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J Biol Chem. 2014 Jun 13;289(24):16936-53. doi: 10.1074/jbc.M114.564385. Epub 2014 Apr 24.

Non-repair pathways for minimizing protein isoaspartyl damage in the yeast Saccharomyces cerevisiae.

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  • 1From the Department of Chemistry and Biochemistry and the Molecular Biology Institute and.
  • 2the Pasarow Mass Spectrometry Laboratory, Neuropsychiatric Institute-Semel Institute for Neuroscience and Human Behavior, UCLA, Los Angeles, California 90095.
  • 3From the Department of Chemistry and Biochemistry and the Molecular Biology Institute and


The spontaneous degradation of asparaginyl and aspartyl residues to isoaspartyl residues is a common type of protein damage in aging organisms. Although the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (EC can initiate the repair of l-isoaspartyl residues to l-aspartyl residues in most organisms, no gene homolog or enzymatic activity is present in the budding yeast Saccharomyces cerevisiae. Therefore, we used biochemical approaches to elucidate how proteins containing isoaspartyl residues are metabolized in this organism. Surprisingly, the level of isoaspartyl residues in yeast proteins (50-300 pmol of isoaspartyl residues/mg of protein extract) is comparable with organisms with protein-l-isoaspartyl (d-aspartyl) O-methyltransferase, suggesting a novel regulatory pathway. Interfering with common protein quality control mechanisms by mutating and inhibiting the proteasomal and autophagic pathways in vivo did not increase isoaspartyl residue levels compared with wild type or uninhibited cells. However, the inhibition of metalloproteases in in vitro aging experiments by EDTA resulted in an ∼3-fold increase in the level of isoaspartyl-containing peptides. Characterization by mass spectrometry of these peptides identified several proteins involved in metabolism as targets of isoaspartyl damage. Further analysis of these peptides revealed that many have an N-terminal isoaspartyl site and originate from proteins with short half-lives. These results suggest that one or more metalloproteases participate in limiting isoaspartyl formation by robust proteolysis.


Aging; Isoaspartyl Residues; Protein Deamidation; Protein Methylation; Protein Stability; Protein Turnover; Protein l-Isoaspartyl Methyltransferase; Proteolytic Enzyme; Yeast Physiology

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