were selected by adding 250 \u0026micro;g/mL hygromycin (Invitrogen, 10687010) or 5 \u0026micro;g/mL puromycin (Invitrogen, A1113803). For KO cell lines, positive clones were isolated through limiting dilution assay, and knockdown and knockout efficiency were determined by Western blot.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConstruction of HAP1-sgHRI-HRI-TurboID cell line.\u003c/b\u003e The pEGFP-N1-HRI-TurboID plasmid was constructed by inserting the CDs sequence of HRI into the 5' edge of the TurboID cDNA sequence. To select positive clones against hygromycin, a homologous arms assay was constructed with a pSMPUW-Hygro vector, and lentiviral packaging was performed. Packaged lentiviruses were added into HAP1-HRI\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells and selected by 250 \u0026micro;g/mL hygromycin (Invitrogen, 10687010). The positive clones were determined by Western blot.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blot.\u003c/b\u003e Cells were washed with cold PBS twice, and ultrasonic protein was extracted on ice in protease and phosphatase inhibitor (Thermo Fisher Scientific, A32959) in RIPA buffer (Beyotime, P0013B) with 1% PMSF (Thermo Fisher, 36978). Lysates were centrifuged at 12000 rpm/min for 10 min. Supernatants were collected and denatured at 95\u0026deg;C for 10 min in a 4 \u0026times; loading buffer. Equal amounts of protein were resolved on 10% electrophoresis acrylamide gel (Bio-Rad, 1610173), transferred to PVDF membranes (Millipore, ISEQ00010), and blocked in 5% nonfat milk (BD, 232100) in TBS-T (50 mM Tris (pH 7.4), 150 mM NaCl, 0.1% Tween20 (Sigma, P7949-500ML) for 2 h at room temperature (RT). In TBS-T, membranes were incubated with primary antibodies overnight at 4\u0026deg;C in 5% bovine serum albumin (BSA)(Millipore, 9048-46-8). Primary antibodies were used in this study as follows: β-tubulin antibody (66240-1-Ig; 1:5000); EIF2A antibody (11170-1-AP; 1:2000); ATF4 antibody (10835-1-AP; 1:1000); LONP1 antibody (15698-1-AP; 1:1000); CLPP antibody (15440-1-AP; 1:1000); HRI antibody (20499-1-AP; 1:1000); TIM23 antibody (11123-1-AP; 1:1000); GOT1 antibody (14886-1-AP; 1:1000); GOT2 antibody (14800-1-AP; 1:1000) were purchased from Proteintech; PERK antibody (3192s; 1:1000); CHOP antibody (D46F1; 1:1000); HSP60 antibody (D6F1; 1:1000); GAPDH antibody (5174; 1:1000); Histone H3 antibody (4499; 1:5000); BIP antibody (3183S; 1:1000); GCN2 antibody (3302S; 1:1000); COX IV antibody (3E11; 1:1000); COX IV antibody (4844; 1:1000); CS antibody (14309; 1:1000); LAMP1 antibody (9091; 1:1000); Streptavidin-HRP antibody (3999s; 1:1000) were purchased from CST; ATF5 antibody (Ab184923; 1:2000); PKR antibody (Ab32506; 1:1000); Phospho-EIF2A antibody (Ser51) (Ab32157; 1:2000) were purchased from Abcam; HSPE1 antibody (Thermo Fisher, PA5-79415; 1:2000); Flag antibody (Sigma-Aldrich, F1804; 1:1000). Membranes were washed with TBS-T three times, 10 min each time. Membranes were incubated in appropriate HRP-conjugated secondary antibodies (Bio-Rad, 170\u0026ndash;6515; 1:5000 and 170\u0026ndash;6516; 1:5000) at 1:10,000 dilution in 5% milk in TBS-T for 2 h at RT. Membranes were washed three times with TBS-T, 10 min each time. Membranes were detected using standard chemiluminescence with ECL (Affinity, KF003) and imaged by GE Healthcare Amersham\u0026trade; Imager 600 in an automatic exposure model to ensure that bands are not saturated. Band intensity was determined using ImageJ software (NIH) and adobe photoshop cc software (Adobe). At least 3 independent experiments were performed.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantitative Real-Time PCR (RT-qPCR).\u003c/b\u003e Total RNA was extracted using RNeasy Plus Universal Mini Kit (50) (QIAGEN, 73404). According to the manufacturer's directions, RNA was converted to cDNA using the ReverTra Ace qPCR RT Master Mix (TOYOBO, FSQ-301). Each reaction includes 2 \u0026micro;L 4\u0026times;DN Master Mix ( with gDNA Remover), RNA template 0.2 \u0026micro;g, and nuclease-free water 5 \u0026micro;L, 37\u0026deg;C incubated 5 min, and then added 5\u0026times;RT Master Mix II 2 \u0026micro;L, 37\u0026deg;C 15 min, 50\u0026deg;C 5 min, 98\u0026deg;C 5 min. RT-qPCR was performed using Blaze TaqTM SYBR Green qPCR Mix2.0 kit (Gene Copoeia, QP043). Each reaction of qPCR contains 2\u0026times;SYBR Green PCR Master Mix 10 \u0026micro;L, forward primer 0.7 \u0026micro;M, reverse primer 0.7 \u0026micro;M, RNase-free water 5.2 \u0026micro;L, template cDNA 2 \u0026micro;L. PCR initial heat activation 95\u0026deg;C 2 min, 95\u0026deg;C, denaturation 5 s, 60\u0026deg;C, extension 10 s, 40 cycles. The primer used in qPCR as following: \u003cem\u003eACTIN\u003c/em\u003e (Human) Forward: 5'-GGACCTGACTGACTACCTCAT-3', Reverse: CGTAGCACAGCTTCTCCTTAAT-3'); \u003cem\u003eLONP1\u003c/em\u003e (Human) (Forward: 5'-TCAATGTCACCCGCAACTAC-3', Reverse: 5'-GAACTCCAGGATGCGTTTCT-3'); \u003cem\u003eCLPP\u003c/em\u003e (Human) (Forward: 5'-CTCTTCCTGCAATCCGAGAG-3', Reverse: 5'-GGATGTACTGCATCGTGTCG-3'); \u003cem\u003eHSP60\u003c/em\u003e (Human) (Forward: 5'-TCCAGGGTTTGGTGACAATAG-3', Reverse: 5'-GATTCAGGGTCAATCCCTCTTC-3'); \u003cem\u003eHSPE1\u003c/em\u003e (Human) (Forward: 5'-GCTGCTGAAACTGTAACCAAAG-3', Reverse: 5'-CTCCACCCTTTCCTTTAGAACC-3'); \u003cem\u003eCHOP\u003c/em\u003e (Human) (Forward: 5'-GTCTAAGGCACTGAGCGTATC-3', Reverse: 5'-CAGGTGTGGTGATGTATGAAGA-3'); \u003cem\u003eATF5\u003c/em\u003e (Human) (Forward: 5'-CCCACCTGACCTGGAAGC-3', Reverse: 5'-CCTCGTTGCGGCAGTAGAT-3'); \u003cem\u003eATF4\u003c/em\u003e (Human) (Forward: 5'-GGAGATAGGAAGCCAGACTACA-3', Reverse: 5'-GGCTCATACAGATGCCACTATC-3'); \u003cem\u003eBIP\u003c/em\u003e (Human) (Forward: 5'-AACCATCCCGTGGCATAAA-3', Reverse: 5' -GGACATACATCAAGCAGTACCA-3'\u0026rsquo;). The sample was analyzed in three biological replicates, and the relative expression values were obtained using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method .\u003c/p\u003e \u003cp\u003e \u003cb\u003eROS detection.\u003c/b\u003e ROS was detected using DCFDA cellular ROS detection assay kit (Abcam, ab113851). The protocol was as follows: HAP1 cells were harvested and seeded in a dark 96-well assay plate (black with clear flat bottom), allowing cells to be attached overnight. Cells were washed once with 1\u0026times;Buffer. Cells were stained with 25 \u0026micro;M DCFDA in 1\u0026times;Buffer for 45 min at 37\u0026deg;C, washed once in 1 \u0026times; Buffer, and treated with GTPP (10 \u0026micro;M), ISRIB (200 nM), and GTPP\u0026thinsp;+\u0026thinsp;ISRIB, DMSO as a negative control, 55 \u0026micro;M TBHP (Tert-Butyl Hydrogen peroxide) as the positive control. Read signals at Ex/Em: 485/535 nm by PerkinElmer VICTOR Nivo multimode plate reader.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTransmission electron microscopy.\u003c/b\u003e HAP1 cells were seeded into a 60 mm dish and treated with GTPP (10 \u0026micro;M) for 2 and 4 h when cells were confluence about 80\u0026thinsp;~\u0026thinsp;90%, with DMSO as the negative control. Method for ultrastructure analysis of mitochondria was performed according to the protocol: fixation: cells were collected and fixed with 2.5% glutaraldehyde at 4\u0026deg;C for 2 h, rinsed with 0.1 mol/l phosphoric acid rinsing solution three times, 15 min each time, fixed with 1% osmium acid for 2 h, rinsed with 0.1mol/L phosphoric acid rinsing solution 3 times, 15 min. Dehydration: dehydrated with 50% of ethanol dehydration for 15 min, dehydrated with different concentrations of ethanol dehydration for 15 min, 70, 90, and 95%, respectively, the above septs were conducted in a fume cupboard, dehydrated with 100% acetone 3 times at RT, 15 min each time. Embedding: cells were embedded with pure acetone along with an embedding solution (2:1) at 37\u0026deg;C for 3 h, embedded with pure acetone along with an embedding solution (1:2) at 37\u0026deg;C for 3 h, and placed in pure embedding solution at 45\u0026deg;C for more than 2 h, and then converted to 60\u0026deg;C. Solidify: cells were placed in a drying oven at 45\u0026deg;C for more than 3 h and at 60\u0026deg;C for 24 h. Sliced by Lycra UC7 ultra-thin microtome to 50\u0026ndash;60 nm. Double stained with 3% uranyl acetate-lead citrate. Cellular ultrastructure was observed and filmed with a JEOL JEM-1400 transmission electron microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA-Seq.\u003c/b\u003e\u0026nbsp;Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample were quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA) with a RIN number\u0026thinsp;\u0026gt;\u0026thinsp;7.0. The final cDNA libraries were constructed using TruSeq\u0026reg; Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA). The 2\u0026times;150 bp paired-end sequencing (PE150) was performed on an Illumina Novaseq\u0026trade; 6000 (LC-Biotechnology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of RNA data.\u003c/b\u003e Fastp software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/OpenGene/fastp\u003c/span\u003e\u003cspan address=\"https://github.com/OpenGene/fastp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to remove the reads that contained adaptor contamination, low-quality bases (Q\u0026thinsp;\u0026le;\u0026thinsp;20, more than 50%), and undetermined bases (more than 10%). Then, sequence quality was also verified using fastp. HISAT2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ccb.jhu.edu/software/hisat2\u003c/span\u003e\u003cspan address=\"https://ccb.jhu.edu/software/hisat2\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to map reads to the reference genome of Homo sapiens Ensembl_release101. The mapped reads of each sample were assembled using StringTie (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ccb.jhu.edu/software/stringtie\u003c/span\u003e\u003cspan address=\"https://ccb.jhu.edu/software/stringtie\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with default parameters. Then, all transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using gffcompare (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/gpertea/gffcompare/\u003c/span\u003e\u003cspan address=\"https://github.com/gpertea/gffcompare/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). RSEM was used to perform gene expression levels by calculating fragments per kilobase million (FPKM). The differentially expressed genes were selected with |log2 Fold Change| \u0026gt; 1 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 by R package DESeq2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bioconductor.org/packages/release/bioc/html/DESeq2.html\u003c/span\u003e\u003cspan address=\"http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eRibo-Seq.\u003c/b\u003e\u0026nbsp;Cycloheximide was added to the cell medium to a final 100 \u0026micro;g/ml concentration to block translation. The resuspended extracts in lysis buffer were transferred into a clean microtube, pipetted several times, and incubated on ice for 10 min. Then cells were triturated 10 times through a 26-G needle. The lysate was centrifuged at 20000 g/min for 10 min at 4\u0026deg;C, and the supernatant was collected. To prepare ribosome footprints (RFs), 7.5 \u0026micro;L of RNase I and 5 \u0026micro;L of DNase I was added to 300 \u0026micro;L of lysate to incubate for 45 min at RT with gentle mixing on a nutator mixer. Nuclease digestion was stopped by adding 10 \u0026micro;L of SUPERase\u0026middot;In RNase inhibitor. Size exclusion columns (Illustra MicroSpin S-400 HR Columns; GE Healthcare; catalog no. 27-5140-01) were equilibrated with 3 ml of polysome buffer by gravity flow and centrifuged at 600 g/min for 4 min at RT. 100 \u0026micro;L of digested RFs were added to the column and centrifuged at 600 g/min for 2 min. Next, 10 \u0026micro;L 10% (wt/vol) SDS was added to the elution, and RFs with a size greater than 17nt were isolated according to the RNA Clean and Concentrator-25 kit (Zymo Research; R1017). rRNA was removed using DNA probes complementary to rRNA sequences. Then RNase H and DNase I were used to digest the probes. RFs were purified using magnet beads (Vazyme). Ribo-seq libraries were constructed using NEBNext\u0026reg; Multiple Small RNA Library Prep Set for Illumina\u0026reg; (catalog no. E7300S, E7300L) after obtaining the ribosome footprints above. PCR products were sequenced using Illumina HiSeq\u0026trade; 2500 (LC-Biotechnology CO., Ltd., Hangzhou, China).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of Ribo-seq data.\u003c/b\u003e Raw reads containing over 50% of low-quality bases or over 10% of N bases were removed. Adapter sequences were trimmed. Reads with lengths between 10\u0026thinsp;~\u0026thinsp;50 bp were retained for subsequent analysis. Bowtie2 was used for mapping reads to the ribosome RNA (rRNA) database. The rRNA removed reads of each sample were mapped to the reference genome by Bowtie2, allowing no mismatches. Reads number in the open reading frame of coding genes was calculated by the software RiboTaper, and the gene expression level was normalized using the FPKM method, the same as the RNA-Seq.\u0026nbsp;To identify differentially translated genes across sample groups, the edgeR package (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rproject.org/\u003c/span\u003e\u003cspan address=\"http://www.rproject.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used. Genes with |log2 Fold Change| \u0026gt; 1 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 in comparison were considered as significant DTGs. DTGs were then subjected to enrichment analysis of GO functions and KEGG pathways.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLiquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.\u003c/b\u003e Cells were treated with G-TPP (10 \u0026micro;M) for 2 and 4 h, respectively, equal volume DMSO was added as the control, and 500 mmol/L Biotin (Sigma, V900418-1G) was added for 30 min before finishing treatment time. The samples were performed by Immunoprecipitation. IP samples were separated by SDS-PAGE, and a gel lane was cut for LC/MS/MS. Briefly, the samples were digested, desalted, and analyzed by Q Exactive high-resolution mass spectrometer (Thermo Scientific) accompanied by Easy-nLC 1200 (Thermo Scientific). Proteome Discover 2.5 software was used to map reads to the reference genome of UniProt-Human and analyze the data.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunoprecipitation.\u003c/b\u003e To investigate the relationship between HRI and MAVS, the pEGFP-N1-MAVS-TurboID plasmid was constructed by inserting the CD sequence of MAVS into the 5' edge of the TurboID cDNA sequence. The pEGFP-N1-MAVS-TurboID plasmid was transfected into HEK 293T cells using Lipofectamine 3000 reagent for 36 h, and cells were treated with G-TPP for 4 h, equal volume DMSO as the control, and 500 mmol/L Biotin (Sigma, V900418-1G) was added for 30 min before finishing treatment time. Rinsed with pre-cooled PBS once, added 400 \u0026micro;L RAPA lysis solution, placed on ice for 30 min to absolutely dissolve, sonicated with cell sonicator for 1 min, 12000 rpm/min, centrifuged for 10 min, and 80 \u0026micro;L of lysate were transferred into a clean 1.5 mL centrifuge tube, and 5\u0026times;loading buffer was added, heated at 98\u0026deg;C, 10 min, as Input samples. 50 \u0026micro;L of Hydrophilic Streptavidin Magnetic beads (NEB, S1421S) were washed with 500 \u0026micro;L lysate buffer, discarded supernatant, and beads were added into remaining cell lysate, 18 rpm/min, 4\u0026deg;C overnight. The cell lysate was washed 3 times, 500 \u0026micro;L each time and 80 \u0026micro;L of lysate buffer were added into 5\u0026times;loading buffer, 98\u0026deg;C heated for 10 min. Input samples and IP samples were performed with Western blot.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCytoplasmic and nuclear extraction test.\u003c/b\u003e HAP1 cells were seeded into a 60 mm dish, and cells were treated with G-TPP (10 \u0026micro;M) for 2 h when cellular confluence was up to 80% and an equal volume of DMSO as a control. Cells were washed twice with pre-cooled PBS after treatment and discarded PBS. The extraction assay was performed according to MinuteTM Cytoplasmic and Nuclear Extraction Kit (Invent Biotechnologies, Inc., SC-003). 500 \u0026micro;L of cytoplasmic extraction buffer were added, placed on ice for 5 min, then transferred into a pre-cooled 1.5 mL microtube, vortexed vigorously for 15 s, and centrifuged for 5 min at top speed in a microcentrifuge at 4\u0026deg;C. The supernatant (cytosol fraction) was transferred into a fresh pre-chilled 1.5 mL microtube. Appropriate amounts of nuclear extraction buffer were added to the pellets, vortexed vigorously for 15 s, incubated microtube on ice for 1 min, and repeated this step 4 times. Immediately transferred the nuclear extract into a pre-chilled filter cartridge with a collection microtube and centrifuged at top speed (14,000\u0026thinsp;~\u0026thinsp;16,000 rpm/min) in a microcentrifuge for 30 s at 4\u0026deg;C. Discarded filter cartridge, Added 5\u0026times;loading buffer in the cytoplasm and the nucleoplasm components, heated at 95\u0026deg;C, 10 min to denature protein, and then performed by Western blot.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDrugs treatment.\u003c/b\u003e G-TPP (synthesized from Chengdu Ruizhi Chemical Research Co. LTD) is dissolved in DMSO, and the final concentration is 10 \u0026micro;M. ISRIB (Med Chem Express) dissolves in DMSO, and the final concentration is 200 nM.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e Data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The significance of the variability between different treatment groups was analyzed by a two-way analysis of variance (ANOVA) test via GraphPad Prism software (version 8.0.2.). \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. The pictures of Western blot were imaged by both Image J software and photoshop cc software.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eG-TPP induces UPR\u003c/b\u003e \u003csup\u003e \u003cb\u003emt\u003c/b\u003e \u003c/sup\u003e \u003cb\u003evia rapid and specific activation of ISR.\u003c/b\u003e To understand the mechanism of UPR\u003csup\u003emt\u003c/sup\u003e with G-TPP treatment, the expression of eIF2α phosphorylation, ATF4, ATF5, and CHOP were detected in different cell lines and primary cells with G-TPP treatment at a concentration of 10 \u0026micro;M. The results showed that the human liver cancer cell lines (SMMC7721 cell lines) and the male chronic myeloid leukemia (HAP1 cell lines) responded well to G-TPP treatment, eIF2α phosphorylation, and the protein expression of ATF4 was significantly up-regulated. Hence, both SMMC7721 and HAP1 cell lines were used for subsequent experiments.\u003c/p\u003e \u003cp\u003eSMMC7721 and HAP1 cells were treated with G-TPP for 2 and 4 h, respectively. The results showed that eIF2α phosphorylation and ATF4 protein were significantly up-regulated but ATF5 expression was not changed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The same results were obtained in MEF cells with G-TPP treatment for 2 and 4 h (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Furthermore, HAP1 cells were treated with G-TPP for 5, 10, 30 min, and 1 h. The Western blot showed that eIF2α phosphorylation and up-expression of ATF4 occurred at about 30 min and 1 h, respectively. However, the expression of ATF5 was still unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This phenomenon also occurred in SMMC7721 cell lines (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). It suggests that UPR\u003csup\u003emt\u003c/sup\u003e is an acute activation process through eIF2α phosphorylation with G-TPP treatment. However, the transcriptional expression of ATF4 was not up-regulated in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), indicating that the ATF4 protein expression-dependent model is activated upon G-TPP-induced UPR\u003csup\u003emt\u003c/sup\u003e. The transcriptional expression of ATF5 was also not influenced in the SMMC7721 cell line (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eWe furtherly wonder what the role of CHOP in G-TPP-induced UPR\u003csup\u003emt\u003c/sup\u003e is. The results showed that CHOP was significantly activated with G-TPP treatment at the transcriptional level (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and protein level in HAP1 cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.D-E), suggesting that CHOP is also a general activation transcriptional factor in response to G-TPP-induced UPR\u003csup\u003emt\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe investigated the expression of mitochondrial stress proteins encoded by nuclear genes with G-TPP treatment. However, the expression of HSP60, HSPE1, LONP1, and CLPP was not changed at protein and transcriptional levels with G-TPP treatment (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.F-G). These results demonstrate that GTPP can induce UPR\u003csup\u003emt\u003c/sup\u003e rapidly through the expression of eIF2α phosphorylation, ATF4, and CHOP. Meanwhile, the expression of BIP, a UPR\u003csup\u003eER\u003c/sup\u003e marker protein was not activated at transcriptional and protein levels (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.H-I), indicating that a mitochondrial special UPR\u003csup\u003emt\u003c/sup\u003e is activated through eIF2α phosphorylation.\u003c/p\u003e \u003cp\u003eFurtherly, HAP1 cells were treated with G-TPP for 4 h and sequenced by the RNA-seq and Ribosome profiling, with DMSO as a control. The data showed that the overall trend of differentially expressed genes (DEGs) differed significantly (|log2FC|\u0026gt;1 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with G-TPP treatment compared to the control group at transcriptional and translational levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.F). Furthermore, the genes in our study and some genes with DEGs were analyzed, and CHOP expression was up-regulated significantly with G-TPP treatment at both levels compared with DMSO treatment. The expression of ATF4 was nearly twice as higher as that of the control group by Ribo-Seq, although there was no significant difference at the transcriptional level, which is consistent with the results verified in our study. However, at both levels, there was no significant difference in the expression of ATF5, HSP60, HSPE1, CLPP, and LONP1. Interestingly, we found that some DEPs were significantly up-regulated, such as HSPs: HSPA1B, HSPA1A, HSPA6, HSPA8 and DNAJB1, cytochrome P450 enzymes (CYP1A1 and CYP1B1), transcriptional factor DDIT4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.G). UPR\u003csup\u003emt\u003c/sup\u003e is a rapid and specific stress response expressing eIF2α phosphorylation, ATF4, and CHOP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExtended Data\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. \u003cb\u003eG-TPP induce ISR rapidly and specifically\u003c/b\u003e. \u003cb\u003eA-C\u003c/b\u003e, western blot analysis of eIF2α-P, eIF2α, ATF4, ATF5 and CHOP level treated with DMSO and G-TPP for indicated timepoint in SMMC7721 cells and MEFs cells, respectively. \u003cb\u003eD-E\u003c/b\u003e, qPCR of ATF4 and ATF5 mRNA in SMMC7721 cells with or without G-TPP treatment (mean of levels relative to untreated\u0026thinsp;\u0026plusmn;\u0026thinsp;s.d.; n\u0026thinsp;=\u0026thinsp;3 biological replicates). \u003cb\u003eF\u003c/b\u003e, western blot analysis of LONP1, CLPP, HSPE1 and HSP60 level upon treatment of HAP1 cells with DMSO and G-TPP for 2 h and 4 h. \u003cb\u003eG\u003c/b\u003e, qPCR of LONP1, CLPP, HSPE1 and HSP60 mRNA in HAP1 cells with or without G-TPP treatment (mean of levels relative to untreated\u0026thinsp;\u0026plusmn;\u0026thinsp;s.d. ; n\u0026thinsp;=\u0026thinsp;3 biological replicates). \u003cb\u003eH\u003c/b\u003e, western blot analysis of eIF2α-P, eIF2α, ATF4 and BIP level treatment of HAP1 cells with DMSO and G-TPP for indicated timepoint. \u003cb\u003eI\u003c/b\u003e, qPCR of BIP mRNA in HAP1 cells with or without G-TPP treatment (mean of levels relative to untreated\u0026thinsp;\u0026plusmn;\u0026thinsp;s.d.; n\u0026thinsp;=\u0026thinsp;3 biological replicates).\u003c/p\u003e \u003cp\u003e \u003cb\u003eUPR\u003c/b\u003e \u003csup\u003e \u003cb\u003emt\u003c/b\u003e \u003c/sup\u003e \u003cb\u003e-induced eIF2α phosphorylation is necessary for the expression of ATF4 and CHOP.\u003c/b\u003e eIF2α phosphorylation, ATF4, and CHOP was induced with G-TPP-induced UPR\u003csup\u003emt\u003c/sup\u003e in our study. Hence, we investigated the relationship between each other. HAP1 cells were treated with ISRIB (ISR inhibitor) for 2 h. The results showed that eIF2α phosphorylation was not inhibited while the protein expression of ATF4 and CHOP were completely decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Meanwhile, the transcriptional expression of CHOP was distinctly downregulated with G-TPP treatment compared to the DMSO group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These results indicate that ISRIB blocks the effects downstream of eIF2α phosphorylation.\u003c/p\u003e \u003cp\u003eFurtherly, eIF2α S51A\u003csup\u003e+/\u0026minus;\u003c/sup\u003e MEFs cells and wild-type (WT) cells were treated with G-TPP for 4 h and DMSO as a control. The results showed that eIF2α phosphorylation was induced in both MEFs WT cells and eIF2α S51A\u003csup\u003e+/\u0026minus;\u003c/sup\u003e cells, while the protein expression of ATF4 and CHOP in eIF2α S51A\u003csup\u003e+/\u0026minus;\u003c/sup\u003e cells was significantly lower than that of WT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Overall, eIF2α phosphorylation is essential for ATF4 and CHOP expression induction.\u003c/p\u003e \u003cp\u003eTo identify whether the ATFS-1 homologous gene exists in mammalian cells, we divided cells into the cytosolic fraction and the nuclear fraction after the G-TPP treatment for 2 h. The Western blot results showed that it was ATF4 trafficked into the nucleus under UPR\u003csup\u003emt\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), suggesting that ATF4 may play a key role in mito-nuclear communication upon UPR\u003csup\u003emt\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo identify whether the expression of CHOP is controlled by ATF4, ATF4 knockdown cells were performed in HAP1 cells, and knockdown efficiency was determined at transcriptional and protein levels, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.E-F). eIF2α phosphorylation was increased in both ATF4 knockdown cells and control cells, and ATF4 protein expression was inhibited in ATF4 knockdown cells compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Protein expression of CHOP was not obtained in ATF4 knockdown cells. Meanwhile, CHOP's transcriptional expression was abolished in ATF4 knockdown cells compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). These results indicate that eIF2α is upstream of ATF4 and CHOP, activates ATF4 traffics into the nucleus, and initiates CHOP expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eROS generation and mitochondrial morphology was unchanged by G-TPP induced UPR\u003c/b\u003e \u003csup\u003e \u003cb\u003emt\u003c/b\u003e \u003c/sup\u003e. We identified that UPR\u003csup\u003emt\u003c/sup\u003e was activated through eIF2α phosphorylation in our study. Hence, we investigated the influence of mitochondrial function and morphology upon UPR\u003csup\u003emt\u003c/sup\u003e through eIF2α phosphorylation. HAP1 cell lines were treated with G-TPP at indicated timepoint, DMSO as a negative control, and TBHP as a positive control. The results showed that the generation of reactive oxygen species (ROS) was not increased with G-TPP treatment compared to the control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In addition, mitochondrial morphology through a transmission electron microscope (TEM) with G-TPP treatment for 2 and 4 h in HAP1 cells. The results showed that mitochondria morphology was normal, and elliptical, with a clear bilayer membrane structure and clear ridge structure with G-TPP treatment compared to the DMSO group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C). Meanwhile, the morphology of ER was not changed with G-TPP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Altogether, activated UPR\u003csup\u003emt\u003c/sup\u003e does not affect mitochondrial morphology and ROS generation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHRI mediates UPR\u003c/b\u003e \u003csup\u003e \u003cb\u003emt\u003c/b\u003e \u003c/sup\u003e \u003cb\u003einduced by mitochondrial unfolded proteins.\u003c/b\u003e We demonstrated that UPR\u003csup\u003emt\u003c/sup\u003e was induced through eIF2α phosphorylation. To identify the key kinase for activation of UPR\u003csup\u003emt\u003c/sup\u003e, four known eIF2α kinases were knocked out individually in both HAP1 cell lines and SMMC7721 cell lines using a CRISPR-Cas9 gene-editing system with single-guided RNAs. Knockout efficiency was confirmed at the protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). In both SMMC7721 and HAP1 cell lines deficient in PERK, PKR, and GCN2, respectively, the protein expression of ATF4 and eIF2α phosphorylation was moderately decreased compared to WT cells, especially the downregulation of ATF4. However, ATF4 protein and eIF2α phosphorylation were inhibited in HRI\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-H and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-H). The transcriptional expression of CHOP was significantly increased in HAP1 cell lines deficient in PERK, PKR, and GCN2 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), respectively. However, the transcriptional expression of CHOP was completely inhibited in HAP1 cell lines deficient in HRI (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-L). The same results were confirmed in SMMC7721 cell lines. The transcriptional expression of CHOP was significantly increased in knockout cells of PERK and PKR (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), respectively, excepting GCN2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and HRI\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003ecells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-L). These results demonstrate that HRI mediates UPR\u003csup\u003emt\u003c/sup\u003e with the accumulation of mitochondrial unfolded proteins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExtended data\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. \u003cb\u003eHRI mediate ISR induced by mitochondrial unfolded proteins\u003c/b\u003e. \u003cb\u003eA-D\u003c/b\u003e, western blot analysis of knockout efficiency of PERK, PKR, GCN2 and HRI in SMMC7721 cells, respectively. \u003cb\u003eE-F\u003c/b\u003e, western blot analysis of eIF2α-P, eIF2α, ATF4 and CHOP level treated with DMSO and G-TPP for 2 h in SMMC7721 wild-type or KO cells of PERK, GCN2, PKR and HRI, respectively. \u003cb\u003eI-L\u003c/b\u003e, qPCR of CHOP mRNA with or without G-TPP treatment in SMMC7721 wild-type or KO cells of PERK, GCN2, PKR and HRI, respectively (mean of levels relative to untreated\u0026thinsp;\u0026plusmn;\u0026thinsp;s.d.; n\u0026thinsp;=\u0026thinsp;3 biological replicates).\u003c/p\u003e \u003cp\u003e \u003cb\u003eReduced mitochondrial protein translation is specifically induced by mitochondrial unfolded protein.\u003c/b\u003e eIF2α phosphorylation inhibits global protein translation, and we suppose that mitochondrial protein translation rather than cytoplasmic protein translation is mainly suppressed to alleviate the mitochondrial load. Cells were divided into the cytoplasm fraction and the nuclear fraction after G-TPP treatment for 4 h in HAP1 cells. The Western blot results showed that the expression of cytochrome-c oxidase subunit IV (COX IV), a subunit of the electron transport chain (ETC) located within the inner membrane, citrate synthase (CS), translocase of the inner membrane channel subunit 23 (TIM23), the major channel of the TIM complex proteins was decreased obviously, and the number of mitochondria proteins input were also decreased significantly with GTPP treatment compared to DMSO treatment. Lysosome-associated membrane protein1 (LAMP1), glutamic-oxaloacetate transaminase1 (GOT1), and GOT2 belong to cytoplasmic proteins. The quantity of LAMP1, GOT1, and GOT2 in the cytosol was not decreased significantly with G-TPP, G-TPP\u0026thinsp;+\u0026thinsp;ISRIB, respectively, compared to DMSO treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results confirmed our speculation that G-TPP-induced UPR\u003csup\u003emt\u003c/sup\u003e causes the translation decrease of mitochondrial proteins and the number of mitochondria proteins imported, while there is no significant effect on the translation of cytoplasmic protein. A pro-survival stress response will be induced by inhibiting mitochondrial protein translation and the number of mitochondria proteins imported to relieve mitochondrial load and promote mitochondrial proteostasis recovery upon UPR\u003csup\u003emt\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMitochondrial recruitment of HRI and interaction with many mitochondrial proteins upon G-TPP inducedUPR\u003c/b\u003e \u003csup\u003e \u003cb\u003emt\u003c/b\u003e \u003c/sup\u003e. In this study, we confirmed that HRI was involved in G-TPP-induced UPR\u003csup\u003emt\u003c/sup\u003e. Hence, we want to know how HRI initiates this process, and we suppose that HRI in the cytosol may migrate to the vicinity of mitochondria and interact with relative proteins. Hence, an EGFP-MAVS-TurboID plasmid was constructed, which contains TurboID proximity labeling. The immunoprecipitant results showed that only HRI was captured nearby of MAVS compared to the other eIF2α kinases, PERK, PKR, and GCN2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). These results suggest that HRI in the cytosol migrates to mitochondrial out member and initiates downstream signal pathway during G-TPP induced UPR\u003csup\u003emt\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFurtherly, we speculate that HRI may play with many proteins to initiate UPR\u003csup\u003emt\u003c/sup\u003e. Hence, recovering the expression of HRI in HAP1 cell lines deficient in HRI was constructed, containing TurboID proximity labeling (HAP1-sgHRI-HRI-TurboID), and the HRI protein expression was confirmed by Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). HAP1-sgHRI-HRI-TurboID cell lines were treated with G-TPP for 2 and 4 h, respectively, and IP and LC-MS/MS were performed. The results showed that 30 proteins with DMSO treatment, 58 proteins with G-TPP treatment for 2 h, and 86 proteins with G-TPP treatment were obtained for 4 h. 28 proteins and 56 proteins were increased with G-TPP treatment for 2 and 4 h, respectively, compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Furthermore, the GO function analysis was screened through a hypergeometric distribution algorithm to show enrichment score (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05), enrichment significance for BP (biological process), CC (cellular component), and MF (molecular function) were analyzed by Fisher's test, and the top 10 terms were listed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), the number of terms differed significantly between G-TPP treatment group and DMSO treatment group. In addition, KEGG signaling pathways analysis also differed significantly with G-TPP treatment compared to the control group. The top 30 terms were shown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). These results suggest that HRI interacts with many proteins during G-TPP-induced UPR\u003csup\u003emt\u003c/sup\u003e and initiate the different cellular function and signaling pathways to combat this cellular response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe mitochondrial-targeted chaperone inhibitor (G-TPP) was used in our study, which induce specially UPR\u003csup\u003emt [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. The study has shown that acute treatment of G-TPP (6 h) was performed for induction of UPR\u003csup\u003emt\u003c/sup\u003e, which induces the up-expression of HSPD1 (1.7 fold), ATF4 (2.1 fold), and CHOP (11.3 fold), particularly the expression of CHOP in Hela cells \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Rapid and specific stress response was also induced with the up-expression of eIF2α phosphorylation at 30 min and ATF4 protein at 1 h, respectively, after G-TPP treatment in our study. Meanwhile, the expression of CHOP was also activated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, E and Extended data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In addition, UPR\u003csup\u003eER\u003c/sup\u003e was not activated at transcriptional and protein levels (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-I). It suggests that UPR\u003csup\u003emt\u003c/sup\u003e is a more rapid and special stress response with pharmacological treatment to restore cellular homeostasis, which is consistent with the previous study that UPR\u003csup\u003eER\u003c/sup\u003e results in UPR\u003csup\u003emt\u003c/sup\u003e and mitochondrial dysfunction in an ATF4-dependent manner, but mitochondrial stressors-induced UPR\u003csup\u003emt\u003c/sup\u003e did not activate UPR\u003csup\u003eER\u003c/sup\u003e in alveolar epithelial cells, such as oligomycin, doxycycline, and antimycin A treatment \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Meanwhile, mitochondrial morphology and the generation of ROS were not changed upon UPR\u003csup\u003emt\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). It is speculated that the activation of UPR\u003csup\u003emt\u003c/sup\u003e aims to promote a cellular survival response and restore mitochondrial proteostasis quickly.\u003c/p\u003e \u003cp\u003eUPR\u003csup\u003emt\u003c/sup\u003e is proposed to be triggered by the accumulation of unfolded or misfolded proteins, and it has been shown that a misfolded mitochondrial matrix protein ∆OTC can trigger UPR\u003csup\u003emt\u003c/sup\u003e in mammalian cells. The evidence-based on activating CHOP and its target genes in Hela cells with the overexpression of ∆OTC, such as Cpn60, Cpn10, mtDnaJ, and ClpP, represents a classical UPR\u003csup\u003emt [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. However, we found that eIF2α phosphorylation, the expression of ATF4 and CHOP are the main regulators with G-TPP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Extended data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B), while chaperone and protease were not up-regulated in our study (Extended data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-G), which consistent with some recent studies that ATF4 and CHOP are main regulators\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUPR\u003csup\u003emt\u003c/sup\u003e is a protein quality control mechanism that strives to achieve mitochondrial proteostasis in the face of misfolded proteins because of the reliance on both the nuclear and mitochondrial genomes, a perturbation of the coordination of these genomes results in a mito-nuclear imbalance\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Mitochondrial protein translation and mitochondrial protein import efficiency rather than cytoplasmic proteins were reduced through eIF2α phosphorylation in our study (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The study has shown that compromised mitochondrial protein import acts as a signal for the activation of UPR\u003csup\u003emt\u003c/sup\u003e, and MTS of ATFS-1 as a sensor for this signal in \u003cem\u003eC. elegans.\u003c/em\u003e ATFS-1 accumulates in the cytosol and traffics into the nucleus, which activates mitochondrial-specific chaperon and protease that promotes fold under stress conditions in \u003cem\u003eC. elegans\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. The mito-nuclear retrograde response was regulated by ATF4 in our study (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), which may play a homologous role with ATFS-1 in the mammal. However, a study has demonstrated that ATF5, homologous to ATFS-1, regulated UPR\u003csup\u003emt\u003c/sup\u003e during mitochondrial dysfunction in mammalian cells \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. We could not detect it, and this discrepancy is unknown.\u003c/p\u003e \u003cp\u003eThe previous finding has confirmed that the transcriptional and protein expression of ATF4 was induced by other mitochondrial stressors, such as Doxycycline, Actinonin, and ionophore carbonyl cyanide-4 (trifluoromethoxy), phenylhydrazone (FCCP), MitoBloCK-6 (MB) and CDDO, these drugs can alter mitochondrial proteostasis \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. In addition, eIF2α phosphorylation and ATF4 pathway were induced by G-TPP treatment in different cells, such as MEFs and Hela cells, and in SMMC7721 and HAP1 cell lines confirmed in our study, it demonstrates that UPR\u003csup\u003emt\u003c/sup\u003e can be induced in different cell lines. In combination, eIF2α phosphorylation, the protein expression of ATF4, and the transcriptional expression of CHOP are induced upon UPR\u003csup\u003emt\u003c/sup\u003e, but it is different from prototypical UPR\u003csup\u003emt\u003c/sup\u003e in worms \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e and mammals \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Meanwhile, some DEPs were determined, such as HSPs: HSPA1B, HSPA1A, HSPA6, HSPA8, and DNAJB1, cytochrome P450 enzymes (CYP1A1 and CYP1B1), transcription factor DDIT4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), which consistent with the previous study that heat shock proteins (HSPA1A, HSPA1B, and DNAJB1) were up-regulated steeply in DELE1- or HRI- knockout cells, and cells treated with ISRIB and CCCP \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. It suggests that an alternative stress response program may be induced, and the mechanism needs further elaboration.\u003c/p\u003e \u003cp\u003eCHOP, another key transcriptional factor of ISR, was activated by ISR through ATF4. CHOP was also induced during mitochondrial stress \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. The study has shown that the translational expression of CHOP was activated in the context of mammalian UPR\u003csup\u003emt\u003c/sup\u003e, such as the knockdown of \u003cem\u003esurf1\u003c/em\u003e and \u003cem\u003eTIM23\u003c/em\u003e, respectively \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Our study found that CHOP was up-regulated at the transcriptional and translational levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E). Both HSPE1 and CLPP are deemed as targets of CHOP. However, we did not detect any change in these genes at the transcriptional and translational levels (Extended data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-G). In addition, CHOP can tune cellular response to different types of stress by initiating apoptotic and non-apoptotic programs \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Hence, the next works should be addressed the role of CHOP in the maintenance of mitochondrial proteostasis.\u003c/p\u003e \u003cp\u003eeIF2α phosphorylation is the main regulator upon UPR\u003csup\u003emt\u003c/sup\u003e. Our study found that the HRI branch of eIF2α kinases is involved in G-TPP-induced UPR\u003csup\u003emt\u003c/sup\u003e. These results are consistent with the previous studies that OMA1 cleaved DELE1 into DELE1-S in the mitochondria and then redistributed to the cytosol, where it interacted with HRI to induce the expression of ATF4 or CHOP in response to mitochondrial stress \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. We expounded the downstream mechanism furtherly that HRI in the cytosol activates eIF2α phosphorylation and the translational expression of ATF4, which traffics into the nucleus and initiates CHOP expression in response to UPR\u003csup\u003emt\u003c/sup\u003e. The activation of eIF2α phosphorylation mainly blocks the decrease of mitochondrial protein translation and the number of mitochondrial proteins imported. Meanwhile, HRI interacts with many proteins in the vicinity of mitochondria screened by MS and initiate downstream molecule through different signaling pathways. However, which protein interacts with HRI needs to be furtherly confirmed.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, we unraveled the mechanism of UPR\u003csup\u003emt\u003c/sup\u003e through eIF2α phosphorylation in the mammal. The schematic diagram was summarized (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), which will provide the theoretical foundation or therapeutic target for disease resulting from UPR\u003csup\u003emt\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Southwest Minzu University Double World-Class Project（XM2023012); Southwest Minzu University Research Startup Funds (16011211013/RQD202100), Natural Science Foundation of Sichuan Province (2022NSFSC0073), CAAS innovation programme fund.\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eX.Q., Z.Z., and Y.L. contributed to the conceptualization of the study. X.Q., Y.Y., and Y.W. were responsible for the methodology design. Y.W., and X.Q. conducted to verify the experimental results. Y.W. and X.Q. wrote the original draft. Z.Z. and Y.L. were involved in writing, reviewing, editing, providing supervision, and funding acquisition. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang L (2016) Mitochondrial purine and pyrimidine metabolism and beyond. Nucleosides Nucleotides Nucleic Acids 35(10\u0026ndash;12):578\u0026ndash;594. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/15257770.2015.1125001\u003c/span\u003e\u003cspan address=\"10.1080/15257770.2015.1125001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWellen KE, Thompson CB (2012) A two-way street: reciprocal regulation of metabolism and signalling. Nat Rev Mol Cell Biol 13(4):270\u0026ndash;276. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nrm3305\u003c/span\u003e\u003cspan address=\"10.1038/nrm3305\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmyrnias I (2021) The mitochondrial unfolded protein response and its diverse roles in cellular stress. Int J Biochem Cell Biol 133:105934. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biocel.2021.105934\u003c/span\u003e\u003cspan address=\"10.1016/j.biocel.2021.105934\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBohovych I, Khalimonchuk O (2016) Sending Out an SOS: Mitochondria as a Signaling Hub. Front Cell Dev Biology 4:109. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fcell.2016.00109\u003c/span\u003e\u003cspan address=\"10.3389/fcell.2016.00109\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeinberg SE, Sena LA, Chandel NS (2015) Mitochondria in the regulation of innate and adaptive immunity. Immunity 42(3):406\u0026ndash;417. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.immuni.2015.02.002\u003c/span\u003e\u003cspan address=\"10.1016/j.immuni.2015.02.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhola PD, Letai A (2016) Mitochondria-Judges and Executioners of Cell Death Sentences. Mol Cell 61(5):695\u0026ndash;704. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.molcel.2016.02.019\u003c/span\u003e\u003cspan address=\"10.1016/j.molcel.2016.02.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGanguly G, Chakrabarti S, Chatterjee U, Saso L (2017) Proteinopathy, oxidative stress and mitochondrial dysfunction: cross talk in Alzheimer's disease and Parkinson's disease. Drug Des Devel Ther 11:797\u0026ndash;810. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2147/DDDT.S130514\u003c/span\u003e\u003cspan address=\"10.2147/DDDT.S130514\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun N, Youle RJ, Finkel T (2016) The Mitochondrial Basis of Aging. Mol Cell 61(5):654\u0026ndash;666. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.molcel.2016.01.028\u003c/span\u003e\u003cspan address=\"10.1016/j.molcel.2016.01.028\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuomalainen A, Battersby BJ (2018) Mitochondrial diseases: the contribution of organelle stress responses to pathology. Nat Rev Mol Cell Biol 19(2):77\u0026ndash;92. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nrm.2017.66\u003c/span\u003e\u003cspan address=\"10.1038/nrm.2017.66\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Q, Wang JH, Ilya VL, Stan S, Michael TR, Nicholas JH (2002) A mitochondrial specific stress response in mammalian cells. EMBO J 21(17):4411\u0026ndash;4419. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/emboj/cdf445\u003c/span\u003e\u003cspan address=\"10.1093/emboj/cdf445\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNargund AM, Pellegrino MW, Fiorese CJ, Baker BM, Haynes CM (2012) Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation. Science 337(6094):587\u0026ndash;590. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.1223560\u003c/span\u003e\u003cspan address=\"10.1126/science.1223560\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaynes CM, Yang Y, Blais SP, Neubert TA, Ron D (2010) The matrix peptide exporter HAF-1 signals a mitochondrial UPR by activating the transcription factor ZC376.7 in C. elegans. Mol Cell 37(4):529\u0026ndash;540. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.molcel.2010.01.015\u003c/span\u003e\u003cspan address=\"10.1016/j.molcel.2010.01.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoutkooper RH, Mouchiroud L, Ryu D, Moullan N, Katsyuba E, Knott G et al (2013) Mitonuclear protein imbalance as a conserved longevity mechanism. Nature 497(7450):451\u0026ndash;457. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nature12188\u003c/span\u003e\u003cspan address=\"10.1038/nature12188\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmrita M, Nargund CJF, Mark W, Pellegrino P, Deng, Cole, Haynes M (2015) Mitochondrial and nuclear accumulation of the transcription factor ATFS-1 promotes OXPHOS recovery during the UPR(mt). Mol Cell 58(1):123\u0026ndash;133. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.molcel.2015.02.008\u003c/span\u003e\u003cspan address=\"10.1016/j.molcel.2015.02.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin Y-F, Schulz AM, Pellegrino MW, Lu Y, Shaham S, Haynes CM (2016) Maintenance and propagation of a deleterious mitochondrial genome by the mitochondrial unfolded protein response. Nature 533(7603):416\u0026ndash;419. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nature17989\u003c/span\u003e\u003cspan address=\"10.1038/nature17989\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFiorese CJ, Schulz AM, Lin YF, Rosin N, Pellegrino MW, Haynes CM (2016) The Transcription Factor ATF5 Mediates a Mammalian Mitochondrial UPR. Curr Biol 26(15):2037\u0026ndash;2043. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cub.2016.06.002\u003c/span\u003e\u003cspan address=\"10.1016/j.cub.2016.06.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuiros PM, Prado MA, Zamboni N, D'Amico D, Williams RW, Finley D et al (2017) Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals. J Cell Biol 216(7):2027\u0026ndash;2045. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1083/jcb.201702058\u003c/span\u003e\u003cspan address=\"10.1083/jcb.201702058\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartinez-Reyes I, Sanchez-Arago M, Cuezva JM (2012) AMPK and GCN2-ATF4 signal the repression of mitochondria in colon cancer cells. Biochem J 444(2):249\u0026ndash;259. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1042/BJ20111829\u003c/span\u003e\u003cspan address=\"10.1042/BJ20111829\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMichel S, Canonne M, Arnould T, Renard P (2015) Inhibition of mitochondrial genome expression triggers the activation of CHOP-10 by a cell signaling dependent on the integrated stress response but not the mitochondrial unfolded protein response. Mitochondrion 21:58\u0026ndash;68. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.mito.2015.01.005\u003c/span\u003e\u003cspan address=\"10.1016/j.mito.2015.01.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePakos-Zebrucka K, Koryga I, Mnich K, Ljujic M, Samali A, Gorman AM (2016) The integrated stress response. EMBO Rep 17(10):1374\u0026ndash;1395. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.15252/embr.201642195\u003c/span\u003e\u003cspan address=\"10.15252/embr.201642195\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaker BM, Nargund AM, Sun T, Haynes CM (2012) Protective coupling of mitochondrial function and protein synthesis via the eIF2α kinase GCN-2. PLoS Genet 8(6):e1002760. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pgen.1002760\u003c/span\u003e\u003cspan address=\"10.1371/journal.pgen.1002760\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBao XR, Ong SE, Goldberger O, Peng J, Sharma R, Thompson DA et al (2016) Mitochondrial dysfunction remodels one-carbon metabolism in human cells. Elife 16(5):e10575. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7554/eLife.10575\u003c/span\u003e\u003cspan address=\"10.7554/eLife.10575\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeske BF, Fusakio ME, Zhou D, Shan J, McClintick JN, Kilberg MS et al (2013) CHOP induces activating transcription factor 5 (ATF5) to trigger apoptosis in response to perturbations in protein homeostasis. Mol Biol Cell 24(15):2477\u0026ndash;2490. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1091/mbc.E13-01-0067\u003c/span\u003e\u003cspan address=\"10.1091/mbc.E13-01-0067\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMunch C, Harper JW (2016) Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation. Nature 534(7609):710\u0026ndash;713. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nature18302\u003c/span\u003e\u003cspan address=\"10.1038/nature18302\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFabienne C, Fiesel EDJ, Hudec R (2017) Wolfdieter Springer. Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy. Oncotarget. 8(63):106233\u0026ndash;106248. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18632/oncotarget.22287\u003c/span\u003e\u003cspan address=\"10.18632/oncotarget.22287\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDingyuan Jiang HC, Xie N, Banerjee S, Liu R, Dai H, Victor J, Thannickal GL (2020) )ATF4 Mediates Mitochondrial Unfolded Protein Response in Alveolar Epithelial Cells. Am J Respir Cell Mol Biol 63(4):478\u0026ndash;489. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1165/rcmb.2020-0107OC\u003c/span\u003e\u003cspan address=\"10.1165/rcmb.2020-0107OC\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSutandy FXR, G\u0026ouml;\u0026szlig;ner I, Tascher G, M\u0026uuml;nch C (2023) A cytosolic surveillance mechanism activates the mitochondrial UPR. Nature 618(7966):849\u0026ndash;854. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41586-023-06142-0\u003c/span\u003e\u003cspan address=\"10.1038/s41586-023-06142-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee-Glover LP, Shutt TE (2023) Mitochondrial quality control pathways sense mitochondrial protein import. Trends Endocrinol Metabolism 2300243\u0026ndash;00246. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tem.2023.11.004\u003c/span\u003e\u003cspan address=\"10.1016/j.tem.2023.11.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRolland SG, Schneid S, Schwarz M, Rackles E, Fischer C, Haeussler S et al (2019)Compromised Mitochondrial Protein Import Acts as a Signal for UPR(mt). Cell Rep 28(7):1659\u0026ndash;1669e1655. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.celrep.2019.07.049\u003c/span\u003e\u003cspan address=\"10.1016/j.celrep.2019.07.049\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFessler E, Eckl EM, Schmitt S, Mancilla IA, Meyer-Bender MF, Hanf M et al (2020)A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol. Nature 579(7799): 433\u0026ndash;437. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41586-020-2076-4\u003c/span\u003e\u003cspan address=\"10.1038/s41586-020-2076-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJovaisaite V, Auwerx J (2015) The mitochondrial unfolded protein response-synchronizing genomes. Curr Opin Cell Biol. 2015 33:74\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ceb.2014.12.003\u003c/span\u003e\u003cspan address=\"10.1016/j.ceb.2014.12.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOliveira AN, Hood DA (2018) Effect of Tim23 knockdown in vivo on mitochondrial protein import and retrograde signaling to the UPR(mt) in muscle. Am J Physiol Cell Physiol 315(4):C516\u0026ndash;C526. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1152/ajpcell.00275.2017\u003c/span\u003e\u003cspan address=\"10.1152/ajpcell.00275.2017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo X, Aviles G, Liu Y, Tian R, Unger BA, Lin YT et al (2020) Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway. Nature 579(7799):427\u0026ndash;432. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41586-020-2078-2\u003c/span\u003e\u003cspan address=\"10.1038/s41586-020-2078-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"mitochondrial unfolded protein response, eIF2α phosphorylation, heme-regulated inhibitor, mitochondrial proteostasis","lastPublishedDoi":"10.21203/rs.3.rs-4121132/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4121132/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMitochondrial unfolded protein response (UPR\u003csup\u003emt\u003c/sup\u003e) is triggered through eIF2α phosphorylation in mammal. However, the mechanisms of UPR\u003csup\u003emt\u003c/sup\u003e activation and the influence on mitochondrial protein translation through eIF2α phosphorylation remain unclear. In this study, we confirmed that UPR\u003csup\u003emt\u003c/sup\u003e was a rapid and specific stress response through eIF2α phosphorylation with pharmacological induction, along with the protein expression of eIF2α phosphorylation, ATF4, and CHOP. Meanwhile, with the up-regulation of some chaperones, cytochrome P450 enzymes, and DDIT4 determined by RNA-Seq and ribosome profiling, eIF2α phosphorylation is essential for expressing ATF4 and CHOP, then ATF4 traffics into the nucleus and initiates CHOP expression. In addition, the generation of ROS and mitochondrial morphology was unchanged under GTPP induced UPR\u003csup\u003emt\u003c/sup\u003e. Furthermore, we unraveled the mechanism that HRI kinase mediates UPR\u003csup\u003emt\u003c/sup\u003e induced with mitochondrial unfolded proteins by CRISPR-Cas9 technology and mitochondrial recruitment of HRI and interaction with other proteins. Meanwhile, we confirmed that mitochondrial protein translation and the number of mitochondrial protein imports were inhibited through eIF2α phosphorylation with the accumulation of mitochondrial unfolded protein. These findings provide the molecular mechanism of UPR\u003csup\u003emt\u003c/sup\u003e and the impact on cellular protein translation, which will offer a novel insights into the functional research of UPR\u003csup\u003emt\u003c/sup\u003e, including its implications for human diseases and pathobiology.\u003c/p\u003e","manuscriptTitle":"Unfolded proteins in the mitochondria activate HRI and shut-down of mitochondrial protein translation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-21 08:59:02","doi":"10.21203/rs.3.rs-4121132/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5ab3040d-ab59-47d8-8107-e97e53bfcefb","owner":[],"postedDate":"March 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-21T18:44:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-21 08:59:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4121132","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4121132","identity":"rs-4121132","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}