Effect of protease inhibitors on protein degradation in rat hepatoma cells I. Effect on general protein degradation

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Biochimica et Biophysica Acta, 3 7 2 ( 1 9 7 4 ) 3 5 8 - - 3 6 5 © Elsevier Scientific Publishing Company,

A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 27537

E F F E C T OF PROTEASE INHIBITORS ON P R O T E I N D E G R A D A T I O N IN RAT HEPATOMA CELLS I. E F F E C T ON G E N E R A L PROTEIN D E G R A D A T I O N ANN McILHINNEY

and BRIGID L.M. HOGAN

Biochemistry Group, School of Biological Sciences, University o f Sussex, Falmer, Brigh ton BN1 9QG, Sussex (U. K. ) (Received May 6th, 1974)

Summary Tosyllysine chloromethyl ketone and tosylphenylalanine chloromethyl ketone in vitro are active-site specific and irreversible inhibitors of trypsin (EC 3.4.21.4) and chymotrypsin (EC. 3.4.21.1) respectively. Using rat hepatoma cells in suspension culture, both inhibitors were found to partially inhibit breakdown of prelabelled cell proteins to amino acids, the effect being greatest in the absence of serum. Protein synthesis in rat hepatoma cells, reticulocytes and reticulyte lysates was also irreversibly inhibited by these compounds. Reduction of ATP levels with antimycin a inhibited protein degradation, but neither tosylphenylalanine chloromethyl ketone nor tosyllysine chloromethyl ketone had any effect on ATP concentration in rat hepatoma cells. These results suggest that the degradation of at least some proteins in animal cells may involve the action of serine protease(s).

Introduction Very little is known a b o u t the mechanism and control of intracellular protein degradation in animal cells, despite its biological importance in determining both the steady state concentrations of different proteins, and the rate at which these change [1,2]. Complete degradation of proteins to acid soluble peptides or amino acids almost certainly involves several proteolytic enzymes of different specificities. The initial cleavage into two or more peptides need not destroy the biological activity of the protein, b u t sooner or later a cleavage will occur which irreversibly inactivates the protein. This step may be carried out b y a specific enzyme tightly b o u n d to the protein, as suggested for N e u r o Abbreviations: Tos-LysCH2C1, tosyllysine c h l o r o m e t h y l ketone; Tos-PheCH2C1 , tosylphenylalanine c h l o r o m e t h y l ketone; HTC, rat h e p a t o m a cells.

359 spora tryptophan synthase (EC 4.2.1.20) [3] and yeast phosphofructokinase

(EC 2.7.1.11) [4], or by a non-specific protease in the cytoplasm. Cleavage may be triggered by removal of an inhibitory protein or factor from the specific protease [ 3], or by a conformational change induced in the protein b y the removal of its substrate or co-factor [5,6], b y phosphorylation, or by random unfolding, exposing a site for attack by either an associated protease or one which is free in the cytoplasm. Following the early cleavage(s), a number of other proteolytic enzymes may degrade the peptides further to amino acids. While earlier (specific) proteases may be important for controlling the activity of individual proteins, the subsequent proteases will help to supply amino acids for recycling into other metabolic pathways. It is therefore possible to study different aspects of protein degradation by looking at either the loss of ability to precipitate with a specific antibody, or of biological activity [8,9] of a specific protein, or the release of acid soluble radioactivity from pre-labelled total cell proteins [8,10]. This latter is usually referred to as "general protein degradation". General protein degradation in cultured animal cells is enhanced by serum starvation and in some cells by amino acid starvation, and is inhibited by serum, and insulin [8,11]. Depletion of ATP levels using sodium azide or 2,4-dinitrophenol will also inhibit this degradation [8]. As far as we are aware there have been no detailed studies on the effect of specifi c inhibitors of proteolytic enzymes on the degradation of general or specific proteins in mammalian cells, although in bacteria the enhanced turnover of proteins due to starvation for a carbon or nitrogen source is inhibited by proteolytic enzyme inhibitors [10]. Tos-Phe CH2 C1 and Tos-LysCH2 C1 both inhibitors of serine proteases [12,13], have been shown to inhibit the cleavage of large Poliovirus -- specific precursor proteins in infected HeLa cells [ 1 4 ] . Tos-PheCH2 C1 also leads to an increase in the size of some newly synthesized protein molecules in uninfected HeLa cells [15] suggesting that post-translational cleavage is inhibited. In this paper we report that both Tos-PheCh2 C1 and Tos-LysCH2 C1 inhibit the degradation of pre-labelled general proteins in rat hepatoma cells in culture, providing evidence that serine proteases may be involved in this process. In the following paper we discuss the effect of these inhibitors on the loss of biological activity of specific proteins. We also show here that both Tos-PheCH2 C1 and Tos-LysCH2 C1 inhibit protein synthesis in HTG cells and reticulocytes, b u t their effect on degradation appears to be independent of their inhibition of protein synthesis. Materials and Methods Cell culture

HTC cells, obtained originally from Dr G.M. Tomkins, were grown in suspension culture in Swims $77 medium (GIBCO) modified to contain 50 mM Tricine (Calbiochem), 3 g/1 glucose, 2 mM glutamine and 10% calf serum (Flow Labs, Irvine, Scotland). The population doubling time was approximately 24 h and cells reached a maximum density of 7--10 " l 0 s cells/ml.

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Amino acid incorporation into proteins This was measured at 37°C using 0.5 pCi/ml [4,5-3H]leucine (51 Ci/ mmole Radiochemical Centre, Amersham). Replicate aliquots (1 ml; 3.5--4.0 • 105 cells) were removed and the cells spun down, washed once with phosphate buffered saline and lysed with sodium laurylsulphate (0.5 ml; 0.5% w/v) followed by addition of 1 M NaOH ( l m l ) and incubation for 20 min at 37°C. Trichloroacetic acid (1.5 ml; 25% w/v) was added and the precipitates were collected onto glass fibre filters for scintillation counting. Degradation of generally labelled cellular proteins HTC cells at a density of 3.5--4.0 • 10 s cells/ml were incubated with 0.5 pCi/ml of L-[4,5 -3 H] leucine for 5--12 h. The cells were harvested, washed with fresh medium and then resuspended in media as described in the figure legends, with 2 mM unlabelled L-leucine. Incubations were at 37°C and duplicate aliquots of 1 ml were pipetted into 0.1 ml of 100% w/v trichloroacetic acid. The samples were allowed to stand at 4°C for at least 30 min and, following centrifugation, the supernatant layers transferred to tubes containing 20 pl of bovine serum albumin (Armour Pharmaceutical Company; 50 mg/ml). After a second centrifugation to remove the carrier protein, 0.3 ml portions of the supernatant were counted in triton-toluene scintillator with an efficiency of about 25%. The a m o u n t of trichloroacetic acid soluble radioactivity at 0 time, amounting to less than 2% of total radioactivity at 0 time, was subtracted from the results, and the acid soluble counts were expressed as the percentage of radioactivity in total protein at 0 time. This was estimated by dissolving the zero time acidprecipitable pellet, after washing it twice with 10% (w/v) trichloroacetic acid in 1 ml of 0.5 M NaOH. A (1.3 ml aliquot was used for estimation of radioactivity. A TP measurements ATP levels were estimated by the fluorometric m e t h o d of Lowry et al. [16], using the equivalent of 6--8 • 10 s cells in an assay volume of 2 ml and a standard ATP range of 0--2.5 nmoles/ml. The results were expressed as nmoles of ATP/mg protein. Reticulocy tes and lysates Rabbit reticulocytes and lysates were prepared and incubated as previously described [17]. Reticulocytes and lysates were incubated in volumes of 1 ml and aliquots were of 50 pl. Trichloroacetic acid precipitates were collected onto glass fibre discs and radioactivity was estimated by gas flow counting. Tos-PheCH2C1 and Tos-LysCH2Cl were obtained from Sigma. TosPheCH 2C1 was dissolved in methanol and was added in a volume of less than 1% of the total incubation volume. Tos-LysCH2 CI was made up in water. An equal volume of methanol or water was added to the controls. Results When HTC cells were grown for 5--12 h in medium containing L-[ 3 H]leucine, then washed and resuspended in medium with excess unlabelled leucine, release of trichlororacetic acid soluble radioactivity continued for at least

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Fig. 1. E f f e c t o f s e r u m s t a r v a t i o n , T o s - P h e C H 2 C1 a n d T o s - L y s C H 2 CI o n d e g r a d a t i o n o f g e n e r a l l y l a b e l l e d cellular p r o t e i n s . H T C c e l l s g r o w n for 5 h i n m e d i u m c o n t a i n i n g L - [ 4 , 5 - 3 H ] l e u c i n e w e r e r e s u s p e n d e d in m e d i u m c o n t a i n i n g 2 m M c o l d l e u c i n e , w i t h o r w i t h o u t s e r u m . P r o t e i n b r e a k d o w n w a s d e t e r m i n e d as described. A. o o c o n t r o l cells w i t h o u t s e r u m ; ¢ "-, cells w i t h o u t s e r u m + 1 0 -4 M T o s PheCH2CI; ~ ~, c o n t r o l c e l l s w i t h s e r u m ; • • , c e l l s w i t h s e r u m + 1 0 -4 M T o s - P h e C H 2 C I . B. ~, c o n t r o l cells w i t h s e r u m ; • ± , cells w i t h s e r u m + 5 • 1 0 ~ M TDS-LysCH2CI; o o control cells without serum; • e c e l l s w i t h o u t + 5 : 1 0 -4 M T o s - L y s C H 2 CI. T C A , t r i c h l o r o a c e t i c acid. Fig. 2. E f f e c t serum. • o

o f v a r i o u s c o n c e n t r a t i o n s o f T o s - P h e C H 2 C I o n p r o t e i n d e g r a d a t i o n in m e d i u m w i t h o u t • , c o n t r o l ; -~ , + 1 0 -4 M T o s - P h e C H 2 C I ; ~ ~ , + 5 • 1 0 -5 M T o s - P h e C H 2 C I ; o, + 10-5 M Tos-PheCH2CI.

2--3 h at an almost linear rate as shown in Fig. 1. With serum present the rate of loss of radioactivity into the trichloroacetic acid soluble fraction varied from 1-3% per hour, but when serum was omitted the rate of protein breakdown increased to 3--5% per h. In cells incubated both with and without serum, protein breakdown was inhibited by the inhibitors of serine proteases, TosPheCH:C1 and Tos-LysCH2C1 (Fig. 1), the degree of inhibition being dependent on the dose (Fig. 2). At 10 -3 M, Tos-PheCHECl is fairly insoluble and at concentrations less than 10-SM this inhibitor has little effect. At 10 -4 M, which was the concentration chosen for most of the experiments described, breakdown was inhibited by 55% in the absence of serum and 34% in its presence after a 3 h incubation. A similar degree of inhibition with TosLysCH2C1 was obtained at a concentration of 5 " 10 -4 M (results not shown). To test the possibility that the rate of protein degradation was being underestimated by the reutilisation of radioactive amino acids released during turnover, protein synthesis was completely blocked by cycloheximide. If the radioactive leucine was not equilibrating rapidly with the excess cold leucine in the medium, inhibiting protein synthesis should increase the rate of release of radioactivity into the trichloroacetic acid-soluble fraction. Cycloheximide (50 pg/ml) did not increase the rate of release of acid soluble radioactivity, but in fact slightly decreased it (by about 9% in cells incubated with serum, and 20% in cells without serum over a period of 3 h) and therefore itself partially inhibited protein breakdown [ 8 ] . Similar results were obtained with 2 pM pactamycin (a concentration which completely inhibits polypeptide chain initiation without affecting elongation [18] in HTC cells).

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l y s a t e in t h e p r e s e n c e

Besides inhibiting protein degradation both Tos-PheCH2C1 and TosLysCH2C1 were found to be effective inhibitors of protein synthesis in HTC cells (Fig. 3) and in the case of Tos-PheCH2C1 this inhibition was irreversible (the reversibility of the inhibition by Tos-LysCH2C1 was not studied). At 10 -4 M, Tos-PheCH2 C1 also inhibited haemoglobin synthesis in reticulocytes by 50% after 3 min (results not shown). To see whether this inhibition of protein synthesis is secondary to an inhibition of protein degradation we looked at the effect of 10-4M TosPheCH2CL on a reticulocyte lysate. We have shown [17] that the reticulocyte lysate system, like the HTC lysate [17,19] is unable to degrade proteins. However, protein synthesis in the reticulocyte was still inhibited by Tos-PheCH 2C1 (Fig. 4) and further studies have indicated that predominantly polypeptide

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Fig. 5. E f f e c t o f A T P d e p l e t i o n o n g e n e r a l p r o t e i n b r e a k d o w n . HTC cells at 5.0 • l0 s/ml were incubated for 5 h with radioactive leucine and then resuspended in medium consisting of an isotonic solution of the inorganic constituents o f t h e m o d i f i e d $ 7 7 m e d i u m ('salts' m e d i u m ) p l u s 2 m M l e u c i n e , o o control + glucose to a concentration o f 3 g/l; "t0 + 7 5 n g / m l a n t i m y c i n a.

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TABLE I E F F E C T O F Tos-PheCH2C1 A N D T o s - L y s C H 2 C l ON A T P L E V E L S I N I T I A L L Y A N D A F T E R A 45 MIN INCUBATION IN COMPLETE MEDIUM WITH SERUM Additions (M)

Initial A T P l e v e l (nmoles/mg protein)

A T P level a f t e r 4 5 m i n (nmoles/mg protein)

None 10 - 4 T o s - P h e C H 2 C1 5 . 1 0 - 4 Tos-LysCH2C1

17.2 14.2 17.4

17.3 14.2 19.7

chain initiation is being affected (McIlhinney, A. and Sampson, unpublished results). One possible explanation of the inhibition of protein synthesis and degradation by Tos-PheCH2CI and Tos-LysCH2C1 could be that they are lowering intracellular ATP levels. In Fig. 5 it can been seen that reducing ATP levels to virtually zero within 15 min b y incubating cells in 'salt medium' without glucose in the presence of 75 ng/ml of antimycin a (an inhibitor of oxidative phosphorylation) slows degradation by without 27%. However, comparison of ATP levels in Tos-PheCH 2 C1 and Tos-LysCH 2 Cl-treated cells with control levels indicated that neither inhibitor was affecting energy production (Table I). Discussion

In the experiments described here the rate of general protein degradation was estimated by measuring the release of radioactive leucine from pre-labelled cell proteins, introducing a possible error due to reutilisation of radioactive amino acids for protein synthesis. If re-utilisation of amino acids was at all significant, the apparent degradation rate would be an underestimate of the actual rate. Gan and Jeffay [20] have reported that under starvation conditions, recycling of amino acids in rat liver can be as much as 90%, and Eagle et al. [21] have observed that animal cells grown in culture with a radioactive amino acid and then resuspended in medium without that amino acid lose very little radioactivity (0.04 to 0.19% per h) into the trichloroacetic-soluble fraction. However, if the cells are resuspended in medium containing unlabelled amino acid in large excess a b o u t 1% of radioactivity is lost per hour. In all the experiments described here, the prelabelled HTC cells were resuspended in medium with a vast excess of non-radioactive leucine and, as Eagle and Piez [22] have reported that the rate of equilibration in mammalian cells in culture between the intracellular amino acid pool and the environment is very rapid in mammalian cells in culture, it seems likely that there is little re-utilisation of amino acids. We have shown here that Tos-PheCH2C1 and Tos-LysCH2C1 partially inhibit general protein breakdown in HTC cells, suggesting that serine proteases are involved in this process. However, before this conclusion can be accepted, several alternative explanations must be eliminated. Firstly, it is possible that Tos-PheCH2C1 and Tos-LysCH2C1 are working indirectly through inhibiting protein synthesis rather than by direct action on the degradative system. TosPheCH2C1 appears to inhibit protein synthesis in bacteria by preventing the

364 formation of a complex between the elongation factor EF-Tu, GTP and aminoacyl t R N A [23]. Highland et a1.[24] have recently reported that 0 . 5 " 1 0 -~ M Tos-PheCH2C1 had no effect on amino acid incorporation in a reconstituted rabbit reticulocyte cell-free system and conclude that there must be some basic difference between bacteria EF-Tu and the mammalian elongation factor, EF1. In direct contrast to this results, we have found that 10 -4 M Tos-PheCH2C1 inhibits protein synthesis in both intact reticulocytes and reticulocyte lysates incubated with heam, and we have evidence that it is predominantly polypeptide chain initiation which is affected (McIlhinney, A. and Sampson, unpublished observations). This would account for the discrepancy between our results and those of Highland et al. since little initiation may have been taking place in their reconstituted cell-free protein synthesizing system. If the action of Tos-PheCH 2C1 and Tos-LysCH 2C1 on protein degradation were entirely through their effect on protein synthesis, it would be expected that other inhibitors of protein synthesis would block degradation to the same extent. However, both 50 pg/ml cycloheximide (which completely inhibits initiation and elongation) and 2 pM pactamycin (which inhibits only initiation) reduce the rate of degradation very much less than either 10 -4 M Tos-PheCH2C1 or 5 " 10 .4 M Tos-LysCH2C1. A second way in which Tos-PheCH 2C1 and Tos-LysCH 2C1 might act indirectly is by lowering ATP levels. This is known to inhibit protein turnover both in bacterial [25,26] and in mammalian cells [8], suggesting that intracellular protein degradation involves an energy-requiring step, such as conformational change in the protein substrate [8,27] or activation of a protease. ATP depletion also inhibits protein synthesis completely [28] and it is possible that this could explain at least part of the inhibition of protein degradation. However, neither Tos-PheCHzC1 nor Tos-LysCH2C1 affect ATP levels in HTC cells (Table I). Rapidly reducing ATP levels to virtually zero with antimycin a inhibits general protein turnover by only 27% (Fig. 5), so that not all protein degradation can be ATP dependent. In fact, antimycin decreased the turnover rate in cells cultured in medium minus amino acids, glucose and serum to the level it would have been in cells growing in complete medium, suggesting that it is the enhanced turnover due to starvation which requires ATP. It is interesting to note that omitting both amino acids and serum from the medium stimulated turnover in HTC cells to a greater extent than omitting serum alone, so that amino acid levels may be important in controlling protein degradation in these cells. Thus, after 3 h, HTC cells incubated with both serum and amino acids lost 7.5% of trichloroacetic acid-precipitable radioactivity to the medium, while cells w i t h o u t serum but with amino acids lost 9.7% and cells with neither lost 12.4% (Figs 1 and 5). This is in contrast to 3T3 and SV3T3 cells where amino acid starvation has been reported to have no effect on protein degradation [29]. We conclude t h a t although a proportion of the inhibition of degradation by Tos-PheCH2C1 and Tos-LysCH~C1 may be due to their effects on protein synthesis, this is not enough to account for the magnitude of the inhibition seen, and implies the direct involvement of serine proteases in the degradation of at least some proteins. The fact that inhibitors of both trypsin and chymotrypsin-like enzymes should inhibit turnover is perhaps not too surprising, since

365 several enzymes with different specifities would be required to degrade a protein completely to its constituent amino acids.

Acknowledgements We would like to thank the Medical Research Council and the Science Research Council for financial support and the Medical Research Council for a studentship for A. McIlhinney. We are grateful to Drs J. Kay and R. Shields for comments on the manuscript.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

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