Proteasome Structures Affected by Ionizing Radiation

Milena Pervan, Keisuke S. Iwamoto, and William H. McBride,

Department of Radiation Oncology, Experimental Division, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California

Abstract of degradation, alterations in either of which, if it is a Exposure of cells to ionizing radiation slows the regulatory protein, could critically affect cellular physiological rate of degradation of substrates through the processes. In recent years, proteasomes, which are large, proteasome. Because the 26S proteasome degrades most cylindrical, multicatalytic enzymatic complexes that are short-lived cellular proteins, changes in its activity might ubiquitous in eukaryotes (1-3), have taken center stage in significantly, and selectively, alter the life span of many intracellular proteolysis as they have increasingly been shown signaling proteins and play a role in promoting the to be responsible for degradation of most native regulatory biological consequences of radiation exposure, such as proteins, as well as those misfolded and damaged. Protea- cell cycle arrest, DNA repair, and apoptosis. Experiments somes can degrade proteins in an ATP-independent manner or were therefore undertaken to identify the radiation target an ATP-dependent manner. The former primarily targets that is associated with the proteasome. Regardless of damaged proteins and peptides, whereas the latter is part of whether they were irradiated before or after extraction the ubiquitin-dependent 26S proteasome pathway that func- and purification from human prostate cancer PC3 cells, tions to control expression of most native proteins. The 26S 26S proteasomes remained intact but showed a rapid 30% proteasome therefore closely regulates short-lived proteins that to 50% dose-independent decrease in their three major mediate basic physiological processes such as cell cycle enzymatic activities following exposure to 1 to 20 Gy. progression (cyclins A, D, and E, p53, p21, p27 mdm2, HIF- There was no effect on 20S proteasomes, suggesting that 1a), DNA transcription (InB/nuclear factor nB, c-myc, c-Jun, the radiation-sensitive target is located in the 19S cap of c-fos, AP-1, STAT-1), DNA repair (DNA-PKcs, rad 23), the 26S proteasome, rather than in the enzymatically apoptosis (p53, p21, mdm2, bcl2, bax, caspase-3), inflamma- active core. Because the base of the 19S cap contains an tion and immunity (InB/nuclear factor nB, tumor necrosis ATPase ring that mediates substrate unfolding, pore factor-R1), and cell growth (epidermal growth factor receptor, opening, and translocation of substrates into the catalytic insulin-like growth factor receptor, and platelet-derived growth chamber, we examined whether the ATPase activity of factor receptor; refs. 4-13). Proteasomes have therefore purified 26S proteasomes was affected. In fact, in vitro evolved to perform not only catabolic but also regulatory irradiation of proteasomes enhanced their ATPase functions. activity. Furthermore, pretreatment with low All proteasomes have a common 20S core that has the concentrations of the free radical scavenger tempol was proteolytically active sites internally sequestered. Four septa- able to prevent both the radiation-induced decrease in meric rings are arranged symmetrically so that two outer rings proteolytic activity and the increase in ATP utilization, of a-type subunits form an antechamber and two inner rings indicating that free radicals are mediators of these of h-type subunits form the proteolytic chamber. Only three, radiation-induced phenomena. Finally, we have shown h5(X), h1(Y) and h2(Z), of the seven different h-type that cell irradiation results in the accumulation of subunits are proteolytically active, being associated with the proteasome substrates: polyubiquitinated proteins and three major activities of the proteasome, namely chymotryp- ornithine decarboxylase, indicating that the observed sin-like activity (cleaving after the COOH-terminal of decrease in proteasome function is physiologically hydrophobic residues), trypsin-like activity (cleaving after relevant. (Mol Cancer Res 2005;3(7):381–90) basic residues), and peptidylglutamyl-hydrolyzing activity (PGPH, cleaving after acidic residues), respectively. Core Introduction proteasomes exist only in a latent state and need to be The levels at which a protein is expressed in a cell is ‘‘activated’’ by binding either a 19S regulatory complex or an determined by a balance between rate of production and rate 11S activator to the a-type rings (14, 15). The 19S regulator is comprised of about 17 distinct subunits with six ATPase subunits in a base adjacent to the core and a lid comprised of non-ATPase subunits. The functions of the lid Received 3/18/05; revised 6/1/05; accepted 6/13/05. Grant support: National Cancer Institute grant CA-87887 (to W.H. McBride). complex are not fully elucidated but it seems to be involved in The costs of publication of this article were defrayed in part by the payment of substrate recognition and binding (16-18), whereas ATPases page charges. This article must therefore be hereby marked advertisement in are, in addition, responsible for processes relating to pore accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: William H. McBride, Department of Radiation Oncology, opening, gating of the translocation channel, substrate unfold- Roy E. Coats Research Laboratories, David Geffen School of Medicine at UCLA, ing and transfer of unfolded proteins to the enzymatically 10833 Le Conte Avenue, Los Angeles, CA 90095-1714. Phone: 310-794-7051; Fax: 310-206-1260. E-mail: [email protected] active sites (19-22). The 11S hexameric activator performs Copyright D 2005 American Association for Cancer Research. similar functions in an ATPase-independent manner. It is

composed of two homologous but not identical subunits, mRNA and protein stability. This study was designed to PA28a and PA28h (23) arranged in a ring (24). IFN-g identify the proteasomal targets that are affected by radiation stimulates expression of the 11S activator (15, 25) and and mediate the slowing of the rate of substrate degradation, substitution of the X, Y and Z constitutive subunits of the which is critical for our understanding of how cells respond to 20S core by LMP7, LMP2 and MECL-1 (26). This substitution radiation challenge. forms what is often called the ‘‘immunoproteasome’’ because it alters substrate cleavage specificity so as to enhance the production of antigenic peptides suitable for presentation by Results MHC class I molecules (27). Pajonk et al. (31, 32) previously described slowing of the When making rapid responses to a changing environment or rate of degradation of substrates specific for proteasome other external stresses, a cell initially relies on posttranscrip- chymotrypsin-like activity following exposure to ionizing tional control mechanisms. Because of its position as a ‘‘master radiation that was independent of dose between 25 cGy and controller’’ of protein degradation, the proteasome is a good 20 Gy. To determine if all major enzymatic activities were candidate for affecting a switch of cells from nonstressed to affected, the rate of degradation of fluorogenic peptides stressed states. Reports that proteasome function is altered specific for chymotrypsin-like, trypsin-like, and PGPH, as following exposure of cells to ionizing and UV radiation, heat well as for tripeptidyl peptidase II, activity was assessed in shock, or oxidative stress are consistent with this hypothesis crude extracts 3 hours after radiation doses ranging from 0.5 (28-30). We have shown that exposure of cells to doses of to 20 Gy (Fig. 1A). A 30% to 50% slowing of the rate of ionizing radiation ranging from 25 cGy to 20 Gy slows the rate degradation of substrates specific for the proteasome, but not at which the proteasomes degrade small fluorogenic peptides for tripeptidyl peptidase II, indicated that inhibition was specific for chymotrypsin-like activity by 30% to 50% (31). limited to the proteasome, but not to any specific enzymatic Radiation is ‘‘alarming’’ to cells and activates mechanisms activity associated with the proteasome. leading to rapid cell cycle arrest, DNA repair, and cell death or The dynamics and the duration of inhibition induced by survival. At least initially it seems likely that these responses ionizing radiation were assessed by measuring chymotrypsin- are controlled posttranscriptionally and involve alterations in like activity of crude PC3 cellular extracts at different time

FIGURE 1. Radiation-induced inhibition of proteasome activity. A. PC3 cells were irradiated with different doses (0.5-20 Gy) and harvested at 3 hours postirradiation. Proteasome activities (chymotrypsin-like, trypsin-like, and PGPH) and tripeptidyl peptidase II activity were assayed using appropriate fluorogenic peptides as described in Materials and Methods. B. Time response: cells were irradiated with 10 Gy, harvested at different times thereafter and extracts were assayed for chymotrypsin-like activity. Asterisks, significant differences as calculated by Student’s t test; *, P < 0.025, n =6.C. Proteasome content in PC3 cells following irradiation: crude extracts from irradiated (10 Gy) and control cells were harvested at indicated times, separated by 10% SDS- PAGE, transferred onto a polyvinylidene difluoride membrane, and immunoblotted with monoclonal antibodies raised against 20S proteasome subunit a6, 11S activator subunit a (PA28 a), and 19S regulator non-ATPase subunit Rnp8 (S12). Equal loading was confirmed by reblotting with anti-a-tubulin.

points over a 24-hour period after exposure to 10 Gy. Ionizing radiation generates free radicals that mediate many Radiation-induced slowing of the rate of substrate degradation of its effects. To test if free radicals mediated radiation- occurred within 15 minutes postirradiation. The activity induced proteasome inhibition and ATPase changes, two free recovered slowly with time and was normal at 24 hours radical scavengers were used—glutathione (GSH), an endog- (Fig. 1B). To ensure that the proteasome content was not altered enous cellular antioxidant, and 4-hydroxy-tempo (tempol), a following cell irradiation, extracts of PC3 cells were separated low–molecular weight nitroxide compound with antioxidant by SDS-PAGE 1 and 3 hours following 10 Gy irradiation and activity—both of which protect cells against oxidative stress immunoblotted with monoclonal antibodies against the 20S caused by ionizing radiation (33, 34). Both, tempol and GSH, proteasome subunit a6, the 11S activator subunit a (PA28 a), inhibited proteasome activity at high doses (Fig. 4A), but only and the 19S proteasome regulator non-ATPase subunit Rnp8 minimally at 10 and 5 mmol/L, respectively. These concen- (Fig. 1C). Irradiation did not alter proteasome expression levels trations were, however, able to prevent proteasome inhibition over the 3-hour time period. caused by irradiation (Fig. 4B) indicating involvement of free Because all three proteasome enzymatic activities were radicals in this radiation effect. Furthermore, 10 mmol/L diminished by irradiation, experiments were undertaken to tempol was able to prevent the increase in ATP usage by the determine if 20S core activity was affected. Cells were 26S proteasome (Fig. 4C), suggesting that radiation-induced irradiated with 0 or 10 Gy and 20S and 26S proteasomes were free radicals also affected ATPase activity. GSH was not separated from crude extracts by velocity sedimentation tested because it interfered directly with luciferase activity centrifugation using continuous 10% to 40% glycerol gradients. (data not shown). 26S proteasomes were detected in fractions 15 to 18 by To determine if the effect of radiation on the 26S degradation of fluorogenic substrates specific for chymotrypsin- proteasome had physiological relevance, we assayed levels like activity in the presence of 2 mmol/L ATP (Fig. 2A). 20S of ubiquitinated proteins following cell irradiation. Because core particle activity was activated by the addition of 0.05% ATP-dependent 26S proteolysis in cells is coupled with the SDS, which also inactivates 26S proteasomes, and was found in ubiquitination process through removal of ubiquitinated fractions 4 to 7. To determine if irradiation directly influenced substrates (35), a decline in 26S proteasome function should proteasome function, fractions containing the 26S and the 20S impair degradation of ubiquitinated proteins. To test this, proteasomes were pooled, total protein concentration was cellular extracts from irradiated and control PC3 cells were equalized, and half the unirradiated samples were irradiated precipitated using agarose-immobilized p62-derived ubiquitin- in vitro with 10 Gy. Irrespective of whether the proteasomes binding domain, separated on SDS-PAGE (10-20% gradient) had been irradiated prior to or after extraction from cells, the and immunoblotted with antiubiquitin antibody. As shown in rate of degradation of fluorogenic substrate by 26S proteasomes Fig. 5A and B, ionizing radiation induced a 50% increase in was slowed by the same extent, whereas 20S proteasome the cytosolic levels of ubiquitinated proteins, suggesting that activity was not affected (Fig. 2B). Furthermore, in vitro irradiation directly affects processing of proteasome intra- irradiation of 26S proteasomes that were purified from cellular substrates through the ubiquitin/26S proteasome irradiated cells did not further increase the extent of inhibition, system. which was similar over the dose range 1 to 20 Gy (Fig. 2C). We further tested the effect of irradiation on degradation The finding that the activity of the 20S enzymatic core particle of the 26S proteasome substrate ornithine decarboxylase was resistant to irradiation indicated that the radiation target(s) (ODC), which does not require ubiquitination (36). This was not within proteolytically active sites but resided in the 19S was done in living cells using an ODC-ZsGreen protein cap associated with the 26S structure. To confirm that the 26S (ZsG) fusion protein reporter system. PC3 cells were tran- purified particles were intact after irradiation, they were siently transfected with ODC-ZsG. Flow-cytometric analysis resolved by native gel electrophoresis followed by functional detected accumulation of the fusion protein 5 hours following staining with 100 mmol/L fluorogenic peptide LLVY-7-amido- irradiation. 4-methylcoumarin (AMC) and protein staining with Coomassie blue. There was no change in the protein level associated with the functional 26S proteasome band after direct proteasome Discussion irradiation and no evidence of the presence of free 19S or 20S In recent years, a picture is emerging of the proteasome as fractions (Fig. 3A), indicating that the proteasome structure a master controller of multiple signal transduction pathways remained intact. whose activity is affected by a variety of agents including We next considered the possibility that radiation induced oxidative stress (37), heat shock (38), ionizing radiation (39), structural or functional changes within the subunits of the UV radiation (28), chemotherapeutic drugs (40), viruses 19S regulator and specifically that the base of the 19S (41-43), and carcinogens (44). This is replacing the regulator consisting of six ATPase subunits, which is previously held view that the proteasome was simply a responsible for opening the 20S proteasome and translocation disposal mechanism for unwanted proteins that played little of substrates into the catalytically active core, might be a role in cellular regulation. Here we confirm our previous target. Proteasome ATP utilization was assessed following observation that chymotrypsin-like activity of the proteasome irradiation using a chemiluminescent luciferase assay to is partially inhibited over a wide range of radiation doses and measure residual ATP. As shown in Fig. 3B, proteasome further show that trypsin-like and PGPH activities of the ATP utilization increased following irradiation, as proteolytic proteasome are similarly affected. This suggests a global activity declined. effect on proteasome function in the cell, even though the

FIGURE 2. Effect of ionizing radiation on 26S but not 20S proteasomes. A. 26S and 20S proteasomes were separated by velocity sedimentation. One milligram of total soluble protein from PC3 cells was applied to the top of the 10% to 40% glycerol gradient. Four hundred – microliter fractions were taken from the top of the gradient and assayed for chymotrypsin-like activity. The 26S proteasome activity was assessed in the presence of 2 mmol/L ATP and the 20S core activity in the presence of 0.05% SDS. B. PC3 cells were irradiated with 0 or 10 Gy. Three hours later, 26S and 20S proteasomes were isolated by glycerol gradient velocity sedimentation centrifugation. Purified proteasomes were then irradiated with 0 or 10 Gy and chymotrypsin-like activity evaluated immediately. CTRL, nonirradiated cells; 10 Gy, irradiated cells; CTRL + 10 Gy, irradiated purified proteasomes from control cells; 10 Gy + 10 Gy, irradiated purified proteasomes from irradiated cells. C. 26S-containing fractions from PC3 cells were pooled, diluted with buffer I to 1% glycerol, irradiated with different radiation doses, and immediately assayed for proteasome chymotrypsin-like activity.

sites of the three enzymatic activities seem to operate fairly We have shown that the radiation effect is limited to the 26S independently, as is seen by the finding that the proteasome proteasome, whereas 20S structures remain active, indicating inhibitor PS-341 completely inhibits chymotrypsin-like activ- that the proteolytic sites are not targets for radiation. There are ity and only partially affects PGPH activity, whereas trypsin- reports that the 26S proteasome is more sensitive than the 20S 1 like activity remains unaffected. core to UV radiation, low dose H2O2, as well as to N-acetyl cysteine (28, 45, 46). It is tempting to hypothesize that slowing the rate of degradation of regulatory proteins through the 26S 1 Pervan, unpublished observations. proteasome initiates a stress alarm in the cell, while continuing to

self-compartmentalized proteases have been reported to substitute for loss of proteasome function in lymphoma cells adapted to grow in the presence of proteasome inhibitors (49, 50). The suggestion is therefore that the abnormally high sensitivity of the proteasome to even low doses of radiation is specific and unique. As with the response to other stress stimuli, the inhibition of proteasome activity by ionizing irradiation is very rapid, probably immediate. Activity recovers within 24 hours. The duration of inhibition seems different for different stressors. For example, inhibition of proteasome activity in human keratinocytes induced by UVA- and UVB-irradiation was much more pronounced at 24 hours than 1 hour postirradiation and lasted at least for 96 hours (28). Proteasome activity in human leukemia K562 cells partially recovers 24 hours after treatment with 1 mmol/L H2O2 but not after 2 mmol/L H2O2 (38), suggesting that the level of oxidative stress may determine the rate of proteasome recovery. It may be particularly prolonged after exposure to agents that cause substantial damage to proteins. This makes even more striking the finding that doses of ionizing radiation that would be expected to cause only perhaps 10,000 ionization events in a cell are as effective as a dose 100- fold larger at causing proteasome inhibition. Even more surprising is the finding that we were able to observe a direct effect of radiation on purified proteasomes, suggesting a chemical rather than biological mechanism of inhibition. This chemical mechanism was mediated through free radicals, which was shown by the addition of free radical scavengers, tempol and GSH. Because these two drugs are often used in radiation experiments, it is noteworthy to mention that changes in redox status by addition of the higher doses of either of the two free radical scavengers used in this study also impaired proteasome activity. This observation was in agreement with previous evidence that alterations in intracellular glutathione metabolism affect proteasome function (51) as does N-acetyl cysteine (46). The implication is that proteasome function is highly sensitive to redox changes and that the proteasome is an exquisitely sensitive sensor of stress that responds rather like a switch to initiate rapid molecular reprogramming. We present evidence that the targets for radiation are located FIGURE 3. Radiation does not cause 26S proteasomes to disassoci- ate but does affect proteasome ATPases. A. Glycerol gradient fractions in the 19S proteasome regulator or at the 19S and 20S junction. containing 26S proteasome were irradiated with 0, 10, and 50 Gy, and Lack of a radiation effect on the core particle excludes the separated by nondenaturing PAGE. The position of the 26S fraction was identified by overlaying the gel with fluorogenic substrate in 100 nmol/L in possibility that proteolytically active sites are the sensitive buffer I (right) and then the gel was stained with Coomassie blue (left) proteasome components. Several years ago, Glickman showed showing that the 26S proteasome did not dissasociate. B. 26S gradient that a complex formed by the 20S proteasome and the base of purified proteasomes were irradiated with 0 or 10 Gy and incubated at 37jC with varying concentrations of ATP. For the standard curve, the the 19S regulator is sufficient for ATP-dependent degradation of same volume of buffer I was added to each sample. The remaining, nonubiquitinated substrates, but it is insufficient for degradation unused ATP was determined after 15 minutes by addition of luciferase/ of ubiquitinated proteins (52). The fluorogenic peptide sub- luciferin reaction. Emitted light was measured immediately using the chemoluminometer. strates used in our experiments do not require ubiquitination; their degradation depends on activation of the core particle which is a role played by the ATPase subunits. We found that allow removal of damaged, potentially toxic proteins through proteasome ATPase activity was increased following irradiation, non-26S proteasomes, although this potential division of labor an effect that was prevented by addition of free radical between different proteasome species may be an oversimplifi- scavengers. Thus, redox changes in the proteasome ATPase cation of the situation as some proteasome functions overlap. environment modify ATPase activity in a manner that decreases The effects of radiation on the activity of purified enzymes are proteolytic processing but increases ATP uptake. Recently, ATP well known and it requires massive doses well above those used hydrolysis has been linked to proteasome disassembly during in this study to inactivate them (47, 48). This is confirmed by the substrate proteolysis (53). This does not seem to happen finding that we didn’t observe any significant alterations in following irradiation in vitro, possibly because of the absence of tripeptidyl peptidase II/tricorn hydrolyzing activity. These giant substrate degradation in our experimental setup.

Presently, very little is known about proteasome ATPases. However, it seems likely that the three-dimensional structure is crucial for proper function of this dynamic complex. Parallel arguments can be drawn between the proteasome and other ATPase-regulated systems, such as the Na+/K+ pump, in which minor structural alterations cause functional inactivation of the pump (54). Our hypothesis is that small conformational changes in the proteasome subunit tertiary structure caused by redox imbalance inactivates a ‘‘clutch’’ mechanism(s) that couples ATP hydrolysis to substrate translocation or gate opening, resulting in a ‘‘revving engine’’ with no accomplishment of useful work. However, future studies are needed to test this hypothesis. Radiation slows degradative rate—it does not stop it completely, at least as assessed using fluorogenic substrates, and this opens the question of whether such an effect is biologically relevant. In support of the fact that this is relevant, we show that partial inhibition is sufficient to affect the intracellular molecular profile as there is a general increase in the level of ubiquitinylated proteins and accumulation of the 26S proteasome substrate ODC. The contention is further supported by the findings of others showing rapid alterations in stability of many proteins that are cleared by the proteasome such as p53, p21, and InBa (55, 56) following irradiation, which has obvious implications for radiation- induced cellular responses. However, many of these stress proteins are controlled by complex molecular interactions and their expression may not depend only on proteasome degradation. For example, we have shown that expression of InBa follows a biphasic pattern (56). It was enhanced at low doses of radiation, as would be expected if proteasome function was inhibited, and was not decreased after higher doses which activate nuclear factor nB. Therefore, partial proteasome inhibition seems to be a state within which radiation-induced transcriptional responses take place in an interactive manner. The longstanding concept of the proteasome as a robust nonspecific degradation machine is rapidly changing. Recent advances in the field imply critical involvement of proteasome in the regulation of many aspects of cellular physiology. Proteasomes show high sensitivity to minimal redox changes caused by irradiation (and other stress stimuli) implying that this pathway should be perceived as a sophisticated and highly sensitive stress sensing mechanism that can rapidly and simultaneously orchestrate multiple cellular processes following exposure to radiation. However, a number of important

FIGURE 4. Free radical scavengers prevent radiation-induced changes in proteasome activity. A. Effect of free radical scavengers on proteasome function. Dose-response curves for treatment of proteasomes with tempol and GSH were generated by measuring chymotrypsin-like activity with Suc-LLVY-AMC. B. 26S gradient purified proteasomes were irradiated (10 Gy) in the presence of 10 mmol/L tempol and 5 mmol/L GSH and chymotrypsin-like activity was determined. Asterisks, significant differences from control, as calculated by Student’s t test; *, P < 0.025; n =3.C. 26S gradient purified proteasomes were irradiated with and without 10 mmol/L tempol, incubated 15 minutes with varying concentrations of ATP, and the amount of the remaining ATP was determined by the addition of luciferase/luciferin. Emitted light was measured immediately using the chemoluminometer.

FIGURE 5. Levels of proteasome substrate changes following radiation. A. Ubiquitinated proteins from PC3 cells were purified by agarose-immobilized p62-derived ubiquitin-binding domain, separated by SDS-PAGE (10-20%), and immunoblotted with anti-ubiquitin antibody (clone FK1). Radiation increased the level of polyubiquitinated proteins. B. Quantified data from (A) are expressed as a percentage of control. C. Expression of ODC-ZsG in unirradiated and irradiated (2Gy) transiently transfected PC3 cells after 5 hours as assayed by flow-cytometry. Radiation increased the level of ODC-ZsG protein.

structural and functional questions regarding the proteasome as Herndon, VA) supplemented with 10% FCS (Sigma-Aldrich, a sensor of radiation effects remain unanswered and answers to Inc., St. Louis, MO) and 1% antibiotic-antimycotic (Medi- some of these questions could reshape our current understand- atech). Cells were used at 70% confluence, with medium ing of events involved in cellular radiobiology. changed 1 day prior to experimentation.

Materials and Methods Drugs, Peptides, and Antibodies Cell Culture Fluorogenic peptides for proteasome chymotrypsin-like Human prostate carcinoma PC3 cells (obtained from activity: Suc-LLVY-AMC (Sigma), trypsin-like activity: Z- American Type Culture Collection, Manassas, VA) were ARR-AMC (Calbiochem, San Diego, CA), PGPH activity: maintained at 37jC and 5% CO2 in DMEM (Mediatech, Inc., Z-LLE-AMC (Sigma), and tricorn peptidase (tripeptidyl

peptidase II) activity: 2-acetylaminofluorene-AMC were Precipitation of Ubiquitinated Proteins dissolved in DMSO as 100 mmol/L stock solutions and Ubiquitinated proteins were precipitated using agarose- stored at À20jC in 200 mL aliquots. Free radical scavengers immobilized, p62-derived ubiquitin-binding domain (Affinity tempol (Sigma) and GSH (Sigma) were freshly prepared for Research Products). PC3 cells were lysed in ice-cold lysis each experiment and diluted with DMEM. Monoclonal buffer [20 mmol/L Tris-HCl (pH 7.4), 1% NP40, 137 mmol/L antibodies against the 20S proteasome subunit a6, the 11S NaCl, 50 Amol/L EDTA, 1 mmol/L PMSF, 1 Amol/L pepstatin activator subunit a (PA28 a) and the 19S proteasome A, 1 Amol/L leupeptin, and 5 mmol/L N-ethylmaleimide] for regulator non-ATPase subunit Rnp8 (Affinity Research 20 minutes at 4jC with occasional rocking. Cells were further Products, Ltd., Exeter, United Kingdom) were aliquoted disrupted by passage through a 21-gauge needle. Extracts and stored at À20jC until used. were spun in the microcentrifuge at 20,000 Â g for 10 minutes at 4jC and the supernatant was transferred into a Cell Irradiation fresh tube. Protein concentration was determined using the Exponentially growing cells were washed with HBSS bicinchoninic acid kit, as described earlier. Cell lysates (Mediatech), detached from plates with trypsin-EDTA solution (protein, 1 mg/mL) were incubated with 25 AL of washed (Mediatech) at 37jC, washed in media, and counted in a ubiquitin binding domain–agarose beads (peptide, 2 mg/mL). hemocytometer. Irradiations were done in tubes using a 137Cs- Following incubation at room temperature for 20 minutes irradiator at a dose rate of about 5 Gy/min. Purified with occasional agitation, unbound proteins were removed proteasomes were irradiated on ice and cells at room and beads were washed thrice with 50 mmol/L Tris, 0.1% temperature. bovine serum albumin (pH 7.5). Bound proteins were eluted in 50 AL of the sample buffer [62.5 mmol/L Tris-HCl (pH 6.8), 2% SDS, 25% glycerol, 0.01% bromophenol blue, 14.4 Preparation of Crude Extracts mmol/L h-mercaptoethanol] and heated to z95jC for 4 to 5 Following drug and/or radiation treatment, cells were washed minutes. Samples were centrifuged at 12,000 Â g for 20 with ice-cold PBS, and buffer I [50 mmol/L Tris-HCl (pH 7.5), seconds to pellet the beads and supernatants were carefully 5 mmol/L MgCl , 2 mmol/L DTT, 2 mmol/L ATP], gently 2 removed. If not used immediately, samples were frozen at scraped off the plates and lysed by three freeze-thaw cycles À20jC for later use. (using liquid nitrogen) in the presence of homogenization buffer [50 mmol/L Tris-HCl (pH 7.5), 5 mmol/L MgCl , 1 mmol/L 2 Western Immunoblotting DTT, 2 mmol/L ATP, 250 mmol/L sucrose]. Samples were Crude cell extracts were prepared in lysis buffer [20 clarified by centrifugation at 4jC for 5 minutes at 1,000 Â g mmol/L Tris-HCl (pH 7.4), 1% NP40, 137 mmol/L NaCl, followed by 25 minutes at 10,000 Â g. Supernatants were 50 Amol/L EDTA, 1 mmol/L PMSF] supplemented with a collected and kept on ice until used. cocktail of protease inhibitors (Sigma). Lysates were centrifuged at 20,000 Â g for 10 minutes at 4jC and Proteasome Purification resulting supernatants were assayed for total protein using 20S and 26S proteasomes were separated using 10% to 40% micro-BCA (Pierce). Twenty to 30 Ag of total protein was glycerol gradients prepared with buffer I. Crude extract diluted with Laemmli sample buffer (Bio-Rad, Richmond, containing 1 mg of total protein was applied to the top of the CA) supplemented with 5% h-mercaptoethanol and separated j gradient and centrifuged at 100,000 Â g for 18 hours at 4 C. by 10% SDS-PAGE. The proteins were transferred onto Fractions of 400 mL were taken starting from the top of polyvinylidene difluoride membrane (Bio-Rad), blocked in gradient. 20S proteasomes were activated using 0.05% SDS. 5% nonfat dry milk, and probed with appropriate primary and 26S proteasomes were maintained and assayed in the presence peroxidase-conjugated secondary antibodies. Protein bands of 2 mmol/L ATP. The presence of proteasomes was indicated were detected with enhanced ECL Plus chemiluminescence by fluorogenic assay using Suc-LLVY-AMC as described detection solution (Amersham Life Science Inc., Piscataway, below. The protein concentration of each fraction was NJ) and blue chemiluminescence was detected using the dual- determined using micro-BCA kit (Pierce, Rockford, IL) fluorescence PhosphorImager Storm 860 (Molecular Dynamics, according to the manufacturer’s protocol. Amersham).

Fluorogenic Assay ATPase Activity Assay Chymotrypsin-like, trypsin-like, and PGPH activity of the ATPase usage by purified 26S proteasomes was determined proteasome and tricorn protease activity were determined by using an ATP determination kit (Molecular Probes, Eugene, their ability to degrade appropriate fluorogenic substrates, as OR). Ten microliters (1 Ag) of irradiated and nonirradiated described before (49). Briefly, serial dilutions of samples proteasomes were incubated for 15 minutes with 100 ALof starting at 100 Ag/mL were made in buffer I with a final volume varying concentrations of ATP diluted in modified buffer I of 100 mL and placed into 96-well black plate (Corning Inc., [50 mmol/L Tris-HCl (pH 7.5), 5 mmol/L MgCl2] and residual New York, NY). Fluorogenic substrate was added to a final activity assayed. A standard curve was prepared using dilutions concentration of 100 Amol/L and the fluorescence of the of ATP with 10 AL of buffer added in place of proteasomes. The released AMC group was measured over a 30-minute period at reaction was started by mixing 80 AL solution [0.5 mmol/L 37jC at 360/430 nm in kinetic mode making one reading every D-luciferin, 1.25 Ag/mL firefly luciferase, 25 mmol/L tricine minute. buffer (pH 7.8), 5 mmol/L MgSO4, 100 Amol/L EDTA and

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Milena Pervan, Keisuke S. Iwamoto and William H. McBride

Mol Cancer Res 2005;3:381-390.

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