Proteasome inhibition by lipofuscin/ceroid during postmitotic aging of fibroblasts

We have studied the effects of hyperoxia and of cell loading with artificia l lipofuscin or ceroid pigment on the postmitotic aging of human lung fibro blast cell cultures. Normobaric hyperoxia (40% oxygen) caused an irreversib le senescence-like growth arrest after about 4 wk and shortened postmitotic life span from 1-1/2 years down to 3 months, During the first 8 wk of hype roxria-induced 'aging', overall protein degradation (breakdown of [S-35]met hionine metabolically radiolabeled cell proteins increased somewhat, but by 12 wk and thereafter overall proteolysis was significantly depressed, in c ontrast, protein synthesis rates were unaffected by 12 wk of hyperoxia. Lys osomal cathepsin-specific activity (using the fluorogenic substrate z-FR-MC A) and cytoplasmic proteasome-specific activity (measured with suc-LLVY-MCA ) both declined by 80% or more over 12 wk. Hyperoxia also caused a remarkab le increase iu lipofuscin/ceroid formation and accumulation over 12 wk, as judged by both fluorescence measurements and FACscan methods. To test wheth er the association between lipofuscin/ceroid accumulation and decreased pro teolysis might be causal, we next exposed cells to lipofuscin/ceroid loadin g under normoxic conditions. Lipofuscin/ceroid-loaded cells indeed exhibite d a gradual decrease in overall protein degradation over 4 wk of treatment, whereas protein synthesis was unaffected. Proteasome specific activity dec reased by 25% over this period, which is important since proteasome is norm ally responsible for degrading oxidized cell proteins. In contrast, an appa rent increase in lysosomal cathepsin activity was actually caused by a larg e increase in the number of lysosomes per cell. To test whether Lipofuscin/ ceroid could in fact directly inhibit proteasome activity, thus causing oxi dized proteins to accumulate, we incubated purified proteasome with lipofus cin/ceroid preparations in vitro. We found that proteasome is directly inhi bited by lipofuscin/ceroid. Our results indicate that an accumulation of ox idized proteins land lipids) such as lipofuscin/ceroid may actually cause f urther increases in damage accumulation during aging by inhibiting the prot easome.