Degradation of Abnormal Proteins in HeLa Cells WALTER F. PROUTY Department of Biochemzstry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvanza 15261
ABSTRACT Canavanine, an arginine analog, is incorporated into HeLa cell protein when cells are incubated in the absence of arginine, and this incorporation can result in the production of nonfunctional enzymes or abnormal proteins. The cells degrade these abnormal proteins up to three times more rapidly than normal cell proteins. The capacity for selective degradation of abnormal proteins is not limited to HeLa cells since human fibroblasts also showed increased degradative rates following exposure to canavanine. In addition, enhanced degradation is not a peculiar property of canavanine incorporation since other amino acid analogs also promoted protein degradation. Thus, mammalian cells have the capacity to recognize and selectively degrade abnormal proteins.
Denaturation of a protein has been proposed as a n early and, perhaps, rate limiting step in intracellular protein degradation (Schimke, '70; Li and Knox, '72; Ballard et al., '74). According to this hypothesis, in vivo denaturation conditions should result in a class of proteins which are degraded rapidly and selectively. In bacteria, abnormal proteins resulting from genetic mutations (Goldschmidt, '70; Platt et al., '70), errors i n translation (Goldberg, '72) or incorporation of either puromycin (Goldberg, '72; Pine, '67a) or amino acid a a l o g s (Pine, '67b; Prouty and Goldberg, '72; Prouty et al., '75) are degraded up to twentyfold faster than normal cell proteins. Thus, prokaryotic cells can degrade selectively abnormal proteins (Goldberg et al., '74). The ability to degrade selectively abnormal proteins would be of great advantage since accumulation of enzymes with altered catalytic capacity could have drastic effects on the functioning of a cell. In this regard, the finding of enzymes with reduced specific activity in old compared to young animals is of interest, since i t may suggest that the production of altered proteins may be a normal part of the aging process (Halliday and Tarrant, '72; Gerschon and Gerschon, '73; Haining and Legen, '73). Such observations provide a basis for one theory of senescence (Orgel, '63). Mouse L cells can degrade selectively altered hypoxanthine guanine phosphoriboJ. CELL. PHYSIOL.,88: 371-382
syltransferase without affecting breakdown of general cell proteins (Capecchi et al., '74). To study whether or not selective degradation of abnormal proteins in mammalian cells is a general phenomenon, HeLa cells were incubated in the presence and absence of amino acid analogs in an attempt to induce formation of large quantities of abnormal proteins. The subsequent fate of these proteins was then measured. The analogs chosen for study are among those which have been shown previously to be incorporated into proteins of mammalian or bacterial cells. MATERIALS AND METHODS
All chemicals were analytical reagent grade. L-Canavanine sulfate was purchased from Calbiochem, and L-(4,5-3H) leucine was obtained from SchwarzIMann. L-Threoa-amino-0-chlorobutryic acid was synthesized from L-allothreonine (purchased from ICN, K and K) as previously described (Konoshita and Umezawa, '51). L("T guanido) Canavanine was purchased from Calatomic and DL-("C) ornithine from Amershaml Searle. HeLa S3 cells (a kind gift of Dr. B. A. Phillips) were grown in suspension culture in Joklik modified Eagle's medium with 5 % calf serum (both purchased from Grand Island Biological Co.) and kept between 4Received Sept. 30, '75. Accepted Dec. 6, '75. This work was supported by NSF grant number GM 41875. A preliminary report of this work h a s been presented (Prouty. W. F. 1975 Fed. Proc. Abs. [No. 2 4 5 2 ] , 3 4 : 651). 1
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8 X 105 cellslml. Cells were tested at monthly intervals and found to be free of mycoplasma contamination by the procedure of Levine ('72). For labeling purposes cells were incubated in 50 ml tubes with screw caps (Bellco Biological Glassware), and stirring of cells was accomplished with an 8 mm magnetic spinning bar. Since this procedure appears to result in some mechanical disruption of cells ( < 1% per hour) experiments were usually not run in excess of six hours. To promote incorporation of amino acid analogs into proteins, cells were washed, collected by centrifugation and suspended at a concentration of 8-14 X lo5 cells/ml in modified Eagles medium, containing 0.04 mM leucine, and 5% dialyzed calf serum, but lacking the amino acid to be replaced by an analog. The analog was added to the medium at the concentrations indicated and the cells incubated for 1530 minutes with continuous stirring before addition of L-(4-5-3H)leucine (0.5-1.0 pCi/ ml) for an additional 30 minutes to 3 hours. Under these conditions, incorporation of radioactivity into hot trichloroacetic acid precipitable material was linear for at least three hours. Protein breakdown was measured as appearance of label in trichloroacetic acid soluble form from cells which were washed to remove unincorporated radioactive amino acid .(i.e. acid soluble radioactivity was
3
less than 2 % acid precipitable radioactivity) and resuspended in Eagle's medium containing 2 mM L-leucine. Increasing the amount of leucine in the chase medium to 10 mM had no effect on the apparent rate of protein breakdown. Inclusion of cycloheximide (100 pglml) with growing cells as a means to prevent reutilization did not further enhance the measured rate of protein breakdown. Thus, 2 mM L-leucine appears adequate to minimize isotopic reutilization. Cycloheximide inhibits protein breakdown in some mammalian systems (Levitan and Webb, '69) and this could complicate the interpretation of the effectiveness of the leucine chase. However, degradation of proteins synthesized in the presence of 'Cvaline, 14C guanido arginine, or I4C guanido-canavanine were the same when measured in the presence or absence of cycloheximide. In addition, cycloheximide appears to have no effect on the rate of inactivation of a specific HeLa protein, ornithine decarboxylase (submitted to J. Cell. Physiol.). Paper electrophoresis was carried out on a Savant High voltage electrophoresis apparatus. Radioactivity on the paper strips was measured on a Packard Radiochromatogram Scanner Model No. 7201. Ornithine decarboxylase activity was measured in 20,000 g supernatant fractions of HeLa lysates (obtained by repeated freezing and thawing of cells) by the evolution of radio-
PROTEIN SYNTHESIS
2CANAY
I
2
-X
3
HOURS Fig. 1 Incorporation of leucine into HeLa cells incubated in the presence of arginine or canavanine. HeLa cells growing at a concentration of 5 X 105 cellslml were centrifuged, washed with Earle's salts and resuspended at a concentration of 1.4 X 106 cells per milliliter i n minimal medium lacking arginine and containing leucine at 0.04 mM. Arginine (0.25 mM) or canavanine (0.25 mM) and 14C leucine (0.5 pCi/ml) was added to a portion of the culture. Incorporation of leucine was determined as the incorporation of radioactive material into hot acid precipitable form and is expressed as microgram incorporated per 106 cells. The radioactive proteins were poured over glass fiber filters and counted in a toluence based scintillaCanavanine, X- - -X. tion fluid. Symbols are: Arginine, 0-0;
373
PROTEIN CATABOLISM IN HeLa CELLS
+
I
ORIGIN
14C
GUANIDO CANAVANINE
L- CANAVA N I N E
-
@
-
ACID SOLUBLE FRACTION OF PRONASE DIGEST
Fig. 2 Paper electrophoresis of amino acids i n HeLa proteins following exposure to (14C) canavanine. HeLa cells were centrifuged, washed a n d resuspended i n medium lacking arginine a s i n figure 1. ( W - g u a n i d o ) Canavanine (2 pg/ml, 5 pCi/ml) was added a n d cells incubated three hours. Cells were centrifuged, washed, resuspended i n 0.01 M Tris, 1 mM Ca C12 then lysed by freezing a n d thawing five times. The mixture w a s centrifuged at 5,000 X g a n d the supernatant was poured over a Sephadex G25 column (1.5 X 5 cm). Samples at the front were collected and incubated for 18 hours with pronase (1 mg/4 mg substrate protein) i n a solution containing 0.03% sodium azide to prevent bacterial or fungal growth. Reaction was stopped by addition of trichloroacetic acid to 5 % , t h e precipitate removed b y centrifugation a n d the acid extracted from the supernatant with diethylether a n d the solution lyophilized. Samples containing (14C) canavanine or L-canavanine sulfate were treated similarly. The lyophilized materials were solubilized in water, spotted on a piece of Whatman No. 4 paper, subjected to electrophoresis (buffer; 1 % acetic acid, 2 % pyridine v/v, 30 Voltslcm, 200 minutes). The dried chromatogram w a s sprayed with ninhydrin and finally scanned for radioactivity. Under these conditions, canavanine moved 15 c m toward the cathode.
into HeLa cell protein is shown in figure 2. HeLa cells were incubated in medium lacking arginine and containing L('4C guanido) canavanine; incorporation of labeled canaRESULTS vanine was linear for a t least six hours. Incorporation of canavanine into Radioactive proteins from a cell lysate were HeLa cells protein then separated from small molecular weight Canavanine is a naturally occurring radioactive material by chromatography on analog of arginine and differs from argi- a Sephadex G25 column. The resultant nine by replacement of a n oxygen atom for protein fraction was subjected to pronase the methylene group at C-5 of the arginine digestion and the acid soluble material molecule. then analyzed by paper electrophoresis (fig. Exposure of HeLa cells to conavanine, 2 ) . The major spot of radioactivity is coinduring arginine starvation, reduces, but cident with the major spot of radioactivity does not abolish, the incorporation of ra- of authentic (14C-guanido) canavanine suldioactive leucine into cell protein (fig. 1). fate and a ninhydrin positive spot repreThat canavanine is, in fact, incorporated senting unlabeled L-canavanine. Similar
active carbon dioxide from (1-14C)ornithine as previously described (Russell and Snyder, '68).
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WALTER F. PROUTY
A HOURS
I00
50
10
5
;.;_ 0
B
I
I
10
20 MINUTES AT 49°C Figure 3
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PROTEIN CATABOLISM IN HeLa CELLS
375
hours, there was no further loss of viability as measured after 20 hours incubation, in accord with previous results (Miedema and Kruse, '66). Thus, loss of cell viability resulting from incubation with canavanine required continuous exposure to the analog. If incorporation of canavanine into proteins affects protein conformation, then proteins synthesized in the presence of canavanine ought to be distinguished from the normal proteins by some criterion such as heat denaturation. Ornithine decarboxylase can be induced in HeLa cells by addition of fresh medium to the culture (Prouty, in preparation). As seen in figure 3A, if cells are exposed to an increasing ratio of canavanine to arginine i n the medium, the amount of functional ornithine decarboxylase induced is diminished. Furthermore, the enzyme synthesized in the presence of the analog has an increased rate of heat inactivation at 49 C and the rate increases as the ratio of canavanine to arginine in the culture medium is increased (fig. 3B). If incorporation of canavanine is a random process, a family of altered proteins would result and these should give complex heat Fig. 3 Induction (A) and heat inactivation (B) of ornithine decarboxylase in the presence of ar- denaturation curves. However, the curves ginine and canavanine. HeLa cells growing at 5 x in figure 3B appear to follow simple first lo5 cells/ml were centrifuged, washed and resusorder decay. A larger number of points for pended at a concentration of 1.1 X 1 0 6 cellslml i n medium lacking arginine. Arginine and cana- a more extended period of time may have vanine were then added to aliquots of the culture revealed complexity in these curves. In any at the concentrations indicated below and at the event exposure to canavanine during syntimes indicated aliquots containing ca. 5 X 106 cellslml were taken for assay of ornithine decar- thesis appears to result i n altered enzymes. Presumably, prolonged exposure to canaboxylase as in MATERIALS AND METHODS. A washed cell pellet was resuspended in 0.15 ml buffer (10 vanine will result in diminished specific mM Tris, pH 7.4, 0.1 mM EDTA, 0.1 mM pyridoxal activity of enzymes critical to cell viability, phosphate, 5 mM dithiothreitol), the cells broken thus causing the loss of cell viability deby freezing and thawing and the enzyme assayed scribed above. in the 20,000 X g supernatant of the broken cell
results were obtained in two other solvent systems. Thus, canavanine is incorporated into HeLa proteins, an observation consistent with results showing incorporation of canavanine into protein of Walker Carcinosarcoma cells (Kruse et al., '59) or hamster cells (Hare, '69). Although exposure to canavanine during arginine starvation slowed the rate of incorporation of '4C-leucine into acid precipitable material (e.g. 0.183 and 1.3 mM canavanine slowed the rate of 14C leucine incorporation into protein by 43 and 5 5 % , respectively, over a period of one hour), it is not known whether or not exposure to the analog inhibited protein synthesis since the specific activity of leucine pools was not measured. Exposure to canavanine for up to six hours did not reduce cell viability, as measured by exclusion of Trypan blue, by more than 5 % . However, prolonged exposure (20 hours) to 1.0 mM canavanine in medium lacking arginine resulted in an almost complete loss of viability. If cells were washed free of canavanine at six
suspension. At 4 hours incubation an aliquot containing ca. 2.5 X lo7 cells was removed from each sample and the supernatant fraction of the broken cell suspension used for the heat inactivation experiment shown in B. Equal aliquots of the supernatant fraction were incubated at 49OC. At the times indicated tubes were removed from the incubator, plunged into a n ice bath, and cold buffer was added to the tube. The fractions were then assayed for remaining ornithine decarboxylase activity a s above. Symbols represent addition of the following concentrations of arginine or canavanine. Symbol
0-0 A-A
0-0 0-0 0-0
Arginine
Can av anine
mM
m M
0.3 0.3
0
0.3 0.15
0.03
0.3 1.o 1.0 1.0
Fate of proteins containing canavanine To measure the degradation of proteins containing canavanine, HeLa cells were suspended in medium lacking arginine but containing radioactive leucine; then either arginine or canavanine was added. After three hours incubation, cells were centrifuged, washed and resuspended in minimum essential medium containing 2 mM leucine to minimize reutilization of label released by proteolysis. Use of the same radioactive label in the two conditions permits more accurately the comparison of the fate of normal and abnormal proteins, since transport of label and effectiveness of chase are very similar in the two condi-
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WALTER F. PROUTY
tions. Proteins containing canavanine are degraded more rapidly than those containing arginine (fig. 4). Similar results were obtained when cells were labeled with ('4C) phenylalanine. Thus, use of leucine does not give rise to a unique result. In addition, proteins synthesized in the presence of ("T) guanido canavanine were degraded more rapidly than those synthesized in the presence of (3H) arginine although the extent of the difference tended to vary probably due to the different effectiveness of the chase in the various procedures. That enhanced degradation was not a nonspecific effect of the presence of unincorporated canavanine is indicated by the unaltered rate of degradation of cells exposed to ar-
z 3
0
D
Y
U
w
a m
s w I-
0
a n
s
HOURS Fig. 4 Degradation of proteins synthesized the presence of arginine or canavanine. HeLa cells were collected by centrifugation and resuspended at a concentration of 1 X 106 cellslml in medium containing either arginine (0.3 mM) or canavanine (2.0 mM) and 0.04 mM *4C leucine (0.5 pCilm1). Cells were incubated one hour at 37O, then centrifuged, washed and resuspended at 8 X 1 0 5 cells/ ml in chase medium as described in MATERIALS A N D METHODS. Canavanine (2 mM) was added to one half of the culture incubated in arginine to test the effects of the drug directly on the degradative process. Protein breakdown was measured in rep licate 0.5 ml aliquots as in MATERIALS AND METHODS. After three hours all three cultures contained over 97% viable cells as determined by their ability to exclude Trypan Blue. Symbols are: Canavanine, A __ A , Arginine, X-X, Arginine, then canavanine, -0.
z 3 0
n Y
U
W
a
m
z W L a a
s HOURS Fig. 5 Effects of incubation with varying amounts of canavanine on subsequent protein breakdown. HeLa cells were centrifuged, washed and resuspended in minimal medium lacking arginine and leucine and incubated for 30 minutes. Aliquots of the cells were then supplemented with (3H)-leucine (0.04 mM, 1 pCi/ml) and either arginine (0.6 mM) or canavanine (0.2, 1.0, or 2.0 mM) and incubated a n additional 30 minutes. Cells were washed and protein breakdown measured as in MATERIALS AND METHODS. After three hours, over 98% of the cells exposed to arginine or 0.2 mM canavanine were viable, while over 92% of cells exposed to higher concentrations of canavanine remained viable.
ginine when canavanine was added to the chase medium (fig. 4). The increased rate of proteolysis is probably due to the degradation of intracellular proteins and not a result of release to the medium of labeled proteins by some cells and subsequent endocytosis and degradation by others. The conditions used in these experiments result in a release of 0.8% prelabeled cell proteins to the medium per hour as measured by the appearance of acid precipitable radioactivity in an 800 X g supernatant fraction. Such release is probably from cell breakage (e.g. by the magnetic stirring bar) or secretion. Furthermore, in the presence of 5 % serum, less than 0.01% of exogenously added radioactive protein (prepared from either E . coli or HeLa cells) was made acid soluble in one hour, presumably by endocytosis and degradation (Ryser, '68, and unpublished results). These data indicate that the majority of label made acid soluble in the present experiments must have originated from intracellular proteins and that the proteins probably did not pass through the medium prior to their degradation.
377
PROTEIN CATABOLISM I N HeLa CELLS
Canavanine probably competes with arginine for charging by the amino acyl tRNA synthetase (Allende and Allende, '64). In fact, in bacteria canavanine appears to replace arginine in growing polypeptides on a random basis (Schachtele et al., '68). Thus increasing the intracellular ratio of canavanine to arginine presumably increases the amount of canavanine incorporated into protein. This would probably increase the quantity of abnormal protein synthesized (fig. 3b) and hence, the rate of subsequent protein degradation. Increasing the concentration of canavanine exposed to cells during starvation for arginine results in a greater subsequent rate of protein degradation (fig. 5). Under optimal conditions tested here, canavanine incorporation stimulated degradation rates by a factor of 2.5-3 over basal levels. In addition, starvation for arginine prior to canavanine addition might be expected to lower the intracellular arginine pool, thus increasing the canavanine to arginine ratio. A ninety minute arginine starvation period prior to exposure to canavanine in-
/ 3? 20
W + 0
a LL
arz
HOURS Fig. 6 Degradation of proteins synthesized in t h e presence of arginine or canavanine after starvation for arginine for 0 or 90 minutes. HeLa cells were collected by centrifugation, washed and resuspended i n complete medium lacking arginine or leucine. At zero or 90 minutes L-leucine, (0.04 mM) a n d either canavanine (0.183 mM) or argin i n e (0.54 mM) w a s added to a portion of the cells. After 15 minutes incubation, L-(3H) leucine (0.5 pCi/ml) was added and incubation continued a n additional 45 minutes. Protein breakdown was determined asdescribed in MATERIALS AND METHODS.
TABLE 1
Effect of exposure of HeLa cells t o various a m i n o acid analogs on subsequent protein degradation Cells exposed to
Conc. m M
Control, normal amino acids Norleucine Seleno-Methionine Ethionine Azatryptophan Threo-amino-chloro butyric acid Canavanine
1.3 2.6 2.6
1.9 1.7 2.0
Protein breakdown i n one hour %
4.8 5.3 4.7 4.9 7.1 12.4 13.7
Effects of exposure to various amino acid analogs on subsequent rates of protein breakdown. HeLa cells were centrifuged, washed and resuspended in medium lacking methionine, arginine, tryptophan or valine and containing leucine a t 0.04 mM. Analogs were added a t the concentration indicated along with 3H leucine (0.5 p C i / ml) and cells incubated three hours Protein breakdown was then measured as in MATERIALS AND METHODS. D e g ~ radation was linear for three hours. In these and several similar experiments, there appeared to be a n inverse relationship between the rate of incorporation of leucine into cell protein and t h e subsequent rate of protein breakdown, e.g., whereas canavanine and azatryptop h a n slowed incorporation of leucine by 50 a n d 30% respectively, norleucine or seleno-methionine did not slow leucine incorporation.
creases the subsequent rate of degradation compared to cells exposed to canavanine but not pre-starved for arginine (fig. 6). These observations indicate that incorporation of canavanine into cell proteins results in proteins which are more rapidly hydrolyzed by the cellular catabolic machinery. Medium manipulations appear to be effective in altering intracellular concentrations of canavanine and arginine, though such concentrations were not measured and the alterations still remain speculative. Generality of increased degradation rate o n exposure to a m i n o acid analogs Enhanced rates of protein breakdown are not peculiar to exposure to canavanine since other amino acid analogs which are known to be incorporated into cell protein (Fowden et al., '67) also stimulate the subsequent rate of protein breakdown (table 1). Various analogs have different effects on protein degradation rates. Whereas, azatryptophan, an analog of tryptophan and threoamino chlorobutyric acid, an analog of valine, stimulated subsequent protein degradation, several analogs of
3 78
WALTER F. PROUTY
methionine (norleucine, selenomethionine, or ethionine) had little or no effect. Other mammalian cells can also degrade selectively proteins containing amino acid analogs. Incorporation of canavanine or threoamino chlorobutyric acid into protein of normal human fibroblasts also resulted in enhanced rates of protein breakdown. Interestingly, conditions in HeLa cells which resulted in a 2.3-fold stimulation of degradation rates over basal level resulted in only a 1.5-fold stimulation of degradation in the fibroblasts (W. Prouty, unpublished results). DISCUSSION
An abundance of evidence suggests that canavanine is not only incorporated into cell proteins, but that it replaces arginine randomly. For example, activation of arginine is competitively inhibited by canavanine with both the purified rat liver arginyl tRNAsynthetase (Allende and Allende, '64) and the E . coli enzyme (Bowman et al., '61). Since the specificity of amino acid incorporation lies in the tRNA charging reaction, any canavanine charged to tRNA would enter proteins in place of arginine in random positions. Canavanine was shown to be incorporated into nuclear globulin proteins in mammalian tumor cells (Bell, '74) and into proteins constituting the hexons of adenovirus infected human embryonic kidney cells (Neurath et al., '70). In E. coli infected with MS2 bacteriophage, a polypeptide corresponding to the coat protein is made in the presence of canavanine, but this polypeptide is nonfunctional in that it does not exert translational control, nor does it participate in phage assembly (Prouty, '75). HeLa cells, like E . coli (Goldberg, '72) have the capacity to degrade rapidly and selectively, proteins containing amino acid analogs. Thus, in both a eukaryotic and prokaryotic cell, incorporation of an amino acid analog which presumably results in an altered, denatured protein can serve as a signal to initiate protein breakdown. These results are in accord with previous studies showing selective degradation of missense mutations in hypoxanthine guanine phosphoribosyl transferase in mouse L cells (Capecchi et al., '74), of mutant hemoglobin molecules (Schaeffer, '73), or of puromycyl peptides in hepatoma cells
(McIlhinney and Hogan, '74). In addition, reticulocytes appear to have the capacity to selectively degrade abnormal hemoglobin molecules resulting from incorporation of an amino acid analog (Rabinowitz and Fisher, '64). Regarding the similarity of the prokaryotic and eukaryotic cells in their ability to recognize abnormal proteins, the anaIogs most effective at stimulating protein degradation in HeLa cells were also most effective in E . coli (Goldberg, '72), while several methionine analogs, which were ineffective in HeLa cells, also had a very slight effect in E . coli. Interestingly, there is also a direct correlation between those analogs which slow growth in bacterial auxotrophs and those which stimulate degradation (Goldberg, '72). Efforts to alter the intracellular canavanine to arginine concentrations by manipulation of the medium or incubation conditions alter the rate of subsequent degradation (figs. 5, 6). Since canavanine appears to be incorporated into protein randomly in place of arginine (Allende and Allende, '64; Boman et al., '61; Schachtele et al., '68) an increase in intracellular canavanine to arginine ratios would increase the amount of abnormal proteins synthesized. However, although the data are consistent with the hypothesis that increased incorporation of canavanine results in an increased rate of protein degradation, intracellular concentrations of the two amino acids were not measured. The present study does not allow a quantitative estimation of the ratio of canavanine to arginine in HeLa protein, but from experiments utilizing (14C guanido) canavanine (0.04 mM) when arginine was absent in the medium, it is estimated that one canavanine moiety was incorporated for every 20 arginine moieties. A pool of arginine molecules appears to be maintained i n these cells even when incubated in arginine free medium. Thus, a functional ornithine decarboxylase with heat inactivation kinetics identical to that of the enzyme induced in control cells is induced even in arginine free medium, although induction is at one-sixth the rate seen in control cells (unpublished results). Since puromycyl peptides are also degraded rapidly, it might be argued that the analogs cause premature termination and thus act like puromycin (McIlhinney and Hogan, '74). How-
PROTEIN CATABOLISM I N HeLa CELLS
ever, analysis of HeLa proteins synthesized in the presence of canavanine indicate that the average molecular weight of the polypeptides is the same or, perhaps, slightly larger than normal cell proteins (in preparation). T h e presence of unincorporated canavanine in the cell had no direct effect on the degradative process in that it did not alter the rate of degradation of proteins synthesized prior to exposure to the analog (fig. 4). Thus, the stimulating effects of canavanine on protein breakdown rates is probably a direct result of the production of abnormal proteins, i.e. substrates for the protein catabolic machinery. It is of interest that maximal rates of degradation as observed here (figs. 5, 6) do not exceed basal rates by more than three fold and represent 12 to 15% of total cell radioactive protein degraded per hour. In contrast, bacterial cells degrade similar proteins at rates exceeding 50% per hour (Goldberg, '72; Prouty and Goldberg, '72; Prouty et al., '75). This difference in maximal degradation rate may reflect a fundamental difference in the catabolic capacity of mammalian and bacterial cells. However, the difference could be an artefact of reutilization which is reported to be very high in HeLa cells, i.e. amino acids arising from protein degradation are preferentially utilized for protein synthesis (Righetti et al., '71). Proteins ingested via endocytosis are known to enter lysosomes and there degraded to acid soluble material (Ryser, '68). In related studies, electron micrographs of HeLa cells exposed to amino acid analogs indicated a marked proliferation of digestive vacuoles in these cells (in preparation). These observations may suggest a common lysosomal site for the degradation of the two types of protein substrate although no direct evidence is available to support this hypothesis. On the other hand, bacteria which also selectively degrade abnormal protein have no such digestive organelle. The basal rates of degradation measured here (4-5% per hour) are much higher than previously published data (1 % per hour) Eagle et al., '59; Eagle et al., '61). These rates are especially high in light of the observations of Righetti et al. ('71) in which high rates of reutilization were demonstrated. Thus, the measured rates
379
could underestimate the actual rates. One possible explanation for the high rates of basal degradation is that there are at least two kinetically distinct populations of proteins in bacterial (Pine, '67a) or mammalian cells (Poole and Wibo, '73). In the present experiments, cells were exposed to radioactive precursor for up to three hours, a period which constitutes a pulse of radioactivity and which selectively labels those proteins with shorter half lives (Buchanan, '61). On the other hand, previous studies indicating a degradation rate of 1% per hour involved study of proteins labeled at least one generation time (ca. 24 hours) and thus, tended to measure the fate of more stable cell proteins. Others have also reported degradation rates in mammalian cells of around 5% per hour for proteins labeled in one to three hour pulses (Poole and Wibo, '73; Capecchi et al., '74). Thus, these results are not inconsistent with earlier studies. Johnson and Kenney ('73) reported that incorporation of amino acid analogs into tyrosine amino transferase altered its heat stability but not its half life. Such results do not contradict the present findings since these authors used immunologic methods to detect the protein and it is possible that this enzyme must be altered enough to destroy its antigenicity before it is recognized as abnormal. I have also found that ornithine decarboxylase can be induced in the presence of a small concentration of canavanine and the resultant molecule is much more susceptible to heat denaturation than the native enzyme. However, the rate of inactivation is unaltered. Thus, the rate of heat denaturation does not necessarily correlate with the rate of degradation. Production of abnormal proteins by incorporation of amino acid analogs has been used in an attempt to magnify a normal cell capacity. Production of abnormal proteins is probably not without analogies in various natural systems. For example, in studies of aging, the specific activities of numerous enzymes are known to decrease with age, i.e. abnormal proteins appear to accumulate as a normal part of the aging process (Gershon and Gershon, '73). Coupled with this observation is the recent finding of increased protein catabolic rates of a specific protein in aging cells (Haining
380
WALTER F. PROUTY
and Legen, '73). In addition, many hemoglobinopothies are the result of production of abnormal hemoglobin molecules (Ranney, '70; Adams et d., '72), and, at least in one instance, diminished quantities of abnormal hemoglobins is the result of selective catabolism (Adams et al., '72). There may also be examples of in vivo denaturation conditions. For example, deamidation of asparagine or glutamine residues can occur via a non-enzymatic process and in vitro deamidation rates of various proteins has been shown to correlate with in vivo rates of degradation (Robinson, '74). It is tempting to speculate that deamidation results in proteins sufficiently altered to be recognized as abnormal and thus result in the selective degradation of those proteins. This idea is also consistent with the concept that denaturation is the rate limiting step in protein degradation (Schimke, '70). In conclusion, mammalian cells have the capacity to recognize and selectively degrade abnormal proteins when they constitute either a minute portion of total cell protein (Capecchi et al., '74) or when produced in bulk form as in these experiments; such selective degradation may serve to protect the cell from harmful effects of accumulation of abberant polypeptides. LITERATURE CITED Adams, J. G., W. P. Winter, D. L. Ricknagel and H. M. Spenser 1972 Biosynthesis of hemoglobin Ann Arbor: Evidence for catabolic and feedback regulation. Science, 176: 1427-1429. Allende, C. C., and J. E. Allende 1964 Purification and substrate specificity of arginyl tRNA synthetase from rat liver. J. Biol. Chem., 239: 1102-1 112. Ballard, F. J., M. F. Hopgood, L. Reshef and R. W. Hanson 1974 Degradation of phosphoenolpyruvate carboxykinase (guanosine tetraphosphate) in uivo and in nitro. Biochem. J., 140: 531-538. Bell, D. 1974 The effect of canavanine on herpes simplex virus replication. J. Gen. Viol., 22: 319-330. Boman, H. G., I. A. Boman and W. K. Maas 1961 In: Biological Structure and Function, Proc. First IUB/IUBS Symp. T. W. Goodwin and 0. Lindberg, eds. Academic Press, Inc., N.Y., p. 297. Buchanan, D. L. 1961 Analysis of continuous dosage isotopic experiments. Arch Biochem. Biophys., 94: 489499. Capeechi, M. R., N. E. Capecchi, S. H. Hughes and G. M. Wahl 1974 Selective degradation of abnormal proteins in mammalian tissue culture cells. Proc. Nat. Acad. Sci. (U.S.A.), 71: 47324736.
Eagle, H., K. A. Piez, R. Fleischmen and V. I. Oyama 1959 Protein turnover i n mammalian cell culture. J. Biol. Chem., 234: 592-603. Eagle, H., K . A. Piez and M. Levy 1961 The intracellular amino acid concentrations required for protein synthesis i n cultured human cells. J. Biol. Chem., 236: 2039-2042. Fowden, L., D. Lewis and H. Tristram 1967 Toxic amino acids: their action as anti-metabolites. In: Adv. in Enzymol. Vol. 29. F. F. Nord, ed. Interscience Publishers, Inc., N.Y., pp. 89-163. Gershon, H., and D. Gershon 1973 Inactive enzyme molecules in aging mice: liver aldolase. Proc. Nat. Acad. Sci., 70: 909-913. Goldberg, A. L. 1972 Degradation of abnormal proteins in E . coli. Proc. Nat. Acad. Sci. (U.S.A.), 69: 422-426. Goldberg, A. L., and J. F. Dice 1974 Intracellular protein degradation in mammalian and bacterial cells. Ann. Rev. Biochem., 43: 835-869. Goldberg, A. L., E. M. Howell, J. B. Li, S. B. Martel and W. F. Prouty 1974 Physiological significance of protein degradation in animal and bacterial cells. Fed. Proc., 33: 1112-1 120. Goldschmidt, R. 1970 In uiuo degradation of nonsense fragments in E. coli. Nature, 228: 1151-1 156. Haining, J. L., and J. S. Legan 1973 Catalase turnover in rat liver and kidney as a function of age. Exp. Gerontol., 8: 25-31. Hare, J . D. 1969 Reversible inhibition of DNA synthesis by the arginine analog canavanine i n hamster and mouse cells in uitro. Exptl. Cell Res., 58: 170-173. Holliday, R., and G. M. Tarrant 1972 Altered enzymes in aging human fibroblasts. Nature, 238 : 26-30. Johnson, R. W., and F. T. Kenney 1973 Regulation of tyrosine amino-transferase in rat liver. J. Biol. Chem., 248: 4528-4531. Konishita, M., and S. Umezawa 1951 Synthesis of three a-amino-p-chlorobutyric acid. J. Chem. Soc. Japan Pur. Chem., 72: 3 8 2 3 8 4 , Kruse, P. F., Jr., P. B. White, H. A. Carter and T. A. McCoy 1959 Incorporation of canavanine into protein of Walker carcinosarcoma 256 cells cultured in uitro. Cancer Res., 19: 122-125. Levine, E. M. 1972 Mycoplasma contamination of animal cell cultures: a simple, rapid detection method. Exp. Cell. Res., 74: 99-109. Levitan, I. B., and T. E. Webb 1969 Regulation of tyrosine amino transferase in the isolated perfused rat liver. J. Biol. Chem., 244: 46844688. Li, J. B., and W. E. Knox 1972 Inactivation of tryptophan oxygenase in uiuo and in uitro. J. Biol. Chem., 247: 7550-7555. McIlhinney, A,, and B. L. M. Hogan 1974 Rapid degradation of puromycyl peptides in hepatoma cells and reticulocytes. Febs. Letts., 40: 297-301. Medina, E., and P. F. Kruse, Jr. 1966 Effect of canavanine on proliferation and metabolism of human cells in nitro. Proc. Sco. Exp. Sci. Med., 121: 1220-1222. Neurath, A. R., F. P. Wiener, B. A. Rubin and R. W. Hartzell 1970 Inhibition of adenovirus replication by canavanine. Biochem. Biophys. Res. Commun., 41: 1509-1517. Orgel, L. E. 1963 The maintenance of the ac-
PROTEIN CATABOLISM IN HeLa CELLS curacy of protein synthesis and its relevance to aging. Proc. Nat. Acad. Sci. (U.S.A.), 49: 517521. Pine, M.J. 1967a Response of intracellular proteolysis to alteration of bacterial protein and the implications in metabolic regulation. J. Bacteriol., 9 3 : 1527-1533. 1967b Intracellular protein breakdown i n the L1210 ascites leukemia. Canc. Res., 27: 522-525. Poole, B., and M. Wibo 1973 Protein degradationin cultured cells. J. Biol. Chem., 248: 62216226. Platt, T., J. H. Miller and K. Weber 1970 In viuo degradation of mutant Lac repressor. Nature, 228: 1154-1156. Prouty, W. F. 1975 Fate of MS2 proteins synthesized i n the presence of amino acid analogs. J. Virol., 16: 1090-1094. Prouty, W. F., and A. L. Goldberg 1972 Inhibitors of protein degradation in E. coli and their physiological effects. J. Biol. Chem., 247: 33413352. Prouty, W. F., M. J. Karnofsky and A. L. Goldberg 1975 Degradation of abnormal proteins in E . coli.J. Biol. Chem.,250: 1112-1122. Rabinowitz, M., and J. M. Fisher 1964 Characteristics of the inhibition ofhemoglobin synthesis
-
381
i n rabbit reticulocytes by threeiu-aminog-chlorobutyric acid. Biochim. Biophys. Acta, 91: 313322. Ranney, H. M. 1970 Clinically important variants of human hemoglobin. N. Eng. J. Med., 282: 144-152. Righetti, P., E. P. Little and G. Wolf 1971 Amino acid reutilization in protein synthesis in HeLa cells. J. Biol. Chem., 246: 57244732. Robinson, A. B. 1974 Evolution and the distribution of elutaminvl and asuaraginvl residues i n proteins. Proc. Nat. Acad: Scir (U.S.A.), 71: 8.8_ 5 4~.~ 88. _ Russell, D. H., and S. H. Snyder 1968 Amine synthesis i n regenerating rat liver. Proc. Nat. Acad. Sci. (U.S.A.), 60: 1420-1427. Ryser, J. 1968 Uptake of protein by mammalian cells: a n underdeveloped area. Science, 159: 390496. Schachtele, C. F., D. L. Anderson and P. Rogers 1968 Mechanism of canavanine death i n E. coli. J. Mol. Biol., 33: 861-872. Schaeffer, J. R. 1973 Structure and synthesis of the unstable hemoglobin sabine. J. Biol. Chem., 248 : 7473-7480. Schimke, R. T. 1970 In: Mammalian Protein Metabolism. Vol. IV. H. N. Munro. New York Academic Press, pp. 177-228.
Note added in proof: While this manuscript was i n preparation, I discovered that similar results were being reported i n work with WI-38 cells: Bradley, M. O., R. T. Schimke and L. Hayflick (1975, submitted for publication) J. Biol. Chem.
ABSTRACT Canavanine, an arginine analog, is incorporated into HeLa cell protein when cells are incubated in the absence of arginine, and this incorporation can result in the production of nonfunctional enzymes or abnormal proteins. The cells degrade these abnormal proteins up to three times more rapidly than normal cell proteins. The capacity for selective degradation of abnormal proteins is not limited to HeLa cells since human fibroblasts also showed increased degradative rates following exposure to canavanine. In addition, enhanced degradation is not a peculiar property of canavanine incorporation since other amino acid analogs also promoted protein degradation. Thus, mammalian cells have the capacity to recognize and selectively degrade abnormal proteins.
Denaturation of a protein has been proposed as a n early and, perhaps, rate limiting step in intracellular protein degradation (Schimke, '70; Li and Knox, '72; Ballard et al., '74). According to this hypothesis, in vivo denaturation conditions should result in a class of proteins which are degraded rapidly and selectively. In bacteria, abnormal proteins resulting from genetic mutations (Goldschmidt, '70; Platt et al., '70), errors i n translation (Goldberg, '72) or incorporation of either puromycin (Goldberg, '72; Pine, '67a) or amino acid a a l o g s (Pine, '67b; Prouty and Goldberg, '72; Prouty et al., '75) are degraded up to twentyfold faster than normal cell proteins. Thus, prokaryotic cells can degrade selectively abnormal proteins (Goldberg et al., '74). The ability to degrade selectively abnormal proteins would be of great advantage since accumulation of enzymes with altered catalytic capacity could have drastic effects on the functioning of a cell. In this regard, the finding of enzymes with reduced specific activity in old compared to young animals is of interest, since i t may suggest that the production of altered proteins may be a normal part of the aging process (Halliday and Tarrant, '72; Gerschon and Gerschon, '73; Haining and Legen, '73). Such observations provide a basis for one theory of senescence (Orgel, '63). Mouse L cells can degrade selectively altered hypoxanthine guanine phosphoriboJ. CELL. PHYSIOL.,88: 371-382
syltransferase without affecting breakdown of general cell proteins (Capecchi et al., '74). To study whether or not selective degradation of abnormal proteins in mammalian cells is a general phenomenon, HeLa cells were incubated in the presence and absence of amino acid analogs in an attempt to induce formation of large quantities of abnormal proteins. The subsequent fate of these proteins was then measured. The analogs chosen for study are among those which have been shown previously to be incorporated into proteins of mammalian or bacterial cells. MATERIALS AND METHODS
All chemicals were analytical reagent grade. L-Canavanine sulfate was purchased from Calbiochem, and L-(4,5-3H) leucine was obtained from SchwarzIMann. L-Threoa-amino-0-chlorobutryic acid was synthesized from L-allothreonine (purchased from ICN, K and K) as previously described (Konoshita and Umezawa, '51). L("T guanido) Canavanine was purchased from Calatomic and DL-("C) ornithine from Amershaml Searle. HeLa S3 cells (a kind gift of Dr. B. A. Phillips) were grown in suspension culture in Joklik modified Eagle's medium with 5 % calf serum (both purchased from Grand Island Biological Co.) and kept between 4Received Sept. 30, '75. Accepted Dec. 6, '75. This work was supported by NSF grant number GM 41875. A preliminary report of this work h a s been presented (Prouty. W. F. 1975 Fed. Proc. Abs. [No. 2 4 5 2 ] , 3 4 : 651). 1
3 72
WALTER F. PROUTY
8 X 105 cellslml. Cells were tested at monthly intervals and found to be free of mycoplasma contamination by the procedure of Levine ('72). For labeling purposes cells were incubated in 50 ml tubes with screw caps (Bellco Biological Glassware), and stirring of cells was accomplished with an 8 mm magnetic spinning bar. Since this procedure appears to result in some mechanical disruption of cells ( < 1% per hour) experiments were usually not run in excess of six hours. To promote incorporation of amino acid analogs into proteins, cells were washed, collected by centrifugation and suspended at a concentration of 8-14 X lo5 cells/ml in modified Eagles medium, containing 0.04 mM leucine, and 5% dialyzed calf serum, but lacking the amino acid to be replaced by an analog. The analog was added to the medium at the concentrations indicated and the cells incubated for 1530 minutes with continuous stirring before addition of L-(4-5-3H)leucine (0.5-1.0 pCi/ ml) for an additional 30 minutes to 3 hours. Under these conditions, incorporation of radioactivity into hot trichloroacetic acid precipitable material was linear for at least three hours. Protein breakdown was measured as appearance of label in trichloroacetic acid soluble form from cells which were washed to remove unincorporated radioactive amino acid .(i.e. acid soluble radioactivity was
3
less than 2 % acid precipitable radioactivity) and resuspended in Eagle's medium containing 2 mM L-leucine. Increasing the amount of leucine in the chase medium to 10 mM had no effect on the apparent rate of protein breakdown. Inclusion of cycloheximide (100 pglml) with growing cells as a means to prevent reutilization did not further enhance the measured rate of protein breakdown. Thus, 2 mM L-leucine appears adequate to minimize isotopic reutilization. Cycloheximide inhibits protein breakdown in some mammalian systems (Levitan and Webb, '69) and this could complicate the interpretation of the effectiveness of the leucine chase. However, degradation of proteins synthesized in the presence of 'Cvaline, 14C guanido arginine, or I4C guanido-canavanine were the same when measured in the presence or absence of cycloheximide. In addition, cycloheximide appears to have no effect on the rate of inactivation of a specific HeLa protein, ornithine decarboxylase (submitted to J. Cell. Physiol.). Paper electrophoresis was carried out on a Savant High voltage electrophoresis apparatus. Radioactivity on the paper strips was measured on a Packard Radiochromatogram Scanner Model No. 7201. Ornithine decarboxylase activity was measured in 20,000 g supernatant fractions of HeLa lysates (obtained by repeated freezing and thawing of cells) by the evolution of radio-
PROTEIN SYNTHESIS
2CANAY
I
2
-X
3
HOURS Fig. 1 Incorporation of leucine into HeLa cells incubated in the presence of arginine or canavanine. HeLa cells growing at a concentration of 5 X 105 cellslml were centrifuged, washed with Earle's salts and resuspended at a concentration of 1.4 X 106 cells per milliliter i n minimal medium lacking arginine and containing leucine at 0.04 mM. Arginine (0.25 mM) or canavanine (0.25 mM) and 14C leucine (0.5 pCi/ml) was added to a portion of the culture. Incorporation of leucine was determined as the incorporation of radioactive material into hot acid precipitable form and is expressed as microgram incorporated per 106 cells. The radioactive proteins were poured over glass fiber filters and counted in a toluence based scintillaCanavanine, X- - -X. tion fluid. Symbols are: Arginine, 0-0;
373
PROTEIN CATABOLISM IN HeLa CELLS
+
I
ORIGIN
14C
GUANIDO CANAVANINE
L- CANAVA N I N E
-
@
-
ACID SOLUBLE FRACTION OF PRONASE DIGEST
Fig. 2 Paper electrophoresis of amino acids i n HeLa proteins following exposure to (14C) canavanine. HeLa cells were centrifuged, washed a n d resuspended i n medium lacking arginine a s i n figure 1. ( W - g u a n i d o ) Canavanine (2 pg/ml, 5 pCi/ml) was added a n d cells incubated three hours. Cells were centrifuged, washed, resuspended i n 0.01 M Tris, 1 mM Ca C12 then lysed by freezing a n d thawing five times. The mixture w a s centrifuged at 5,000 X g a n d the supernatant was poured over a Sephadex G25 column (1.5 X 5 cm). Samples at the front were collected and incubated for 18 hours with pronase (1 mg/4 mg substrate protein) i n a solution containing 0.03% sodium azide to prevent bacterial or fungal growth. Reaction was stopped by addition of trichloroacetic acid to 5 % , t h e precipitate removed b y centrifugation a n d the acid extracted from the supernatant with diethylether a n d the solution lyophilized. Samples containing (14C) canavanine or L-canavanine sulfate were treated similarly. The lyophilized materials were solubilized in water, spotted on a piece of Whatman No. 4 paper, subjected to electrophoresis (buffer; 1 % acetic acid, 2 % pyridine v/v, 30 Voltslcm, 200 minutes). The dried chromatogram w a s sprayed with ninhydrin and finally scanned for radioactivity. Under these conditions, canavanine moved 15 c m toward the cathode.
into HeLa cell protein is shown in figure 2. HeLa cells were incubated in medium lacking arginine and containing L('4C guanido) canavanine; incorporation of labeled canaRESULTS vanine was linear for a t least six hours. Incorporation of canavanine into Radioactive proteins from a cell lysate were HeLa cells protein then separated from small molecular weight Canavanine is a naturally occurring radioactive material by chromatography on analog of arginine and differs from argi- a Sephadex G25 column. The resultant nine by replacement of a n oxygen atom for protein fraction was subjected to pronase the methylene group at C-5 of the arginine digestion and the acid soluble material molecule. then analyzed by paper electrophoresis (fig. Exposure of HeLa cells to conavanine, 2 ) . The major spot of radioactivity is coinduring arginine starvation, reduces, but cident with the major spot of radioactivity does not abolish, the incorporation of ra- of authentic (14C-guanido) canavanine suldioactive leucine into cell protein (fig. 1). fate and a ninhydrin positive spot repreThat canavanine is, in fact, incorporated senting unlabeled L-canavanine. Similar
active carbon dioxide from (1-14C)ornithine as previously described (Russell and Snyder, '68).
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WALTER F. PROUTY
A HOURS
I00
50
10
5
;.;_ 0
B
I
I
10
20 MINUTES AT 49°C Figure 3
I
30
PROTEIN CATABOLISM IN HeLa CELLS
375
hours, there was no further loss of viability as measured after 20 hours incubation, in accord with previous results (Miedema and Kruse, '66). Thus, loss of cell viability resulting from incubation with canavanine required continuous exposure to the analog. If incorporation of canavanine into proteins affects protein conformation, then proteins synthesized in the presence of canavanine ought to be distinguished from the normal proteins by some criterion such as heat denaturation. Ornithine decarboxylase can be induced in HeLa cells by addition of fresh medium to the culture (Prouty, in preparation). As seen in figure 3A, if cells are exposed to an increasing ratio of canavanine to arginine i n the medium, the amount of functional ornithine decarboxylase induced is diminished. Furthermore, the enzyme synthesized in the presence of the analog has an increased rate of heat inactivation at 49 C and the rate increases as the ratio of canavanine to arginine in the culture medium is increased (fig. 3B). If incorporation of canavanine is a random process, a family of altered proteins would result and these should give complex heat Fig. 3 Induction (A) and heat inactivation (B) of ornithine decarboxylase in the presence of ar- denaturation curves. However, the curves ginine and canavanine. HeLa cells growing at 5 x in figure 3B appear to follow simple first lo5 cells/ml were centrifuged, washed and resusorder decay. A larger number of points for pended at a concentration of 1.1 X 1 0 6 cellslml i n medium lacking arginine. Arginine and cana- a more extended period of time may have vanine were then added to aliquots of the culture revealed complexity in these curves. In any at the concentrations indicated below and at the event exposure to canavanine during syntimes indicated aliquots containing ca. 5 X 106 cellslml were taken for assay of ornithine decar- thesis appears to result i n altered enzymes. Presumably, prolonged exposure to canaboxylase as in MATERIALS AND METHODS. A washed cell pellet was resuspended in 0.15 ml buffer (10 vanine will result in diminished specific mM Tris, pH 7.4, 0.1 mM EDTA, 0.1 mM pyridoxal activity of enzymes critical to cell viability, phosphate, 5 mM dithiothreitol), the cells broken thus causing the loss of cell viability deby freezing and thawing and the enzyme assayed scribed above. in the 20,000 X g supernatant of the broken cell
results were obtained in two other solvent systems. Thus, canavanine is incorporated into HeLa proteins, an observation consistent with results showing incorporation of canavanine into protein of Walker Carcinosarcoma cells (Kruse et al., '59) or hamster cells (Hare, '69). Although exposure to canavanine during arginine starvation slowed the rate of incorporation of '4C-leucine into acid precipitable material (e.g. 0.183 and 1.3 mM canavanine slowed the rate of 14C leucine incorporation into protein by 43 and 5 5 % , respectively, over a period of one hour), it is not known whether or not exposure to the analog inhibited protein synthesis since the specific activity of leucine pools was not measured. Exposure to canavanine for up to six hours did not reduce cell viability, as measured by exclusion of Trypan blue, by more than 5 % . However, prolonged exposure (20 hours) to 1.0 mM canavanine in medium lacking arginine resulted in an almost complete loss of viability. If cells were washed free of canavanine at six
suspension. At 4 hours incubation an aliquot containing ca. 2.5 X lo7 cells was removed from each sample and the supernatant fraction of the broken cell suspension used for the heat inactivation experiment shown in B. Equal aliquots of the supernatant fraction were incubated at 49OC. At the times indicated tubes were removed from the incubator, plunged into a n ice bath, and cold buffer was added to the tube. The fractions were then assayed for remaining ornithine decarboxylase activity a s above. Symbols represent addition of the following concentrations of arginine or canavanine. Symbol
0-0 A-A
0-0 0-0 0-0
Arginine
Can av anine
mM
m M
0.3 0.3
0
0.3 0.15
0.03
0.3 1.o 1.0 1.0
Fate of proteins containing canavanine To measure the degradation of proteins containing canavanine, HeLa cells were suspended in medium lacking arginine but containing radioactive leucine; then either arginine or canavanine was added. After three hours incubation, cells were centrifuged, washed and resuspended in minimum essential medium containing 2 mM leucine to minimize reutilization of label released by proteolysis. Use of the same radioactive label in the two conditions permits more accurately the comparison of the fate of normal and abnormal proteins, since transport of label and effectiveness of chase are very similar in the two condi-
376
WALTER F. PROUTY
tions. Proteins containing canavanine are degraded more rapidly than those containing arginine (fig. 4). Similar results were obtained when cells were labeled with ('4C) phenylalanine. Thus, use of leucine does not give rise to a unique result. In addition, proteins synthesized in the presence of ("T) guanido canavanine were degraded more rapidly than those synthesized in the presence of (3H) arginine although the extent of the difference tended to vary probably due to the different effectiveness of the chase in the various procedures. That enhanced degradation was not a nonspecific effect of the presence of unincorporated canavanine is indicated by the unaltered rate of degradation of cells exposed to ar-
z 3
0
D
Y
U
w
a m
s w I-
0
a n
s
HOURS Fig. 4 Degradation of proteins synthesized the presence of arginine or canavanine. HeLa cells were collected by centrifugation and resuspended at a concentration of 1 X 106 cellslml in medium containing either arginine (0.3 mM) or canavanine (2.0 mM) and 0.04 mM *4C leucine (0.5 pCilm1). Cells were incubated one hour at 37O, then centrifuged, washed and resuspended at 8 X 1 0 5 cells/ ml in chase medium as described in MATERIALS A N D METHODS. Canavanine (2 mM) was added to one half of the culture incubated in arginine to test the effects of the drug directly on the degradative process. Protein breakdown was measured in rep licate 0.5 ml aliquots as in MATERIALS AND METHODS. After three hours all three cultures contained over 97% viable cells as determined by their ability to exclude Trypan Blue. Symbols are: Canavanine, A __ A , Arginine, X-X, Arginine, then canavanine, -0.
z 3 0
n Y
U
W
a
m
z W L a a
s HOURS Fig. 5 Effects of incubation with varying amounts of canavanine on subsequent protein breakdown. HeLa cells were centrifuged, washed and resuspended in minimal medium lacking arginine and leucine and incubated for 30 minutes. Aliquots of the cells were then supplemented with (3H)-leucine (0.04 mM, 1 pCi/ml) and either arginine (0.6 mM) or canavanine (0.2, 1.0, or 2.0 mM) and incubated a n additional 30 minutes. Cells were washed and protein breakdown measured as in MATERIALS AND METHODS. After three hours, over 98% of the cells exposed to arginine or 0.2 mM canavanine were viable, while over 92% of cells exposed to higher concentrations of canavanine remained viable.
ginine when canavanine was added to the chase medium (fig. 4). The increased rate of proteolysis is probably due to the degradation of intracellular proteins and not a result of release to the medium of labeled proteins by some cells and subsequent endocytosis and degradation by others. The conditions used in these experiments result in a release of 0.8% prelabeled cell proteins to the medium per hour as measured by the appearance of acid precipitable radioactivity in an 800 X g supernatant fraction. Such release is probably from cell breakage (e.g. by the magnetic stirring bar) or secretion. Furthermore, in the presence of 5 % serum, less than 0.01% of exogenously added radioactive protein (prepared from either E . coli or HeLa cells) was made acid soluble in one hour, presumably by endocytosis and degradation (Ryser, '68, and unpublished results). These data indicate that the majority of label made acid soluble in the present experiments must have originated from intracellular proteins and that the proteins probably did not pass through the medium prior to their degradation.
377
PROTEIN CATABOLISM I N HeLa CELLS
Canavanine probably competes with arginine for charging by the amino acyl tRNA synthetase (Allende and Allende, '64). In fact, in bacteria canavanine appears to replace arginine in growing polypeptides on a random basis (Schachtele et al., '68). Thus increasing the intracellular ratio of canavanine to arginine presumably increases the amount of canavanine incorporated into protein. This would probably increase the quantity of abnormal protein synthesized (fig. 3b) and hence, the rate of subsequent protein degradation. Increasing the concentration of canavanine exposed to cells during starvation for arginine results in a greater subsequent rate of protein degradation (fig. 5). Under optimal conditions tested here, canavanine incorporation stimulated degradation rates by a factor of 2.5-3 over basal levels. In addition, starvation for arginine prior to canavanine addition might be expected to lower the intracellular arginine pool, thus increasing the canavanine to arginine ratio. A ninety minute arginine starvation period prior to exposure to canavanine in-
/ 3? 20
W + 0
a LL
arz
HOURS Fig. 6 Degradation of proteins synthesized in t h e presence of arginine or canavanine after starvation for arginine for 0 or 90 minutes. HeLa cells were collected by centrifugation, washed and resuspended i n complete medium lacking arginine or leucine. At zero or 90 minutes L-leucine, (0.04 mM) a n d either canavanine (0.183 mM) or argin i n e (0.54 mM) w a s added to a portion of the cells. After 15 minutes incubation, L-(3H) leucine (0.5 pCi/ml) was added and incubation continued a n additional 45 minutes. Protein breakdown was determined asdescribed in MATERIALS AND METHODS.
TABLE 1
Effect of exposure of HeLa cells t o various a m i n o acid analogs on subsequent protein degradation Cells exposed to
Conc. m M
Control, normal amino acids Norleucine Seleno-Methionine Ethionine Azatryptophan Threo-amino-chloro butyric acid Canavanine
1.3 2.6 2.6
1.9 1.7 2.0
Protein breakdown i n one hour %
4.8 5.3 4.7 4.9 7.1 12.4 13.7
Effects of exposure to various amino acid analogs on subsequent rates of protein breakdown. HeLa cells were centrifuged, washed and resuspended in medium lacking methionine, arginine, tryptophan or valine and containing leucine a t 0.04 mM. Analogs were added a t the concentration indicated along with 3H leucine (0.5 p C i / ml) and cells incubated three hours Protein breakdown was then measured as in MATERIALS AND METHODS. D e g ~ radation was linear for three hours. In these and several similar experiments, there appeared to be a n inverse relationship between the rate of incorporation of leucine into cell protein and t h e subsequent rate of protein breakdown, e.g., whereas canavanine and azatryptop h a n slowed incorporation of leucine by 50 a n d 30% respectively, norleucine or seleno-methionine did not slow leucine incorporation.
creases the subsequent rate of degradation compared to cells exposed to canavanine but not pre-starved for arginine (fig. 6). These observations indicate that incorporation of canavanine into cell proteins results in proteins which are more rapidly hydrolyzed by the cellular catabolic machinery. Medium manipulations appear to be effective in altering intracellular concentrations of canavanine and arginine, though such concentrations were not measured and the alterations still remain speculative. Generality of increased degradation rate o n exposure to a m i n o acid analogs Enhanced rates of protein breakdown are not peculiar to exposure to canavanine since other amino acid analogs which are known to be incorporated into cell protein (Fowden et al., '67) also stimulate the subsequent rate of protein breakdown (table 1). Various analogs have different effects on protein degradation rates. Whereas, azatryptophan, an analog of tryptophan and threoamino chlorobutyric acid, an analog of valine, stimulated subsequent protein degradation, several analogs of
3 78
WALTER F. PROUTY
methionine (norleucine, selenomethionine, or ethionine) had little or no effect. Other mammalian cells can also degrade selectively proteins containing amino acid analogs. Incorporation of canavanine or threoamino chlorobutyric acid into protein of normal human fibroblasts also resulted in enhanced rates of protein breakdown. Interestingly, conditions in HeLa cells which resulted in a 2.3-fold stimulation of degradation rates over basal level resulted in only a 1.5-fold stimulation of degradation in the fibroblasts (W. Prouty, unpublished results). DISCUSSION
An abundance of evidence suggests that canavanine is not only incorporated into cell proteins, but that it replaces arginine randomly. For example, activation of arginine is competitively inhibited by canavanine with both the purified rat liver arginyl tRNAsynthetase (Allende and Allende, '64) and the E . coli enzyme (Bowman et al., '61). Since the specificity of amino acid incorporation lies in the tRNA charging reaction, any canavanine charged to tRNA would enter proteins in place of arginine in random positions. Canavanine was shown to be incorporated into nuclear globulin proteins in mammalian tumor cells (Bell, '74) and into proteins constituting the hexons of adenovirus infected human embryonic kidney cells (Neurath et al., '70). In E. coli infected with MS2 bacteriophage, a polypeptide corresponding to the coat protein is made in the presence of canavanine, but this polypeptide is nonfunctional in that it does not exert translational control, nor does it participate in phage assembly (Prouty, '75). HeLa cells, like E . coli (Goldberg, '72) have the capacity to degrade rapidly and selectively, proteins containing amino acid analogs. Thus, in both a eukaryotic and prokaryotic cell, incorporation of an amino acid analog which presumably results in an altered, denatured protein can serve as a signal to initiate protein breakdown. These results are in accord with previous studies showing selective degradation of missense mutations in hypoxanthine guanine phosphoribosyl transferase in mouse L cells (Capecchi et al., '74), of mutant hemoglobin molecules (Schaeffer, '73), or of puromycyl peptides in hepatoma cells
(McIlhinney and Hogan, '74). In addition, reticulocytes appear to have the capacity to selectively degrade abnormal hemoglobin molecules resulting from incorporation of an amino acid analog (Rabinowitz and Fisher, '64). Regarding the similarity of the prokaryotic and eukaryotic cells in their ability to recognize abnormal proteins, the anaIogs most effective at stimulating protein degradation in HeLa cells were also most effective in E . coli (Goldberg, '72), while several methionine analogs, which were ineffective in HeLa cells, also had a very slight effect in E . coli. Interestingly, there is also a direct correlation between those analogs which slow growth in bacterial auxotrophs and those which stimulate degradation (Goldberg, '72). Efforts to alter the intracellular canavanine to arginine concentrations by manipulation of the medium or incubation conditions alter the rate of subsequent degradation (figs. 5, 6). Since canavanine appears to be incorporated into protein randomly in place of arginine (Allende and Allende, '64; Boman et al., '61; Schachtele et al., '68) an increase in intracellular canavanine to arginine ratios would increase the amount of abnormal proteins synthesized. However, although the data are consistent with the hypothesis that increased incorporation of canavanine results in an increased rate of protein degradation, intracellular concentrations of the two amino acids were not measured. The present study does not allow a quantitative estimation of the ratio of canavanine to arginine in HeLa protein, but from experiments utilizing (14C guanido) canavanine (0.04 mM) when arginine was absent in the medium, it is estimated that one canavanine moiety was incorporated for every 20 arginine moieties. A pool of arginine molecules appears to be maintained i n these cells even when incubated in arginine free medium. Thus, a functional ornithine decarboxylase with heat inactivation kinetics identical to that of the enzyme induced in control cells is induced even in arginine free medium, although induction is at one-sixth the rate seen in control cells (unpublished results). Since puromycyl peptides are also degraded rapidly, it might be argued that the analogs cause premature termination and thus act like puromycin (McIlhinney and Hogan, '74). How-
PROTEIN CATABOLISM I N HeLa CELLS
ever, analysis of HeLa proteins synthesized in the presence of canavanine indicate that the average molecular weight of the polypeptides is the same or, perhaps, slightly larger than normal cell proteins (in preparation). T h e presence of unincorporated canavanine in the cell had no direct effect on the degradative process in that it did not alter the rate of degradation of proteins synthesized prior to exposure to the analog (fig. 4). Thus, the stimulating effects of canavanine on protein breakdown rates is probably a direct result of the production of abnormal proteins, i.e. substrates for the protein catabolic machinery. It is of interest that maximal rates of degradation as observed here (figs. 5, 6) do not exceed basal rates by more than three fold and represent 12 to 15% of total cell radioactive protein degraded per hour. In contrast, bacterial cells degrade similar proteins at rates exceeding 50% per hour (Goldberg, '72; Prouty and Goldberg, '72; Prouty et al., '75). This difference in maximal degradation rate may reflect a fundamental difference in the catabolic capacity of mammalian and bacterial cells. However, the difference could be an artefact of reutilization which is reported to be very high in HeLa cells, i.e. amino acids arising from protein degradation are preferentially utilized for protein synthesis (Righetti et al., '71). Proteins ingested via endocytosis are known to enter lysosomes and there degraded to acid soluble material (Ryser, '68). In related studies, electron micrographs of HeLa cells exposed to amino acid analogs indicated a marked proliferation of digestive vacuoles in these cells (in preparation). These observations may suggest a common lysosomal site for the degradation of the two types of protein substrate although no direct evidence is available to support this hypothesis. On the other hand, bacteria which also selectively degrade abnormal protein have no such digestive organelle. The basal rates of degradation measured here (4-5% per hour) are much higher than previously published data (1 % per hour) Eagle et al., '59; Eagle et al., '61). These rates are especially high in light of the observations of Righetti et al. ('71) in which high rates of reutilization were demonstrated. Thus, the measured rates
379
could underestimate the actual rates. One possible explanation for the high rates of basal degradation is that there are at least two kinetically distinct populations of proteins in bacterial (Pine, '67a) or mammalian cells (Poole and Wibo, '73). In the present experiments, cells were exposed to radioactive precursor for up to three hours, a period which constitutes a pulse of radioactivity and which selectively labels those proteins with shorter half lives (Buchanan, '61). On the other hand, previous studies indicating a degradation rate of 1% per hour involved study of proteins labeled at least one generation time (ca. 24 hours) and thus, tended to measure the fate of more stable cell proteins. Others have also reported degradation rates in mammalian cells of around 5% per hour for proteins labeled in one to three hour pulses (Poole and Wibo, '73; Capecchi et al., '74). Thus, these results are not inconsistent with earlier studies. Johnson and Kenney ('73) reported that incorporation of amino acid analogs into tyrosine amino transferase altered its heat stability but not its half life. Such results do not contradict the present findings since these authors used immunologic methods to detect the protein and it is possible that this enzyme must be altered enough to destroy its antigenicity before it is recognized as abnormal. I have also found that ornithine decarboxylase can be induced in the presence of a small concentration of canavanine and the resultant molecule is much more susceptible to heat denaturation than the native enzyme. However, the rate of inactivation is unaltered. Thus, the rate of heat denaturation does not necessarily correlate with the rate of degradation. Production of abnormal proteins by incorporation of amino acid analogs has been used in an attempt to magnify a normal cell capacity. Production of abnormal proteins is probably not without analogies in various natural systems. For example, in studies of aging, the specific activities of numerous enzymes are known to decrease with age, i.e. abnormal proteins appear to accumulate as a normal part of the aging process (Gershon and Gershon, '73). Coupled with this observation is the recent finding of increased protein catabolic rates of a specific protein in aging cells (Haining
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WALTER F. PROUTY
and Legen, '73). In addition, many hemoglobinopothies are the result of production of abnormal hemoglobin molecules (Ranney, '70; Adams et d., '72), and, at least in one instance, diminished quantities of abnormal hemoglobins is the result of selective catabolism (Adams et al., '72). There may also be examples of in vivo denaturation conditions. For example, deamidation of asparagine or glutamine residues can occur via a non-enzymatic process and in vitro deamidation rates of various proteins has been shown to correlate with in vivo rates of degradation (Robinson, '74). It is tempting to speculate that deamidation results in proteins sufficiently altered to be recognized as abnormal and thus result in the selective degradation of those proteins. This idea is also consistent with the concept that denaturation is the rate limiting step in protein degradation (Schimke, '70). In conclusion, mammalian cells have the capacity to recognize and selectively degrade abnormal proteins when they constitute either a minute portion of total cell protein (Capecchi et al., '74) or when produced in bulk form as in these experiments; such selective degradation may serve to protect the cell from harmful effects of accumulation of abberant polypeptides. LITERATURE CITED Adams, J. G., W. P. Winter, D. L. Ricknagel and H. M. Spenser 1972 Biosynthesis of hemoglobin Ann Arbor: Evidence for catabolic and feedback regulation. Science, 176: 1427-1429. Allende, C. C., and J. E. Allende 1964 Purification and substrate specificity of arginyl tRNA synthetase from rat liver. J. Biol. Chem., 239: 1102-1 112. Ballard, F. J., M. F. Hopgood, L. Reshef and R. W. Hanson 1974 Degradation of phosphoenolpyruvate carboxykinase (guanosine tetraphosphate) in uivo and in nitro. Biochem. J., 140: 531-538. Bell, D. 1974 The effect of canavanine on herpes simplex virus replication. J. Gen. Viol., 22: 319-330. Boman, H. G., I. A. Boman and W. K. Maas 1961 In: Biological Structure and Function, Proc. First IUB/IUBS Symp. T. W. Goodwin and 0. Lindberg, eds. Academic Press, Inc., N.Y., p. 297. Buchanan, D. L. 1961 Analysis of continuous dosage isotopic experiments. Arch Biochem. Biophys., 94: 489499. Capeechi, M. R., N. E. Capecchi, S. H. Hughes and G. M. Wahl 1974 Selective degradation of abnormal proteins in mammalian tissue culture cells. Proc. Nat. Acad. Sci. (U.S.A.), 71: 47324736.
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PROTEIN CATABOLISM IN HeLa CELLS curacy of protein synthesis and its relevance to aging. Proc. Nat. Acad. Sci. (U.S.A.), 49: 517521. Pine, M.J. 1967a Response of intracellular proteolysis to alteration of bacterial protein and the implications in metabolic regulation. J. Bacteriol., 9 3 : 1527-1533. 1967b Intracellular protein breakdown i n the L1210 ascites leukemia. Canc. Res., 27: 522-525. Poole, B., and M. Wibo 1973 Protein degradationin cultured cells. J. Biol. Chem., 248: 62216226. Platt, T., J. H. Miller and K. Weber 1970 In viuo degradation of mutant Lac repressor. Nature, 228: 1154-1156. Prouty, W. F. 1975 Fate of MS2 proteins synthesized i n the presence of amino acid analogs. J. Virol., 16: 1090-1094. Prouty, W. F., and A. L. Goldberg 1972 Inhibitors of protein degradation in E. coli and their physiological effects. J. Biol. Chem., 247: 33413352. Prouty, W. F., M. J. Karnofsky and A. L. Goldberg 1975 Degradation of abnormal proteins in E . coli.J. Biol. Chem.,250: 1112-1122. Rabinowitz, M., and J. M. Fisher 1964 Characteristics of the inhibition ofhemoglobin synthesis
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i n rabbit reticulocytes by threeiu-aminog-chlorobutyric acid. Biochim. Biophys. Acta, 91: 313322. Ranney, H. M. 1970 Clinically important variants of human hemoglobin. N. Eng. J. Med., 282: 144-152. Righetti, P., E. P. Little and G. Wolf 1971 Amino acid reutilization in protein synthesis in HeLa cells. J. Biol. Chem., 246: 57244732. Robinson, A. B. 1974 Evolution and the distribution of elutaminvl and asuaraginvl residues i n proteins. Proc. Nat. Acad: Scir (U.S.A.), 71: 8.8_ 5 4~.~ 88. _ Russell, D. H., and S. H. Snyder 1968 Amine synthesis i n regenerating rat liver. Proc. Nat. Acad. Sci. (U.S.A.), 60: 1420-1427. Ryser, J. 1968 Uptake of protein by mammalian cells: a n underdeveloped area. Science, 159: 390496. Schachtele, C. F., D. L. Anderson and P. Rogers 1968 Mechanism of canavanine death i n E. coli. J. Mol. Biol., 33: 861-872. Schaeffer, J. R. 1973 Structure and synthesis of the unstable hemoglobin sabine. J. Biol. Chem., 248 : 7473-7480. Schimke, R. T. 1970 In: Mammalian Protein Metabolism. Vol. IV. H. N. Munro. New York Academic Press, pp. 177-228.
Note added in proof: While this manuscript was i n preparation, I discovered that similar results were being reported i n work with WI-38 cells: Bradley, M. O., R. T. Schimke and L. Hayflick (1975, submitted for publication) J. Biol. Chem.
