Journal: Science Advances

Article Title: Temporal control of the integrated stress response by a stochastic molecular switch

doi: 10.1126/sciadv.abk2022

Figure Lengend Snippet: ( A ) Schematic representation of the parameters and reactions included in the mathematical model. The stress-sensing module represents the activation of stress kinases. PKR is activated by binding to dsRNA (viral stress, p-PKR*), HRI by arsenite treatment (oxidative stress, HRI*), and PERK by thapsigargin (ER stress, PERK*). Active stress kinases signal to the decision module, where upon crossing a p-eIF2α level threshold will trigger SG formation (SG-On). Elevated p-eIF2α levels activate the recovery module consisting of the GADD34 negative feedback loop, i.e., ppp15R1a promoter activation (P OFF to P ON ) with time delay (clock symbol), GADD34 transcription (mGADD34), and protein synthesis (GADD34). In turn, GADD34 dephosphorylates eIF2α and thereby resumes translation. Gray arrow, basal eIF2α dephosphorylation by CReP, the constitutive regulatory subunit of PP1. Ø, degradation. ( B and C ) Absolute quantification of eIF2α and PKR mean molecule numbers in Huh7 YFP-TIA1 cells, in the absence and presence of IFN-α. (B) Representative quantitative Western blot analysis of eIF2α and PKR. (C) Estimated eIF2α and PKR mean molecule number per cell (±SD). Number of repeats ( n ) and statistical significance compared to untreated cells (untr.) are indicated; **** P < 0.0001. ( D and E ) Determination of protein half-lives by CHX pulse experiments and Western blot analyses. (D) PKR and eIF2α half-lives ( n = 3). (E) GADD34 ( n = 4). Black line, best nonlinear fit.

Article Snippet: Lysates ( n = 8) containing 20 μg of total protein were spiked with different amounts of recombinant GST-tagged eIF2α (Abnova, 5/10/15/22/33/50/75 ng), recombinant GST-tagged PKR kinase domain (Abcam, 1/2/3/4.4/6.7/10/12.5 ng), or protein lysis buffer.

Techniques: Activation Assay, Binding Assay, De-Phosphorylation Assay, Western Blot

Journal: Science Advances

Article Title: Temporal control of the integrated stress response by a stochastic molecular switch

doi: 10.1126/sciadv.abk2022

Figure Lengend Snippet: ( A to C ) Activation of PKR in Huh7 YFP-TIA1 cells transfected with increasing amounts of 200-bp dsRNA ( n = 3). (A) Representative Western blot analysis of p-PKR and p-eIF2α expression levels. Expression levels of β-actin served as loading control. The percentage of p-eIF2α was analyzed by Phos-tag polyacrylamide gel. (B) Shown are quantifications of mean p-PKR expression levels (±SD) normalized to the loading control and relative to untreated cells (top) and quantifications of the mean p-eIF2α percentage (±SD). Statistical significance is indicated compared to untreated cells. (C) The presence of SGs in transfected cells was analyzed by fluorescence microscopy (for each condition, n > 100). Shown are mean percentages ± SD. Statistical significance is indicated compared to untreated cells. * P < 0.05, ** P < 0.01. ( D to F ) Induction of oxidative stress in Huh7 YFP-TIA1 cells by treatment with increasing concentrations of arsenite for 45 min ( n = 3). (D) Representative Western blot and Phos-tag analyses. Shown are mean percentages ± SD of p-eIF2α (E) and SG-positive cells (for each condition, n > 100) (F). Statistical significance is indicated compared to untreated cells; **** P < 0.0001. ( G ) Dose-response analysis and determination of the p-eIF2α level that results in formation of SGs in 50% of cells upon treatment with arsenite [related to (F); n = 3] and thapsigargin (related to fig. S4, E to H; n = 3).

Article Snippet: Lysates ( n = 8) containing 20 μg of total protein were spiked with different amounts of recombinant GST-tagged eIF2α (Abnova, 5/10/15/22/33/50/75 ng), recombinant GST-tagged PKR kinase domain (Abcam, 1/2/3/4.4/6.7/10/12.5 ng), or protein lysis buffer.

Techniques: Activation Assay, Transfection, Western Blot, Expressing, Control, Fluorescence, Microscopy

Journal: Science Advances

Article Title: Temporal control of the integrated stress response by a stochastic molecular switch

doi: 10.1126/sciadv.abk2022

Figure Lengend Snippet: ( A ) In vitro PKR kinase assay. His-tagged PKR and His-tagged eIF2α were incubated with increasing molarities of 200-bp dsRNA ( n = 3). The top panels show representative Western blot analyses of p-PKR and p-eIF2α levels. Silver staining of proteins in the gel served as loading control. Quantifications of mean levels relative to untreated control ± SD are shown in the bottom panels. Statistical significance is indicated compared to untreated; * P < 0.05, ** P < 0.01. ( B to E ) Computational prediction of PKR activation by dsRNA. (B) Overview of the different steps tested in PKR model development. (C) Differences in the chi-square (Δχ 2 ) and corrected Akaike information criterion (ΔAICc) to the optimal PKR activation model variant (variant 3, see figs. S6 and S7). The model describing PKR dimerization upon binding to dsRNA was significantly improved by considering PKR cooperative binding to dsRNA (ΔAICc > 200). Cis and trans reactions did not improve the cooperativity model (ΔAICc = 18.4). (D) Overview of the optimal model variant. PKR monomers reversibly bind to PKR on dsRNA in a cooperative manner and form active PKR oligomers (dsR:PKRoligo). (E) Best model fits of p-PKR levels to in-cell (related to ) and in vitro kinase assays ( n = 500 multistart optimization runs). a.u., arbitrary units.

Article Snippet: Lysates ( n = 8) containing 20 μg of total protein were spiked with different amounts of recombinant GST-tagged eIF2α (Abnova, 5/10/15/22/33/50/75 ng), recombinant GST-tagged PKR kinase domain (Abcam, 1/2/3/4.4/6.7/10/12.5 ng), or protein lysis buffer.

Techniques: In Vitro, Kinase Assay, Incubation, Western Blot, Silver Staining, Control, Activation Assay, Variant Assay, Binding Assay

Journal: Science Advances

Article Title: Temporal control of the integrated stress response by a stochastic molecular switch

doi: 10.1126/sciadv.abk2022

Figure Lengend Snippet: ( A ) FISH analysis. Top: Representative still images of uninfected and HCV-infected cells treated with IFN-α. HCV (+) ssRNA genomes, GADD34 transcripts, and total polyA-tailed mRNAs were detected by FISH. Outlined in red, SG-positive cell; outlined in white, unstressed cell. White circles indicate single transcripts. Scale bars, 20 μm. Bottom: GADD34 mean transcript levels ± SD. Statistical significance and the number of analyzed cells ( n ) are indicated at the top of the graph; ** P < 0.001, **** P < 0.0001. ( B ) Model best fits ( n = 2500 multistart optimization runs) to the percentage of SG-positive cells experimentally measured in arsenite or thapsigargin titration. ( C ) Computational simulations of dose-response curves for p-eIF2α, GADD34 mRNA, and protein and SG-positive cells in the population at steady state. Shown are percentages of maximal values as a function of different kinase activities. The reference kinase activity (10 0 ) results in 50% SG-positive cells (intermediate stress). Kinase activities <10 −1 , low to moderate stress; >2, high stress. ( D ) Model prediction: behavior of the GADD34 negative feedback loop parameters (promoter activity, mRNA, and protein) after stress release. The percentage of their maximum response over time is shown. Estimated decay processes { t 1/2 , Prom. ≈ 256 min; mRNA ≈ 200 min [from ]; protein ≈ 37 min}. ( E ) Mean expression levels ± SD of GADD34 pre-mRNA and mature mRNA upon thapsigargin treatment ( n = 3). ( F ) Model prediction: behavior of p-eIF2α levels and number of SG-positive cells after a second 1-hour stress pulse applied at different times after stress release. Phases I to III, levels of cell protection against a second stress pulse.

Article Snippet: Lysates ( n = 8) containing 20 μg of total protein were spiked with different amounts of recombinant GST-tagged eIF2α (Abnova, 5/10/15/22/33/50/75 ng), recombinant GST-tagged PKR kinase domain (Abcam, 1/2/3/4.4/6.7/10/12.5 ng), or protein lysis buffer.

Techniques: Infection, Titration, Activity Assay, Expressing

Journal: Science Advances

Article Title: Temporal control of the integrated stress response by a stochastic molecular switch

doi: 10.1126/sciadv.abk2022

Figure Lengend Snippet: ( A ) Computational simulations of two consecutive 1-hour stress pulses interspaced by a 5-hour recovery period. Shown is a range of stress kinase activities (stress intensity) varying between 10-fold lower (min) and 10-fold higher (max) than the reference kinase activity leading to 50% SG-positive cells. Color plots show the behavior of ppp1r15a promoter activity, concentrations of GADD34 mRNA and protein, and percentages of p-eIF2α and SG-positive cells. Graphs at the bottom reflect the behavior of the abovementioned components for one chosen stress intensity (black line, moderate stress). ( B ) Experimental validation of the predictions shown in (A). Huh7 cells were subjected to a first heat shock (HS1) at 42°C for 1 hour and immediately transferred at 37°C for recovery. Cells were harvested at the indicated time points after the first or the second heat shock (HS2) ( n = 2). Cells treated with 2 μM thapsigargin for 1 and 6 hours served as reference. Shown are representative Western blot and Phos-tag gel analyses. Bottom panels show the quantification of GADD34 expression levels normalized to the loading control and relative to untreated cells as well as the percentage of p-eIF2α. ( C ) Model prediction: behavior of the SG response and GADD34 negative feedback loop components over a 24-hour time period upon continuous stress. The color of the curves reflects different kinase activity levels. ( D ) During the adaptation to a stress stimulus, depending on PKR activity, the expression of GADD34 will result in dose-response curve shifts (hysteresis) in the percentage of p-eIF2α (top) and of SG-positive cells (bottom). The shift of the dose-response curve depends on the stress duration and intensity and is reverted after stress relief. Blue and red shared areas indicate 1σ confidence intervals of estimated PKR activities in HCV and HCV + IFN-α experiments.

Article Snippet: Lysates ( n = 8) containing 20 μg of total protein were spiked with different amounts of recombinant GST-tagged eIF2α (Abnova, 5/10/15/22/33/50/75 ng), recombinant GST-tagged PKR kinase domain (Abcam, 1/2/3/4.4/6.7/10/12.5 ng), or protein lysis buffer.

Techniques: Activity Assay, Western Blot, Expressing, Control

Journal: eLife

Article Title: The switch-like expression of heme-regulated kinase 1 mediates neuronal proteostasis following proteasome inhibition

doi: 10.7554/eLife.52714

Figure Lengend Snippet: ( A ) Genetic deletion (KO) or inhibition of the eIF2α kinases GCN2, PERK or PKR did not rescue the inhibition of protein synthesis elicited by proteasome inhibition. (wt vs. kinase KO or inhibition: GCN2, unpaired t-test p≤0.0001, PERK, unpaired t-test p≤0.001 and PKR p≤0.01, respectively, n = 2 experiments, two biological replicates per KO). Error bars = SD. ( B ) Representative puromycylation western blot showing that genetic deletion of HRI kinase rescues the inhibition of protein synthesis. Molecular weight markers from top-to-bottom are 250, 150, 100, 75, 50, 37, 25, 20, and 15 kD. ( C ) Analysis of the experiment shown in B. The amount of PI-induced protein synthesis inhibition was significantly reduced in the HRI KO (unpaired t-test on wt PI vs KO. PI p≤0.0001, wt control vs. PI p≤0.0001, KO. control vs. PI p≤0.05, n = 5 experiments, error bars SD. ( D ) Metabolic labeling (magenta; using puromycylation, see Materials and methods) of the global nascent protein pool in cultured hippocampal neurons following proteasome inhibition showing that HRI KO neurons exhibit less PI-induced inhibition of protein synthesis. Scale bar = 100 μm. ( E ) Analysis of experiments like that shown in D. Protein synthesis in hippocampal neurons was significantly reduced in the HRI KO following proteasome inhibition (unpaired t-test on wt control vs. k.o. control p>0.05, wt PI vs k.o. PI p≤0.01, wt control vs. PI p≤0.0001, KO control vs. PI p≤0.05. n = 3 experiments, wt control n = 1104, wt-PI n = 955, ko control = 1457, ko-PI n = 1052 neurons). ( F ) Analysis of eIF2α phosphorylation in response to proteasome inhibition. PI treatment led to a significant increase in peIF2α levels. (unpaired t-test on wt control vs. PI: p≤0.01, t-test on HRI KO control vs. PI: p≤0.05. two experiments –see . ( G ) RNA-seq data from hippocampal slices indicate that HRI mRNA is the most abundant amongst the 4 eIF2α kinases ( H ) Representative fluorescence in situ hybridization image detecting HRI mRNA in neuronal somata and dendrites. Scale bar = 20 μm for somata and 10 µm for dendrites-See . Figure 3—source data 1. Source data for . Figure 3—source data 2. Source data for .

Article Snippet: 200 ng of recombinant HRI (SignalChem) or 500 ng of recombinant eIF2α (abcam) were incubated with 2 µg of purified 20S proteasome (Enzo) in 20 mM Hepes pH 7.4/2 mM EDTA/1 mM EGTA for the indicated times at 37°C.

Techniques: Inhibition, Western Blot, Molecular Weight, Labeling, Cell Culture, RNA Sequencing Assay, Fluorescence, In Situ Hybridization

Journal: eLife

Article Title: The switch-like expression of heme-regulated kinase 1 mediates neuronal proteostasis following proteasome inhibition

doi: 10.7554/eLife.52714

Figure Lengend Snippet: ( A ) In vitro eIF2α phosphorylation assay by HRI; 100 and 200 pm of hemin were added to the reaction mixture (500 ng eIF2α and 3,3 ng of HRI), inhibiting the activity of the kinase (unpaired t-test control vs hemin 100pm p≤0.0001, control vs hemin 200pm p≤0.01). ( B ) In vitro eIF2α phosphorylation assay by HRI; a constant amount of eIF2α was added for each reaction combined with increasing amounts of HRI as indicated (See Materials and methods). Increasing amounts of HRI protein lead to significantly higher levels of eIF2α phosphorylation in hippocampal lysates, compared to blood lysates (the lysates were cleared of protein by heating 10 min at 60 ° C) (unpaired t-test 0.05 ng HRI Blood vs hippocampus p≥0.05, 0.1 ng HRI Blood vs hippocampus p≥0.05, 0.33 ng HRI Blood vs hippocampus p≤0.05, 66 ng HRI Blood vs hippocampus p≤0.05, n = 3 experiments). ( C ) Western blot analysis of experiments testing whether hemin (heme complexed with Fe 3+ ) can mitigate the effects of PI. Hemin significantly reduced the PI-induced protein synthesis inhibition. (PI vs. control, p≤0.0001; PI vs. PI +hemin, p≤0.01 Error bars = SD, three experiments) ( D ) Representative images showing that HA-HRIexpressing neurons (arrow) (green; anti-HA labeling) exhibit lower levels of protein synthesis (magenta) when compared to neighboring neurons, which do not express HA-HRI. Also shown are somata and dendrites (labeled with an anti-MAP2 antibody) and nuclei (labeled with DAPI). Scale bar = 50 μm. ( E ) Quantification of somatic protein synthesis levels in neurons expressing or lacking HA-HRI; HA-HRI positive neurons exhibited significantly lower levels of protein synthesis, (p≤0.0001, HA negative neurons n = 852 and HA positive neurons n = 253, unpaired t-test). Figure 6—source data 1. Source data for .

Article Snippet: 200 ng of recombinant HRI (SignalChem) or 500 ng of recombinant eIF2α (abcam) were incubated with 2 µg of purified 20S proteasome (Enzo) in 20 mM Hepes pH 7.4/2 mM EDTA/1 mM EGTA for the indicated times at 37°C.

Techniques: In Vitro, Phosphorylation Assay, Activity Assay, Western Blot, Inhibition, Labeling, Expressing

Journal: eLife

Article Title: The switch-like expression of heme-regulated kinase 1 mediates neuronal proteostasis following proteasome inhibition

doi: 10.7554/eLife.52714

Figure Lengend Snippet: Correlation of the level of HA-HRI expression (using a transfected construct) and the corresponding nascent protein signal (obtained using brief puromycin labeling) in the same neuron. As expected, the correlation between the expression of HRI kinase and the inhibition in protein synthesis was not linear, the best fit corresponds to a power line. This is consistent with the assumption that one molecule of HRI will lead to the phosphorylation of multiple molecules of substrate. Given this dependence, small increases in HRI expression will exert the greatest impact on protein synthesis within a low to intermediate range: when expression is not too low to impact eIF2α phosphorylation, but also not too high because then most protein synthesis is already shut off. Consequently, it is possible that a 30% increase in HRI expression will decrease protein synthesis by more than 50%.

Article Snippet: 200 ng of recombinant HRI (SignalChem) or 500 ng of recombinant eIF2α (abcam) were incubated with 2 µg of purified 20S proteasome (Enzo) in 20 mM Hepes pH 7.4/2 mM EDTA/1 mM EGTA for the indicated times at 37°C.

Techniques: Expressing, Transfection, Construct, Labeling, Inhibition

Journal: eLife

Article Title: The switch-like expression of heme-regulated kinase 1 mediates neuronal proteostasis following proteasome inhibition

doi: 10.7554/eLife.52714

Figure Lengend Snippet:

Article Snippet: 200 ng of recombinant HRI (SignalChem) or 500 ng of recombinant eIF2α (abcam) were incubated with 2 µg of purified 20S proteasome (Enzo) in 20 mM Hepes pH 7.4/2 mM EDTA/1 mM EGTA for the indicated times at 37°C.

Techniques: Transfection, Construct, Recombinant, Software, Generated, Sequencing, TaqMan Assay

Journal: Asian Journal of Andrology

Article Title: Expression and function of HSP110 family in mouse testis after vasectomy

doi: 10.4103/1008-682X.164197

Figure Lengend Snippet: Vasectomy induces changes in mRNA and protein levels of HSP110 family members. ( a ) The level of HSP110 mRNA in the mouse testis was determined by RT-PCR. ( b ) Quantification of ORP150 and HSPA4l mRNA in vasectomized and control mice relative to β-actin. Control, one representative of sham-operated control (note that sham-operated controls at each time point postvasectomy had the same expression level for each HSP110 gene). *Statistically significant difference for vasectomy versus sham operation of the same day ( P < 0.05). Results of one representative experiment of two others. ( c ) Expression of HSP110 in mouse testes at different time points postvasectomy detected by Western blot. GAPDH was assessed as an internal loading control. Control, one representative of sham-operated control (note that sham-operated controls at each time point postvasectomy had the same expression level for each HSP110 protein). Results of one representative experiment of two others.

Article Snippet: The primary antibodies used were: GAPDH (sc-25778: Santa Cruz Biotechnology, Santa Cruz, CA, USA); HSPA4 (sc-365366: Santa Cruz Biotechnology, Santa Cruz, CA, USA); HSPA4l, ORP150, and HSPH1 (ARP53693, ARP46716, ARP53693: Aviva Systems Biology, Beijing, China); phospho-JNK, phospho-eIF2α, JNK, eIF2α, GRP78, and GADD153 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Expressing, Western Blot