20S Proteasome Prevents Aggregation of Heat-Denatured Proteins

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Biomacromolecules 2004, 5, 1465-1469

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20S Proteasome Prevents Aggregation of Heat-Denatured Proteins without PA700 Regulatory Subcomplex Like a Molecular Chaperone Mihiro Yano, Yasuko Koumoto, Yukiko Kanesaki, Xueji Wu, and Hiroshi Kido* Division of Enzyme Chemistry, Institute for Enzyme Research, The University of Tokushima, Tokushima 770-8503, Japan Received January 16, 2004; Revised Manuscript Received March 26, 2004

The eukaryotic 20S proteasome is the multifunctional catalytic core of the 26S proteasome, which plays a central role in intracellular protein degradation. Association of the 20S core with a regulatory subcomplex, termed PA700 (also known as the 19S cap), forms the 26S proteasome, which degrades ubiquitinated and nonubiquitinated proteins through an ATP-dependent process. Although proteolytic assistance by this regulatory particle is a general feature of proteasome-dependent turnover, the 20S proteasome itself can degrade some proteins directly, bypassing ubiquitination and PA700, as an alternative mechanism in vitro. The mechanism underlying this pathway is based on the ability of the 20S proteasome to recognize partially unfolded proteins. Here we show that the 20S proteasome recognizes the heat-denatured forms of model proteins such as citrate synthase, malate dehydrogenase. and glyceraldehydes-3-phosphate dehydrogenase, and prevents their aggregation in vitro. This process was not followed by the refolding of these denatured substrates into their native states, whereas PA700 or the 26S proteasome generally promotes their reactivation. These results indicate that the 20S proteasome might play a role in maintaining denatured and misfolded substrates in a soluble state, thereby facilitating their refolding or degradation. Introduction The proteasome plays an essential role in the degradation of many cellular proteins through a highly regulated mechanism.1,2 The eukaryotic 20S proteasome has been found to be a catalytic component of the 26S proteasome. This core particle is a 700 kDa, cylinder-shaped protease arranged in a stack of four heptameric rings that contain two different types of subunits. The outer rings are composed of R-subunits, and the inner ones are composed of catalytic β-subunits, respectively. The 20S proteasome has proteolytic active sites in a central chamber,3,4 cleaving a broad range of peptide bonds in abnormal or short-lived proteins.5 In the multistep mechanism underlying protein degradation, it is commonly assumed that the regulatory particle, the 20-subunit complex containing six ATPases that bind to one or both of the terminal rings of the 20S proteasome, recognizes ubiquitinated or nonubiquitinated substrates as a prerequisite for their proteolysis6 and then unfolds and successively translocates them through the narrow gate to the catalytic sites of the 20S proteasome.7 Thus, the binding of the regulatory particle to the 20S core is the most unique feature for the effective function of the proteasome. On the other hand, it has been suggested that the 20S proteasome 8 and 26S prpteasome exist in dynamic equilibrium in the eukaryotic cytosol.9 Furthermore, the 20S proteasome by itself can degrade some nonubiquitinated proteins such as p21WAF/CIP, 10 scrambled bovine pancreatic ribonuclease (scRNAase),11 and several * To whom correspondence should be addressed. Phone: +81-886-337423. Fax: +81-886-33-7425. E-mail: [email protected].

oxidized proteins12,13 in a PA700-independent manner in vitro, although PA700 enhances the degradation of substrate proteins. These substrates, which are susceptible for the 20S proteasome degradation, generally have a loosely folded structure.14 Recently, binding activity of the misfolded protein was detected for the regulatory particle of the proteasome.15,16 In these studies, PA700 or the 26S proteasome acts as a defining molecular chaperone that preferentially binds to nonnative nonubiquitinated proteins, thereby preventing their aggregation and promoting their refolding to native structures.17 The recognition and degradation of nonubiquitinated proteins by the 26S proteasome may reflect this chaperone function of the regulatory complex. On the other hand, to obtain an important clue for a better understanding of regulatory particle-independent proteolysis, we have examined the potential function of the 20S proteasome as a molecular chaperone, assisting the recruitment of substrates to catalytic sites directly. First, we recently found that the purified 20S proteasome exhibits ATP-ADP exchange activity, other than proteolytic activities, in vitro.18 In this study, surprisingly, the addition of ADP to the assay system resulted in a substantially enhanced rate of ATP hydrolysis, whereas the ATPase activity of the 20S proteasome determined in the absence of ADP is generally hard to detect. Although similar findings have been shown with molecular chaperones such as Hsp7019,20 and 14-3-3 protein,21 it remained poorly understood how this activity correlates with the function of a molecular chaperone. Second, to test the direct interaction of the 20S proteasome with the nonnative structure of a protein, we assessed the effect of the

10.1021/bm049957a CCC: $27.50 © 2004 American Chemical Society Published on Web 05/04/2004

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20S proteasome on several heat-denatured, model proteins in vitro. Our data demonstrated that the 20S proteasome by itself recognizes these misfolded proteins and prevents their aggregation in a dose dependent manner, although this process is not followed by refolding of a denatured substrate into its native state. These observations may provide a novel insight into the mechanism underlying direct degradation of nonubiquitinated substrates by the 20S proteasome. Materials and Methods Materials. Firefly luciferase was obtained from Promega. Malate dehydrogenase (MDH), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), and citrate synthase (CS) were purchased from Roche. ATP, ADP, and ECL detection kit were purchased from Amersham Pharmacia Biotech. Proteinase K (PK), BSA, and all other reagents were obtained from Sigma. Clasto-lactacystin was purchased from Calbiochem. All protein substrates and the 20S proteasome were dialyzed against 100 mM Hepes-KOH buffer before use. Purification of Human and Rat 20S Proteasome. The 20S proteasome was purified from human lymphoblastoma cell line Molt-4, clone 8 cells and rat liver, as described previously.18 Briefly, Molt-4, clone 8 cells (6 × 109 cells) or rat livers (190 g) were homogenized in 10 mM Tris-HCl buffer, pH 7.0, containing 135 mM NaCl, and then the homogenate was centrifuged at 100 500 × g for 1 h at 4 °C. The supernatant brought to pH 5.5 by acetic acid was centrifuged at 25 000 × g for 20 min, and then polyethylene glycerol 6000 was added to the supernatant to 10%. The resulting precipitate was dissolved in 25 mM Tris-HCl, pH 6.5 and then subjected on a TSKgel DEAE 3SW column (Toyo Soda Co., Tokyo Japan). The proteasome with an amidolytic activity of succinyl-LLVY-4-methyl-coumaryl7-amide as a substrate was eluted with 200 mM Tris-HCl buffer, pH 6.5, and then subjected on a hydroxyapatite column. The proteasome eluted with 200 mM potassium phosphate buffer, pH 6.7, was further subjected to HPLC on a TSKgel G3000 SW column with 25 mM ammonium formate buffer, pH 5.5, containing 1 M urea. Aggregation Assay by Centrifugation. Firefly luciferase (2.2 µM) was incubated with or without the 20S proteasome (0.56 µM) in a buffer (100 mM Hepes-KOH, pH 8.0, containing 10 mM DTT, 5 mM ATP and 5 mM MgCl2) at 42 °C for various times. The reaction mixtures were centrifuged at 15,000 × g for 10 min. Supernatants and precipitates with 10% trichloroacetic acid were analyzed by SDS/PAGE. The amounts of protein bands stained with Coomassie Brilliant Blue R-250 were quantified by densitometry.22 Activity and Reactivation of Luciferase. Luciferase (0.1 µM) was incubated in the absence or presence of the 20S proteasome (0.3 µM) in refolding buffer (25 mM HepesKOH, pH 8.0, containing 100 mM DTT, 50 mM KCl, 5 mM ATP, 5 mM MgCl2 and 1 mg/mL BSA) at 42 °C for various times, and then aliquots were withdrawn, diluted 50-fold, and measured for luciferase activity using a Promega luciferase assay system and a Promega luminometer. Reactivation was initiated by a temperature shift to 30 °C after

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incubation for 10 min at 42 °C.23 At various times, aliquots were removed and luciferase activity was measured. Light Scattering Assay. MDH (100 nM) or GAPDH (125 nM) was incubated with various amounts of the 20S proteasome at 47 °C (for MDH) or 45 °C (for GAPDH) in a thermostated quartz cuvette. Aggregation was monitored continuously as the increase in turbidity at 320 nm (for MDH) or 340 nm (for GAPDH).24 The reaction buffer (100 mM Hepes-KOH, pH 7.5) was first preincubated at 47 or 45 °C, and then the 20S proteasome was added. After the potential change in light scattering due to the 20S proteasome was monitored for 5 min, each substrate was diluted into the reaction mixture, and then light scattering was followed with a F-2000 fluorometer. As control experiments, equivalent amount of BSA to 20S proteasome was added in the reaction mixture. Assay for Proteinase K Susceptibility. CS (200 nM) was preincubated with clasto-lactacystin in 40 mM Hepes-KOH buffer, pH 7.5, in the presence or absence of the 20S proteasome (12.5 nM) at room temperature for 15 min and further incubated at 43 °C for various times up to 30 min. PK was then added to the reaction mixture at a final concentration of 10 µg/mL (0.36 µM) and allowed to incubate on ice for 5 min. Reactions were terminated with 2 mM phenylmethylsulfonyl fluoride (PMSF), then separated by SDS/PAGE followed by silver staining. Results 20S Proteasome Protects Luciferase from Aggregation but Does Not Promote Its Refolding. The purified 20S proteasome from Molt-4, clone 8 cells and rat livers on SDS/ PAGE gave typical multiple bands in the range of 21-31 kDa, and no protein bands corresponding to PA700 subunits were observed (Figure 1A). Luciferase was reported as a chaperone substrate, because it tends to aggregate upon thermal denaturation and its folding status has been characterized in some detail.25 Thus, the aggregation and refolding of denatured luciferase provide a profitable relevant in vitro model system for studying the interactions of misfolded substrates with the 20S proteasome. To determine whether the thermal unfolding of luciferase is accompanied by aggregation, luciferase was incubated at 42 °C for various times in the absence or presence of the 20S proteasome, and then its aggregation was determined by centrifugation, followed by SDS/PAGE analysis of the resulting pellets and supernatants. In the absence of the 20S proteasome, after incubation for 30 min, luciferase was barely detectable in the supernatants, and more than 90% of the luciferase was precipitated as an aggregated form (Figure 1B). In contrast, in the presence of the 20S proteasome during heat treatment (the molar ratio of the 20S proteasome to luciferase was equal to 0.33), almost all of the luciferase remained soluble and its aggregated form was little observed, with no decrease in the amount of total luciferase in the assay mixture (Figure 1B). These results demonstrate that the 20S proteasome prevents the aggregation of heat-denatured luciferase without degradation. This effect was not enhanced by the addition of ATP.

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Figure 2. 20S proteasome inhibits the thermal aggregation of MDH and GAPDH. A. MDH at the concentration of 100 nM was incubated at 47 °C either alone (b) or in the presence of 25 nM (2), 50 nM (O),100 nM of the 20S proteasome (4), or 100 nM of BSA (9). At the times indicated, samples were monitored for the apparent absorbance at 320 nm, which is indicative of light scattering due to MDH aggregation. B. GAPDH at the concentration of 125 nM was heattreated at 45 °C in the presence or absence of increasing amounts of the 20S proteasome. Aggregation of GAPDH alone (b), GAPDH with 25 nM (2), 35 nM (O), and 50 nM of the 20S proteasome (4), or GAPDH with 50 nM of BSA (9). The aggregation of GAPDH was monitored at 340 nm. Relative scattering is expressed in arbitrary units. Results are representative of three independent experiments.

Figure 1. Effects of the 20S proteasome on luciferase in thermal aggregation and inactivation. A. The silver-stained electrophoretic patterns on SDS/PAGE of the human (lane 1) and rat (lane 2) 20S proteasome. B. Thermal aggregation of luciferase at 42 °C, in the absence (O) or presence (b) of the 20S proteasome (ratio of the 20S proteasome to luciferase, 0.33), was assessed. After incubation for various times, the reaction mixtures were centrifuged and the supernatants were subjected to SDS/PAGE followed by Coomassie blue staining. The amounts of soluble luciferase were quantified by densitometry and presented as the percentages of total amounts of luciferase. C. Kinetics for luciferase inactivation was analyzed in the absence (O) or presence (b) of the 20S proteasome (ratio of the 20S proteasome to luciferase, 3.0) during the heat treatment at 42 °C. Error bars represent the standard deviation from three replicate trials.

Inactivation of lucifarase at 42 °C was monitored by measuring the remaining activity of the luciferase. The enzyme activity was decreased at 42 °C in a time-dependent manner, and it was little detected after incubation for 25 min in the absence of the 20S proteasome in the reaction mixture (Figure 1C). In contrast, the 20S proteasome slightly prevented the rate of inactivation and no enzyme activity was observed after incubation for 40 min after incubation even at a higher concentration of the 20S proteasome (ratio of the 20S proteasome to luciferase, up to 3.0), as shown in Figure 1C. In addition, after inactivation of luciferase for

10 min at 42 °C in the absence or presence of the 20S proteasome, reactivation was followed with a temperature shift to 30 °C for various times. The 20S proteasome, however, was unable to refold the heat-denatured luciferase (data not shown). These results suggest that the 20S proteasome prevents the aggregation of heat-denatured protein, and the other chaperone system(s) is required for subsequent refolding process. 20S Proteasome Prevents the Aggregation of Other Misfolded Proteins. To confirm of the chaperone-like activity of the 20S proteasome, we further examined the effects of the 20S proteasome on other misfolded proteins, such as MDH and GAPDH, which are often thermal aggregation-sensitive. MDH is a homodimer, and GAPDH is a homotetramer; when heated at 45 °C (for MDH) or 47 °C (GAPDH), the populations of the partially folded conformations of both proteins, leading to aggregation.23 The inhibitory effects of the 20S proteasome on these protein aggregations were monitored by light-scattering using a spectrometer. Increasing amounts of the 20S proteasome significantly prevented the heat-induced aggregation of MDH and GAPDH (Figure 2, parts A and B). In contrast, 100 nM/ BSA instead of 20S proteasome did not prevent the aggregation of MDH and GAPDH. The decrease in turbidity of MDH or GAPDH in the mixture was not due to the degradation by the 20S proteasome (data not shown). These results indicate that the 20S proteasome interacts with a variety of unfolded proteins to prevent their aggregation, whereas direct evidence of binding between them has not been to detect.

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the anti-aggregation for CS without incorporation into its interior cavity. Discussion

Figure 3. 20S proteasome changes susceptibility of the heatdenatured CS to PK digestion. After native CS (200 nM) was preincubated in buffer containing 200 µM clasto-lactacystin, it was heat-treated for various times at 43 °C in the absence (A) or presence (B) of the 20S proteasome (25 nM), and then samples were digested with PK (10 µg/mL) on ice for 5 min as indicated. After stopping the digestion by 2 mM PMSF, the reaction mixtures were subjected to SDS-PAGE followed by silver staining.

Misfolded Protein Is not Incorporated into the Internal Cavity of the 20S Proteasome at the Interaction Stage. To address the interaction of the 20S proteasome with denatured substrate proteins, PK protection assay was undertaken in vitro. As a model substrate, we chose CS, because this enzyme is thermal aggregation-sensitive and a well-suited target for studying the influence of most chaperone families on it.26 When thermal aggregation of CS was monitored as the turbidity at 500 nm for 15 min at 43 °C, the 20S proteasome prevented its aggregation in a dosedependent manner (data not shown). After thermal treatment of CS at 43 °C for various times up to 30 min in the presence or absence of the 20S proteasome, the CS was further treated with PK at 0 °C for 5 min. As shown in Figure 3, parts A and B, native CS was not degraded by PK despite the presence or absence of the 20S proteasome. Heat-denatured CS, however, was susceptible to PK digestion in the initial phase of heat treatment for 5-10 min and then nonsusceptible to digestion in the advanced aggregation phase of heat treatment for 15-30 min (Figure 3A). On the other hand, the 20S proteasome induced the heat-denatured CS hypersensitive to PK, even in the advanced phase after 15 min (Figure 3B). These results in combination with the effect of the 20S proteasome on prevention of the aggregation of heat-denatured CS suggest that the 20S proteasome shows

In the proteasome-dependent protein turnover system, the 26S proteasome degrades both ubiquitin-conjugated proteins and certain nonubiquitinated proteins in an ATP-dependent manner.1,27 In this process, it is widely known that substrate proteins should first bind to PA700, which recruits them into the catalytic 20S core, where they are degraded. Thus, it has been believed that the 20S proteasome never functions as an isolated enzyme, but rather only when bound to regulatory proteins in vivo, although it can be activated as to unfolded polypeptides in vitro. However, the recent observation that PA700 is not an absolute requirement for proteasomemediated degradation was demonstrated for the turnover of some substrates. The 20S proteasome can selectively degrade a range of different oxidized proteins in a PA700-independent manner. Although the molecular basis of the degradation of oxidative proteins remained unclear, a recent study on oxidized calmodulin (CaM) indicated that the signal for CaM recognition by the 20S proteasome is the secondary structural loss induced by either oxidation or calcium binding.14 Interestingly, the degradation of oxidized CaM results in digestion into multiple large fragments, suggesting that the 20S proteasome recognizes CaM in the incompletely unfolded state but that the global unfolding of CaM is not required for its targeting to the 20S proteasome. Another direct reaction of the 20S proteasome to a substrate is known in the turnover of p21WAF/CIP. The cyclin-dependent kinase (cdk) inhibitor p21 Cip was first reported to be a nonubiquitinated substrate because its mutant form lacking all the target lysine residues for ubiquitylation was degraded in a proteasome-dependent manner.28 It is noteworthy that the 20S proteasome directly recognizes p21WAF/CIP through the C8 R-subunit in the outer ring and degrades it without PA700.10 These observations indicate that ubiquitinindependent degradation by the 20S proteasome may be more important than generally assumed, although it is uncertain whether the 20S proteasome is active without binding to regulatory proteins in cells. In this work, we have shown that the 20S proteasome preferentially recognizes several heat-denatured proteins and protects them as to irreversible aggregation in vitro. This is, to the best of our knowledge, the first such demonstration, because the 20S proteasome has never been reported to inhibit the aggregation of misfolded proteins, whereas PA700 apparently exhibited several chaperone-like properties including the ability to prevent their aggregation in previous studies.15,16 However, our findings differ from theirs in some important ways. In fact, the 20S proteasome required a much higher protein:substrate ratio for its effect than in the case of PA700. Furthermore, the 20S proteasome failed to promote the refolding of a denatured protein into the native state. Therefore, the prevention of aggregation may simply reflect the ability of the 20S proteasome to interact with an unfolded protein, unlike known chaperones. The effects of the 20S proteasome on misfolded proteins might lead to a

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maintenance of substrate proteins in an unfolded conformation for subsequent proteasomal degradation or refolding in conjunction with other chaperones including PA700. In addition, since partially folded proteins including ones with misfolded conformations tend to aggregate,29 and the degradation of these species should be difficult without an antiaggregation factor,30 these abilities are probably critical components of the concerted action of the 20S proteasome by which it binds unfolded substrates to inhibit the competing aggregation reaction, thereby facilitating their entry into the catalytic site without PA700. However, the precise mechanisms of anti-aggregation by the 20S proteasome of heatinduced unfolded substrates and the molecular basis of the interaction between the 20S proteasome and unfolded substrates remain to be resolved. What are the biological implications of the findings demonstrated here? Once a cell is exposed to several stresses such as heat, loss of the native structure probably often generates unfolded states of substrate proteins. When unfolded, nonubiquitylated molecules meet the free 20S proteasome, and it is interesting to consider the alternative possibility that they bypass PA700 and become targeted directly to the 20S proteasome, although full activation may be achieved through the attachment of the regulatory complex. However, it remains unknown if incomplete unfolded substrates are degraded by the 20S proteasome directly, how these proteins reach their active sites, and if they pass through the narrow entry port of the 20S proteasome. Further studies on the actions of the 20S proteasome toward unfolded proteins are currently in progress and likely to clarify the mechanism underlying direct proteolysis by the 20S proteasome. Acknowledgment. This work was supported, in part, by a Grant-in-Aid (14570121) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan References and Notes (1) Coux, O.; Tanaka, K.; Goldberg, A. L. Annu. ReV. Biochem. 1996, 65, 801-847. (2) DeMartino, G. N.; Slaughter, C. A. J. Biol. Chem. 1999, 274, 2212322126.

Biomacromolecules, Vol. 5, No. 4, 2004 1469 (3) Groll, M.; Ditzel, L.; Lo¨we, J.; Stock, D.; Bochtler, M.; Bartunik, H. D.; Huber, R. Nature 1997, 386, 463-471. (4) Baumeister, W.; Walt, J.; Zu¨hl, F.; Seemu¨ller, E. Cell 1998, 92, 367380. (5) Orlowski, M.; Wilk, S. Arch. Biochem. Biophys. 2000, 383, 1-16. (6) DeMartino, G. N.; Proske, R. G.; Moomaw, C. R.; Strong, A. A.; Song, X.; Hisamatu, H.; Tanaka, K.; Slaughter, C. A. J. Biol. Chem. 1996, 271, 3112-3118. (7) Larsen, C. N.; Finley, D. Cell 1997, 91, 431-434. (8) Rechsteiner, M.; Hoffman, L.; Dubiel, W. J. Biol. Chem. 1993, 268, 6065-6068. (9) Peter, J. M. Trends Biochem. Sci. 1994, 19, 377-382. (10) Touitou, R.; Richardson, J.; Bose, S.; Nakanishi, M.; Rivett, J.; Allday, M. J. EMBO J. 2001, 20, 2367-2375. (11) Liu, C.-W.; Millen, L.; Roman, T. B.; Xiong, H.; Gilbert, H. F.; Novia, R.; DeMartino, G. N.; Thomas, P. J. J. Biol. Chem. 2002, 277, 26815-26820. (12) Wang, R.; Chait, B. T.; Wolf, I.; Kohanski, R. A.; Cardozo, C. Biochemistry 1999, 38, 14573-14581. (13) Lasch, P.; Petras, T.; Ullrich, O.; Backmann, J.; Naumann, D.; Grune, T. J. Biol. Chem. 2001, 276, 9492-9502. (14) Ferrington, D. A.; Sun, H.; Murray, K. K.; Costa, J.; Williams, T. D.; Bigelow, D. J.; Squier, T. C. J. Biol. Chem. 2001, 276, 937943. (15) Braun, B. C.; Glickman, M.; Kraft, R.; Dahlmann, B.; Kloetzel, P. M.; Finley, D.; Schmidt, M. Nat. Cell Biol. 1999, 1, 221-226. (16) Strickland, E.; Hakala, K.; Thomas, P. J.; DeMartino, G. N. J. Biol. Chem. 2000, 275, 5565-5572. (17) Bukau, B.; Horwich, A. L. Cell 1998, 92, 351-366. (18) Yano, M.; Mori, S.; Kido, H. J. Biol. Chem. 1999, 274, 3437534382. (19) Hiromura, M.; Yano, M.; Mori, H.; Inoue, M.; Kido, H. J. Biol. Chem. 1998, 273, 5435-5438. (20) Wu, X.; Yano, M.; Washida, H.; Kido, H. Biochem. J. 2004, 378, 793-799. (21) Yano, M.; Mori, S.; Niwa, Y.; Inoue, M.; Kido, H. FEBS Lett. 1997, 419, 244-248. (22) Minami, Y.; Ho¨hfeld, J.; Ohtsuka, K.; Hartl, F. U. J. Biol. Chem. 1996, 271, 19617-19624. (23) Lee, G. J.; Vierling, E. Methods Enzymol. 1998, 290, 350-365. (24) Lee, G. J.; Roseman, A. M.; Saibil, H. R.; Vierling, E. EMBO J. 1997, 16, 659-671. (25) Schroder, H.; Langer, T.; Hartl, F.-U.; Bukau, B. EMBO J. 1993, 12, 4137-4144. (26) Buchner, J.; Grallert, H.; Jakob, U. Methods Enzymol. 1998, 290, 323-338. (27) Voges, D.; Zwickl, P.; Baumeister, W. Annu. ReV. Biochem. 1999, 68, 1015-1068. (28) Sheaff, R. J.; Singer, J. D.; Swanger, J.; Smitherman, M.; Roberts, J. M.; Clurman B. E. Mol. Cell 2000, 5, 403-410. (29) Hartl, F. U.; Hayer-Hartl, M. Science 2002, 295, 1852-1858. (30) Wicker, S.; Maurizi, M. R.; Gottesman, S. Science 1999, 286, 888-1893.

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