Proteasome Structures Affected by Ionizing Radiation

Abstract

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. (Mol Cancer Res 2005;3(7):381 -90)

Epidermal Growth Factor Receptor vIII Expression in U87 Glioblastoma Cells Alters Their Proteasome Composition, Function, and Response to Irradiation

“…In U87EGFRvIII cells, NPI0052 induced six subunits, including core constitutive subunits but not immunosubunits. The ability of NPI0052, but not irradiation, to induce constitutive core subunits may be related to the fact that the drug inactivates these subunits, whereas irradiation acts on the 19S cap (20). The inability of NPI0052 to induce immunoproteasome production in U87EGFRvIII cells is consistent with the fact that these subunits are poorly expressed in such cells (Fig.…”

“…Previous work from our laboratory showed that irradiation decreases proteasome function within minutes and that activity recovers over the next 24 hours (20). The mechanism of recovery is not known, but in this study, 2 Gy irradiation increased the expression levels for proteasome subunits at 6 hours in U87 cells and by 24 hours, proteasome subunit mRNAs were restored to preradiation levels.…”

“…This observation was dose independent with maximum inhibition even at low doses. The inhibition of 26S proteasome function was correlated to increased expression of IkB-a, a substrate of the 26S proteasome at low doses of radiation (19), and a general increase in ubiquitinated proteins (20). Remarkably, the activities of purified proteasome preparations irradiated on ice were also reduced (19,20), indicating that the proteasome is a direct target of radiation.…”

“…The inhibition of 26S proteasome function was correlated to increased expression of IkB-a, a substrate of the 26S proteasome at low doses of radiation (19), and a general increase in ubiquitinated proteins (20). Remarkably, the activities of purified proteasome preparations irradiated on ice were also reduced (19,20), indicating that the proteasome is a direct target of radiation. Further study showed that only 26S, not 20S or 20S +11S, proteasomes are affected by radiation and its inhibition is ATP dependent, suggesting that ATPase subunits of the 19S cap are probable mediators of the response (20).…”

“…Remarkably, the activities of purified proteasome preparations irradiated on ice were also reduced (19,20), indicating that the proteasome is a direct target of radiation. Further study showed that only 26S, not 20S or 20S +11S, proteasomes are affected by radiation and its inhibition is ATP dependent, suggesting that ATPase subunits of the 19S cap are probable mediators of the response (20).…”