Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part I - effects of proliferative senescence

Oxidized and cross-linked proteins tend to accumulate in aging cells. Decli ning activity of proteolytic enzymes, particularly the proteasome, has been proposed as a possible explanation for this phenomenon, and direct inhibit ion of the proteasome by oxidized and cross-linked proteins has been demons trated in vitro. We have further examined this hypothesis during both proli ferative senescence (this paper) and postmitotic senescence (see the accomp anying paper, ref 1) of human BJ fibroblasts. During proliferative senescen ce, we found a marked decline in all proteasome activities (trypsin-like ac tivity, chymotrypsin-like activity, and peptidyl-glutamyl-hydrolyzing activ ity) and in lysosomal cathepsin activity. Despite the loss of proteasome ac tivity, there was no concomitant change in cellular levels of actual protea some protein (immunoassays) or in the steady-state levels of mRNAs for esse ntial proteasome subunits. The decline in proteasome activities and lysosom al cathepsin activities was accompanied by dramatic increases in the accumu lation of oxidized and cross-linked proteins. Furthermore, as proliferation stage increased, cells exhibited a decreasing ability to degrade the oxida tively damaged proteins generated by an acute, experimentally applied oxida tive stress. Thus, oxidized and cross-linked proteins accumulated rapidly i n cells of higher proliferation stages. Our data are consistent with the hy pothesis that proteasome is progressively inhibited by small accumulations of oxidized and cross-linked proteins during proliferative senescence until late proliferation stages, when so much proteasome activity has been lost that oxidized proteins accumulate at ever-increasing rates. Lysosomes attem pt to deal with the accumulating oxidized and cross-linked proteins, but de clining lysosomal cathepsin activity apparently limits their effectiveness. This hypothesis, which may explain the progressive intracellular accumulat ion of oxidized and cross-linked proteins in aging, is further explored dur ing postmitotic senescence in the accompanying paper (1).