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Specific SKN-1/Nrf stress responses to perturbations in translation elongation and proteasome activity.

Li X, Matilainen O, Jin C, Glover-Cutter KM, Holmberg CI, Blackwell TK - PLoS Genet. (2011)

Bottom Line: In contrast, when translation elongation is impaired, SKN-1 does not upregulate proteasome genes, and UPS activity is then reduced.SKN-1 therefore has a critical tissue-specific function in increasing proteasome gene expression and UPS activity under normal conditions, as well as when the UPS system is stressed, but mounts distinct responses when protein synthesis is perturbed.The data suggest that SKN-1 may increase longevity, not only through its well-documented role in boosting stress resistance, but also through contributing to protein homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Joslin Diabetes Center, Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
SKN-1, the Caenorhabditis elegans Nrf1/2/3 ortholog, promotes both oxidative stress resistance and longevity. SKN-1 responds to oxidative stress by upregulating genes that detoxify and defend against free radicals and other reactive molecules, a SKN-1/Nrf function that is both well-known and conserved. Here we show that SKN-1 has a broader and more complex role in maintaining cellular stress defenses. SKN-1 sustains expression and activity of the ubiquitin-proteasome system (UPS) and coordinates specific protective responses to perturbations in protein synthesis or degradation through the UPS. If translation initiation or elongation is impaired, SKN-1 upregulates overlapping sets of cytoprotective genes and increases stress resistance. When proteasome gene expression and activity are blocked, SKN-1 activates multiple classes of proteasome subunit genes in a compensatory response. SKN-1 thereby maintains UPS activity in the intestine in vivo under normal conditions and promotes survival when the proteasome is inhibited. In contrast, when translation elongation is impaired, SKN-1 does not upregulate proteasome genes, and UPS activity is then reduced. This indicates that UPS activity depends upon presence of an intact translation elongation apparatus; and it supports a model, suggested by genetic and biochemical studies in yeast, that protein synthesis and degradation may be coupled processes. SKN-1 therefore has a critical tissue-specific function in increasing proteasome gene expression and UPS activity under normal conditions, as well as when the UPS system is stressed, but mounts distinct responses when protein synthesis is perturbed. The specificity of these SKN-1-mediated stress responses, along with the apparent coordination between UPS and translation elongation activity, may promote protein homeostasis under stress or disease conditions. The data suggest that SKN-1 may increase longevity, not only through its well-documented role in boosting stress resistance, but also through contributing to protein homeostasis.

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Knockdown of SKN-1 or TEFs, but not TIFs, reduces UPS activity in the intestine.(A) Representative images of Pvha-6::UbG76V-Dendra2 and Pvha-6::Dendra2 reporters in animals exposed to control, skn-1, eef-1A.1 (TEF) or eif-1(TIF) RNAi. Red fluorescence derives from substrate that was present at the time of photoconversion. Bar: 20 µm. In (A–C), bar graphs depict the percentages of green and red fluorescence compared to either the initial value (t = 0 for green) or point of photoconversion (t = C for red) for UbG76V-Dendra2 and Dendra2, respectively (± SEM). (B,C) Summary of UbG76V-Dendra2 and Dendra2 imaging in intestinal cells after 9 hours in response to TEF and TIF RNAi, respectively. Significant differences relative to control RNAi (red fluorescence after 9 hours) are indicated with **P<0.01, *P<0.05.
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pgen-1002119-g005: Knockdown of SKN-1 or TEFs, but not TIFs, reduces UPS activity in the intestine.(A) Representative images of Pvha-6::UbG76V-Dendra2 and Pvha-6::Dendra2 reporters in animals exposed to control, skn-1, eef-1A.1 (TEF) or eif-1(TIF) RNAi. Red fluorescence derives from substrate that was present at the time of photoconversion. Bar: 20 µm. In (A–C), bar graphs depict the percentages of green and red fluorescence compared to either the initial value (t = 0 for green) or point of photoconversion (t = C for red) for UbG76V-Dendra2 and Dendra2, respectively (± SEM). (B,C) Summary of UbG76V-Dendra2 and Dendra2 imaging in intestinal cells after 9 hours in response to TEF and TIF RNAi, respectively. Significant differences relative to control RNAi (red fluorescence after 9 hours) are indicated with **P<0.01, *P<0.05.

Mentions: As our data suggested that skn-1 contributes to proteasome gene expression, particularly when proteasome activity is impaired, we used a novel in vivo assay to investigate whether SKN-1 is important for UPS activity under normal conditions [40]. We generated a strain (Pvha-6::UbG76V-Dendra2) in which the intestine-specific promoter vha-6 drives expression of a photoswitchable green-to-red fluorescent protein (Dendra2) that is fused to a non-hydrolyzable ubiquitin moiety (UbG76V) [40], [41]. By monitoring this fusion protein after photoconversion, we could assess ubiquitin-dependent protein degradation activity in living animals [40]. In control RNAi animals, at 9 hours after photoconversion the levels of red-fluorescing intestinal UbG76V-Dendra2 had been reduced to 40% of that present just after photoconversion, but a control Dendra2 that lacked UbG76V was still stable (Figure 5A, upper left panels). UbG76V-Dendra2 degradation was dramatically inhibited by RNAi against the proteasome genes pbs-5, rpn-2, or rpt-4, indicating that this degradation required the proteasome (Figure 5B and S4C). Together, the data show that this intestinal UbG76V-Dendra2 protein is degraded by the UPS.


Specific SKN-1/Nrf stress responses to perturbations in translation elongation and proteasome activity.

Li X, Matilainen O, Jin C, Glover-Cutter KM, Holmberg CI, Blackwell TK - PLoS Genet. (2011)

Knockdown of SKN-1 or TEFs, but not TIFs, reduces UPS activity in the intestine.(A) Representative images of Pvha-6::UbG76V-Dendra2 and Pvha-6::Dendra2 reporters in animals exposed to control, skn-1, eef-1A.1 (TEF) or eif-1(TIF) RNAi. Red fluorescence derives from substrate that was present at the time of photoconversion. Bar: 20 µm. In (A–C), bar graphs depict the percentages of green and red fluorescence compared to either the initial value (t = 0 for green) or point of photoconversion (t = C for red) for UbG76V-Dendra2 and Dendra2, respectively (± SEM). (B,C) Summary of UbG76V-Dendra2 and Dendra2 imaging in intestinal cells after 9 hours in response to TEF and TIF RNAi, respectively. Significant differences relative to control RNAi (red fluorescence after 9 hours) are indicated with **P<0.01, *P<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111486&req=5

pgen-1002119-g005: Knockdown of SKN-1 or TEFs, but not TIFs, reduces UPS activity in the intestine.(A) Representative images of Pvha-6::UbG76V-Dendra2 and Pvha-6::Dendra2 reporters in animals exposed to control, skn-1, eef-1A.1 (TEF) or eif-1(TIF) RNAi. Red fluorescence derives from substrate that was present at the time of photoconversion. Bar: 20 µm. In (A–C), bar graphs depict the percentages of green and red fluorescence compared to either the initial value (t = 0 for green) or point of photoconversion (t = C for red) for UbG76V-Dendra2 and Dendra2, respectively (± SEM). (B,C) Summary of UbG76V-Dendra2 and Dendra2 imaging in intestinal cells after 9 hours in response to TEF and TIF RNAi, respectively. Significant differences relative to control RNAi (red fluorescence after 9 hours) are indicated with **P<0.01, *P<0.05.
Mentions: As our data suggested that skn-1 contributes to proteasome gene expression, particularly when proteasome activity is impaired, we used a novel in vivo assay to investigate whether SKN-1 is important for UPS activity under normal conditions [40]. We generated a strain (Pvha-6::UbG76V-Dendra2) in which the intestine-specific promoter vha-6 drives expression of a photoswitchable green-to-red fluorescent protein (Dendra2) that is fused to a non-hydrolyzable ubiquitin moiety (UbG76V) [40], [41]. By monitoring this fusion protein after photoconversion, we could assess ubiquitin-dependent protein degradation activity in living animals [40]. In control RNAi animals, at 9 hours after photoconversion the levels of red-fluorescing intestinal UbG76V-Dendra2 had been reduced to 40% of that present just after photoconversion, but a control Dendra2 that lacked UbG76V was still stable (Figure 5A, upper left panels). UbG76V-Dendra2 degradation was dramatically inhibited by RNAi against the proteasome genes pbs-5, rpn-2, or rpt-4, indicating that this degradation required the proteasome (Figure 5B and S4C). Together, the data show that this intestinal UbG76V-Dendra2 protein is degraded by the UPS.

Bottom Line: In contrast, when translation elongation is impaired, SKN-1 does not upregulate proteasome genes, and UPS activity is then reduced.SKN-1 therefore has a critical tissue-specific function in increasing proteasome gene expression and UPS activity under normal conditions, as well as when the UPS system is stressed, but mounts distinct responses when protein synthesis is perturbed.The data suggest that SKN-1 may increase longevity, not only through its well-documented role in boosting stress resistance, but also through contributing to protein homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Joslin Diabetes Center, Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
SKN-1, the Caenorhabditis elegans Nrf1/2/3 ortholog, promotes both oxidative stress resistance and longevity. SKN-1 responds to oxidative stress by upregulating genes that detoxify and defend against free radicals and other reactive molecules, a SKN-1/Nrf function that is both well-known and conserved. Here we show that SKN-1 has a broader and more complex role in maintaining cellular stress defenses. SKN-1 sustains expression and activity of the ubiquitin-proteasome system (UPS) and coordinates specific protective responses to perturbations in protein synthesis or degradation through the UPS. If translation initiation or elongation is impaired, SKN-1 upregulates overlapping sets of cytoprotective genes and increases stress resistance. When proteasome gene expression and activity are blocked, SKN-1 activates multiple classes of proteasome subunit genes in a compensatory response. SKN-1 thereby maintains UPS activity in the intestine in vivo under normal conditions and promotes survival when the proteasome is inhibited. In contrast, when translation elongation is impaired, SKN-1 does not upregulate proteasome genes, and UPS activity is then reduced. This indicates that UPS activity depends upon presence of an intact translation elongation apparatus; and it supports a model, suggested by genetic and biochemical studies in yeast, that protein synthesis and degradation may be coupled processes. SKN-1 therefore has a critical tissue-specific function in increasing proteasome gene expression and UPS activity under normal conditions, as well as when the UPS system is stressed, but mounts distinct responses when protein synthesis is perturbed. The specificity of these SKN-1-mediated stress responses, along with the apparent coordination between UPS and translation elongation activity, may promote protein homeostasis under stress or disease conditions. The data suggest that SKN-1 may increase longevity, not only through its well-documented role in boosting stress resistance, but also through contributing to protein homeostasis.

Show MeSH
Related in: MedlinePlus