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The interplay between protein L-isoaspartyl methyltransferase activity and insulin-like signaling to extend lifespan in Caenorhabditis elegans.

Khare S, Linster CL, Clarke SG - PLoS ONE (2011)

Bottom Line: We demonstrate that reducing the levels of the DAF-16 downstream transcriptional effector of the IIS pathway by RNA interference reduces the lifespan extension resulting from PCM-1 overexpression.Using quantitative real-time PCR analysis, we show the up-regulation of DAF-16-dependent stress response genes in the PCM-1 overexpressor animals compared to wild-type and pcm-1 mutant nematodes under mild thermal stress conditions.Although we observe a higher accumulation of damaged proteins in pcm-1 mutant nematodes, the basal level of isoaspartyl residues detected in wild-type animals was not reduced by PCM-1 overexpression.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
The protein L-isoaspartyl-O-methyltransferase functions to initiate the repair of isomerized aspartyl and asparaginyl residues that spontaneously accumulate with age in a variety of organisms. Caenorhabditis elegans nematodes lacking the pcm-1 gene encoding this enzyme display a normal lifespan and phenotype under standard laboratory growth conditions. However, significant defects in development, egg laying, dauer survival, and autophagy have been observed in pcm-1 mutant nematodes when deprived of food and when exposed to oxidative stress. Interestingly, overexpression of this repair enzyme in both Drosophila and C. elegans extends adult lifespan under thermal stress. In this work, we show the involvement of the insulin/insulin-like growth factor-1 signaling (IIS) pathway in PCM-1-dependent lifespan extension in C. elegans. We demonstrate that reducing the levels of the DAF-16 downstream transcriptional effector of the IIS pathway by RNA interference reduces the lifespan extension resulting from PCM-1 overexpression. Using quantitative real-time PCR analysis, we show the up-regulation of DAF-16-dependent stress response genes in the PCM-1 overexpressor animals compared to wild-type and pcm-1 mutant nematodes under mild thermal stress conditions. Additionally, similar to other long-lived C. elegans mutants in the IIS pathway, including daf-2 and age-1 mutants, PCM-1 overexpressor adult animals display increased resistance to severe thermal stress, whereas pcm-1 mutant animals survive less long under these conditions. Although we observe a higher accumulation of damaged proteins in pcm-1 mutant nematodes, the basal level of isoaspartyl residues detected in wild-type animals was not reduced by PCM-1 overexpression. Our results support a signaling role for the protein L-isoaspartyl methyltransferase in lifespan extension that involves the IIS pathway, but that may be independent of its function in overall protein repair.

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Quantification of methyl-accepting protein substrates in C. elegans extracts for the recombinant human L-isoaspartyl methyltransferase at 20°C.Protein extracts from N2 and pcm-1 (qa201) nematode strains were prepared from a mixed stage nematode population after liquid culture for 5 days at 20°C and incubated with [3H]AdoMet and without or with recombinant human L-isoaspartyl methyltransferase as described in the Methods section. Base-labile methyl esters were quantified in wild-type (N2 - hrPCM, dashed line with open rectangles; N2 + hrPCM, solid line with closed rectangles) and pcm-1 (qa201) mutant (pcm-1 (qa201) - hrPCM, dashed line with open triangles; pcm-1 (qa201) + hrPCM, solid line with closed triangles) strains after polypeptide fractionation by SDS polyacrylamide gel electrophoresis. The migration positions of the molecular weight markers in kDa are indicated by arrows (Biorad SDS-PAGE Standards Catalog #161-0317). Base-labile [3H]-methyl ester groups present in peak 22 (not visible in figure) for N2 + hrPCM and pcm-1 (qa201) + hrPCM are 7285 cpm and 3683 cpm, respectively. Similar results were obtained in three independent experiments; one representative experiment is shown here and two others in Figure S4.
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pone-0020850-g004: Quantification of methyl-accepting protein substrates in C. elegans extracts for the recombinant human L-isoaspartyl methyltransferase at 20°C.Protein extracts from N2 and pcm-1 (qa201) nematode strains were prepared from a mixed stage nematode population after liquid culture for 5 days at 20°C and incubated with [3H]AdoMet and without or with recombinant human L-isoaspartyl methyltransferase as described in the Methods section. Base-labile methyl esters were quantified in wild-type (N2 - hrPCM, dashed line with open rectangles; N2 + hrPCM, solid line with closed rectangles) and pcm-1 (qa201) mutant (pcm-1 (qa201) - hrPCM, dashed line with open triangles; pcm-1 (qa201) + hrPCM, solid line with closed triangles) strains after polypeptide fractionation by SDS polyacrylamide gel electrophoresis. The migration positions of the molecular weight markers in kDa are indicated by arrows (Biorad SDS-PAGE Standards Catalog #161-0317). Base-labile [3H]-methyl ester groups present in peak 22 (not visible in figure) for N2 + hrPCM and pcm-1 (qa201) + hrPCM are 7285 cpm and 3683 cpm, respectively. Similar results were obtained in three independent experiments; one representative experiment is shown here and two others in Figure S4.

Mentions: In three independent experiments, we found significantly higher levels of base-labile [3H]-methyl ester groups in pcm-1 (qa201) mutant extracts compared to wild-type extracts prepared from nematodes grown at 20°C (Figs. 4 and S4) for proteins of molecular weights ranging from about 25 to 200 kDa. However, for reasons that presently remain unclear, in two other experiments of the same type we did not observe as significant of an increase in protein damage in the pcm-1 mutant (Figure S5, top panel). For the two assays performed on extracts derived from nematodes grown at 25°C, we did not observe significantly higher isoaspartyl levels in pcm-1 mutant protein extracts than in wild-type extracts (Figure S5, bottom panel). Further work is necessary to understand the reason underlying the variability of these results, but they indicate that pcm-1 mutant animals display a tendency to accumulate higher protein isoaspartyl levels. As pcm-1 mutant C. elegans do not present any lifespan phenotype at either 20°C or 25°C, these results indicate that isoaspartyl damage might not affect lifespan in nematodes. In all of our labeling experiments we generally observed a major peak of radioactive methyl group incorporation at 21 kDa, but the level of this incorporation seemed to be independent of PCM-1 activity.


The interplay between protein L-isoaspartyl methyltransferase activity and insulin-like signaling to extend lifespan in Caenorhabditis elegans.

Khare S, Linster CL, Clarke SG - PLoS ONE (2011)

Quantification of methyl-accepting protein substrates in C. elegans extracts for the recombinant human L-isoaspartyl methyltransferase at 20°C.Protein extracts from N2 and pcm-1 (qa201) nematode strains were prepared from a mixed stage nematode population after liquid culture for 5 days at 20°C and incubated with [3H]AdoMet and without or with recombinant human L-isoaspartyl methyltransferase as described in the Methods section. Base-labile methyl esters were quantified in wild-type (N2 - hrPCM, dashed line with open rectangles; N2 + hrPCM, solid line with closed rectangles) and pcm-1 (qa201) mutant (pcm-1 (qa201) - hrPCM, dashed line with open triangles; pcm-1 (qa201) + hrPCM, solid line with closed triangles) strains after polypeptide fractionation by SDS polyacrylamide gel electrophoresis. The migration positions of the molecular weight markers in kDa are indicated by arrows (Biorad SDS-PAGE Standards Catalog #161-0317). Base-labile [3H]-methyl ester groups present in peak 22 (not visible in figure) for N2 + hrPCM and pcm-1 (qa201) + hrPCM are 7285 cpm and 3683 cpm, respectively. Similar results were obtained in three independent experiments; one representative experiment is shown here and two others in Figure S4.
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Related In: Results  -  Collection

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pone-0020850-g004: Quantification of methyl-accepting protein substrates in C. elegans extracts for the recombinant human L-isoaspartyl methyltransferase at 20°C.Protein extracts from N2 and pcm-1 (qa201) nematode strains were prepared from a mixed stage nematode population after liquid culture for 5 days at 20°C and incubated with [3H]AdoMet and without or with recombinant human L-isoaspartyl methyltransferase as described in the Methods section. Base-labile methyl esters were quantified in wild-type (N2 - hrPCM, dashed line with open rectangles; N2 + hrPCM, solid line with closed rectangles) and pcm-1 (qa201) mutant (pcm-1 (qa201) - hrPCM, dashed line with open triangles; pcm-1 (qa201) + hrPCM, solid line with closed triangles) strains after polypeptide fractionation by SDS polyacrylamide gel electrophoresis. The migration positions of the molecular weight markers in kDa are indicated by arrows (Biorad SDS-PAGE Standards Catalog #161-0317). Base-labile [3H]-methyl ester groups present in peak 22 (not visible in figure) for N2 + hrPCM and pcm-1 (qa201) + hrPCM are 7285 cpm and 3683 cpm, respectively. Similar results were obtained in three independent experiments; one representative experiment is shown here and two others in Figure S4.
Mentions: In three independent experiments, we found significantly higher levels of base-labile [3H]-methyl ester groups in pcm-1 (qa201) mutant extracts compared to wild-type extracts prepared from nematodes grown at 20°C (Figs. 4 and S4) for proteins of molecular weights ranging from about 25 to 200 kDa. However, for reasons that presently remain unclear, in two other experiments of the same type we did not observe as significant of an increase in protein damage in the pcm-1 mutant (Figure S5, top panel). For the two assays performed on extracts derived from nematodes grown at 25°C, we did not observe significantly higher isoaspartyl levels in pcm-1 mutant protein extracts than in wild-type extracts (Figure S5, bottom panel). Further work is necessary to understand the reason underlying the variability of these results, but they indicate that pcm-1 mutant animals display a tendency to accumulate higher protein isoaspartyl levels. As pcm-1 mutant C. elegans do not present any lifespan phenotype at either 20°C or 25°C, these results indicate that isoaspartyl damage might not affect lifespan in nematodes. In all of our labeling experiments we generally observed a major peak of radioactive methyl group incorporation at 21 kDa, but the level of this incorporation seemed to be independent of PCM-1 activity.

Bottom Line: We demonstrate that reducing the levels of the DAF-16 downstream transcriptional effector of the IIS pathway by RNA interference reduces the lifespan extension resulting from PCM-1 overexpression.Using quantitative real-time PCR analysis, we show the up-regulation of DAF-16-dependent stress response genes in the PCM-1 overexpressor animals compared to wild-type and pcm-1 mutant nematodes under mild thermal stress conditions.Although we observe a higher accumulation of damaged proteins in pcm-1 mutant nematodes, the basal level of isoaspartyl residues detected in wild-type animals was not reduced by PCM-1 overexpression.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
The protein L-isoaspartyl-O-methyltransferase functions to initiate the repair of isomerized aspartyl and asparaginyl residues that spontaneously accumulate with age in a variety of organisms. Caenorhabditis elegans nematodes lacking the pcm-1 gene encoding this enzyme display a normal lifespan and phenotype under standard laboratory growth conditions. However, significant defects in development, egg laying, dauer survival, and autophagy have been observed in pcm-1 mutant nematodes when deprived of food and when exposed to oxidative stress. Interestingly, overexpression of this repair enzyme in both Drosophila and C. elegans extends adult lifespan under thermal stress. In this work, we show the involvement of the insulin/insulin-like growth factor-1 signaling (IIS) pathway in PCM-1-dependent lifespan extension in C. elegans. We demonstrate that reducing the levels of the DAF-16 downstream transcriptional effector of the IIS pathway by RNA interference reduces the lifespan extension resulting from PCM-1 overexpression. Using quantitative real-time PCR analysis, we show the up-regulation of DAF-16-dependent stress response genes in the PCM-1 overexpressor animals compared to wild-type and pcm-1 mutant nematodes under mild thermal stress conditions. Additionally, similar to other long-lived C. elegans mutants in the IIS pathway, including daf-2 and age-1 mutants, PCM-1 overexpressor adult animals display increased resistance to severe thermal stress, whereas pcm-1 mutant animals survive less long under these conditions. Although we observe a higher accumulation of damaged proteins in pcm-1 mutant nematodes, the basal level of isoaspartyl residues detected in wild-type animals was not reduced by PCM-1 overexpression. Our results support a signaling role for the protein L-isoaspartyl methyltransferase in lifespan extension that involves the IIS pathway, but that may be independent of its function in overall protein repair.

Show MeSH