We have studied the effects of
hyperoxia and of cell loading with artificial
lipofuscin or
ceroid pigment on the postmitotic aging of human lung fibroblast cell cultures. Normobaric
hyperoxia (40%
oxygen) caused an irreversible 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
hyperoxia-induced 'aging', overall protein degradation (breakdown of [(35)S]
methionine metabolically radiolabeled cell
proteins) increased somewhat, but by 12 wk and thereafter overall proteolysis was significantly depressed. In contrast,
protein synthesis rates were unaffected by 12 wk of
hyperoxia. Lysosomal
cathepsin-specific activity (using the
fluorogenic substrate z-FR-MCA) and cytoplasmic
proteasome-specific activity (measured with
suc-LLVY-MCA) both declined by 80% or more over 12 wk.
Hyperoxia also caused a remarkable increase in
lipofuscin/
ceroid formation and accumulation over 12 wk, as judged by both fluorescence measurements and FACscan methods. To test whether the association between
lipofuscin/
ceroid accumulation and decreased proteolysis might be causal, we next exposed cells to
lipofuscin/
ceroid loading under normoxic conditions.
Lipofuscin/
ceroid-loaded cells indeed exhibited a gradual decrease in overall protein degradation over 4 wk of treatment, whereas
protein synthesis was unaffected.
Proteasome specific activity decreased by 25% over this period, which is important since
proteasome is normally responsible for degrading oxidized cell
proteins. In contrast, an apparent increase in lysosomal
cathepsin activity was actually caused by a large increase in the number of lysosomes per cell. To test whether
lipofuscin/
ceroid could in fact directly inhibit
proteasome activity, thus causing oxidized
proteins to accumulate, we incubated purified
proteasome with
lipofuscin/
ceroid preparations in vitro. We found that
proteasome is directly inhibited by
lipofuscin/
ceroid. Our results indicate that an accumulation of oxidized
proteins (and
lipids) such as
lipofuscin/
ceroid may actually cause further increases in damage accumulation during aging by inhibiting the
proteasome.