Carboxyl Methylation of Deamidated Calmodulin Increases Its Stability in Xenopus Oocyte Cytoplasm: IMPLICATIONS FOR PROTEIN REPAIR

Abstract

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.