Repair of age-damaged proteins catalyzed by PCMT. Isoaspartyl residues arise spontaneously during the aging of proteins by deamidation of asparaginyl residues and the isomerization of aspartyl residues via a succinimidyl intermediate. Protein isoaspartyl residues serve as substrates for PCMT, which initiates the repair of the polypeptide backbone via reformation of the succinimidyl intermediate.

Ubiquitous expression of the Pcmt gene in Drosophila embryos. In situ hybridization of dechorionated stage-12 embryos was performed as described in Materials and Methods. Embryos are oriented with the anterior end of the embryo toward the top of the figure. The dorsal (A) and side (B) views of embryos hybridized with antisense probes are shown as well as the side view of an embryo hybridized with a sense probe (C). The positions of the embryonic brain (b), midgut (mg), ventral nerve cord (vnc), and amnioserosa (as) are indicated.

Overexpression of the UAS-Pcmt gene using an hsp70-GAL4 driver. Non-Cy female progeny of a cross between an hsp70-GAL4 driver strain (Bloomington stock 2077) and an X-chromosome UAS-Pcmt responder strain were exposed to five 15-min treatments at 34°C with the treatment separated by 15-min rest intervals at room temperature (●). Extracts were prepared from flies at various times after heat shock and analyzed for PCMT activity (8). PCMT-specific activities were measured in extracts of flies from the homozygous X-chromosome responder strain subjected to a similar heat-shock regimen (○).

(A) Expression of PCMT in actin-Pcmt flies. Adult progeny of a cross between the actin 5C-GAL4 driver strain (Bloomington stock 4414) and the X-chromosome UAS-Pcmt responder strain were maintained at 25 (○) or 29°C (●). For the 29°C group, progeny were transferred from 25 to 29°C within several hours after adult eclosion. Activities were normalized to PCMT levels measured in flies of the homozygous parental responder strain maintained at the same temperatures for the same period. (B and C) PCMT overexpression driven by the actin5C promoter extends the lifespan of flies at 29°C. (B) Survival at 25°C. ●, survival of actin-Pcmt flies; ○, survival of the parental X-chromosome UAS-Pcmt responder strain. (C) Survival at 29°C. The meanings of ● and ○ are the same as described in B. ▴, survival of actin5C-GAL4 bearing progeny from an outcross between the driver strain and w¹¹¹⁸/w¹¹¹⁸ flies.

(A) Expression of PCMT in hsp-Pcmt flies at 29°C. Progeny of a cross between the hsp70-GAL4 driver and UAS-Pcmt responder strains were transferred from 25 to 29°C within several hours after adult eclosion. PCMT activities were measured in extracts prepared from flies after various days of culture. Activities were normalized to PCMT levels measured in flies of the homozygous parental responder strain maintained at 29°C for the same period. (B and C) PCMT overexpression driven by the hsp70 promoter extends the lifespan of flies at 29°C. Female flies were maintained in vials in groups of 10 and transferred to new vials every 4 days. One hundred female flies were used for each lifespan measurement. The data points shown for X- or second-chromosome responder lines represent means of three or two independent experiments, respectively. (B) Survival at 25°C. ○, survival of homozygous responder flies carrying an insertion of the UAS-Pcmt transgene on the X. □, survival of homozygous responder flies carrying an insertion of the UAS-Pcmt transgene on the second chromosome. ●, survival of hsp-Pcmt (overexpressing) flies using an X-linked UAS-Pcmt transgene. (C) Survival at 29°C. The meainings of ● and ○ are the same as described in B. ■, survival of overexpressing hsp-Pcmt flies using a second-chromosome UAS-Pcmt transgene; ▴, survival of hsp70-GAL4 bearing progeny from an outcross between the driver strain and w¹¹¹⁸/w¹¹¹⁸ flies.