Exposure of cells to ionizing radiation slows the rate of degradation of substrates through the
proteasome. Because the
26S proteasome degrades most short-lived cellular
proteins, changes in its activity might significantly, and selectively, alter the life span of many signaling
proteins and play a role in promoting the
biological consequences of radiation exposure, such as cell cycle arrest, DNA repair, and apoptosis. Experiments were therefore undertaken to identify the radiation target that is associated with the
proteasome. Regardless of whether they were irradiated before or after extraction and purification from human
prostate cancer PC3 cells, 26S proteasomes remained intact but showed a rapid 30% to 50% dose-independent decrease in their three major enzymatic activities following exposure to 1 to 20 Gy. There was no effect on 20S proteasomes, suggesting that the radiation-sensitive target is located in the 19S cap of the
26S proteasome, rather than in the enzymatically active core. Because the base of the 19S cap contains an
ATPase ring that mediates substrate unfolding, pore opening, and translocation of substrates into the catalytic chamber, we examined whether the
ATPase activity of purified 26S proteasomes was affected. In fact, in vitro irradiation of proteasomes enhanced their
ATPase activity. Furthermore, pretreatment with low concentrations of the
free radical scavenger tempol was able to prevent both the radiation-induced decrease in proteolytic activity and the increase in
ATP utilization, indicating that
free radicals are mediators of these radiation-induced phenomena. Finally, we have shown that cell irradiation results in the accumulation of
proteasome substrates: polyubiquitinated
proteins and
ornithine decarboxylase, indicating that the observed decrease in
proteasome function is physiologically relevant.