Carboxyl methylation of deamidated calmodulin increases its stability in Xenopus oocyte cytoplasm. Implications for protein repair.
The widely distributed protein-L-isoaspartate(D-aspartate) O-methyltransferase (PIMT; EC 2.1.1.77) is postulated to play a role in the repair or metabolism of damaged cellular proteins containing L-isoaspartyl residues derived primarily from the spontaneous deamidation of protein asparaginyl residues. To evaluate the functional consequence of PIMT-catalyzed methylation on the stability of isoaspartyl-containing proteins in cells, Xenopus laevis oocytes were microinjected with both deamidated and nondeamidated forms of recombinant chicken calmodulin (CaM) containing a hemagglutinin (HA) epitope at its N terminus. Processing of HA-CaM was monitored by electrophoretic analysis and Western blotting of oocyte extracts. The experiments indicate that deamidated HA-CaM is degraded after microinjection, while nondeamidated HA-CaM is stable. Kinetic analysis is consistent with the entry of microinjected HA-CaM into two intracellular pools with distinct hydrolytic stabilities. The larger, more stable pool may consist of HA-CaM bound to the heterogeneous pool of oocyte CaM binding proteins detected by an overlay procedure. Enzymatic methylation of deamidated HA-CaM with purified PIMT prior to injection results in its stabilization. Conversely, inhibition of endogenous oocyte PIMT with sinefungin, a nonhydrolyzable analog of S-adenosylhomocysteine, increases the rate of deamidated HA-CaM degradation. These results are consistent with a role for PIMT-catalyzed methylation in the repair of damaged cellular proteins.
Department of Biology, Boston College, Chestnut Hill, Massachusetts 02167-3811, USA.
G
G
Szymanska
J D
JD
Leszyk
C M
CM
O'Connor
D000577:Amides / Q000378:metabolism; D000818:Animals; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002645:Chickens; D003593:Cytoplasm; D005260:Female; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008745:Methylation; D009865:Oocytes; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014981:Xenopus
D000577:Amides; D002147:Calmodulin; D006388:Hemagglutinins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase