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

Abstract

Oxidized and cross-linked proteins tend to accumulate in aging cells. Declining activity of proteolytic enzymes, particularly the proteasome, has been proposed as a possible explanation for this phenomenon, and direct inhibition of the proteasome by oxidized and cross-linked proteins has been demonstrated in vitro. We have further examined this hypothesis during both proliferative senescence (this paper) and postmitotic senescence (see the accompanying paper, ref 1 ) of human BJ fibroblasts. During proliferative senescence, we found a marked decline in all proteasome activities (trypsin-like activity, chymotrypsin-like activity, and peptidyl-glutamyl-hydrolyzing activity) and in lysosomal cathepsin activity. Despite the loss of proteasome activity, there was no concomitant change in cellular levels of actual proteasome protein (immunoassays) or in the steady-state levels of mRNAs for essential proteasome subunits. The decline in proteasome activities and lysosomal cathepsin activities was accompanied by dramatic increases in the accumulation of oxidized and cross-linked proteins. Furthermore, as proliferation stage increased, cells exhibited a decreasing ability to degrade the oxidatively damaged proteins generated by an acute, experimentally applied oxidative stress. Thus, oxidized and cross-linked proteins accumulated rapidly in cells of higher proliferation stages. Our data are consistent with the hypothesis 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 attempt to deal with the accumulating oxidized and cross-linked proteins, but declining lysosomal cathepsin activity apparently limits their effectiveness. This hypothesis, which may explain the progressive intracellular accumulation of oxidized and cross-linked proteins in aging, is further explored during postmitotic senescence in the accompanying paper (1).

Age-Associated Changes in Histology and Gene-Expression Profile in the Rat Ventral Prostate

“…In contrast, the ubiquitin-26S proteasome plays a much smaller role in oxidized protein degradation but is pivotally involved in the degradation of cellular signaling proteins, including the various transcriptional factors. In agreement with our findings, declines in proteasome activity of human fibroblasts (Ly et al, 2000;Sitte et al, 2000aSitte et al, , 2000b, pulmonary arterial-endothelial cells (Merker et al, 2001), and gastrocnemius muscle of the mouse during senescence have been noted (Pang et al, 2002). Taken together, a reduction in expression of genes encoding key proteasomal proteins is apparently a common phenomenon in cellular aging.…”

Central Role of the Proteasome in Senescence and Survival of Human Fibroblasts

“…A number of studies have demonstrated that impairment of proteasome function is associated with cellular senescence; however, the available data are fragmented and contradictory (15)(16)(17)(18)(19)(20)(21)42). To understand the involvement of the proteasome, we have taken a detailed molecular and biochemical approach of WI38 fibroblasts undergoing replicative senescence.…”

Repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers premature senescence through the TGF-β1 signaling pathway

“…Exposure of HDFs to hyperoxia under 40% O 2 for several weeks results in SIPS. When the exposure to 40% O 2 is prolonged well after the establishment of SIPS (up to 12 weeks), the proteasome activity sharply decreases (Sitte et al, 2000). In order to test whether proteasome activity also decreases in SIPS induced by acute subcytotoxic stress, we assayed the 20S proteasomal enzymatic activities in four models of SIPS.…”