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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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Importance of SKN-1 for the proteasome bounce-back response.(A) Relative mRNA levels of endogenous proteasome subunit mRNAs in N2 animals fed with control (L4440) or skn-1 RNAi bacteria. All P<0.005 compared to control, except where N = not significant. Error bars indicate SEM. (B) skn-1 RNAi slightly decreases rpt-5p::GFP expression, particularly in the intestine, under normal conditions. Representative projection images show all z-stacks in the left panels, and z-stacks through the intestine on the right. Here and in (C), 2-day-old adults were used for imaging, boundaries of the intestine are indicated by dashed lines, and quantification and statistics are listed in Table S6. (C) skn-1 RNAi blocks the bounce-back response to proteasomal subunit knockdown. Representative confocal projection images of pas-5 or rpn-2 RNAi effects on the rpn-11p::GFP transcriptional reporter are shown, with z-stacks through the intestine or muscles displayed for double RNAi experiments. Abbreviations: M, body-wall muscle; SIM, stomatointestinal muscle. (D) Impaired bounce-back response in skn-1 mutants. Endogenous proteasome subunit mRNAs were detected by qRT-PCR in wild-type (N2) or skn-1(zu135) animals that had been fed with proteasome subunit RNAi bacteria. A paired t test (two-tailed) was employed to compare wild-type (N2) and skn-1(zu135) animals. An unpaired t test (two-tailed) was used to compare proteasome subunit vs control RNAi in N2 animals. Compared to N2 control, all P<0.05. mRNA levels were normalized to tba-1 (α-tubulin).
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pgen-1002119-g004: Importance of SKN-1 for the proteasome bounce-back response.(A) Relative mRNA levels of endogenous proteasome subunit mRNAs in N2 animals fed with control (L4440) or skn-1 RNAi bacteria. All P<0.005 compared to control, except where N = not significant. Error bars indicate SEM. (B) skn-1 RNAi slightly decreases rpt-5p::GFP expression, particularly in the intestine, under normal conditions. Representative projection images show all z-stacks in the left panels, and z-stacks through the intestine on the right. Here and in (C), 2-day-old adults were used for imaging, boundaries of the intestine are indicated by dashed lines, and quantification and statistics are listed in Table S6. (C) skn-1 RNAi blocks the bounce-back response to proteasomal subunit knockdown. Representative confocal projection images of pas-5 or rpn-2 RNAi effects on the rpn-11p::GFP transcriptional reporter are shown, with z-stacks through the intestine or muscles displayed for double RNAi experiments. Abbreviations: M, body-wall muscle; SIM, stomatointestinal muscle. (D) Impaired bounce-back response in skn-1 mutants. Endogenous proteasome subunit mRNAs were detected by qRT-PCR in wild-type (N2) or skn-1(zu135) animals that had been fed with proteasome subunit RNAi bacteria. A paired t test (two-tailed) was employed to compare wild-type (N2) and skn-1(zu135) animals. An unpaired t test (two-tailed) was used to compare proteasome subunit vs control RNAi in N2 animals. Compared to N2 control, all P<0.05. mRNA levels were normalized to tba-1 (α-tubulin).

Mentions: We first investigated the extent to which skn-1 is required for proteasome gene expression under normal conditions. The 26S proteasome consists of at least 32 subunits in C. elegans, including 19S ATPases involved in substrate unfolding (rpt-1∼6), other 19S subunits (rpn-1∼12), 20S α-rings (pas-1∼7) and 20S β-rings (pbs-1∼7) [11]. We examined how skn-1 RNAi affected the expression of the endogenous proteasome subunit genes rpt-3, rpn-12, pas-4 and pbs-6, which represent the four subunit classes above. Each of these genes is a predicted SKN-1 target at which at least four canonical SKN-1 binding sites lie within 1 kb upstream of the translation initiation codon, and SKN-1::GFP was detected by ChIP [20], [22] (data not shown). In whole animals skn-1 RNAi slightly decreased the expression of each gene, except for rpt-3 (Figure 4A). We also examined expression of transcriptional reporters in which proteasome promoters are fused to GFP. RNAi against skn-1 slightly decreased expression of reporters for rpt-5, rpn-11 and pas-5, particularly in the intestine, but did not detectably affect rpn-2 or pbs-4 (Figure 4B and 4C, Figure S4A and S4B, Table S6). The data suggest that under normal conditions SKN-1 contributes to but is apparently not essential for the expression of many proteasome subunit genes.


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)

Importance of SKN-1 for the proteasome bounce-back response.(A) Relative mRNA levels of endogenous proteasome subunit mRNAs in N2 animals fed with control (L4440) or skn-1 RNAi bacteria. All P<0.005 compared to control, except where N = not significant. Error bars indicate SEM. (B) skn-1 RNAi slightly decreases rpt-5p::GFP expression, particularly in the intestine, under normal conditions. Representative projection images show all z-stacks in the left panels, and z-stacks through the intestine on the right. Here and in (C), 2-day-old adults were used for imaging, boundaries of the intestine are indicated by dashed lines, and quantification and statistics are listed in Table S6. (C) skn-1 RNAi blocks the bounce-back response to proteasomal subunit knockdown. Representative confocal projection images of pas-5 or rpn-2 RNAi effects on the rpn-11p::GFP transcriptional reporter are shown, with z-stacks through the intestine or muscles displayed for double RNAi experiments. Abbreviations: M, body-wall muscle; SIM, stomatointestinal muscle. (D) Impaired bounce-back response in skn-1 mutants. Endogenous proteasome subunit mRNAs were detected by qRT-PCR in wild-type (N2) or skn-1(zu135) animals that had been fed with proteasome subunit RNAi bacteria. A paired t test (two-tailed) was employed to compare wild-type (N2) and skn-1(zu135) animals. An unpaired t test (two-tailed) was used to compare proteasome subunit vs control RNAi in N2 animals. Compared to N2 control, all P<0.05. mRNA levels were normalized to tba-1 (α-tubulin).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111486&req=5

pgen-1002119-g004: Importance of SKN-1 for the proteasome bounce-back response.(A) Relative mRNA levels of endogenous proteasome subunit mRNAs in N2 animals fed with control (L4440) or skn-1 RNAi bacteria. All P<0.005 compared to control, except where N = not significant. Error bars indicate SEM. (B) skn-1 RNAi slightly decreases rpt-5p::GFP expression, particularly in the intestine, under normal conditions. Representative projection images show all z-stacks in the left panels, and z-stacks through the intestine on the right. Here and in (C), 2-day-old adults were used for imaging, boundaries of the intestine are indicated by dashed lines, and quantification and statistics are listed in Table S6. (C) skn-1 RNAi blocks the bounce-back response to proteasomal subunit knockdown. Representative confocal projection images of pas-5 or rpn-2 RNAi effects on the rpn-11p::GFP transcriptional reporter are shown, with z-stacks through the intestine or muscles displayed for double RNAi experiments. Abbreviations: M, body-wall muscle; SIM, stomatointestinal muscle. (D) Impaired bounce-back response in skn-1 mutants. Endogenous proteasome subunit mRNAs were detected by qRT-PCR in wild-type (N2) or skn-1(zu135) animals that had been fed with proteasome subunit RNAi bacteria. A paired t test (two-tailed) was employed to compare wild-type (N2) and skn-1(zu135) animals. An unpaired t test (two-tailed) was used to compare proteasome subunit vs control RNAi in N2 animals. Compared to N2 control, all P<0.05. mRNA levels were normalized to tba-1 (α-tubulin).
Mentions: We first investigated the extent to which skn-1 is required for proteasome gene expression under normal conditions. The 26S proteasome consists of at least 32 subunits in C. elegans, including 19S ATPases involved in substrate unfolding (rpt-1∼6), other 19S subunits (rpn-1∼12), 20S α-rings (pas-1∼7) and 20S β-rings (pbs-1∼7) [11]. We examined how skn-1 RNAi affected the expression of the endogenous proteasome subunit genes rpt-3, rpn-12, pas-4 and pbs-6, which represent the four subunit classes above. Each of these genes is a predicted SKN-1 target at which at least four canonical SKN-1 binding sites lie within 1 kb upstream of the translation initiation codon, and SKN-1::GFP was detected by ChIP [20], [22] (data not shown). In whole animals skn-1 RNAi slightly decreased the expression of each gene, except for rpt-3 (Figure 4A). We also examined expression of transcriptional reporters in which proteasome promoters are fused to GFP. RNAi against skn-1 slightly decreased expression of reporters for rpt-5, rpn-11 and pas-5, particularly in the intestine, but did not detectably affect rpn-2 or pbs-4 (Figure 4B and 4C, Figure S4A and S4B, Table S6). The data suggest that under normal conditions SKN-1 contributes to but is apparently not essential for the expression of many proteasome subunit genes.

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