We have investigated the causal factors behind the age-related oxidation of proteins during arrest of cell proliferation. A proteomic approach demonstrated that protein oxidation in non-proliferating cells is observed primarily for proteins being produced in a number of aberrant isoforms. Also, these cells exhibited a reduced translational fidelity as demonstrated by both proteomic analysis and genetic measurements of nonsense suppression. Mutants harboring hyperaccurate ribosomes exhibited a drastically attenuated protein oxidation during growth arrest. In contrast, oxidation was augmented in mutants with error-prone ribosomes. Oxidation increased concomitantly with a reduced rate of translation, indicating that the production of aberrant, and oxidized proteins, is not the result of titration of the co-translational folding machinery. The age-related accumulation of the chaperones, DnaK and GroEL, was drastically attenuated in the hyperaccurate rpsL mutant, demonstrating that the reduced translational fidelity in growth-arrested cells may also be a primary cause for the induction of the heat shock regulon. The data point to an alternative way of approaching the causal factors involved in protein oxidation in eukaryotic G(0) cells.

The relationship between the number of randomly accumulated mutations in a genome and fitness is a key parameter in evolutionary biology. Mutations may interact such that their combined effect on fitness is additive (no epistasis), reinforced (synergistic epistasis) or mitigated (antagonistic epistasis). We measured the decrease in fitness caused by increasing mutation number in the bacterium Salmonella typhimurium using a regulated, error-prone DNA polymerase (polymerase IV, DinB). As mutations accumulated, fitness costs increased at a diminishing rate. This suggests that random mutations interact such that their combined effect on fitness is mitigated and that the genome is buffered against the fitness reduction caused by accumulated mutations. Levels of the heat shock chaperones DnaK and GroEL increased in lineages that had accumulated many mutations, and experimental overproduction of GroEL further increased the fitness of lineages containing deleterious mutations. These findings suggest that overexpression of chaperones contributes to antagonistic epistasis.

Protein carbonylation is an irreversible oxidative modification that increases during organism aging and bacterial growth arrest. We analyzed whether the heat shock regulon has a role in defending Escherichia coli cells against this deleterious modification upon entry into stationary phase. Providing the cell with ectopically elevated levels of the heat shock transcription factor, sigma32, effectively reduced stasis-induced carbonylation. Separate overproduction of the major chaperone systems, DnaK/DnaJ and GroEL/GroES, established that the former of these is more important in counteracting protein carbonylation. Deletion of the heat shock proteases Lon and HslVU enhanced carbonylation whereas a clpP deletion alone had no effect. However, ClpP appears to have a role in reducing protein carbonyls in cells lacking Lon and HslVU. Proteomic immunodetection of carbonylated proteins in the wild-type, lon, and hslVU strains demonstrated that the same spectrum of proteins displayed a higher load of carbonyl groups in the lon and hslVU mutants. These proteins included the beta-subunit of RNA polymerase, elongation factors Tu and G, the E1 subunit of the pyruvate dehydrogenase complex, isocitrate dehydrogenase, 6-phosphogluconate dehydrogenase, and serine hydroxymethyltranferase.

In embryonic stem cells, removal of oxidatively damaged proteins is triggered upon the first signs of cell fate specification but the underlying mechanism is not known. Here, we report that this phase of differentiation encompasses an unexpected induction of genes encoding the proteasome activator PA28αβ (11S), subunits of the immunoproteasome (20Si), and the 20Si regulator TNFα. This induction is accompanied by assembly of mature PA28-20S(i) proteasomes and elevated proteasome activity. Inhibiting accumulation of PA28α using miRNA counteracted the removal of damaged proteins demonstrating that PA28αβ has a hitherto unidentified role required for resetting the levels of protein damage at the transition from self-renewal to cell differentiation.

SummaryIn organisms with a soma-germ demarcation, the germline must be 'preserved' such that harmful damage is not transmitted to the offspring. Keeping the progeny free of damage may be achieved by gametes enjoying elevated, and ⁄ or more functional, homeostatic maintenance systems. This possibility was approached here by testing whether the soma and maturating oocytes (eggs) dissected from female Drosophila melanogaster in reproductive ages display differential capacities for protein quality control and whether these capacities change during aging and mating. Eggs exhibited a high capacity to prevent protein aggregation, strong capacity for 26S proteasome-dependent degradation and reduced levels of oxidatively damaged (carbonylated) proteins compared to the soma. The capacity to prevent protein aggregation was not affected in either soma or eggs by age and ⁄ or mating, while the 26S proteasome capacity declined in the soma but was maintained in the eggs of aged females. However, the levels of carbonylated proteins increased with age in both soma and eggs, and this increase was more pronounced in females allowed to mate continuously. Furthermore, the levels of carbonylated proteins in the eggs of mated flies correlated negatively with the propensity of the eggs to develop into an adult fly. In young flies, mating caused a decrease in 26S proteasome capacity and an increase in protein carbonylation in the soma, but not in the eggs. These results are in line with trade-off theories of aging where aging is considered a consequence of investment in reproduction over somatic maintenance.