Journal: The Journal of Cell Biology

Article Title: Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals

doi: 10.1083/jcb.201702058

Figure Lengend Snippet: ATF4 is activated through the ISR. (A) Western blot analysis showing the increased phosphorylation of eIF2α (Ser51) upon 6 h of treatment with the different mitochondrial stressors. Bottom, ratio between P-eIF2α and eIF2α total levels. (B and C) mRNA expression analysis of ATF4 and its target genes, CHOP (DDIT3) , ASNS , CHAC1 , PCK2 , and the ER stress marker BIP , upon 6 h of treatment with the different mitochondrial stressors and the ER stressor tunicamycin (Tn at 2.5 µg/ml) in HeLa cells, together with the inhibitor of the integrated stress response (ISRIB at 500 nM). (D) Boxplots showing an increase in basal and ATP-dependent respiration of HeLa cells treated with 500 nM of ISRIB for 24 h. OCR: oxygen consumption rate. (E) mRNA expression analysis of eIF2α kinases upon knock down with specific shRNAs. Data are presented as mean ± SEM of two independent shRNAs for each gene. Statistical differences were calculated compared with pLKO1. (F) mRNA expression analysis of ATF4 and some of its target genes upon knock down of the eIF2α kinases and 6 h of treatment with FCCP. Data are presented as mean ± SEM of two independent shRNAs for each gene. No statistical differences were found between the FCCP treated conditions. All experiments were independently performed at least two times, using triplicates for each condition; data are presented as mean ± SEM of a representative experiment; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Acti, actinonin; Dox, doxycycline; MB, MitoBloCK-6.

Article Snippet: The following primary antibodies were used: mouse anti–β-actin (sc-47778; Santa Cruz Biotechnology, Inc.); mouse anti-HSP90 (BD Biosciences, 610418); OXPHOS antibody cocktail (mouse mAbs, ab110413; Abcam); mouse anti-HSP60 (ADI-SPA-806; Enzo Life Sciences); mouse anti-CLPP (WH0008192M1-100; Sigma-Aldrich); mouse anti-HSPA9 (ABIN361739; Antibodies online); rabbit anti-LONP1 (HPA002192; Sigma-Aldrich); rabbit anti-OTC (sc-102051; Santa Cruz Biotechnology, Inc.); mouse anti-OPA1 (BD, 612606); rabbit anti-CREB-2 (ATF4, sc-200; Santa Cruz Biotechnology, Inc.); phospho-eIF2α (Ser51) rabbit mAb (9721; Cell Signaling Technology); eIF2α rabbit mAb (9722; Cell Signaling Technology); and α tubulin (T5168; Sigma-Aldrich).

Techniques: Western Blot, Phospho-proteomics, Expressing, Marker, Knockdown

Journal: The Journal of Cell Biology

Article Title: Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals

doi: 10.1083/jcb.201702058

Figure Lengend Snippet: Genetic link of ATF4 and mitochondrial stress in human and mouse populations. (A and B) Multitissue correlation network analysis of transcript levels across (A) 49 human tissues in GTEx and (B) 16 mouse tissues in the BXD genetic reference population. Nodes represent the transcripts and the width of the ties among nodes indicate the probability to show a significant positive correlation in all tissues analyzed. The human network shows a tighter clustering likely caused by the higher number of tissues and samples per tissues. (C) Heatmap representing the KEGG pathway analysis of the top negative-correlated genes across 49 tissues using GTEx data for ATF4 , CEBPB , DDIT3/CHOP , and NCOR1 (used as positive control). Color key represents the negative logarithm base 10 of the p-value of each pathway obtained in the analysis. (D) Expression quantitative trait locus (eQTL) analysis of Atf4 transcript level in the prefrontal cortex. The yellow mark represents the Atf4 gene locus on chromosome 15, whereas the red mark represent the position of a trans-eQTL for Atf4 expression levels on chromosome 1 (Chr1). (E) eQTL mapping of Atf4 transcript levels across several tissues identifies a common and strong trans-eQTL on chromosome 1 (170–180 Mb). (F) Representation of the chromosome 1 locus (170–180 Mb) containing 142 genes, 6 of which have nonsynonymous substitutions, and only one gene, Fh1 , encodes a mitochondrial protein. Fh1 contains a nonsynonymous sequence variant (A296T; rs32536342 ) that segregates in the BXDs. This sequence variant regulates the expression levels of Fh1 , which in turn regulates Atf4 expression. (G) mRNA expression analysis of HeLa cells after knockdown of fumarate hydratase (shFH) and treatment with monomethyl fumarate at 2.5 mM for 24 h. Data are presented as mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (H) Enrichment score plot from GSEA using microarray data from renal cysts of mice with renal tubule specific inactivation of Fh1 ( Fh1 −/− ; GSE29988 ). The list of mitochondrial stress genes (mt-stress genes; Table S5) was used as the gene set of interest. FDR q-val: false discovery rate adjusted p-value; NES, normalized enrichment score; NOM p-val, nominal p-value. (I) Scheme summarizing our working hypothesis. Mitochondrial stress stimulates the phosphorylation of the eIF2α, which inhibits cytosolic translation and activates the ATF4 pathway. At the same time, mitochondrial stress also reduces the expression of MRPs to inhibit mitochondrial translation and protect mitochondrial function.

Article Snippet: The following primary antibodies were used: mouse anti–β-actin (sc-47778; Santa Cruz Biotechnology, Inc.); mouse anti-HSP90 (BD Biosciences, 610418); OXPHOS antibody cocktail (mouse mAbs, ab110413; Abcam); mouse anti-HSP60 (ADI-SPA-806; Enzo Life Sciences); mouse anti-CLPP (WH0008192M1-100; Sigma-Aldrich); mouse anti-HSPA9 (ABIN361739; Antibodies online); rabbit anti-LONP1 (HPA002192; Sigma-Aldrich); rabbit anti-OTC (sc-102051; Santa Cruz Biotechnology, Inc.); mouse anti-OPA1 (BD, 612606); rabbit anti-CREB-2 (ATF4, sc-200; Santa Cruz Biotechnology, Inc.); phospho-eIF2α (Ser51) rabbit mAb (9721; Cell Signaling Technology); eIF2α rabbit mAb (9722; Cell Signaling Technology); and α tubulin (T5168; Sigma-Aldrich).

Techniques: Positive Control, Expressing, Sequencing, Variant Assay, Knockdown, Microarray, Phospho-proteomics

Journal: Genome Biology

Article Title: A rare codon-based translational program of cell proliferation

doi: 10.1186/s13059-020-1943-5

Figure Lengend Snippet: Distinct codon usages are associated with transcriptome-wide translation efficiencies in different proliferative states. a Mean (± s.e.m.) percentage of wild-type NIH-3T3 cells in the G1 and G2/M phases of the cell cycle, as determined by Hoechst staining for DNA content, for cells grown in either 1%, 2%, 5%, or 10% FCS ( n = 3). b Representative western blot depicting the levels of phosphorylated eIF2α (p-eIF2α), total eIF2α, and the loading control tubulin, for cells growing in either 1%, 2%, 5%, or 10% FCS, as well as in two control conditions triggering stress responses (thapsigargin (TG) and hydrogen peroxide (H 2 O 2 )). c Mean (± s.e.m.) level of p-eIF2α normalized by total eIF2α estimated from western blots, for cells growing in either 1%, 2%, 5%, or 10% FCS, as well as in two control conditions triggering stress responses (thapsigargin (TG) and hydrogen peroxide (H 2 O 2 )) ( n = 3). d Scatter plot of the log2 fold changes in mRNA and ribosome protected fragments (RPF) in cells grown in 10% relative to 1% FCS. Shown are the mean values computed for each transcript from mRNA ( n = 4) and RPF ( n = 3) replicates. Transcripts upregulated and downregulated at ribosome density level (RD, defined as RPF fold change /mRNA fold change ), i.e., changed more than 50% in either direction, are shown in red and blue, respectively. Shown are the Pearson correlation coefficient and respective P value. The dashed line indicates equal change in mRNA and RPF levels. e Scatter plot of per codon scores among genes that are differentially expressed between G2/M and G1 cell cycle phases and genes with differential ribosome density (RD, defined as RPF fold change /mRNA fold change ) when cells are grown in 10% relative to 1% FCS. Shown are the Pearson correlation coefficient and respective P value. The dashed line indicates the linear regression between the two estimates. Boxplots showing the distribution of log2 fold changes in mRNA ( f ) and RD ( g ) between cells grown in 10% relative to 1% FCS, for genes with preferential expression in the G1 and G2/M cell cycle phases. Shown are the P values determined by the non-parametric Mann-Whitney U test. Boxes extend from the 25th to 75th percentiles (inter-quartile range (IQR)), horizontal lines represent the median, and whiskers indicate the lowest and highest datum within 1.5*IQR from the lower and upper quartiles, respectively. h Scatter plot of the log2 fold changes in mRNA and protein levels in cells grown in 10% relative to 1% FCS. Shown are the mean values computed for each transcript/protein from mRNA-seq ( n = 3) and proteomics ( n = 3) data. Transcripts upregulated and downregulated at translational level (defined as the residuals of the linear regression between Protein fold change and mRNA fold change ), i.e., changed more than 50% in either direction, are shown in red and blue, respectively. Shown are also the Pearson correlation coefficient and respective P value. The dashed line indicates the linear regression between the two measurements. i Scatter plot of per codon scores among genes that are differentially expressed between G2/M and G1 cell cycle phases and differential translated genes (see above) when cells are grown in 10% relative to 1% FCS. Shown are also the Pearson correlation coefficient and respective P value. The dashed line indicates the linear regression between the two estimates. j Correlation between codon scores of genes that are differentially expressed between G2/M and G1 cell cycle phases and differential translated genes (see above) estimated from cells growing in 10% relative to either 1%, 2%, or 5% FCS

Article Snippet: The following antibodies were used in the western blot analysis: Phospho-eIF2α (Ser51) Antibody (Cell Signaling #9721), eIF2 α Antibody (Cell Signaling #9722), and α-Tubulin Antibody (Merck Millipore #CP06).

Techniques: Staining, Western Blot, Expressing, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Misfolding, altered membrane distributions, and the unfolded protein response contribute to pathogenicity differences in Na,K-ATPase ATP1A3 mutations

doi: 10.1074/jbc.RA120.015271

Figure Lengend Snippet: Relevant pathways of the unfolded protein response (UPR). This diagram shows only a few of the components of the UPR but highlights three central elements that were examined here. The UPR begins with the recognition of luminal misfolded proteins by the chaperone BiP (GRP78), known to interact with both α and β Na,K-ATPase subunits during biosynthesis in Xenopus oocytes . The UPR initially activates defensive programs to expand the folding capacity of the ER. ( Left ) Activated IRE1α has a cytoplasmic nuclease activity needed to splice the XPB1 mRNA to XBP1s, changing its reading frame so that it encodes a master transcription factor for the defensive arm of the UPR. ( Middle ) Activated PERK phosphorylates an essential translation initiation factor, eIF2α. This attenuates translation, reducing the stress on the ER and making it possible for its resources to be redirected to defensive adaptations . ( Right ) If aggregated proteins nonetheless accumulate, the UPR activates apoptosis . The pathway utilizes the BCL-2 family proteins that regulate the formation of mitochondrial pores ( black circle ) by BAX and BAK to release cytochrome c . Current models hold that BAX and BAK are activated directly by members of one arm of the BCL-2 family (the direct activators), in response to various signals. Apoptosis is constitutively restrained, however, by BCL-2 itself, which binds and blocks BAX and BAK. For apoptosis to proceed, BCL-2 needs to be sequestered ( crosshatch ) by binding to BAD or other members of the sensitizer arm of the BCL-2 family. BAD binding to BCL-2 is attenuated by phosphorylation by a variety of prosurvival kinases ( , ). Dephosphorylation of BAD by protein phosphatase (PPase), for example, by calcineurin, the Ca 2+ -activated phosphatase, will activate proapoptotic signaling . In sum, dephosphorylation of BAD, at Ser99 in this case, is an indication that BAD is free to inactivate BCL-2, making it more likely that BAX and BAK will respond to direct activators, i.e. , sensitizing the cell to apoptosis.

Article Snippet: Antibodies against eIF2α (5324), phospho-eIF2α (9721), and phospho-BAD (4366) were from Cell Signaling Technologies (Danvers, MA, USA).

Techniques: Activity Assay, Binding Assay, Phospho-proteomics, De-Phosphorylation Assay

Journal: The Journal of Biological Chemistry

Article Title: Misfolding, altered membrane distributions, and the unfolded protein response contribute to pathogenicity differences in Na,K-ATPase ATP1A3 mutations

doi: 10.1074/jbc.RA120.015271

Figure Lengend Snippet: Unfolded protein response signaling responses to mutation. A , the spliced mRNA of the transcription factor XBP1s is an early unfolded protein response marker. Although 5 h of thapsigargin treatment (TG) gave a robust response, the apparent twofold increase in the spliced mRNA with chronic tet induction when compared with α3WT grown without tet or between α3WT and either of the mutation-expressing cells was not statistically significant. RQ stands for relative quantification and is the fold-change relative to the calibrator (actin), 2 -ddCt . The data are means ± SEM for 4, 6, 5, and 2 experiments. B , eIF2α phosphorylation was measured in lysates of cultures that were grown chronically in tet, or in tet + ouabain to inhibit endogenous Na,K-ATPase α1. The representative blot was stained first for phospho-eIF2α ( top ) and then stained again for actin as a loading control. Both images are shown. C , BAD phosphorylation measured in the same samples. The blot shown was the same as in B including the eIF2α and actin stain, but BAD runs at lower molecular weight and was stained after phospho-eIF2α and actin. The graphs for both B and C show the means ± SEM from four independent experiments. ∗ p < 0.01, ∗∗ p < 0.001, ∗∗∗ p < 0.0001. Ouabain appeared to protect the cells from dephosphorylation of BAD.

Article Snippet: Antibodies against eIF2α (5324), phospho-eIF2α (9721), and phospho-BAD (4366) were from Cell Signaling Technologies (Danvers, MA, USA).

Techniques: Mutagenesis, Marker, Expressing, Quantitative Proteomics, Phospho-proteomics, Staining, Control, Molecular Weight, De-Phosphorylation Assay

Journal: Molecular Metabolism

Article Title: Hepatic regulation of VLDL receptor by PPARβ/δ and FGF21 modulates non-alcoholic fatty liver disease

doi: 10.1016/j.molmet.2017.12.008

Figure Lengend Snippet: Increased Fgf21 expression in liver of Pparβ/δ- null mice attenuates VLDLR abundance. A, Oil Red O and hematoxylin-eosin staining of livers from male wild-type (WT) and Pparβ/δ- null mice injected intraperitoneally with IgG (9 μg/mouse) or a neutralizing antibody (Ab) (9 μg/mouse) against FGF21 together with DMSO or tunicamycin (Tunic) (3 mg kg −1 body weight). Scale bar: 100 μm. Mice were sacrificed at 14 h after treatment. B, Liver triglyceride levels. C, Vldlr mRNA abundance. ***p < 0.001, **p < 0.01 and *p < 0.05 vs. Pparβ/δ- null mice treated with IgG and DMSO. ## p < 0.01 and # p < 0.05 vs. Pparβ/δ- null mice treated with neutralizing antibody against FGF21 and DMSO. † p < 0.05 vs. Pparβ/δ- null mice treated with IgG and tunicamycin. Liver triglyceride levels (D) and Vldlr mRNA abundance (E) in the liver from WT and Fgf21 −/− mice. Data are presented as the mean ± S.D. (n = 5 per group). ***p < 0.001, **p < 0.01 and *p < 0.05 vs. wild-type mice. Immunoblot analyses of VLDLR, total and phospho-eIF2α and ATF4 (F) and total and phospho-Nrf2 (G) were performed in liver lysates. Data are presented as the mean ± S.D. (n = 5 per group). ***p < 0.001, **p < 0.01 and *p < 0.05 vs. wild-type mice.

Article Snippet: Western blot analysis was performed using antibodies against VLDLR (sc-18824), Nrf2 (sc-722), Nqo1 (sc-393736), ATF4 (sc-200) (Santa Cruz), VLDLR (AF2258) (R&D system), eIF2α (9722), phospho-eIF2α (Ser51) (9721), IgG control (2729S) (Cell Signaling Technology Inc., Danvers, MA), actin (A5441) (Sigma–Aldrich, Madrid, Spain).

Techniques: Expressing, Staining, Injection, Western Blot

Journal: Molecular Metabolism

Article Title: Hepatic regulation of VLDL receptor by PPARβ/δ and FGF21 modulates non-alcoholic fatty liver disease

doi: 10.1016/j.molmet.2017.12.008

Figure Lengend Snippet: Recombinant FGF21 protein attenuates the increase in VLDLR levels caused by ER stress. Human Huh-7 hepatocytes were incubated with DMSO (vehicle, control, CT), tunicamycin (TUNIC) (1 μg/ml) or tunicamycin plus recombinant human FGF21 (1 μg/ml) and the mRNA abundance of VLDLR (A) and CHOP (B) and the protein levels of VLDLR and total and phospho-eIF2α (C) were assessed (n = 3 independent experiments). ***p < 0.001 vs . CT cells. # p < 0.05 vs . tunicamycin-treated cells. Analysis of the hepatic levels of VLDLR in mice injected intraperitoneally with vehicle or recombinant mouse FGF21 together with DMSO or tunicamycin. Data are presented as the mean ± S.D. (n = 5 per group). C, Vldlr and Chop mRNA abundance. D, Immunoblot analyses of hepatic VLDLR. ***p < 0.001, **p < 0.01 and *p < 0.05.

Article Snippet: Western blot analysis was performed using antibodies against VLDLR (sc-18824), Nrf2 (sc-722), Nqo1 (sc-393736), ATF4 (sc-200) (Santa Cruz), VLDLR (AF2258) (R&D system), eIF2α (9722), phospho-eIF2α (Ser51) (9721), IgG control (2729S) (Cell Signaling Technology Inc., Danvers, MA), actin (A5441) (Sigma–Aldrich, Madrid, Spain).

Techniques: Recombinant, Incubation, Control, Injection, Western Blot

Journal: Molecular Metabolism

Article Title: Hepatic regulation of VLDL receptor by PPARβ/δ and FGF21 modulates non-alcoholic fatty liver disease

doi: 10.1016/j.molmet.2017.12.008

Figure Lengend Snippet: Proposed mechanisms by which PPARβ/δ regulates VLDLR levels and hepatic steatosis. Pparβ/δ deficiency may result in an increase in VLDLR levels and hepatic steatosis through several mechanisms. The activation of HRI caused by Pparβ/δ deficiency (reference ) and by activators of this kinase (BTdCPU) may increase the levels of VLDLR through the eIF2α-ATF4 pathway. ER stress can also activate the eIF2α-ATF4 pathway leading to an increase in the expression of VLDLR and FGF21. This hormone suppresses the eIF2α-ATF4 pathway through a negative feedback mechanism and thereby it also regulates the levels of VLDLR. ER stress also enhances the activity of Nrf2, a transcription factor reported to upregulate the expression of Fgf21 (reference ). Fructose feeding increases the levels of ROS and the activity of Nrf2 providing a mechanism for the increase of the levels of VLDLR. All these mechanisms may result in an increase in the levels of VLDLR causing hepatic steatosis. TG: triglyceride.

Article Snippet: Western blot analysis was performed using antibodies against VLDLR (sc-18824), Nrf2 (sc-722), Nqo1 (sc-393736), ATF4 (sc-200) (Santa Cruz), VLDLR (AF2258) (R&D system), eIF2α (9722), phospho-eIF2α (Ser51) (9721), IgG control (2729S) (Cell Signaling Technology Inc., Danvers, MA), actin (A5441) (Sigma–Aldrich, Madrid, Spain).

Techniques: Activation Assay, Expressing, Activity Assay