Activation of the cell death program by nitric oxide involves inhibition of the proteasome.

The ubiquitin/proteasome pathway mediates the degradation of many short-lived proteins that are critically involved in the regulation of cell proliferation and cell death, including the tumor suppressor protein p53. Accumulation of p53 and induction of apoptosis in RAW 264.7 macrophages in response to nitric oxide are well established. However, the molecular mechanisms involved in nitric oxide-induced p53 accumulation are unknown. Here we show that, similar to nitric oxide, treatment of macrophages with specific proteasome inhibitors, including clastolactacystin-beta-lactone, induces p53 accumulation and apoptosis, suggesting that nitric oxide may affect the activity of the proteasome. In support of this hypothesis, both exposure of cells to S-nitrosoglutathione and stimulation of endogenous nitric oxide production by lipopolysaccharide/interferon-gamma treatment result in inhibition of proteasome activity as measured in vitro by the degradation of the proteasome-specific substrate succinyl-Leu-Leu-Val-Tyr-4-methylcoumarin-7-amide. Moreover, chemically diverse nitric oxide donors interfere with proteasome-mediated degradation of polyubiquitinated p53 in vitro. These data imply that nitric oxide-induced apoptosis and accumulation of p53 are, at least in part, mediated by inhibition of the proteasome.

The ubiquitin/proteasome pathway mediates the degradation of many short-lived proteins that are critically involved in the regulation of cell proliferation and cell death, including the tumor suppressor protein p53. Accumulation of p53 and induction of apoptosis in RAW 264.7 macrophages in response to nitric oxide are well established. However, the molecular mechanisms involved in nitric oxide-induced p53 accumulation are unknown. Here we show that, similar to nitric oxide, treatment of macrophages with specific proteasome inhibitors, including clastolactacystin-␤-lactone, induces p53 accumulation and apoptosis, suggesting that nitric oxide may affect the activity of the proteasome. In support of this hypothesis, both exposure of cells to S-nitrosoglutathione and stimulation of endogenous nitric oxide production by lipopolysaccharide/interferon-␥ treatment result in inhibition of proteasome activity as measured in vitro by the degradation of the proteasomespecific substrate succinyl-Leu-Leu-Val-Tyr-4-methylcoumarin-7-amide. Moreover, chemically diverse nitric oxide donors interfere with proteasome-mediated degradation of polyubiquitinated p53 in vitro. These data imply that nitric oxide-induced apoptosis and accumulation of p53 are, at least in part, mediated by inhibition of the proteasome.
Nitric oxide (NO) 1 emerges as a universal pathophysiological mediator in the cardiovascular, immune, and nervous systems. NO is catalytically produced by different NO synthase isoforms that convert L-arginine to NO and stoichiometric amounts of citrulline (1)(2)(3). Once activated, NO synthase isoforms produce not only NO, the primary reaction product, but also those species that are derived from oxidation, reduction, or adduction of NO in physiological milieus, including S-nitrosothiols, peroxynitrite, and transition metal adducts (4,5). Constitutive versus inducible isozymes account for low versus high level NO output systems and allow a rough correspondence between toxic and homeostatic functions of the molecule (6). Cytostatic and/or toxic actions of NO may play important roles in the pathology of tissue or cell destruction (7).
It is widely believed that NO-induced cell death is, at least in part, mediated via apoptosis. Treatment of cells with NO donors or activation of inducible NO synthase resulted in apoptotic alterations in several cell culture systems (8 -10). Furthermore, NO-induced apoptosis was reported to involve activation of the tumor suppressor protein p53, activation of caspases, and altered expression of Bcl-2 family members (11)(12)(13)(14). For RAW 264.7 macrophages, accumulation of the p53 protein resembled an early apoptotic marker, and p53 antisense experiments suggested that, in this cellular system, NOinduced apoptosis is at least partially dependent on p53 (15). However, the molecular mechanisms by which NO induces p53 accumulation remained unclear.
Under normal growth conditions, wild-type p53 is rapidly degraded, which probably is required to tightly control its growth-suppressive properties (16). In response to a variety of stimuli, including DNA damage, hypoxia, or ribonucleotide depletion, p53 can be activated, which generally results in cell cycle arrest or apoptosis of the affected cells (17). Concomitant with its activation, intracellular p53 levels increase significantly, and it is commonly assumed that p53 levels, at least in part, are regulated by the rate of degradation. There is increasing evidence that the ubiquitin/proteasome system (18 -20) plays a major role in p53 degradation (21)(22)(23)(24)(25). Furthermore, in support of the notion that up-regulation of p53 is important for the activation of its growth-suppressive properties, it was reported that treatment of cells with proteasome-specific inhibitors induces both p53 accumulation and apoptosis (26,27). However, since inhibition of the proteasome evoked apoptosis also in p53-negative HL-60 cells, inhibition of the proteasome can result in the induction of p53-dependent as well as of p53-independent apoptotic pathways (26).
Since initiation of NO-induced apoptosis and p53 accumulation are closely associated and since NO affects the activity of various proteins (28 -31), we put the hypothesis forward that NO directly blocks the activity of the proteasome, which in turn results in an increase in p53 levels. Therefore, we analyzed p53 accumulation in response to specific proteasome inhibitors and NO donors in vivo and measured the effect of NO on proteasome-mediated degradation of an artificial peptide substrate and of polyubiquitinated p53 in vitro. This revealed that, similar to proteasome inhibitors, NO directly interferes with proteasome activity, indicating that NO, at least in part, affects intracellular signaling pathways by blocking proteasome-mediated protein degradation. valine), E-64, 1,1-diethyl-2-hydroxy-2-nitrosohydrazine sodium, S-nitroso-N-acetylpenicillamine, and LPS (Escherichia coli serotype 0127: B8) were purchased from Sigma (Deisenhofen, Germany). The fluoropeptide Suc-LLVY-AMC was from Bachem Biochemica (Heidelberg, Germany). The NO synthase inhibitor L-NAME was from Alexis (Grü nberg, Germany). Recombinant murine IFN-␥, ATP, and ATP␥S were obtained from Roche Molecular Biochemicals (Mannheim, Germany). Clastolactacystin-␤-lactone was from Calbiochem (Bad Soden, Germany).
Cell Culture-The mouse monocyte/macrophage cell line RAW 264.7 was maintained in RPMI 1640 medium supplemented with 100 units/ml penicillin, 100 g/ml streptomycin, and 10% heat-inactivated fetal calf serum (complete RPMI 1640 medium). All experiments were performed using complete RPMI 1640 medium. GSNO was dissolved in water. The fluoropeptide Suc-LLVY-AMC, MG-132, MG-115, and clastolactacystin-␤-lactone were dissolved in Me 2 SO. E-64 was dissolved in water/ethanol (1:1, v/v), and calpain inhibitor II was dissolved in ethanol. The various reagents were added to complete RPMI 1640 medium as indicated. For all experiments, appropriate solvent control experiments were performed.
Quantitation of DNA Fragmentation-DNA fragmentation was measured with the diphenylamine assay as reported (32). Briefly, cells were scraped off the culture plates; resuspended in 250 l of 10 mM Tris-HCl and 1 mM EDTA, pH 8.0 (TE buffer); and lysed by the addition of 250 l of buffer containing 5 mM Tris-HCl, 20 mM EDTA, pH 8.0, and 0.5% Triton X-100 for 30 min at 4°C. Intact chromatin (pellet) was then separated from DNA fragments (supernatant) by centrifugation for 15 min at 13,000 ϫ g. Pellets were resuspended in 500 l of TE buffer, and samples were precipitated overnight at 4°C by adding 500 l of 10% trichloroacetic acid. DNA was pelleted by centrifugation (4000 ϫ g, 10 min), and the supernatant was discarded. After the addition of 300 l of 5% trichloroacetic acid, samples were boiled for 15 min. DNA contents were quantitated using diphenylamine (33). The percentage of fragmented DNA was calculated as the ratio of the DNA content in the supernatant to the DNA content in the pellet.
Fluorescence-based Determination of Proteasome Activity-Fluorescence-based determination of proteasome activity was performed as described (34). Briefly, cells were treated with GSNO, MG-132, LPS/ IFN-␥, E-64, and clastolactacystin-␤-lactone, respectively, or with the respective solvents as a control. Upon treatment, cells were washed with phosphate-buffered saline, and the protein extracts were prepared in 100 mM HEPES, 10% sucrose, and 0.1% CHAPS. Then, 50 M Suc-LLVY-AMC (dissolved in 10% Me 2 SO) was incubated with the respective protein extract (100 g) in 5 mM MgCl 2 , 5 mM ATP, 50 mM Tris-HCl, pH 7.8, 20 mM KCl, and 5 mM MgOAc in a total volume of 200 l. After 1 h at 37°C, the reaction was terminated by the addition of 200 l of a solution containing 0.1 M sodium borate, pH 9.0, in ethanol/water (144:16). Degradation of the fluoropeptide Suc-LLVY-AMC was assessed by measuring the fluorescence of aminomethylcoumarin at 460 nm (excitation at 365 nm) using a SpectraFluor fluorometer (Tecan, Crailsheim, Germany). Results are the means Ϯ S.D. (two-tailed Student's t test) of five independent experiments.
In Vitro Degradation of Polyubiquitinated p53-Human wild-type p53 was generated by in vitro transcription-translation in wheat germ extract (Promega, Mannheim, Germany) in the presence of [ 35 S]methionine according to the manufacturer's instructions. As a source of the HPV-16 E6 oncoprotein, E6 was expressed as a glutathione S-transferase fusion protein in E. coli DH5␣ and purified as described (35). E6-AP was expressed and prepared from Sf9 cells infected with a recombinant baculovirus encoding E6-AP as described (35). The ubiquitin-activating enzyme (E1) and the ubiquitin-conjugating enzyme UbcH7 were expressed in E. coli BL21(DE3) using the pET expression system as described previously (36).
To generate polyubiquitinated p53, 1 l of in vitro translated p53 was incubated in the presence of ϳ10 ng of purified baculovirus-expressed E6-AP, 10 ng of glutathione S-transferase-E6 fusion protein, 50 ng of E1, 50 ng of UbcH7, and 6 g of ubiquitin (Sigma) in 20-l volumes. In addition, reactions contained 25 mM Tris-HCl, pH 7.6, 100 mM NaCl, 2 mM ATP or 2 mM ATP␥S (Roche Molecular Biochemicals) as indicated, 4 mM MgCl 2 , and 1 mM dithiothreitol. After 90 min at 25°C, the reactions were stopped by the addition of an SDS-containing buffer. Alternatively, proteasome-mediated degradation of polyubiquitinated p53 was measured by adding 60 l of a mixture containing 7 l of rabbit reticulocyte lysate (Promega), 4 mM ATP, and 8 mM MgCl 2 in 25 mM Tris-HCl, pH 7.6, and 50 mM NaCl. Upon further incubation for 90 min at 37°C, the total reaction mixtures were electrophoresed on 10% SDS-polyacrylamide gels, and 35 S-labeled p53 was detected by fluorography. Where indicated, rabbit reticulocyte lysate was incubated for 15 min with the respective NO donor prior to addition to polyubiquitinated p53.

NO-mediated p53 Accumulation and Apoptotic Cell
Death-To address the possibility that accumulation of p53 in RAW 264.7 macrophages upon NO treatment is linked to inactivation of the proteasome, we initially corroborated the observation that p53 protein accumulates in these cells in response to the NO donor GSNO (9,37,38). As shown in Fig. 1, treatment of cells with GSNO resulted in a significant increase in p53 levels within 2 h after its addition. After 4 h, p53 levels reached a plateau, whereas in unstimulated cells no or very little p53 was detected.
Previous studies established that treatment of cells with a combination of LPS and IFN-␥ promotes endogenous NO formation that causes p53 accumulation and apoptosis, whereas inhibitors of inducible NO synthase, such as L-NAME, preserve cell integrity by blocking NO formation and thus p53 accumulation and apoptosis (37). To test whether GSNO treatment also results in apoptosis, we performed quantitative DNA fragmentation analysis using the diphenylamine assay (Table I). Indeed, in response to 1 mM GSNO, we observed significant endonuclease-mediated DNA damage in RAW 264.7 macrophages after 8 h. These results underscore the ability of NO to promote apoptotic cell death and further support the notion that p53 accumulation represents an early marker for cell death in this cellular system.
Inhibition of the Proteasome Promotes p53 Accumulation and Apoptosis-Inhibition of proteasome activity has been linked to p53 accumulation and initiation of apoptosis in various experimental systems (27,39). To study the effect of proteasome inhibitors on p53 expression in macrophages, we exposed RAW 264.7 cells to several established proteasome-blocking agents (Fig. 2, A and B). As expected, inhibitors such as MG-132 and MG-115 dose-dependently promoted p53 accumulation, with maximal responses at 10 M MG-132 and 50 M MG-115 ( Fig. 2A).
We went on to analyze the effect of proteasome inhibitors on DNA fragmentation (Table I) tently were ϳ5%. The notion that inhibition of proteasome activity induces apoptosis in RAW 264.7 cells was further supported by the use of the proteasome-specific inhibitor clastolactacystin-␤-lactone (40) and, as a control, by the use of calpain inhibitor II or E-64, a nonspecific cysteine protease inhibitor. As shown in Fig. 2B, clastolactacystin-␤-lactone led to p53 accumulation in RAW 264.7 macrophages. In contrast, E-64 and calpain inhibitor II at commonly used concentrations failed to induce p53 accumulation, thus pointing to the unique role of the proteasome system in p53 degradation. Moreover, the addition of clastolactacystin-␤-lactone resulted in apoptosis of the treated cells as measured by DNA fragmentation, although a similar effect was not observed for calpain inhibitor II and E-64 (Table I). Taken together, these data demonstrate that, similar to NO, inhibition of the proteasome results in p53 accumulation and apoptosis in RAW 264.7 macrophages, indicating that protein degradation plays an important role in the regulation of programmed cell death of macrophages.
NO-mediated Inhibition of the Proteasome-To determine whether NO has a direct effect on proteasome activity, we analyzed the ability of cell extracts to degrade the fluoropeptide Suc-LLVY-AMC (41), which was reported to be a preferred substrate of the 20 S proteasome (34,42,43). Proteolytic degradation of Suc-LLVY-AMC releases the fluorescent aminomethylcoumarin portion that serves as an indicator of proteolytic activity. Exposure of RAW 264.7 macrophages for 4 h to 1 mM GSNO resulted in a reduction of proteasome activity by ϳ70% (Fig. 3), whereas treatment with the proteasome inhibitors MG-132 and clastolactacystin-␤-lactone blocked the formation of aminomethylcoumarin almost completely. In contrast, treatment with the nonspecific cysteine protease inhibitor E-64 did not alter the efficiency of Suc-LLVY-AMC degradation.
To test whether endogenously produced NO interferes with proteasome activity, RAW 264.7 macrophages were treated with LPS/IFN-␥. After 24 h, cell extracts were prepared, and their ability to degrade Suc-LLVY-AMC was determined. This revealed that LPS/IFN-␥ treatment blocked peptide cleavage by ϳ40% compared with cell extracts derived from untreated control cells (Fig. 3). Furthermore, this effect was not observed in the additional presence of L-NAME (Fig. 3), demonstrating that the observed reduction in proteasome activity was due to endogenous NO production. Based on these results and the fact that degradation of the fluoropeptide Suc-LLVY-AMC does not require ubiquitination prior to degradation, we conclude that endogenously produced NO or exogenously supplied NO directly inhibits the catalytic activity of the 20 S proteasome.
NO Inhibits Proteasome-mediated Degradation of p53 in Vitro-Unlike degradation of peptides by the 20 S proteasome, 26 S proteasome-mediated degradation is ATP-dependent, and in addition, most natural substrates of the 26 S proteasome, including p53, need to be modified by the covalent attachment of multiple moieties of ubiquitin prior to their recognition as proteolytic substrates (18,(21)(22)(23)(24). Similarly, it was previously shown that the HPV-16 E6 oncoprotein targets p53 for degradation via the ubiquitin/proteasome system. In this system, E6 recruits the ubiquitin-protein ligase E6-AP to p53, resulting in the formation of polyubiquitinated forms of p53 (35). Polyubiquitinated p53 is then recognized by the 26 S proteasome and degraded. Although the E6-facilitated ubiquitination/degradation of p53 is not of physiological relevance with respect to HPV-negative cells, including RAW 264.7 macrophages, this system may be suitable to directly assess the effect of NO on the ability of the 26 S proteasome to degrade polyubiquitinated proteins. To test this possibility, polyubiquitinated p53 was generated in the presence of E6, E6-AP, and the basic components of the ubiquitin conjugation system (Fig. 4A, lanes 3 and  4). Then, as a source of the 26 S proteasome, rabbit reticulocyte lysate was added in the presence of ATP or ATP␥S, a nonhydrolyzable ATP analog that cannot be used as an energy source by the 26 S proteasome. As expected, in the presence of ATP, polyubiquitinated p53 was efficiently degraded (Fig. 4A, lane  5). In contrast, degradation was not observed in the presence of  ATP␥S, showing that degradation in this system is mediated by the 26 S proteasome. The accumulation of unmodified forms of p53 in the presence of both ATP␥S and rabbit reticulocyte lysate is most likely explained by the notion that polyubiquitinated forms of p53 are converted into unmodified forms of p53 by de-ubiquitinating enzymes that are present in reticulocyte lysate.
Having shown that polyubiquitinated p53 represents an efficient substrate for the 26 S proteasome in vitro, we then analyzed the effect of GSNO on proteasome activity in this system (Fig. 4B). This revealed that treatment of reticulocyte lysate with GSNO inhibits p53 degradation (Fig. 4B, lanes 4  and 5). The inhibitory effect of NO was almost maximal at 0.5 mM GSNO, whereas at 200 M GSNO, degradation of p53 was not significantly affected (data not shown). Furthermore, NOinduced inhibition of proteasome-mediated degradation resulted in the accumulation of unmodified p53, indicating that, under the conditions used, the activity of de-ubiquitinating enzymes is not significantly affected by GSNO. In this context, it should be noted that, probably due to the presence of an apparently high p53 de-ubiquitinating activity in RAW 264.7 cell extracts, it was not possible to test if NO treatment had a direct effect on the 26 S proteasome in RAW 264.7 cells (data not shown).
Finally, to establish inhibition of the proteasome system by NO donors irrespective of the chemical structure of the individual NO-delivering compounds, we tested NO donors such as 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine sodium, N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine, and S-nitroso-N-acetylpenicillamine (Fig. 5). All NO donors blocked p53 degradation with a similar efficiency. Taken together, these data strongly indicate that, at least in vitro, NO interferes with p53 degradation by direct inhibition of the 26 S proteasome.

DISCUSSION
The growth-suppressive properties of p53 can be activated in response to a variety of stimuli, including DNA damage. Activation of p53 generally results in growth arrest or in apoptosis of the affected cells, and with respect to DNA damage, it is commonly believed that p53 activation contributes to prevent the propagation of cells that acquired genomic mutations (12,17,44). Concomitant with its activation, intracellular p53 levels increase significantly, and there is strong evidence that p53 levels, at least in part, are regulated by its turnover rate.
Similarly, the action of endogenous or exogenous NO to initiate apoptosis has been linked to p53 accumulation by several independent studies (37, 44 -46). However, the molecular mechanism(s) of NO-induced p53 accumulation remained elusive. In this study, we obtained evidence that NO can directly inhibit the activity of the 20 S proteasome as well as that of the 26 S proteasome. Since p53 was shown to be a substrate of the ubiquitin/proteasome system (21)(22)(23)(24), this provides a likely mechanism for NO-induced p53 accumulation. This possibility is further supported by the finding that specific proteasome inhibitors led to p53 accumulation and apoptosis in RAW 264.7 macrophages with an efficiency similar to NO-releasing compounds.
The observation that cleavage of Suc-LLVY-AMC was significantly decreased in extracts derived from cells treated with NO donors suggests that NO directly interferes with the proteolytic activity of the 20 S proteasome. This hypothesis is supported by the fact that the proteolytic activity of the 26 S proteasome is inhibited by NO treatment in vitro as shown by the inability of NO-treated reticulocyte lysate to degrade polyubiquitinated p53 (Figs. 4 and 5). For several enzymes, it has been established that, in response to NO, their activity is inhibited by covalent modification of thiol groups (28 -31, 47, 48). This appears to be also a likely mechanism for NO-induced proteasome inhibition since modification of thiol groups is considered to inhibit proteasome activity (49).
Besides inhibition of the proteasome, it seems possible that NO interferes also with the activity of the ubiquitin conjugation system since E6/E6-AP-mediated ubiquitination of p53 was completely inhibited in the presence of NO donors (data not shown). However, at present, it is unclear if the presence of NO interferes with binding of the E6⅐E6-AP complex to p53 (which is a prerequisite step for p53 ubiquitination) or with the activities of the ubiquitin-activating enzyme and the ubiquitinconjugating enzyme that are involved in p53 ubiquitination in this particular system. This possibility was not further addressed since E6⅐E6-AP-mediated p53 ubiquitination is not of physiological relevance for p53 degradation in RAW 264.7 cells. In this context, it will be interesting to see if NO also interferes with Mdm2-mediated ubiquitination of p53 since Mdm2 seems to be involved in p53 degradation in normal (i.e. HPV-negative) cells (23)(24)(25).
RAW 264.7 macrophages have been shown to produce NO via inducible NO synthase in response to LPS/IFN-␥ treatment FIG. 3. Proteasome activity determination. The rate of Suc-LLVY-AMC degradation was measured in protein extracts derived from RAW 264.7 macrophages exposed to 1 mM GSNO for 4 h, a combination of LPS (10 g/ml) and IFN-␥ (100 units/ml) in the presence or absence of 1 mM L-NAME for 24 h, and a 10 M concentration of the proteasome inhibitor MG-132 or clastolactacystin-␤-lactone (CL-␤-L) for 4 h. As a control, RAW 264.7 cells were treated with the cysteine protease inhibitor E-64. Relative proteasome activity is expressed in comparison to an unstimulated control (100% control activity). Fluorescence was determined as described under "Experimental Procedures" using a SpectraFluor fluorometer (Ex 365 / Em 460 ). Data shown represent the means Ϯ SD of three individual experiments. **, p Ͻ 0.01 versus control; ***, p Ͻ 0.05 versus control. (37,50,51). Since treatment with LPS/IFN-␥ also resulted in decreased proteasome activity, this indicates that NO is indeed a natural negative regulator of the proteasome. This is further supported by the finding that the effect of LPS/IFN-␥ cannot be observed in the additional presence of L-NAME, an inhibitor of NO synthase. Since the ubiquitin/proteasome system is a major pathway for the degradation of cytosolic and nuclear proteins, this may indicate that the half-lives of many cytosolic and nuclear proteins increase upon NO treatment. Alternatively, it seems possible that the breakdown of distinct substrates of the proteasome such as p53 may be more sensitive to proteasome inhibition than the bulk of short-lived proteins. However, additional experiments will be required to address this possibility.
Since inhibition of the proteasome by NO most likely does not affect only p53 stability, it seems possible that also the turnover of other important regulators of apoptosis and/or components of the cell cycle machinery are influenced by NO. For instance, our data may provide an explanation for the recently published observation that NO blocks NF-B activation (28,31). Since NF-B activation requires proteolytic digestion of the inhibitor IB, inhibition of proteasome activity by NO should inhibit IB degradation and thus activation of NF-B. Another example may be the pro-apoptotic protein Bax, which was recently shown to be degraded by the proteasome (52). Under conditions of NO-induced programmed cell death, the amount of Bax is up-regulated (53,54), either as a result of p53 accumulation, which is known to operate as an inducer of bax gene expression, or as a consequence of NO-mediated proteasome inhibition. Since NO initiates apoptosis in p53-negative cells as well (55), our proposal that NO inhibits proteasome function provides a reasonable explanation for the fact that NO can induce apoptosis irrespective of the cellular p53 status. Finally, the influence of endogenously generated NO on proteasome activity may further highlight the role of NO during immune suppression if one considers the essential role of the proteasome in antigen presentation (56,57).
Our data confirm previous reports showing that proteasome inhibitors induce apoptosis (26,27,58). With a few exceptions, it appeared that dividing cells responded to proteasome inhibitors by activation of apoptosis, whereas in nondividing cells, the same inhibitors revealed anti-apoptotic effects (58), indicating that the individual effect of proteasome inhibitors is determined by the proliferative status of a cell. Interestingly, the ability of NO to initiate apoptosis is also divergent. Although NO formation causes apoptosis in multiple systems (46,FIG. 4. Degradation of polyubiquitinated p53 in vitro. To generate polyubiquitinated p53, in vitro translated 35 S-labeled p53 was incubated in the presence of the HPV-E6 oncoprotein, E6-AP, recombinant ubiquitin-activating enzyme, ubiquitin-conjugating enzyme UbcH7, and ubiquitin for 90 min at 25°C (for details, see "Experimental Procedures"). Then, as a source of 26 S proteasome, rabbit reticulocyte lysate was added, and the reaction mixtures were incubated for an additional 90 min at 37°C. Finally, the whole reaction mixtures were separated by SDS-polyacrylamide gel electrophoresis, and the unmodified form of p53 (p53) and the polyubiquitinated forms of p53 (ub-p53) were detected by fluorography. A, E6-facilitated ubiquitination of p53 was performed in the presence of ATP (lanes [3][4][5] or the nonhydrolyzable ATP analog ATP␥S (lanes 6 and 7). Reactions were either stopped prior to the addition of reticulocyte lysate (lanes 3 and 6) or 90 min after the addition of rabbit reticulocyte lysate (lanes 5 and 7) as indicated. Note that under the conditions used, neither ubiquitination nor degradation of p53 could be observed in the absence of the HPV E6 oncoprotein (lanes 1 and 2). B, prior to addition to polyubiquitinated p53, rabbit reticulocyte lysate was pretreated for 15 min with GSNO as indicated.  59 -61), it was reported to block programmed cell death in others (59,62). It will be challenging to determine whether, with respect to apoptosis and cell survival, the individual response of cells to NO is reflected in the different response of cells to inhibition of the proteasome.