Protein Denaturation during Heat Shock and Related Stress

THEJOURNALOF BIOLOGICAL

VOl. 264, No. 18, Issue of June 25, pp. 10487-104921989 Printed in ii.S.A.

CHEMISTRY

Q 1989 by The American Society for Biochemistry and Molecular Biology, Inc

Protein Denaturation during Heat Shock and Related Stress ESCHERICHIA COW @-GALACTOSIDASEAND PHOTINUS PYRALIS MOUSE CELLS*

LUCIFERASE INACTIVATION IN

(Received for publication, July 18, 1988)

Van Trung Nguyen, Michel Morange, and Olivier Bensaude From the Groupe de Biologie Moldculaire du Stress, Institut Pasteur,25 rue du Dr. Roux, 75724 Paris Ceder 15, France

In an attempt to question the toxic effect of heat shock and related stress, we have studied the activity of reporter enzymes during stress. Escherichia coli j3galactosidase and Photinus pyralis luciferase were synthesized in mouse and Drosophila cells after transfection of the corresponding genes. Both enzymes are rapidly inactivated during hyperthermia. The corresponding polypeptides are not degraded but become insoluble even in the presence of non-ionic detergents. The heat inactivationis more dramatic in vivowithin the living cell than in vitro, in a detergent-free crude cell lysate. The extentof enzyme inactivation at a given temperature depends on the cell type in which the enzyme is expressed. Luciferase is inactivated at lower temperatures within Drosophila cells than withinmouse.cells, whereas &galactosidase is inactivated at higher temperatures in E. coli than inmouse cells. A ”priming” heat shock confers a transient increased resistance (thermotolerance) of cells against a second “challenging” heat shock. Enzyme inactivation during heat shock or exposure of the cells to ethanol isattenuated in heat shock-primed cells. A comparable thermoprotection is raised by a priming heatshock for both luciferaseactivityandprotein synthesis. Thus, the study of reporter enzyme inactivation is a promising tool for understanding the molecular basis of the toxicity of heat shock and related stress as well as the mechanisms leading to thermotolerance.

inactivations could be a consequence of either denaturation and/or of some critical posttranslational modifications during heat shock. Several posttranslational modifications occurring during heat shock are documented. The phosphorylation of nuclear proteins accompanies the changes in chromatin structure occurring during heat stress (14). The phosphorylation states of proteins such as theeucaryotic translation initiation factors eIF-2 and eIF-4 are altered by heat stress (15) and leads to their inactivation. Ubiquitination is another posttranslational modification which increases during heatshock: it is associated with protein degradation (16-18). Accumulation of the heat shock proteins induced by a nonlethal “priming” heat stress correlates with a transient increased cell survival and an accelerated recovery of the altered functions after a second “challenging” heat shock (19, 20). The cells have acquired “thermotolerance.” Thermotolerance is a well-described phenomenon. Cells primed by a nonlethal heat shock acquire transiently an increased resistance to a second challenging heat shock. Cell survival, cloning efficiency, and protein synthesis afterthe second challenging stress are higher in the primed cells. However,little is known about the mechanisms of this protection. Complex cellular properties such as protein synthesis (21), nucleolar morphology (ZZ), intermediate filament network distribution (23), and RNA splicing (24) are less heat shock inactivated in heat shock-primed cells. It has been suggested that the heatshock proteins coded by the heat shock genes might protect the cells either through the prevention of thermaldenaturation of “normal” proteins (25) or through the rapid elimination of “abnormal” proteins andaggregates that they might form (26, A sublethal heat shock has pleiotropic effects on cells: it 27). While we were studying the heat driven expression of inactivates the “normal”pattern of genetic expression at different levels (transcription, splicing, andtranslation of transfected genes controlled by heat shock promoters, we mRNAs into proteins), and it alters the cellular morphology. noticed that activities of control reporter enzymes synthesized Meanwhile, it activates the transcription of the heat shock from supposedly stress-insensitive promoterswere depressed genes and stabilizes the corresponding mRNAs which are after heat stress. This effect was repeatedly observed and was selectively translated; it also temporarily increases cell resist- particularly dramatic when the luciferase coding sequence ance and survival after a second thermal challenge (thermo- was used. Therefore, we thought it might be helpful to follow the activity of exogenous well-characterized enzymes in order tolerance) (1-4). I n vivo, several enzymes have been described as being to interpret theeffects of heat shock on proteins. Thus, either inactivated during hyperthermia: DNA polymerases ( 5 ) , the Escherichia coli @-galactosidaseor the Photinus pyralis Poly(ADP-ribose) synthetase (6), Na+/K+-ATPase (7), Ca2+- luciferase coding sequences were introduced into mouse cells, ATPase (8),and NADPH oxidase (9). Moreover, binding of and the fate of the corresponding proteins was followedduring insulin (lo), epidermal growth factor ( l l ) , steroids (12, 13), stress. In this paper we show that the heat shock inactivation of to their respective receptors is inhibited by heat shock. These reporter enzymes is attenuated in thermotolerant cells. This *This workwas supported by Association pour la Recherche makes it an attractive system for studying the effects of stress Contre le Cancer Grant ARC 6250 and Direction des Recherches on proteins i n vivo and themechanism of thermotolerance.

Etudes et Techniques Grant Delegation Ginerale pour I’Armement 87-124. The costs of publication of this article were defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

EXPERIMENTALPROCEDURES

Plasmids-PlasmidpCH 110carries the &galactosidase gene under the control of the SV-40 early promoter (28).pAGO carries the Herpes

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Enzyme Inactivation during Heat Shock

virus thymidine kinase gene with its own promoter (29). Plasmid pRSV/L carries a cDNA coding for P. pyralis luciferase under control of the Rous sarcoma virus LTR promoter (30). Plasmid pRSV/L was kindly provided by Dr. J. De Wet, University of California, San Diego. Plasmid pRSV/@-galwas constructed and kindly provided by Dr. P. Herbomel, Institut Pasteur, Paris.It carries the @-galactosidase gene from pCH 110 under control of the Rous sarcoma virus LTR promoter. Cells-Since transfection might alter the cell metabolism in some unknown and irreproducible way and since an exhaustive study would require many experiments, we mostly used mouse cells permanently expressing p-galactosidase or luciferase after integration into the genome of the corresponding coding sequences under control of constitutive viral promoters. LZ1 cells were kindly provided by Dr. J.-F. Nicolas of the Institut Pasteur. They were isolated after cotransfection of BALBc 3T3 mouse fibroblasts by pCH 110 and pSVTKneo (31),which drives the constitutive expression of neomycin resistance, followed by G418 selection and cloning. RL3 cells were isolated after cotransfection of Ltk-aprt- mouse fibroblasts by pRSV/L and pAGO followed by HAT selection and cloning. Transient expression experiments were performed with Ltk- mouse cells (1D clone) transfected by the calcium phosphate procedure. All mouse cells were propagated in Dulbeccomodified Eagle's medium supplemented with 10% fetal calf serum. Drosophila Schneider cells were transfected with plasmid pRSV/L by the calcium phosphate procedure and analyzed 48 h after transfection. Heat Shocks-Heat shocks were performed by immersion of the tissue culture dishes in a water-bath; temperature regulated with 0.1 "Caccuracy are specified in thetext. For a given experiment equal amounts of cells were distributed in the desired number of tissue culture dishes. The heat shocks were generally performed with lo6 exponential growing cells in 9.6 cm2dishes. Cells Lysis and Enzyme Analysis-After rinsing with phosphatebuffered saline, mouse cells were lysed and scraped off the culture dish with a rubber policeman in Tris buffer (50 mM Tris-HC1, pH 7.4,50 mM sodium chloride, 8 mM magnesium chloride, 0.2 mM phenylmethylsulfonyl fluoride (dilution from a 0.2 M dimethyl sulfoxide stock solution), 0.5% 2-mercaptoethanol). The suspended cells were disrupted with 20 strokes in an ice-cold Dounce glass homogenizer. Alternatively cells werelysed in Triton buffer (Tris buffer supplemented with 1% Triton) in which no mechanical disruption was required. The lysates were fractionated by 5 min of centrifugation at 10,000 X g at 4 "C giving a pellet and a supernatant. Bacterial cells (200 p1 of a suspension at 0.8 optical density unit at 550 nm) were lysed in 200 pl of Tris buffer supplemented with 0.4% of SDS' and 40 p1 of chloroform. @-Galactosidaseactivity was measured through the hydrolysis of o-nitrophenyl-@-D-galactopyranoside by determination of optical density at 420 nm. Luciferase activity was measured with a scintillation counter as previously described (32). Immunologicals-Anti-P-galactosidase rabbit antibody was kindly provided by Dr. A. Ullmann from the InstitutPasteur. Anti-luciferase rabbit antibody was kindly provided by Dr. G. A. Keller from the University of California, San Diego. Anti-rabbit IgG monoclonal antibody labeled with alkaline phosphatase was from Promega. Protein A-Sepharose was from Pharmacia LKB Biotechnology Inc. and 1'" protein A from Amersham C o p . Immurwadsorbtion of 0-Galactosidase-Approximately 5 X lo6 LZ1 cells in 28-cm2dishes were labeled in methionine-free medium supplemented with 200 pCi/ml of [35S]methi~nine. After 3 h of labeling, the medium was replaced by standard medium, and the cells were immediately heat shocked at 45 "C (times areindicated in the legend to Fig. 2). As controls, one dish was lysed immediately after labeling and another one was chased at 37 "C for 1 h before lysis. Cells were lysed immediately after heat shock in Triton X-100 buffer and the 10,000 X g supernatant was diluted with one volume of RIPA buffer two times (300 RIM NaCl, 2 mM EDTA, 20 mM Tris-HC1, pH 8, 1% deoxycholate, 2% Nonidet P-40, 0.2% sodium dodecyl sulfate, 0.5% 2-mercaptoethanol), incubated with protein A-Sepharose and then centrifuged for 1 min at 10,000 x g to discard the protein A. One p1 of anti-(3-galactosidaseantibody was then added to 500 pl of supernatant. After 1 h at room temperature,protein A-Sepharose was added. The mixture was incubated 30 min at room temperature and centrifuged at 10,000 X g. The protein A pellet was washed with RIPA once, extracted in SDS buffer (125 mM Tris-HC1, pH 6.8, 2%

' The abbreviation used is: SDS, sodium dodecyl sulfate.

sodium dodecyl sulfate, 5% 2-mercaptoethanol, 10% glycerol) heated at 95 'C and loaded on a 10% SDS-polyacrylamide gel. Western Blots-Cells were lysed either in SDS buffer or in Triton lysis buffer. The Tritonlysates were centrifuged for 10 min a t 10,000 X g. The pellets dissolved in SDS buffer and the supernatantsdiluted in an equal volume of 2 X SDS buffer were submitted to polyacrylamide gel electrophoresis (care was takento load on each lane material corresponding to the same amount of cells, approximately lo6).Aliquots of the Triton extracts were used for enzymatic activity measurements. Proteins from the gelwere electrotransferred onto nitrocellulose. The nitrocellulose filter was soaked in 5% milk powder, 50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% sodium azide (TBS milk buffer). The anti-@-galactosidase antibody was used a t a 1/2000 dilution. The anti-luciferase rabbit polyclonal antibody was used at a 1/200 dilution. After three washes in TBS milk, detection was performed either with '"I-protein A (0.05 pCi/ml) and autoradiography or with alkaline phosphatase-labeled anti-mouse IgG monoclonal antibody (Promega) at a 1/2000 dilution, used with the procedure recommended by the supplier. RESULTS

Most of the results reported in this section were obtained with cells expressing permanently the reporter enzymes; these cells allowed easy quantitative studies. However, similar results were also obtained with cells expressing transiently the reporter enzymes after transfectionwith the appropriate plasmids. Heat Shock Inactivation of P. pyralis Luciferase and E. coli P-Galactosidase within Mouse Celk"RL3 cells are mouse fibroblasts L cells having the luciferase gene integrated under the control of the Rous sarcoma virus LTR promoter. They permanently synthesize a functional luciferase protein the activity of which waseasily quantified in cell lysates. At 37 "C, after inhibition of protein synthesis by 10 wg/ml of cycloheximide, luciferase activity faded away with a 200 min half-life (data not shown). Incubation of these cells at 42 "C prior to lysis lead to the disappearance of the luciferase activity following a first order kinetics corresponding to a half-life of 4 min (Fig. 1A). The rapid inactivation at 42 "C might have been due to some rearrangement at this temperature of the peroxisomes, the organelles into which luciferase is targeted (33). Therefore, we examined another reporter protein, the very wellstudied P-galactosidase from E. coli. In mouse cells,it is found mostly in the cytoplasm as revealed by in situ activity histochemical staining (data not shown), and no particular interaction with cell organelles has been reported unless specific target signals had been added to the protein sequence. LZ1 cells are mouse BALBc3T3 fibroblasts which have integrated the P-galactosidase gene under the control of the SV-40 early viral promoter. At 37 "C,after inhibition of protein synthesis by 10 pg/ml of cycloheximide, no significant changes in enzyme activity were detected over a 10-h period (datanot shown). However, when cells were incubated at 45 "C,the enzyme activity decreased rapidly following a first order kinetics with a 35 min half-life (Fig. 1B). The heat inactivationsof both enzymes were moredramatic in vivo within living cells than in uitro. In the case of 8galactosidase, incubation at 45 "C of a crude detergent-free lysate from LZ1 cells did not lead to measurable loss of activity (Fig. 1B) in agreement with previous studies of thermal stability of the pure enzyme (34). In the case of luciferase, incubation at 42 "Cof a crude detergent-free lysate from RL3 cells lead to a lower inactivation rate (18 min half-life) than within the living cells (4 min half-life) (Fig. 1A). The Loss of Enzyme Activities in the Supernatants Is ACcounted for by Precipitation of the Enzymes into Insoluble Structures-The loss of enzyme activity might have been due

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Enzyme Inactivation during Heat Shock

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FIG. 1. A, in vivo loss of luciferase activity in RL6 cells heated a t 42 "C (0).In vitro loss of luciferase activity a t 42 "C within a crude detergent-free RL6 cell lysate (M).B, in vivo loss of P-galactosidase activityin LZ1cells heated at 45 "C (0).In vitro stability of 8galactosidase a t 45 "C within a crude detergent-free LZ1 cell lysate (m). Enzymaticactivitiesinnonheated cell extractsaretakenas 100%.Exponential curve fittings are drawn.

E

FIG. 2. Effect of heat shock on fl-galactosidase molecules. LZ1 cells were labeled with [~""S]methioninefor 3 h and lysed immediatelyafterheat shock. The heat shock was performed innonradioactive culture media a t 45 "C for 15 min ( l a n e I ) , 30 min ( l a n e 2), 45 min (lane 3 ) . Non-heat-shocked control cells were lysed immediately after labeling (0) or chased for 1 h a t 37 "C (0'). A, autoradiograph of the electrophoresed Triton supernatantsfrom methionine labeled cells; the arrows point toa 130-kDaprotein which comigrates with @-galactosidase and fades away in heat-shocked cell supernatants. B, the above supernatants were incubated with anti-8galactosidase antibody and protein A-Sepharose. The fractionretained by protein A-Sepharose waselectrophoresed and autoradiographed. Western blots of extracts from unlabeled LZ1 cells were probed by anti-&galactosidase antibody followed by '*'I-protein A recognition. C, total SDS extracts. D, Triton supernatants. E, Triton pellets. All three C-E, pictures are derived from the same autoradiograph.

to protein degradation. This hypothesis was tested through a n immunochemical approach. LZ1 cells expressing @-galactosidasewere first heavily labeled with [35S]methionine and chased a t 37 or a t 45 "Cprior to lysis in Triton buffer. Samples of the 10,000 x g supernatants were loaded on a polyacrylamide gel for SDS electro- insoluble in the Triton lysis buffer. A significant amount of phoresis. It canbe noted inFig. !?.A,that the lanes correspond-P-galactosidase was detected after electrophoresis and Westing to cells chased at either 37 or 45 "Care almost identical ern blotting of the 10,000 X g pellet from heated cell lysates except for the disappearance from heated cell extracts of a (Fig. 2E). This is in contrast to what is found for cells growing band a t 130 kDa(which comigrateswith @-galactosidase). a t 37 "C where most of the @-galactosidase is soluble and Samples of the same supernatantswere incubated with anti- where a minor amount of the enzyme in the pellet left after @-galactosidase antibodies and adsorbed on protein A (Fig. Triton extraction is detected only after overexposure (data 2B). A decreased amount of @-galactosidase (which could be not shown). quantitated by autoradiograph scanning)was found in lysates Similar resultswere obtained when lysatesof cells expressfrom heat-treated cells. This decrease accounted for the ening luciferase were probed on Western blots with an antizymatic activity loss. luciferase serum (Fig. 3). The fall in enzymatic activity during The decrease in the amount of @-galactosidasefound in the heat-shocked cell extracts could not be explained by protein incubation of cells a t 42 "C corresponded to adecreased degradation. Extracts from heated or control LZ1 cells lysed amount of luciferase in the 10,000 X g Triton supernatant in SDS buffer were electrophoresed, blotted onto nitrocellu- (lanes 3 and 4 ) and to anincrease in the pellet (lunes 5 and 6).No changes appeared in the total SDS extracts (lanes 1 lose membranes, and probed with the anti-@-galactosidase antibody followed by '251-protein A recognition (Fig. 2C). No and 2). Insolubilization of both enzymes resulted from the heat changes in total @-galactosidase amounts were detected. We of extraction. then tried to rule out the hypothesis according to which some treatmentratherthanfromtheconditions Identical observations were obtained with both enzymes reconformational change after the heatshock would hide some critical epitopes in a fraction of the @-galactosidasemolecules. gardless of the cell lysis protocol, i.e. either through scraping Thus, in order to unmask such epitopes, unlabeled10,000 x culture dishes with Tritonlysis buffer or by cell disruption in g Triton supernatants denatured in SDS, were electropho- detergent-free buffer using aglass homogenizer (data not resed, transferred onto nitrocellulose, and probed by anti-@- shown). A Priming Heat Shock Attenuatesthe Loss of Enzyme galactosidase antibody followed by '"I-protein A. A decreased amount of @-galactosidase wasdetected in lysates from heat- Actiuities duringu Chulknging Stress-RL3 cells were primed shocked cells (Fig. 2 0 ) . Again, the decrease in protein recog- by heat shock for 15 min a t 45 "C and allowed to recover 20 nized by antibodies accountsfor the loss of enzymatic activity. h before the challenging heat shock. In such nonlethal conLZ1 heated cells, @-galactosidase becomes ditions these cells recovered 20 h after priming heat shock It appears that in

Enzyme Inactivationduring Heat Shock

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FIG. 3. Effect of heat shock on luciferase molecules. Mouse Lcellsexpressingluciferase48h aftertransfection with plasmid pRSV/L were analyzed by Western blot (the experiments were performedwith transiently expressing transfected mousecells which synthesized much higher amounts of enzyme than the constitutively expressing cells). Extracts from control (lanes 1, 3, and 5 ) or cells heat-shocked 30 min a t 42 'C (lanes 2 , 4 , and 6)were probed with an anti-luciferase antibody and revealed by an alkaline phosphataseconjugated anti-mouse antibody. Lanes 1 and 2 correspond to SDS extracts, lanes 3 and 4 to Triton supernatants, and lanes 5 and 6 to Triton pellets.

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FIG. 5. Influence of the stress severity on luciferase inactivation with or without priming heat shock. Control (0)and primed (+) RL3 cells were challenged by 15 min of exposure to various temperatures( A )or by 30 min exposure to increasing percentage of ethanol in the media ( B ) . Inboth cases, priming was achieved 20 h prior to the challenge by 15 min of heat shock a t 45 "C. Activities are expressed as a percentage of the mean nonchallenged activity.

primed LZ1 cells (Fig. 4B). Forms of stress other than heat shock, such as ethanol, result in a similar cell response. We found that aswith heat shock, incubation of cells with ethanol also leads to luciferase inactivation (Fig. 5B). The ethanol concentrations whichefficiently inactivate luciferase in 30 min are thosecommonly known to induce heat shock protein synthesis (20). Suchethanoltreatmentis alsoknown to induce the thermotolerant state. In this paper, we show the reverse observation: a priming heat shock resulted in a protection of luciferase activity when cells were incubated in 0 6 200 40 80 ethanol containing media (Fig. 5B). This brings further supTIME, min. port to models suggesting similar cellular targets for heat or FIG. 4. Thermoprotection of enzyme activity in heat primedethanol shocks. cells. A, luciferase activity was challenged a t 42 "C in control RL3 Protein synthesis is a complex cellular activity inactivated (0) and primed RL3 (+). B, P-galactosidase activity was challenged by heat shock but well known to be protected by priming heat a t 45 "C in control (0) and primed (W) LZ1 cells. In both cases, shocks (21). A priming heat shock of RL3 cells led to an priming was achieved 20 h prior to the challenge by 15 min heat shock a t 45 "C. Activities are expressed as a percentage of the mean efficient protection of protein synthesis (estimatedfrom [3sS] methionine incorporation) against a challenging heat shock. nonchallenged activity. Was there any correlation between the protection of this luciferase activities similar in magnitude to that of control natural process and the protection of reporter enzyme activunshocked cells. Spectacular protection of luciferase activity ity? To answer this questionwe had touse different challengagainst heat inactivation was observed in primed RL3 cells ing conditions for luciferase activity and protein synthesis to compared with control cells (Figs. 4A and 5A). The kinetics optimize the observation of the thermoprotection. A 15 min of luciferase heat inactivation in primed cells were found to challenge a t 42 "C was optimum to study luciferase inactivation but hardly affected protein synthesis, whereas a 15 min be much slower than in controlcells. The phenomenon of acquired thermoprotection of an en- challenge at 45 "C was optimum to study protein synthesis zyme against heat inactivation can be generalized. Heat in- inactivationbut completely inactivated luciferaseactivity activation a t 45 "C of P-galactosidase was also attenuated in even in primed cells. Thus, we decided to use different but

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Enzyme Inactivation during Heat Shock specific challenges for each activity and to compare the protective effect promoted by the same priming heat shock. This comparison could be achieved by plotting the thermoprotection ratio ( R ) of activity remaining after the second challenging heat shock versus a characteristic of the priming heat shock: R=

primed and challenged activity . prlmed but not challenged activity

In the firstexperiment, a constantpriming time (15min) was used and the temperature was varied (Fig. 6A), while in a second experiment the priming temperature was kept at 45 "C and the priming time was varied (Fig. 6B). The evolution of priming protection on protein synthesis was comparable to the evolution of priming protection on luciferase activity. Thermoprotection of luciferase activity developed with milder shocks than thermoprotection of protein synthesis. If the different heat shock proteins have distinct functions in thermoprotection, this difference might be a consequence of the noncoordinate heat shock protein synthesis (the spectrum of proteins induced by mild or by severe shocks differs). T h e Loss of Enzyme Activities Depends on theCell TypeThe reporter enzyme inactivation kinetics are different in control or thermotolerant cells; they were expected to differ in distinct cell types. In order to circumvent the possibility of generating different proteinsbecause of cloning artifacts, the various cell types to be compared were transfected with the same plasmids. Furthermore, we took advantage of the ability 1.0

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FIG. 7. Luciferase activity in Drosophila cell lysates after heat shock. 48 h aftertransfection with plasmid pRSV/L, Schneider cells were heat shocked at 37 "C during various times and lysed immediately after. Luciferase activity in non-heat-shocked cells is taken as 100%. The exponential curve fitting is drawn.

of the Rous sarcoma virus LTR topromote gene transcription in both eucaryotic and procaryotic cells (47). The kinetics of enzyme inactivation at a given temperature was found to depend on the cell expressing the enzyme. For instance, luciferase has a 200 min half-life at 37 "C in mouse cells transfected with plasmid pRSV/L, but is inactivated by 50% within 8 min at 37 "C in Drosophila cells transfected with the same plasmid (Fig. 7). 37 "C is a standard heatshock temperature for Drosophila cells. In contrast, in lac- E . coli cells transfected with plasmid pRSV/@-gal,no changes in @-galactosidaseactivity were detected during very severe heat shock conditions (up to 30 min at 50 "C), while a 50% decrease in activity was observed during heating 30 min at 45 "C mouse Ltk- cells transfected with the same plasmid (data not shown). DISCUSSION

Both luciferase and @-galactosidaseare inactivated following a first order kinetics during heat shock or exposure of cells to ethanol. The heat shock temperatures optimal for 36 38 40 42 44 46 studying the inactivation of these enzymes are different, 42 "C TEMP, "C for luciferase and 45 "C for @-galactosidase.In contrast to 42 "C, 45 "C is a rather severe temperature for mouse cells. However, a 15-min heat shock at this temperaturewhich leads to significant @-galactosidaseinactivation andinsolubilization does not produce detectable cell death. We find the inactivated enzymes inaTriton-insoluble fraction. This lack of solubility does not seem to involve the formation of interprotein disulfide bridges since the proteins remain soluble in SDS and had similar apparent electrophoretic mobility in the presence or in the absence of 2-mercaptoethanol (data not shown). The protein insolubilization folR 0s lowing aheat shock seems to be a general phenomenon reported for a subset of endogenous cellular proteins: the nuclear matrix proteins (35),the c-myc proteins (36, 37), the interferon-induced dsRNA-dependent protein kinase (48)and 0.0I the small heat shock proteins (38). 30 0 10 20 There are several lines of evidence that abnormal or denaT I M E , min. tured proteins areresponsible for the induction of heat shock FIG. 6. Comparison of the thermoprotection ratio for protein synthesis (0)and for luciferase activity (*) in RL3 cells protein synthesis (39, 40). However, the concept of abnormal produced by the same priming heat shocks. The ratio R is plotted protein is unclear. The heat shock inactivation of enzymes uersm the priming temperature (15 min exposure) ( A ) or versus the reported in this paper may help to specify the concept of priming time (at 45 "C) ( B ) .R = (primed and challenged activity)/ abnormal proteins. (primed but not challenged activity). Cells were allowed20 h recovery The enzyme inactivation might be some consequence of an at 37 "C after the priming stress. Protein synthesis was challenged altered metabolism. It has been suggestedthat oxygen radicals by a 15-min heat shock at 45 "C and measured by incorporation of [35S]meth10nineinto trichloroacetic acid-precipitable material during are produced during heat shock and related stresses (41-44). a 3-h labeling period immediately following the challenging stress. In agreement with this hypothesis, it has been shown that Luciferase activity was challenged by a 15-min heat shock at 42 "C glutathione (45) protects cells against the toxic effects of heat. and measured immediately after the challenging stress. However, if oxidative stress may induce heat shock protein

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Heat Inactivation during Enzyme

synthesis in some cases it does not seem to be responsible for heat shock protein synthesis during aheat shock (46). Alternatively, one would expect that thephysical effects of heat could be directly responsible for enzyme inactivation. However, the enzymes were found to be more stable in vitro in a cell lysate than within a living cell. But the bulk solvent properties and/or theprotein concentrationsof the cytoplasm might be more destabilizing than theconditions which prevail in our lysis buffer. In a first attempt to determine such a solvent influence on the temperature of inactivation, we performed two experiments testing salt and protein concentration effects. In fact, while we found no difference for the luciferase in vitro inactivation kinetics at 42 "C in buffers containing either 50 or 250 mM NaC1, the 10-fold dilution of a luciferase containing crude cell extract resulted in a 2-fold decrease of the inactivation rate (data notshown). At a given temperature, the enzyme inactivation kinetics depends on the cell type in which it is expressed. Although it is an insect enzyme, luciferase appears less stable in fly cells than in mouse cells. The heat shock response is triggered at lower temperature in flies than in mammals. This supports the idea that heat shock alters either the metabolism or the solvent conditions within the cells. On the other hand, the better heat stability of 0-galactosidase within bacterial cells might be a consequence of an eucaryote-specific posttranslational modification. A priming heat shock renders the cells more resistant to a following challenging stress. It appears to be a rule that lesions that occur in cells after heat shock are minimized in the thermotolerant cells (23). Thus, thermotolerant cells would be more resistant and recover faster because they are less damaged. Here we show that reporter enzymes are less damaged. Since we were dealing with proteins completely exotic with regard to the studied mouse cells, we have ruled out the possibility of specifically designed protective cell functions. An attractive hypothesis is that the heat shock proteins would nonspecifically stabilize soluble protein structures (25) by binding to hydrophobic domains of temporarily denatured proteins thuspreventing the formation of heat-induced insoluble aggregates (26, 27). Acknowledgments-We are much indebted to Dr. Patrick Ziarczyk from the Centre National de la Recherche Scientifique UA 1135 for Drosophila cells transfection, to Drs. Robin Anderson, Marie FranGoise Dubois, Moise Pinto, Agnbs Ullmann, and all the members of the Groupe de Biologie Molkculaire du Stress for discussions, and to Dr. Malcolm Buckle for critical reading of the manuscript. REFERENCES 1. Lindquist, S. (1986) Annu. Rev. Biochem. 55,1151-1191 2. Burdon, R. H. (1986) Biochem. J. 240,313-324 3. Carper, S. W., Duffy, J. J., and Gerner, E. W. (1987) Cancer Res. 47,5249-5255 4. Welch, W. J., and Suhan, J. P. (1985) J. Cell Biol. 1 0 1 , 11981211 5. Spiro, I. J., Denman, D. L., and Dewey, W. C. (1982) Rad. Res. 89,134-149 6. Nolan, N. L., and Kidwell, W. R. (1982) Rad. Res. 90,187-203 7. Anderson, R. L. B., and Hahn, G. M. (1985) Rad. Res. 102,314323 8. Cheng, K. K., Hui, S. W., and Lepock, J. R. (1987) Cancer Res. 47,1255-1262

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