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Atypical protein isoaspartyl residues arise spontaneously during the aging process from the deamidation of protein asparaginyl residues and the isomerization of protein aspartyl residues. These abnormal residues are modified in cells by a strongly conserved protein carboxyl methyltransferase (PCMT) as a first step in a repair pathway. Because a decline in cellular repair mechanisms is hypothesized to contribute to senescence, we determined whether increased PCMT activity was correlated with enhanced longevity. Two ubiquitous promoters were used with the binary GAL4-UAS system to drive PCMT overexpression in Drosophila melanogaster. Flies expressing PCMT activity under the regulation of either the hsp70 or actin5C promoter had enzyme activities that were 3-or 7-fold higher, respectively, than control flies at 29°C. Correlated with the observed increases in PCMT activities, such flies lived on average 32-39% longer than control flies. Lifespan extension was not observed at 25°C with either hsp70-or actin5C-driven expression, indicating a temperature-dependent effect on longevity. We conclude that protein repair is an important factor in the determination of lifespan under certain environmental conditions. PCMT activity may become limiting under mild stress conditions that accelerate rates of protein damage.

The ubiquitin-proteasome pathway is believed to selectively degrade post-synthetically damaged proteins in eukaryotic cells. To study this process we used calmodulin (CaM) as a substrate because of its importance in cell regulation and because it acquires isoaspartyl residues in its Ca 2؉ -binding regions both in vivo and after in vitro "aging" (incubation for 2 weeks without Ca 2؉ ). When microinjected into Xenopus oocytes, in vitro aged CaM was degraded much faster than native CaM by a proteasome-dependent process. Similarly, in HeLa cell extracts aged CaM was degraded at a higher rate, even though it was not conjugated to ubiquitin more rapidly than the native species. Ca 2؉ stimulated the ubiquitination of both species, but inhibited their degradation. Thus, for CaM, ubiquitination and proteolysis appear to be dissociated. Accordingly, purified muscle 26 S proteasomes could degrade aged CaM and native Ca 2؉ -free (apo) CaM without ubiquitination. Addition of Ca 2؉ dramatically reduced degradation of the native molecules but only slightly reduced the breakdown of the aged species. Thus, upon Ca 2؉ binding, native CaM assumes a non-degradable conformation, which most of the agedamaged species cannot assume. Thus, flexible conformations, as may arise from age-induced damage or the absence of ligands, can promote degradation directly by the proteasome without ubiquitination.

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