Cell Stress & Chaperones (2000) 5 (3), 181–187 Q Cell Stress Society International 2000 Article no. csac. 2000.167
Cycloheximide- and puromycininduced heat resistance: different effects on cytoplasmic and nuclear luciferases Annemieke A. Michels,1,* Bart Kanon,1 Antonius W.T. Konings,1 Olivier Bensaude,2 and Harm H. Kampinga1 Department of Radiobiology, Faculty of Medical Sciences, University of Groningen, The Netherlands Ge´ne´tique Mole´culaire, CNRS UMR 8541, Ecole Normale Supe´rieure, Paris Cedex 05, France
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Abstract Inhibition of translation can result in cytoprotection against heat shock. The mechanism of this protection has remained elusive so far. Here, the thermoprotective effects of the translation inhibitor cycloheximide (CHX) and puromycin were investigated, using as reporter firefly luciferase localized either in the nucleus or in the cytoplasm. A short preincubation of O23 cells with either translation inhibitor was found to attenuate the heat inactivation of a luciferase directed into the cytoplasm, whereas the heat sensitivity of a nuclear-targeted luciferase remained unaffected. After a long-term CHX pretreatment, both luciferases were more heat resistant. Both the cytoplasmic and the nuclear luciferase are protected against heat-induced inactivation in thermotolerant cells and in cells overexpressing heat shock protein (Hsp)70. CHX incubations further attenuated cytoplasmic luciferase inactivation in thermotolerant and in Hsp70 overexpressing cells, even when Hsp70-mediated protection was saturated. It is concluded that protection by translation inhibition is unlikely due to an increase in the pool of free Hsps normally engaged in translation and released from the nascent polypeptide chains on the ribosomes. Rather, a decrease in nascent chains and thermolabile polypeptides may account for the heat resistance promoted by inhibitors of translation.
INTRODUCTION
Heat-induced protein denaturation and aggregation are supposed to be the cause of heat-induced cell death (Laszlo 1992; Kampinga 1993; Parsell and Lindquist 1993). Cells exposed to a priming heat shock become more resistant to a challenging heat shock. This thermotolerant state correlates with an increase in the expression of heat shock proteins (Hsps) (Parsell and Lindquist 1994; Li et al 1995). The best evidence for a link between (reduced) protein damage, Hsp expression, and thermotolerance comes from data in yeast. A functional Hsp104 was found to be essential for thermotolerance and repair of protein damage (Parsell et al 1994; Lindquist and Kim Correspondence to: Harm H. Kampinga, Tel: 131 50 3632903/2911; Fax: 131 50 3632913; E-mail:
[email protected]. *Current address: Ge´ne´tique Mole´culaire, CNRS UMR 8541, Ecole Normale Supe´rieure, Paris Cedex 05, France Received 20 April 1999; Revised 8 February 2000; Accepted 9 February 2000.
1996). In mammalian cells, protection against protein damage by Hsps and cellular resistance to heat also seem to be linked. Overexpression of Hsp70 attenuates heatinduced protein damage (Stege et al 1994; Michels et al 1997; Nollen et al 1999) and increases cell survival after heat shock (Angelidis et al 1991; Li et al 1991; Stege et al 1994; Nollen et al 1999). Interestingly, inhibition of protein synthesis by cycloheximide (CHX) and puromycin (PUR) also confers heat resistance but without an increase in the expression of Hsps (Lee and Dewey 1986, 1987; Lee et al 1987). The mechanism of such heat protection is still unclear. To investigate the mechanism of protection against heat shock mediated by translation inhibitors, we followed the heat inactivation of a reporter enzyme. Firefly luciferase was targeted either to the cytoplasm (cyt-luciferase) or to the nucleus (nuc-luciferase) (Michels et al 1995). Exposure to CHX or PUR shortly before and during heat shock was found to reduce the rate of cyt-luciferase heat inactivation, 181
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whereas the rate of nuc-luciferase inactivation was only marginally affected. Luciferase inactivation is attenuated in thermotolerant (Nguyen et al 1989; Michels et al 1995) and in Hsp70 overexpressing cells (Michels et al 1997; Nollen et al 1999). Translation inhibition further decreased the rate of luciferase inactivation in both cases, even when Hsp70-mediated protection was maximal. After long-term pretreatment with CHX heat inactivation of both cyt-luciferase and nuc-luciferase was attenuated. The protection by translation inhibitors via a decrease of thermolabile nascent chains and polypeptides is suggested.
48C, briefly washed with ice-cold phosphate-buffered saline (PBS), and subsequently lysed in 500 mL of buffer A (25 mM H3PO4/Tris, pH 7.8, 10 mM MgCl2, 1% Triton X-100, 15% glycerol, 1 mM ethylenediaminetetraacetic acid) containing 0.5% 2-mercaptoethanol. The lysates were kept frozen at 2208C before measurement of luciferase activity. Luciferase activities were measured in a Berthold Lumat 9501 for 10 seconds following the addition of substrates (1.25 mM ATP and luciferin (Sigma) at 87 mg/mL in buffer A). In vitro luciferase inactivation
MATERIALS AND METHODS Plasmids and cloning techniques
Plasmid pRSVLL/V (De Wet et al 1987) encoding for a cytoplasmic-localized firefly luciferase (cyt-luciferase) was kindly provided by Dr S. Subramani (University of California, San Diego, USA). The nuclear-targeted luciferase (nuc-luciferase) was encoded by plasmid pRSVnlsLL/V (Michels et al 1995). Plasmid pCMV70 (Michels et al 1997) was used to express the human inducible Hsp70 (Hunt and Morimoto 1985).
Recombinant firefly luciferase (Boehringer) was diluted to 100 nM in buffer B (25 mM HEPES, pH 7.6, 5 mM MgAc, 50 mM KCl, 5 mM 2-mercaptoethanol, 300 nM bovine serum albumin, and 1 mM ATP). CHX (20 mg/ mL) or PUR (100 mg/mL) was added. Fifty-microliter aliquots of these dilutions were transferred to a waterbath at 378C and preincubated for 15 minutes before being transferred together at 428C. After heating, the tubes were immersed in ice-chilled water and the remaining luciferase activities were measured as described above. Western blotting
Cell culture, transfection, heat shock, and cell lysis
O23 hamster fibroblasts were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (Gibco). Cells (1.5 3 105) were seeded in a 25-cm2 culture dish. They were transfected after 24 hours with either 2 mg pRSVLL/V or pRSVnlsLL/V following the standard calcium phosphate method (Kriegler 1990). In some experiments, cells were cotransfected with pCMV70 (1 mg, unless otherwise indicated). In all transfection experiments the total plasmid quantity was kept equal to 10 mg/25-cm2 dish by addition of plasmid pSP64 (Promega). One day after transfection, the transfected cells were treated with trypsin and distributed equally into cell culture tubes (Nunc). Forty tubes (1 3 104 cells/ tube) were seeded out of a 25-cm2 culture dish, and 20 mM MOPS was added to the medium to increase its buffering capacity. Heat shocks were given 2 days after transfection. The tubes were preincubated in a water bath at 378C and transferred to a second water bath at the indicated temperature (60.18C). The transfer took less than 3 seconds and was taken as time 0. The activity before heat shock was taken as 100%. The medium was replaced at the indicated time intervals before heat shock by medium containing CHX (20 mg/mL) or PUR (100 mg/mL). These concentrations completely inhibited 3H-leucine incorporation into trichloroacetic acid–precipitated proteins after labeling for 2 hours at 378C (data not shown). Immediately after treatment, the cells were cooled to Cell Stress & Chaperones (2000) 5 (3), 181–187
Western blotting was done as described before (Michels et al 1997; Nollen et al 1999). In short, cells were suspended in PBS, sonicated, lysed by addition of 23 sample buffer, and boiled for 5 minutes prior to loading on 12.5% sodium dodecyl sulfate-polyacrylamide gels. After Western blotting, Hsp70 was detected using an anti-Hsp70 mouse monoclonal antibody (C92F3A-5, Stressgen). Binding of anti-mouse secondary antibodies (Amersham) was detected by chemiluminescence (ECL, Amersham). RESULTS Inhibition of protein synthesis attenuates luciferase heat inactivation in the cytoplasm and not in the nucleus
To investigate the effect of translation inhibitors on protein heat inactivation, O23 cells were transiently transfected with pRSVLL/V, encoding for cyt-luciferase or pRSVnlsLL/V, encoding for nuc-luciferase. Both enzymes were rapidly inactivated at 438C (Fig 1). Preincubating the cells for 30 minutes with either CHX or PUR significantly attenuated cyt-luciferase heat inactivation. However, nucluciferase was only marginally protected. The CHX-mediated protection against heat-induced cell killing is known to be dependent on the duration of inhibitor preincubation before the heat shock (Lee and Dewey 1986, 1987; Lee et al 1987; Armour et al 1988;
Translation inhibition and cytoplasmic and nuclear protection
Fig 1. Effect of CHX or PUR on luciferase inactivation in situ. O23 cells transiently transfected with pRSVnlsLL/V (nuc-luciferase: A) or pRSVLL/V (cyt-luciferase: B) were heated at 438C in the absence (circles) or presence of either CHX at 20 mg/mL (triangles) or PUR at 100 mg/mL (squares) 30 minutes before and during heat shock. Luciferase activity is shown as a percentage of the activity before heat shock.
Borrelli et al 1992). Therefore, a time-course experiment was performed (Fig 2). The cyt-luciferase was promptly protected by CHX preincubation. In contrast, such pretreatment did not result in significant protection of nucluciferase. However, prolonged preincubation further slowly increased the heat resistance of cyt-luciferase and brought about a significant thermoprotection to nuc-luciferase. Removal of CHX during heat shock resulted in near to complete lack of protection (data not shown). Thus, treatment with translation inhibitors during heating results in attenuation of luciferase heat inactivation with a higher efficiency in the cytoplasm than in the nucleus. Furthermore, CHX and PUR had no influence on the heat-inactivation rates in vitro (Fig 3). Thus, the protective action of CHX and PUR was not due to an interaction between the inhibitors and the luciferases. It is therefore likely a consequence of the translational arrest.
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Fig 2. Time kinetics of CHX-mediated protection of luciferase against heat inactivation. O23 cells transiently transfected with pRSVnlsLL/V (nuc-luciferase: A) or pRSVLL/V (cyt-luciferase: B) were incubated with CHX (20 mg/mL) for 0–120 minutes before heating for 20 minutes at 438C with the drug present during heating. Luciferase activity is shown as a percentage of the activity before heat shock. The closed symbols indicate the remaining luciferase activity after a heat treatment at 438C for 20 minutes of non-CHX pretreated controls.
Inhibition of protein synthesis attenuates cytoplasmic luciferase heat inactivation in Hsp70-overexpressing cells and in thermotolerant cells
An increase in the level of the Hsp70 chaperone protected both the cyt- and nuc-luciferases (Fig 4A,B) as described before (Michels et al 1997). Inhibition of the translation machinery might increase the pool of free chaperones and as such protect luciferase. One might then expect that overexpression of Hsp70 would buffer the protective effect by the inhibitors. However, a short-term pretreatment with CHX further protected the cyt-luciferase in Hsp70overexpressing cells (Fig 4B). Furthermore, a short-term CHX pretreatment had no effect on nuc-luciferase (Fig 4A). To evaluate an influence of the Hsp70 concentration on the CHX-mediated superprotection, the level of Hsp70 Cell Stress & Chaperones (2000) 5 (3), 181–187
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Fig 3. Effect of CHX or PUR on luciferase inactivation in vitro. Recombinant luciferase was diluted in buffer, without translation inhibitor (circles) or with CHX (20 mg/mL) (triangles) or PUR (100 mg/mL) (squares). Luciferase was heated at 428C and luciferase activity was measured. Luciferase activity is shown as a percentage of the starting activity.
was modulated. Hsp70 expression increased upon transfecting cells with increasing plasmid pCMV70 concentrations (Fig 5A). At low Hsp70 expression, a pCMV70 concentration-dependent protection of cyt-luciferase was observed but, above a certain level of HSP70 expression (corresponding to transfection with 0.5 mg of plasmid), the further increments in Hsp70 expression did not further increase the protection of cyt-luciferase (Fig 5B). A short-term CHX pretreatment further protected cyt-luciferase from heat inactivation even under conditions, where the Hsp70-mediated protection of luciferase was saturated (Fig 5B). Hsp70 is not the only chaperone that might function as a thermoprotectant. Therefore, the effect of translation inhibition was assessed in thermotolerant cells (TT) in which the heat-inducible Hsps were elevated in response to a priming heat shock. O23 cells, transfected with pRSVLL/V or pRSVnlsLL/V were made thermotolerant by a heat shock of 20 minutes at 448C followed by a 16hour recovery at 378C. Both the cyt- and the nuc-luciferases were protected from heat inactivation in TT cells (Fig 6A,B) as found previously (Nguyen et al 1989; Michels et al 1995; Nollen et al 1999). Again, a short-term CHX pretreatment increased the protection of the cyt-luciferase, whereas that of the nuc-luciferase was only marginally enhanced in the TT cells (Fig 6A,B). Long-term preincubation with CHX further attenuated cyt-luciferase inactivation in thermotolerant cells and in cells overexpressing Hsp70 at levels where the Hsp70-mediated protection of luciferase was saturated (Fig 7). This superprotection inCell Stress & Chaperones (2000) 5 (3), 181–187
Fig 4. Effect of CHX on luciferase inactivation in cells cotransfected with Hsp70 O23 cells were transiently transfected with pRSVnlsLL/V (nuc-luciferase: A) or pRSVLL/V (cyt-luciferase: B) were either cotransfected with pCMV70 (Hsp70: squares) or pSP64 (control: circles). The cells were heated at 438C in the absence (closed symbols) or presence (open symbols) of CHX (20 mg/mL) added 30 minutes before and present during heat shock. Luciferase activity is shown as a percentage of the activity before heat shock.
creased steadily with the duration of the CHX pretreatment. Taken together, our results demonstrate that the thermal stability of a reporter enzyme can be considerably increased by the combination of Hsp70 overexpression or thermotolerance and inhibition of protein translation. DISCUSSION
This study demonstrates that inhibition of protein synthesis enhances the heat stability of firefly luciferase in vivo. As the translational inhibitors do not stabilize luciferase in vitro, the thermoprotection caused by CHX and PUR treatment is indirect and most likely a result of the inhibition of translation. Two phases of protection could be distinguished. In the initial phase, thermoprotection developed promptly after the addition of the inhibitors. This phase only concerned the luciferase localized in the cytoplasm but not the luciferase localized in the nucleus.
Translation inhibition and cytoplasmic and nuclear protection
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Fig 5. Titration of Hsp70 expression and luciferase inactivation: effects of CHX. O23 cells were transiently transfected with pRSVLL/ V and cotransfected with increasing quantities of pCMV70. (A) Western analysis of Hsp70 expression after transfection with the indicated plasmid concentrations. (B) Luciferase activity after a heat treatment of cells at 438C for 20 minutes is plotted as function of Hsp70 expression. This was measured by densitometric analysis of the Western blots in panel A and expressed in arbitrary units (a.u.). Cells were heated either in the absence (closed symbols) or presence (open symbols) of CHX (20 mg/mL) added 30 minutes before and present during heat shock. Luciferase activity is shown as a percentage of the activity before heat shock.
In contrast, the heat resistance of both enzymes increased gradually over longer periods of pretreatment. The slow increase in luciferase protection is consistent with the cell survival data that show that prolonging the inhibition of translation before heat shock increases the magnitude of thermoprotection (Lee and Dewey 1986, 1987; Lee et al 1987; Armour et al 1988; Borrelli et al 1992). Noteworthy is the fact that Borrelli and coworkers (Armour et al 1988; Borrelli et al 1992) did find protection against nuclear protein damage upon prolonged incubations with CHX consistent with the timing after which a significant protection of nuc-luciferase is observed. An increased Hsp70 chaperone level slows down the heat inactivation of firefly luciferase in vivo (Michels et al 1997; Nollen et al 1999). Chaperones like Hsc70 interact with nascent polypeptide chains during protein synthesis (Beckmann et al 1990; Frydman et al 1994). Thus, inhibition of translation may increase their availability. This pool of free chaperones might increase the cell’s capacity to attenuate heat denaturation of proteins, including the luciferases studied here. Hsc70 has indeed been reported to be released from nascent chains upon incubation with
Fig 6. Effect of CHX on luciferase inactivation in thermotolerant O23 cells transiently transfected with pRSVnlsLL/V (nuc-luciferase: A) or pRSVLL/V (cyt-luciferase: B) were made thermotolerant by a preheat shock of 20 min at 448C, followed by a 16-hour period of recovery at 378C (TT: triangles) or left untreated (control: circles). The cells were heated at 438C in the absence (closed symbols) or presence (open symbols) of CHX (20 mg/mL) added 30 minutes before and present during heat shock. Luciferase activity is shown as a percentage of the activity before heat shock.
PUR (Hansen et al 1994), and both Hsp70 and its cognate Hsc70 refold luciferase in vitro (Freeman et al 1995). However, there is a limit above which increasing the concentration of Hsp70 did not improve the heat resistance of cyt-luciferase; yet, CHX further increased its heat resistance (Figs 4 and 5). Although Hsc70 is slightly more efficient than Hsp70 in vitro, it is unlikely that once the heat protection brought by Hsp70 was saturated, an increase in free Hsc70 would have an additional effect. Thus, our data argue against a heat protection brought by the release of chaperones from the translation machinery. Alternatively, it has been suggested that inhibition of translation could deplete the cells of nascent polypeptides Cell Stress & Chaperones (2000) 5 (3), 181–187
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erase. Unstable proteins are likely present in both the nuclear and cytoplasmic compartment; thus, long-term translation inhibition is expected to affect both nuc- and cyt-luciferases. Indeed, in contrast to the short-term preincubations, long-term preincubations with CHX protected the nuc- as well as the cyt-luciferase. In summary, this report demonstrates that an increase in heat-inducible proteins/chaperones is not required to attenuate heat-induced protein damage in living cells. A decline in heat-sensitive peptides may account for the heat resistance promoted by inhibitors of translation. ACKNOWLEDGMENT
Fig 7. Time kinetics of CHX-mediated protection of luciferase against heat inactivation in Hsp70 overexpressing and in thermotolerant cells. O23 cells transiently transfected with pRSVLL/V (cytluciferase) were either cotransfected with pCMV70 (Hsp70: squares) or pSP64 (control: circles). Also, pSP64-cotransfected cells were made thermotolerant by a priming heat shock of 20 minutes at 448C, followed by a 16-hour period of recovery at 378C (TT: triangles). CHX (20 mg/mL) was added 0–120 minutes before heating for 20 minutes at 438C with the drug present during heating. Luciferase activity is shown as a percentage of the activity before heat shock. The closed symbols indicate the remaining luciferase activity after a heat treatment at 438C for 20 minutes of non-CHX pretreated controls.
that could potentiate the aggregation of matured proteins (Armour et al 1988; Borelli et al 1991, 1992; Beckmann et al 1992). Due to their partially unfolded state, nascent polypeptides may trap unfolded luciferase conformers and thereby accelerate luciferase inactivation. Inhibition of protein synthesis rapidly eliminates the nascent chains and may therefore prevent trapping of unfolded luciferase molecules in the cytoplasm. Translation arrest is expected to occur within minutes of exposure to the inhibitors and consistently cyt-luciferase, but not nuc-luciferase, was rapidly heat protected by inhibiting translation shortly before and during heat shock. Long-term incubations with CHX further increased the heat resistance of the cyt-luciferase and also resulted in heat resistance of the nuc-luciferase. A further increase in the protective effect on cyt-luciferase is also seen in cells expressing saturating levels of Hsp70 or in TT cells. Thus, increases in the free pool of chaperones as a result of translation arrest are also unlikely to be the cause for the protective effect of CHX on long-term pretreatments Upon prolonged arrest of protein synthesis, short-lived proteins are eliminated. One can speculate that such proteins are heat labile. As thermolabile proteins may also trap unfolded luciferase conformers, a reduction in thermolabile proteins might reduce coaggregation with lucifCell Stress & Chaperones (2000) 5 (3), 181–187
This work was supported by the Dutch Cancer Society (grant RUG 94-830), the European Community (grant CHRX-CT93-0260), and the Association pour la Recherche sur le Cancer (grant ARC no 6250). REFERENCES Angelidis CE, Lazaridis I, Pagoulatos GN. 1991. Constitutive expression of heat-shock protein 70 in mammalian cells confers thermoresistance. Eur J Biochem 199: 35–39. Armour EP, Lee YJ, Corry PM, Borrelli MJ. 1988. Protection from heat-induced protein migration and DNA repair inhibition by cycloheximide. Biochem Biophys Res Commun 157: 611–617. Beckmann RP, Lovett M, Welch WJ. 1992. Examining the function and regulation of hsp70 in cells subjected to metabolic stress. J Cell Biol 117: 1137–1150. Beckmann RP, Mizzen LA, Welch WJ. 1990. Interaction of Hsp70 with newly synthesized proteins: implications for protein folding and assembly. Science 248: 850–854. Borrelli MJ, Stafford DM, Rausch CM, Lepock JR, Lee YJ, Corry PM. 1992. Reduction of levels of nuclear-associated protein in heated cells by cycloheximide, D2O, and thermotolerance. Radiat Res 131: 204–213. Borrelli MJ, Lee YJ, Frey HE, Ofenstein JP, Lepock JR. 1991. Cycloheximide increases the thermostability of proteins in Chinese hamster ovary cells. Biochem Biophys Res Commun 177: 575–581. De Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S. 1987. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7: 725–737. Freeman BC, Myers MP, Schumacher R, Morimoto RI. 1995. Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ-1. EMBO J 15: 2281–2292. Frydman J, Nimmesgern E, Ohtsuka K, Hartl FU. 1994. Folding of nascent polypeptide chains in a high molecular mass assembly with molecular chaperones. Nature 370: 111–117. Hansen WJ, Lingappa VR, Welch WJ. 1994. Complex environment of nascent polypeptide chains. J Biol Chem 269: 26610–26613. Hunt C, Morimoto R. 1985. Conserved features of eukaryotic hsp70 genes revealed by comparison with the nucleotide sequence of human hsp70. Proc Natl Acad Sci USA 82: 6455–6459. Kampinga HH. 1993. Thermotolerance in mammalian cells: protein denaturation, aggregation and stress proteins. J Cell Sci 104: 11– 17. Kriegler M. 1990. Gene Transfer and Expression. A Laboratory Manual. Stockton Press, New York, 96–98.
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