Abstract
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 2.1.1.77) 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.
Highlights
Isoaspartyl damage is a common spontaneous protein modification repaired by the protein isoaspartyl methyltransferase (PCMT)
PCMT activity and homologous proteins have been previously observed in many animals, plants, fungi, archaea, and bacteria, PCMT was found to be absent in Amphidinium carterae [49], Bacillus subtilis (Gram-positive bacteria) [50], Mycoplasma genitalium (Gram-positive bacteria) [51], Synedra ulna [49], and S. cerevisiae (Fig. 2)
Previous work investigating the importance of isoaspartyl repair focused on several key model organisms, including E. coli (28 –30), Arabidopsis thaliana (79 – 81), C. elegans [32,33,34,35, 41, 53], D. melanogaster [31, 82], and mice (7, 36 –39)
Summary
Isoaspartyl damage is a common spontaneous protein modification repaired by the protein isoaspartyl methyltransferase (PCMT). The spontaneous degradation of asparaginyl and aspartyl residues to isoaspartyl residues is a common type of protein damage in aging organisms. Asparaginyl and aspartyl residues are hot spots for damage due to the favorable nucleophilic attack on the side chain carbonyl group by the peptide backbone nitrogen atom of the following residue (see Fig. 1) This reaction leads to the formation of an unstable succinimidyl intermediate that non-enzymatically hydrolyzes into either L-aspartyl or, to a greater extent, the abnormal L-isoaspartyl residue (8 –11). PCMT knock-out animals exhibit an increase in isoaspartyl damage in urinary peptides over time [36, 42] These data suggest that one or more proteolytic pathways can participate in the degradation and excretion of isoaspartyl-containing species in parallel with the methylation repair pathway. We characterize the isoaspartyl-containing polypeptides found in S. cerevisiae and provide evidence for a metalloprotease that limits isoaspartyl damage
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