title: Targeted gene disruption of the Caenorhabditis elegans L-isoaspartyl protein repair methyltransferase impairs survival of dauer stage nematodes.
abstract: The methylation of abnormal L-isoaspartyl residues by protein L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77) can lead to their conversion to L-aspartyl residues. For polypeptides damaged by spontaneous reactions that generate L-isoaspartyl residues, these steps represent a protein repair pathway that can limit the accumulation of potentially detrimental proteins in the aging process. We report here the construction and the characterization of an animal model deficient in this methyltransferase. We utilized Tc1-transposon-mediated mutagenesis in the nematode Caenorhabditis elegans to construct a homozygous excision mutant lacking exons 2-5 of the pcm-1 gene encoding this enzyme. Nematodes carrying this deletion exhibited no detectable L-isoaspartyl methyltransferase activity. These worms demonstrated normal morphology and behavior and adult mutant nematodes exhibited a normal lifespan. However, the survival of dauer-phase mutants was diminished by 3.5-fold relative to wild-type dauers after 50 days in the dauer phase. The fitness of the pcm-1 deletion nematodes was reduced by about 16% relative to that of wild-type nematodes as measured by the ability of these mutants to compete reproductively against a wild-type population. We found that the absence of the functional methyltransferase gene leads to a modest accumulation of altered protein substrates in aged dauer worms. However, in the viable fraction of these dauer worms, no differences were seen in the levels of altered substrate proteins in the parent and methyltransferase-deficient worms, suggesting that the enzyme in wild-type cells does not efficiently catalyze the repair of spontaneously damaged proteins.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles 90095-1569, USA.
authors.forename: R M
authors.initials: RM
authors.lastname: Kagan
authors.forename: A
authors.initials: A
authors.lastname: Niewmierzycka
authors.forename: S
authors.initials: S
authors.lastname: Clarke
mesh_terms: D000483:Alleles; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D002874:Chromosome Mapping; D004251:DNA Transposable Elements; D012734:Disorders of Sex Development; D004591:Electrophoresis, Polyacrylamide Gel; D005298:Fertility; D018390:Gene Targeting; D006801:Humans; D007814:Larva / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D008136:Longevity; D008745:Methylation; D016254:Mutagenesis, Insertional; D010455:Peptides / Q000378:metabolism; D016133:Polymerase Chain Reaction; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D004251:DNA Transposable Elements; D010455:Peptides; D011994:Recombinant Proteins; D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase