17 research outputs found
Kinetics of PMK-1 activation.
<p>Relative level of PMK-1 phosphorylation at the kinase activating residues (Y180/182) and total PMK-1 protein levels in L4/YA worms treated with 5 mM sodium arsenite, 38 μM juglone, or 7 mM acrylamide for up to four hours. Band intensities relative to β-tubulin are given below each pair of blots with control arbitrarily set to 1.</p
<i>skr-1/2</i> is required for juglone and arsenite resistance but not for SKN-1 accumulation or PMK-1 phosphorylation.
<p>(A) Survival of <i>eri-1</i> worms fed control, <i>skn-1</i>, or <i>skr-1/2</i> RNAi on 125 μM juglone after a 2 h pre-treatment at 38 μM. ***P<0.001 compared to control RNAi as determined by Log-rank (Mantel-Cox) test. <i>n</i> = 98–109 worms from a single trial; statistics and results from two other trials are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006361#pgen.1006361.s010" target="_blank">S9 Fig</a>. (B) Survival of N2 worms fed control, <i>skn-1</i>, or <i>skr-1/2</i> RNAi on 10 mM arsenite. ***P<0.001 compared to control RNAi as determined by Log-rank (Mantel-Cox). <i>n</i> = 140–170 worms from a single trial; statistics and results from two other trials are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006361#pgen.1006361.s010" target="_blank">S9 Fig</a>. (C) Survival of worms with enhanced SKN-1 activity exposed to a range of juglone concentrations for 16 h. <i>skr-1/2(RNAi)</i> did not significantly decrease survival at any concentration in any strain. <i>n</i> = 4 independent trials of 12–78 worms per condition and trial. (D) Animals with integrated SKN-1b/c::GFP were fed with either control or <i>skr-1/2(RNAi)</i> and treated with 38 μM juglone for 3 h and accumulation of head SKN-1b/c::GFP was scored. Representative fluorescence micrographs of head SKN-1b/c::GFP are shown with arrows marking GFP. <i>n =</i> 45 to 54 worms. (E) Phosphorylation of PMK-1 was measured in worms fed control or <i>skr-1/2</i> dsRNA and exposed to 5 mM sodium arsenite for 1 h. PMK-1 phosphorylation levels were normalized to β-tubulin and then to control (without stressor); β-tubulin was detected on the same blots after stripping. Total PMK-1 was measured on the same lysates in a different blot and also normalized to β-tubulin. Values are mean plus standard error for densitometry of protein bands from <i>n =</i> 3 replicates of >1,000 worms. Representative immunoblot bands are shown, ***P<0.001 relative to control. There was no significant effect of <i>skr-1/2(RNAi)</i>.</p
PMK-1 activation and its requirement for SKN-1 transcriptional responses.
<p>(A) Relative level of PMK-1 phosphorylation at the kinase activating residues (Y180/182) and total PMK-1 protein levels in L4/YA worms treated with 5 mM sodium arsenite for 1 h, 35 mM paraquat for 2 h, 38 μM juglone for 3 h, or 7 mM acrylamide for 4 h. PMK-1 phosphorylation and protein levels were normalized to β-tubulin, which was detected on the same blot after stripping; histograms represent mean plus standard error of densitometry from <i>n</i> = 4 replicates of ~1,000 worms. ***P<0.001 compared to corresponding control as determined by Student’s T-test. Representative Western blot images are shown. (B) Fold changes in <i>gst-4</i>, <i>gst-10</i>, <i>gst-12</i>, <i>gst-30</i>, and <i>gcs-1</i> mRNA levels relative to N2 control in L4/YA N2 wildtype and <i>pmk-1</i>(<i>km25</i>) mutant worms treated with 5 mM sodium arsenite or 38 μM juglone for 1 h, worms were fed either control RNAi or <i>skn-1</i> RNAi from L1. Histograms represent mean plus standard error of <i>n</i> = 4 replicates of 200–300 worms. All genes were induced significantly by arsenite or juglone (P<0.001); *P<0.05, *** P<0.001 compared to N2;<i>control(RNAi)</i>, ‡ P<0.001 compared to <i>pmk-1(km25);control(RNAi)</i>.</p
<i>skr-1/2</i> is required for induction of SKN-1 dependent detoxification genes.
<p>(A) <i>Pgst-4</i>::<i>GFP</i> fluorescence scoring and representative fluorescence micrographs of worms fed with control or <i>skr-1/2</i> dsRNA after exposure to 5 mM sodium arsenite for 1 h (recovered for 3 h on NGM agar to induce GFP), 35 mM paraquat for 2 h (recovered for 2 h), 38 μM juglone for 3 h (recovered for 1 h), or 7 mM acrylamide for 4 h (no recovery). <i>n</i> = 70–89 worms from 3 independent trials, *P<0.05, ***P<0.001 as determined by Chi-Square test. (B) Relative <i>Pgst-4</i>::<i>GFP</i> fluorescence measured by a plate reader after a 6 h exposure to a range of arsenite concentrations; all values are normalized to <i>control(RNAi)</i> with no arsenite. <i>n</i> = 4–8 wells of worms in a 384 well plate; P<0.001 for <i>skr-1/2(RNAi)</i> and <i>skn-1(RNAi)</i> versus <i>control(RNAi)</i> at all concentrations except for the lowest. (C) Fold changes in mRNA of <i>gst-4</i>, <i>gst-10</i>, <i>gst-12</i>, and <i>gst-30</i> relative to control (no stressors) in worms with control, <i>skn-1</i>, or <i>skr-1/2(RNAi)</i> after exposure to 38 μM juglone for 3 h. mRNA levels were normalized to <i>rpl-2;</i> values are means plus standard error of <i>n</i> = 4 replicates of 200–400 worms. All genes were induced significantly by juglone (P<0.001); ***P<0.001 compared to <i>control (RNAi)</i>.</p
New <i>gst-4</i> regulators identified through genome wide RNAi screening function upstream of <i>wdr-23</i> and <i>skn-1</i>.
<p>Animals integrated with <i>Pgst-4</i>::<i>GFP</i> were fed bacteria producing dsRNA to positive hits obtained from the RNAi screen and exposed to juglone. (A) <i>Pgst-4</i>::<i>GFP</i> fluorescence was scored and representative images are shown. For <i>Pgst-4</i>::<i>GFP</i> scoring, low refers to little to no GFP signals observed throughout the worm, medium refers to GFP signals observed at the anterior and posterior ends of the worm, and high refers to GFP signals observed throughout the body. <i>n</i> = 50–118 worms. (B) <i>Pgst-4</i>::<i>GFP</i> reporter scoring in either a <i>wdr-23</i> loss of function (<i>tm1817</i>) or a <i>skn-1</i> gain of function allele (<i>k1023</i>) mutant background. <i>n</i> = 54–75 worms. *P<0.05, **P<0.01 and ***P<0.001 relative to corresponding control as determined by Chi-Square tests; note that <i>pad-1(RNAi)</i> in <i>skn-1(k1023)</i> could not be tested for significance because it had zero worms with low or medium fluorescence.</p
SKR-1 is expressed broadly and interacts with WDR-23.
<p>(A) Fluorescence and differential interference contrast micrographs showing cytoplasmic and nuclear (arrow) expression of SKR-1::GFP in the intestine (QV254). (B) SKR-1 interacts with WDR-23. HEK293 cells were co-transfected with V5-SKR-1 fusion protein along with either GST only vector (pDEST27), GST-SKN-1c fusion protein, or GST-WDR23a fusion protein. Complexes were captured with GSH beads and interactions with SKR-1 were determined by immunoblotting with anti-V5 mAb. Co-pulldown of V5-SKR-1 with GST-WDR-23a was also detected in a separate independent trial. (C-D) Total WDR-23::GFP fluorescence (normalized to RFP) and percentage of worms with visible nuclear WDR-23::GFP fluorescence with and without <i>skr-1/2(RNAi)</i>. *P<0.05 and **P<0.01. Arrows point to hypodermal nuclei and asterisks mark neuronal cells.</p
SPIN
<div><p>SKN-1/Nrf are the primary antioxidant/detoxification response transcription factors in animals and they promote health and longevity in many contexts. SKN-1/Nrf are activated by a remarkably broad-range of natural and synthetic compounds and physiological conditions. Defining the signaling mechanisms that regulate SKN-1/Nrf activation provides insights into how cells coordinate responses to stress. Nrf2 in mammals is regulated in part by the redox sensor repressor protein named Keap1. In <i>C</i>. <i>elegans</i>, the p38 MAPK cascade in the intestine activates SKN-1 during oxidative stress by promoting its nuclear accumulation. Interestingly, we find variation in the kinetics of p38 MAPK activation and tissues with SKN-1 nuclear accumulation among different pro-oxidants that all trigger strong induction of SKN-1 target genes. Using genome-wide RNAi screening, we identify new genes that are required for activation of the core SKN-1 target gene <i>gst-4</i> during exposure to the natural pro-oxidant juglone. Among 10 putative activators identified in this screen was <i>skr-1/2</i>, highly conserved homologs of yeast and mammalian Skp1, which function to assemble protein complexes. Silencing of <i>skr-1/2</i> inhibits induction of SKN-1 dependent detoxification genes and reduces resistance to pro-oxidants without decreasing p38 MAPK activation. Global transcriptomics revealed strong correlation between genes that are regulated by SKR-1/2 and SKN-1 indicating a high degree of specificity. We also show that SKR-1/2 functions upstream of the WD40 repeat protein WDR-23, which binds to and inhibits SKN-1. Together, these results identify a novel p38 MAPK independent signaling mechanism that activates SKN-1 <i>via</i> SKR-1/2 and involves WDR-23.</p></div
Effect of elevated glycerol levels on hypertonic stress-induced aggregation of endogenous proteins.
<p><i>A:</i> Effect of increasing NaCl concentrations on motility in control, acclimated, <i>osm-11</i> and acclimated <i>gpdh-1; gpdh-2</i> worms. <i>gpdh-1; gpdh-2</i> mutants lack functional GPDH-1 and GPDH-2 enzymes resulting in greatly reduced glycerol accumulation under hypertonic stress conditions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034153#pone.0034153-Lamitina2" target="_blank">[12]</a>. (<i>n</i> = 5–18 experiments with 15–60 worms/experiment). <i>B:</i> Left panel, relative insoluble protein in acclimated <i>gpdh-1; gpdh-2</i> worms maintained in either 200 mM NaCl or exposed to 500 mM NaCl for 4 h. Insoluble protein was quantified as a fraction of total protein and is plotted relative to that observed in worms maintained on 200 mM NaCl. (<i>n</i> = 3 experiments with 4000–5000 worms/experiment). Right panel, examples of SDS-PAGE gels of total and detergent insoluble (insol.) proteins isolated from acclimated <i>gpdh-1; gpdh-2</i> worms maintained in 200 mM NaCl or exposed to 500 mM NaCl. <i>C:</i> Left panel, relative insoluble protein in <i>osm-11</i> worms grown under control conditions (51 mM NaCl) or exposed to 700 mM NaCl for 4 h. Insoluble protein was quantified and plotted in the same manner as described in <i>B</i>. (<i>n</i> = 3 samples of 4000–5000 worms/sample). *P<0.03 compared to animals maintained on 51 mM NaCl. Right panel, examples of SDS-PAGE gels of total and detergent insoluble (insol.) proteins isolated from <i>osm-11</i> worms exposed to 51 or 700 mM NaCl.</p
Effect of elevated glycerol levels on aging-induced aggregation of Q35::YFP.
<p><i>A:</i> Whole worm glycerol levels in controls worms, worms acclimated to 200 mM NaCl and <i>osm-11</i> mutant animals. (<i>n</i> = 4 samples of ∼4000 worms/sample). <i>B:</i> Time course of aging-induced Q35::YFP aggregation in control, acclimated and <i>osm-11</i> mutant animals. (<i>n</i> = 7 experiments with 10–15 worms/experiment).</p
SKN-1b/c::GFP can accumulate in the head without <i>pmk-1</i>.
<p>(A) Scoring of SKN-1b/c::GFP was conducted as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006361#pgen.1006361.g002" target="_blank">Fig 2</a>. **P<0.01 and ***P<0.001 relative to the corresponding controls without arsenite as determined by Chi-Square tests, †P<0.05 and ††P<0.01 relative to the arsenite exposed wildtype worms as determined by Chi-Square tests, <i>n =</i> 51–92. (B) Representative images of SKN-1b/c::GFP. Arrows mark head GFP and arrowheads mark intestinal nuclei.</p