Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part II - aging of nondividing cells

Oxidized/cross-linked intracellular protein materials, known as ceroid pigm ent, age pigment, or lipofuscin, accumulate in postmitotic tissues. It is u nclear, however, whether diminishing proteolytic capacities play a role in the accumulation of such oxidized intracellular proteins. Previous studies revealed that the proteasome is responsible for the degradation of most oxi dized soluble cytoplasmic and nuclear proteins and, we propose, for the pre vention of such damage accumulations. The present investigation was underta ken to test the changes in protein turnover, proteasome activity, lysosome activity, and protein oxidation status during the aging of nondividing cell s. Since the companion paper shows that both proteasome activity and the ov erall protein turnover decline during proliferative senescence whereas the accumulation of oxidized proteins increases significantly, we decided to us e the same human BJ fibroblasts, this time at confluency, at different PD l evels (including those that are essentially postmitotic) to investigate the same parameters under conditions where cells do not divide. We find that t he activity of the cytosolic proteasome declines dramatically during senesc ence of nondividing BJ fibroblasts. The peptidyl-glutamyl-hydrolyzing activ ity was particularly affected. This decline in proteasome activity was acco mpanied by a decrease in the overall turnover of short-lived (radiolabeled) proteins in the nondividing BJ fibroblasts. On the other hand, no decrease in the actual cellular proteasome content, as judged by immunoblots, was f ound. The decline in the activity of the proteasome was also accompanied by an increased accumulation of oxidized proteins, especially of oxidized and cross-linked material. Unlike the loss of lysosomal function seen in our a ccompanying studies of proliferative senescence (1), however, the present s tudy of hyperoxic senescence in nondividing cells actually revealed marked increases in lysosomal cathepsin activity in all but the very 'oldest' post mitotic cells. Our comparative studies of proliferating (1)and nonprolifera ting (this paper) human BJ fibroblasts reveal a good correlation between th e accumulation of oxidized/cross-linked proteins and the decline in proteas ome activity and overall cellular protein turnover during in vitro senescen ce, which may predict a causal relationship during actual cellular aging.