Proc. Nati. Acad. Sci. USA Vol. 76, No. 2, pp. 710-713, February 1979 Biochemistry
Degradation of abnormal proteins in intact mouse reticulocytes: Accumulation of intermediates in the presence of bestatin (protein turnover/abnormal hemoglobin/protease inhibitors/aminopeptidases)
VIOLETA BOTBOL AND OSCAR A. SCORNIK Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755
Communicated by Paul C. Zamecnik, November 27,1978
termediates, possibly because they affect more readily the first than the subsequent steps in proteolysis. Proteinases, however, are usually not sufficient for the complete breakdown of proteins to amino acids. Their action often results in small peptide fragments, hydrolysis of which requires the complementary effect of peptidases (1). We were interested to see whether inhibition of peptidases could interfere with this process, permitting the accumulation of low molecular weight intermediates. We chose for this study the degradation of abnormal globin in reticulocytes (17-20), because it represents primarily the degradation of a single protein of known amino acid sequence, and because cell-free systems capable of degrading this protein have been described (4, 10, 11). Bestatin, (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoylL-leucine, isolated from Streptomyces olivoreticuli, is a competitive inhibitor of mammalian aminopeptidase B and leucine aminopeptidase (21). It has been administered to mice for several days without ill effects (22), and it has been shown to have little effect on the degradation of proteins to trichloroacetic acid-soluble products by isolated rat hepatocytes (23). In this paper, we report that the addition of bestatin to a suspension of mouse reticulocytes degrading abnormal globin results in the accumulation of degradation products, apparently a mixture of di- and tripeptides or both.
ABSTRACT Incubation of intact mouse reticulocytes with bestatin (a competitive inhibitor of aminopeptidases) produced the accumulation of low molecular weight intermediates in the degradation of puromycinyl-peptides or analog-containing proteins that had been pulse labeled with L[1-_4C]leucine. A large fraction of the radioactive protein was degraded to trichloroacetic acid-soluble products within 10 min. In the presence of bestatin (0.5 mg/ml), one-fourth of these products appeared to be dipeptides, tripeptides, or both: they were resistant to ninhydrin at acid pH (a treatment that decarboxylates only free amino acids) except after intensive acid hydrolysis, and they eluted from a Sephadex -10 column with an apparent average size of 300 daltons. These radioactive products did not appear if incorporation of the tracer was prevented by prior treatment with cycloheximide, demonstrating that they originated from polypeptide precursors. Thus, a peptidase inhibitor has been successfully used in the production of low molecular weight intermediates in the in vivo degradation of cellular proteins. The mechanisms involved in the degradation of cell proteins are not yet well understood. A variety of intracellular proteolytic enzymes have been identified (1, 2), and cell-free systems capable of degrading proteins to amino acids (2-5) have been described, but it is difficult to decide which of them, if any, is responsible for the process as it occurs in vivo. Furthermore, it is not certain whether cellular proteins of eukaryotes are broken down within lysosomes (3, 6-9) or in the soluble phase of the cytoplasm (4, 10, 11), or if different classes of proteins are degraded by independent mechanisms (12, 13). Particularly important questions about this process could be asked if it were possible to isolate and identify intermediates, defined as fragments of proteins, the degradation of which has been initiated but not yet completed at the time of their extraction. One could then ask where in cells do the intermediates accumulate, or whether the degradation of a protein in a given cell-free system proceeds through the same sequence of reactions as in vivo. Unfortunately, the demonstration of intermediates in the in vio degradation of proteins has been elusive. Small amounts of low molecular weight intermediates have been reported in Escherichia coli, after partial disruption of the cell structure with toluene treatment (14) or as a result of multiple mutations affecting proteolytic enzymes (15). Another E. coli mutation resulted in the accumulation of a large molecular weight protein which appeared to be an intermediate in the degradation of 3-galactosidase (16). Comparable genetic manipulations have not yet been accomplished in mammalian cells. Proteinase inhibitors could conceivably be used to study accumulation of degradation intermediates. A number of these inhibitors have been shown to affect the degradation of cellular proteins in mammalian cells (12, 13), but we know of no report in which treatment has led to significant accumulation of in-
EXPERIMENTAL PROCEDURE Materials. L-[1-_4C]Leucine, L-[4,5-3H]leucine, Protosol, Aquasol, Biofluor and Econofluor were obtained from New England Nuclear; hemin, puromycin, cycloheximide, blue dextran, and Dowex 50X8-400 (H+ form), from Sigma; Bactopeptone from Difco; Sephadex G-10, particle size 40-120,um, from Pharmacia; 1-acetyl-2-phenylhydrazine, from Eastman Kodak; L-threo-a-amino-13-chlorobutyric acid, from Calbiochem. Bestatin was kindly provided by Hamao Umezawa, Microbial Chemistry Research Foundation (Tokyo, Japan). It was dissolved in 0.9% NaCl at 5 mg/ml and stored frozen. Reticulocytes. Reticulocytosis was induced in adult male CD1 mice (Charles River Breeding Laboratories) by five daily intraperitoneal injections of 1-acetyl-2-phenylhydrazine (2.8 mg/100 g of body weight). Four days after the last injection the animals were bled by decapitation. The cells were collected in heparinized tubes, separated from the plasma, and washed by three centrifugations (800 X g, 5 min) in 140 mM NaCl/5 mM KC1/1.5 mM magnesium acetate. The buffy coat containing leukocytes was removed. Each mouse yielded 0.3 to 0.4 ml of packed cells, approximately 80% of which were reticulocytes, identified by staining with new methylene blue. Labeling of Puromycinyl-Peptides. Packed cells (0.3 ml) were suspended in 1.7 ml of ice-cold Krebs-Ringer bicarbonate, supplemented with glucose, albumin, and amino acids (24). Leucine was adjusted to 0.02 mM and freshly dissolved hemin was added at a concentration of 0.1 mM. The suspension was
The publication costs of this article were defrayed in part by page charge payments. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
710
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Botbol and Scornik shaken at 370C in a 95%. 02/5% CO2 atmosphere for 10 min. Then, 3 ,uCi (1 Ci = 3.7 X 1010 becquerels) of L-[1-14C]leucine (55 ,tCi/tumol) was added, followed 20 s later by 0.2 ml of 10 mM puromycin (in 0.9% NaCi). One minute after the addition of puromycin, the incubation was stopped by combining the suspension with 0.2 ml of cycloheximide (15 mg/ml in 0.9% NaCI) and 10 ml of ice-cold 0.9% NaCI containing 2 mM Ieucine. The cells were centrifuged at 0°C and washed six times with the saline/leucine solution. The final pellet was suspended in 4.7 ml of the supplemented Krebs-Ringer bicarbonate containing 2 mM leucine. Labeling of Analog-Containing Protein. The packed cells were suspended as in the previous protocol, except that valine was omitted and the valine analog L-threo-a-amino-f3chlorobutyric acid was added at a final concentration of 1 mM. The cells were incubated for 5 min, at which time 3s,uCi of L[1-14C]ieucine was added. One minute later, the incubation was stopped as in the previous protocol. Degradation of Abnormal Proteins. A suspension of cells containing labeled abnormal proteins was prepared by either of the protocols described above; 0.1-ml aliquots were incubated at 37°C in a 95% 02/5% CO2 atmosphere for various intervals; the incubation was stopped with the addition of 0.6 ml of icecold 10% trichloroacetic acid, and the resulting precipitate was centrifuged down. A 0.3-ml portion of the supernatant was mixed with 0.05 ml of ninhydrin (90 mg/ml in ethanol), incubated for 1 hr at 900C, and shaken vigorously with 25 Al of 0.15 M NaHCO3 to eliminate the resulting 04CO2. Another 0.3-ml portion was treated in the same way, except that ninhydrin was omitted. Each portion was mixed with 10 ml of Aquasol and its radioactivity was determined by liquid scintillation spectrometry. The radioactivity in the first portion is referred to as ninhydrin-resistant radioactivity; that of the second portion, as total acid-soluble radioactivity. Protein radioactivity was measured in unincubated 0.1-ml aliquots after they were mixed with carrier liver homogenate (10 mg of tissue wet weight), precipitated with hot trichloroacetic acid, and extracted with organic solvents (25). The counting efficiency in each condition was determined with internal standards. Bestatin, at a concentration in the sample of 0.5 mg/ml, did not interfere with the decarboxylation by ninhydrin of added L-[1-14C]leucine (not shown). Sephadex Chromatography of the Ninhydrin-Resistant Material. Cell suspensions containing abnormal protein were incubated in the presence of bestatin (as will be described under Results and Discussion); 0.2 ml of the suspension was mixed with 0.6 ml of ice-cold 10% trichloroacetic acid and the precipitate was removed by centrifugation. The supernatant, with 10 mg of Bactopeptone (as carrier) and 0.1 ml of ninhydrin (90
>
711
100-
0 60
5
4-'
I-
40
/0 OL0
A
Incubation time, min FIG. 1. Accumulation of ninhydrin-resistant acid-soluble radioactivity in the presence of bestatin. A suspension of cells containing either labeled puromycinyl-peptides (Left} or analog-containing globin (Right) was prepared. The cell suspensions were incubated at 37°C for the times indicated, in the presence or absence of bestatin (0.5 mg/ml). The following parameters were measured: total trichloroacetic acid-soluble radioactivity, no bestatin; 0, total trichloroacetic acid-soluble radioactivity, with bestatin; A, ninhydrin-resistant radioactivity, no bestatin; 0, ninhydrin-resistant radioactivity, with bestatin. Also shown on the Left is the total acid-soluble radioactivity (o) in a cell suspension labeled in the same manner but lacking puromycin or the valine analog. ,,
mg/ml in ethanol) was heated at 90°C for 1 hr. A light brown precipitate formed and was removed by centrifugation. The supernatant was diluted with water to 3 ml and extracted thrice with 3 ml of water-saturated diethyl ether. The aqueous phase was percolated through a column (3 X 0.6 cm) of Dowex 50X8 (H+ form), and the column was washed with 5 ml of water. The radioactivity was eluted with 2 ml of 1 M NH40H, followed by 1 ml of water, evaporated to dryness in vacuo, and redissolved in 0.1 ml of chromatography buffer (sodium phosphate, pH 7, 0.1 M/NaCI, 0.05 M/mercaptoethanol, 0.1%/sodium dodecyl sulfate, 0.01%/urea, 6 M/and thymol, 0.1%); 10 ,u of 7% blue dextran and 0.01 ,uCi of L-[4,5-3H]leucine (10 ,uCi/ gAmol) were also added. The sample was chromatographed in a column (21 X 0.8 cm) of Sephadex G-10 equilibrated in chromatography buffer. The eluate was collected in 0.43-ml fractions, which were mixed with Biofluor and assayed for radioactivity. Recovery of the radioactivity through this procedure in three experiments was 55-68%. In one of these experiments the losses were traced as follows: 5% in the ether extraction, 19% in the Dowex step (of which half was not retained and the rest failed to elute), and 15% in the Sephadex chromatography. The results of the chromatography were not corrected for this recovery.
Table 1. Appearance of ninhydrin-resistant, acid-soluble radioactivity depends on the prior incorporation of radioactivity into protein
Radioactivity, dpm/0.1 ml cell suspension Acid-soluble, after 15 min NinhydrinProtein resistant Total (initial)
Conditions 310 1071 2426 a. Puromycinyl-peptides degraded in the presence of bestatin 28 1346 2426 b. Same as a, but bestatin omitted 6 4 128 c. Same as a, but cycloheximide added before the tracer A suspension of cells containing labeled puromycinyl-peptides was prepared and aliquots were incubated in the presence (a) or absence (b) of bestatin (0.5 mg/ml). With another portion of the cells (c), cycloheximide was added 60 s before the tracer (rather than 80 s afterward); the cells were exposed to the labeled leucine for 80 s and then washed and suspended as in the regular procedure. Aliquots of this suspension were incubated in the presence of bestatin. All values represent the average of triplicate aliquots. Counting efficiency was 74%.
712
Biochemistry: Botbol and Scornik
RESULTS AND DISCUSSION Abnormal proteins that were produced in intact reticulocytes by the effect of puromycin (Fig. 1 left) or the valine analog L-threo-a-amino--1-chlorobutyric acid (Fig. 1 right) were pulse-labeled with carboxy-labeled leucine. The cells were washed free of the tracer and incubation was continued. A large portion of the radioactive protein was rapidly degraded to trichloroacetic acid-soluble products, largely the free amino acids. This we know because the radioactivity disappeared after treatment with ninhydrin at acid pH, which decarboxylates only free amino acids (26). Addition of bestatin (0.5 mg/ml) to the incubation mixture had little effect on the rate at which the abnormal proteins were degraded to acid-soluble products but changed the nature of these products, one-fourth of which became now ninhydrin resistant (Fig. 1). In the experiment presented in Fig. 2, we observed the effects of different concentrations of the peptidase inhibitor. When the results were examined in a double reciprocal plot, they fit a straight line extrapolating to a maximum possible accumulation of ninhydrin-resistant material equivalent to 55% of the total acid-soluble radioactivity. It is not surprising that a fraction of all small peptides (the other 45%) resulting from endoproteolytic attack of the globin molecules may be degraded to amino acids by bestatin-insensitive enzymes. Bestatin is known to inhibit aminopeptidase B and leucine aminopeptidase, but not aminopeptidase A or endopeptidases (21).
40r
Proc. Natl. Acad. Sci. USA 76 (1979) Table 2. Acid hydrolysis of the ninhydrin-resistant material
_ -
-
+
+
+
A suspension of cells containing radioactive puromycinyl-peptides was prepared as described under Experimental Procedure (except that the tracer was twice the usual specific radioactivity). Aliquots (0.1 ml) were incubated for 15 min in the presence of bestatin (0.5 mg/ml). Each aliquot then received 0.6 ml of 10% trichloroacetic acid. The total trichloroacetic acid-soluble radioactivity (first line) and the ninhydrin-resistant fraction (second line) were determined as explained under Experimental Procedure. The trichloroacetic acid extract of a second aliquot was combined with 1 vol of concentrated HCl and incubated under reduced pressure for 20 hr at 1061C. This hydrolyzed sample was then evaporated to dryness and redissolved in 0.6 ml of 10% trichloroacetic acid. The total radioactivity (third line) and ninhydrin-resistant fraction (fourth line) were determined as before. Counting efficiency was 74%.
We believe that the acid-soluble radioactivity resistant to ninhydrin represented peptide intermediates in the degradation of the abnormal proteins, on the basis of the following evidence. (i) There was a precursor-product relationship between the labeling of the protein and the appearance of the ninhydrinresistant radioactivity. When cycloheximide was added before the labeled leucine, so that incorporation of the tracer into protein was prevented, further incubation in the presence of V0 594 342
\4 4
A 20
30
10
20 *, C._0J
0.5
C, mg/ml
0.3-
131
'I
10
-0
OL
"
I,_ 1
4.'
I
0
0
1/A
2683 607 2432 36
+
30F
OL 0 0.05
Radioactivity, dpm/0.1 ml cell suspension
Treatment Acid hydrolysis Ninhydrin
20
I
-
%
0.2 -
-I
%
0.1I4
1/C, ml/mg
PIG. 2. Dependence of ninhydrin-resistant radioactivity on bestatin concentration. A suspension of cells containing labeled puromycinyl-peptides was incubated with bestatin at 1.0, 0.5, 0.05, or 0 mg/ml. After 15 min the ninhydrin-resistant and the total acidsoluble radioactivities were determined. The percentage A of the acid-soluble radioactivity that became ninhydrin-resistant is plotted as a function of the bestatin concentration C (Upper). Also, the value at 0 mg/ml was subtracted from the average of the duplicates, and the reciprocal of the resulting value was plotted against the reciprocal of the bestatin concentration (Lower). The values fit a straight line extrapolating on the ordinate to 0.018, the reciprocal of 55%.
5
6
7
8
Effluent volume, ml FIG. 3. Size distribution of the ninhydrin-resistant material. Cells containing labeled puromycinyl-peptides (Upper) or analog-containing protein (Lower) were incubated in the presence of bestatin, 0.5 mg/ml (20 min in first case, and 15 min in the second). The size distribution of the ninhydrin-resistant (0) material was examined by chromatography on a Sephadex G-10 column. The position of the blue dextran (V0) and the elution profile of the internal standard of [3H]leucine (Mr 131) (0) are also shown. The positions of raffinose (Mr 594) and sucrose (Mr 342) were determined separately; their peaks are indicated by arrows and identified by their molecular weights.
Biochemistry: Botbol and Scornik Table 3. Retention by the cells of the ninhydrin-resistant material
Ninhydrin-resistant radioactivity, dpm/0.1 ml cell suspension Incubations Total Cells Medium 370C, 15 min 310 256 54 370C, 15min;0C, 120min 226 167 58 A suspension of cells containing radioactive puromycinyl-peptides was prepared, and 0.1-ml aliquots were incubated in the presence of bestatin (0.5 mg/ml) at the indicated temperatures. At the end of the incubation, duplicate aliquots were diluted with 0.6 ml of ice-cold 0.9% NaCl and the cells were separated from the medium by centrifugation. The medium received 0.05 ml of 100% trichloroacetic acid. Other duplicate (undiluted) aliquots received 0.6 ml of 10% trichloroacetic acid. The acid precipitate was removed by centrifugation in all aliquots and the ninhydrin-resistant radioactivity was determined in the supernatant. The values are the average of both aliquots. The fraction contained in the cells is calculated as the difference between the other two values. Counting efficiency was 74%.
bestatin yielded very little ninhydrin-resistant radioactivity in the acid-soluble extract (Table 1). (ii) Treatment with ninhydrin at acid pH decarboxylates only free amino acids (26). If the radioactivity of the ninhydrin-resistant material represents carboxy-labeled leucine, of which either the carboxylic C or the a-amino N is engaged in peptide bonds, these bonds should be broken by intensive acid hydrolysis, releasing the radioactivity in a ninhydrin-sensitive form. This was indeed the case (Table 2). (iii) The ninhydrin-resistant material accumulated in the presence of bestatin during the degradation of puromycinylpeptides (Fig. 3 upper) or analog-containing proteins (Fig. 3 lower), eluted from a Sephadex G-10 column as an approximately symmetrical peak with an apparent size distribution averaging about 300 daltons. This molecular mass is consistent with leucine-containing di- and tripeptides, or both, and could be expected from the substrate specificity of the enzymes known to be inhibited by bestatin (21). The radioactivity must represent a heterogeneous mixture; the sequences of a and f chains of mouse hemoglobin (27) contain a total of 34 leucine residues, which could possibly integrate 28 different di- and 83 different tripeptide sequences. These intermediates are largely retained by the cells; this was shown in an experiment in which the reticulocytes were separated from the suspension by centrifugation. After incubation for 15 min at 37°C, the cells contained more than 80% of the ninhydrin-resistant material. The distribution was similar even after storage of the cell suspension at 0°C for 2 hr (Table 3). Thus a peptidase inhibitor has been successfully used in the production of low molecular weight peptide intermediates in the in vivo degradation of cellular proteins. Further characterization of the intermediates and studies of their subcellular localization should provide valuable insight into the pathway(s) of degradation of abnormal proteins.
Proc. Natl. Acad. Sci. USA 76 (1979)
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We are grateful to Dr. Hamao Umezawa for a generous gift of bestatin and Dr. Mary Lee S. Ledbetter for critical revision of the manuscript. This research was supported by Grant AM 13336 of the National Institutes of Health. 1. Barrett, -A. J., ed. (1977) Proteinases in Mammalian Cells and Tissues (North-Holland, Amsterdam). 2. Hanson, H., Ansorge, S. & Bohley, P., eds. (1977) Acta Biol. Med. Ger. 36, 1503-1968. 3. Mortimore, G. E. & Ward, W. F. (1976) in Lysosomes in Biology and Pathology, eds. Dingle, J. T. & Dean, R. T. (North-Holland, Amsterdam), Vol. 5, pp. 157-184. 4. Etlinger, J. D. & Goldberg, A. L. (1977) Proc. Natl. Acad. Sci. USA 74, 54-58. 5. Holzer, H. (1978) in Symposium on Protein Turnover and Lysosomal Function, eds. Segal, H. & Doyle, D. (Academic, New York), pp. 305-314. 6. de Duve, C. & Wattiaux, R. (1966) Annu. Rev. Physiol. 28, 435-492. 7. Dean, R. T. (1977) Acta Biol. Med. Ger. 36, 1815-1820. 8. Khairallah, E. A., Airhart, J., Bruno, M. K., Puchalsky, D. & Khairallah, L. (1977) Acta Biol. Med. Ger. 36, 1735-1745. 9. Rudek, D. E., Dien, P. Y. & Schneider, D. L. (1978) Biochem. Biophys. Res. Commun. 82,342-347. 10. Ciehanover, A., Hod, Y. & Hershko, A. (1978) Biochem. Biophys. Res. Commun. 81, 1100-1105. 11. Daniels, R. S. & Hipkiss, A. R. (1978) Biochem. Soc. Trans. 6, 623-625. 12. Goldberg, A. L. & St. John, A. C. (1976) Annu. Rev. Biochem. 45, 747-803. 13. Ballard, F. J. (1977) Essays Biochem. 13, 1-37. 14. Pine, M. J. (1975) in Intracellular Protein Turnover, eds. Schimke, R. T. & Katunuma, N. (Academic, New York), pp. 65-76. 15. Miller, C. G. (1975) Annu. Rev. Microbiol. 29,485-504. 16. Kowit, J. D. & Goldberg, A. L. (1977) J. Biol. Chem. 252, 8350-8357. 17. Rabinovitz, M. & Fisher, J. M. (1961) Biochem. Biophys. Res. Commun. 6, 449-453. 18. Morris, A., Arlinghaus, R., Favelukes, S. & Schweet, R. (1963) Biochemistry 2, 1084-1090. 19. McIlhinney, A. & Hogan, B. L. M. (1974) FEBS Lett. 40,297301. 20. DeSimone, J., Kleve, L., Longley, M. A. & Schaeffer, J. (1974) Biochem. Biophys. Res. Commun. 57,248-254. 21. Umezawa, H. & Aoyagi, T. (1977) in Proteinases in Mammalian Cells and Tissues, ed. Barrett, A. J. (North-Holland, Amsterdam), pp. 637-662. 22. Umezawa, H. (1977) Acta Biol. Med. Ger. 36, 1899-1915. 23. Hopgood, M. F., Clark, M. G. & Ballard, F. J. (1977) Biochem. J. 164, 399-407. 24. Scornik, 0. A., Schadel, B. F., Franze-Fernandez, M. T. & Botbol, V. (1978) Fed. Proc. Fed. Am. Soc. Exp. Biol. 37, 1332
(abstr.). 25. Scornik, 0. A. (1974) J. Biol. Chem. 249,3876-3883. 26. Van Slyke, D. D., Dillon, R. T., MacFadyen, D. A. & Hamilton, P. (1941) J. Biol. Chem. 141, 627. 27. Dayhoff, M., ed. (1972) Atlas of Protein Sequence and Structure
(National Biomedical Research Foundation, Washington, DC), Vol. 5, pp. D-58, D-69.
Degradation of abnormal proteins in intact mouse reticulocytes: Accumulation of intermediates in the presence of bestatin (protein turnover/abnormal hemoglobin/protease inhibitors/aminopeptidases)
VIOLETA BOTBOL AND OSCAR A. SCORNIK Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755
Communicated by Paul C. Zamecnik, November 27,1978
termediates, possibly because they affect more readily the first than the subsequent steps in proteolysis. Proteinases, however, are usually not sufficient for the complete breakdown of proteins to amino acids. Their action often results in small peptide fragments, hydrolysis of which requires the complementary effect of peptidases (1). We were interested to see whether inhibition of peptidases could interfere with this process, permitting the accumulation of low molecular weight intermediates. We chose for this study the degradation of abnormal globin in reticulocytes (17-20), because it represents primarily the degradation of a single protein of known amino acid sequence, and because cell-free systems capable of degrading this protein have been described (4, 10, 11). Bestatin, (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoylL-leucine, isolated from Streptomyces olivoreticuli, is a competitive inhibitor of mammalian aminopeptidase B and leucine aminopeptidase (21). It has been administered to mice for several days without ill effects (22), and it has been shown to have little effect on the degradation of proteins to trichloroacetic acid-soluble products by isolated rat hepatocytes (23). In this paper, we report that the addition of bestatin to a suspension of mouse reticulocytes degrading abnormal globin results in the accumulation of degradation products, apparently a mixture of di- and tripeptides or both.
ABSTRACT Incubation of intact mouse reticulocytes with bestatin (a competitive inhibitor of aminopeptidases) produced the accumulation of low molecular weight intermediates in the degradation of puromycinyl-peptides or analog-containing proteins that had been pulse labeled with L[1-_4C]leucine. A large fraction of the radioactive protein was degraded to trichloroacetic acid-soluble products within 10 min. In the presence of bestatin (0.5 mg/ml), one-fourth of these products appeared to be dipeptides, tripeptides, or both: they were resistant to ninhydrin at acid pH (a treatment that decarboxylates only free amino acids) except after intensive acid hydrolysis, and they eluted from a Sephadex -10 column with an apparent average size of 300 daltons. These radioactive products did not appear if incorporation of the tracer was prevented by prior treatment with cycloheximide, demonstrating that they originated from polypeptide precursors. Thus, a peptidase inhibitor has been successfully used in the production of low molecular weight intermediates in the in vivo degradation of cellular proteins. The mechanisms involved in the degradation of cell proteins are not yet well understood. A variety of intracellular proteolytic enzymes have been identified (1, 2), and cell-free systems capable of degrading proteins to amino acids (2-5) have been described, but it is difficult to decide which of them, if any, is responsible for the process as it occurs in vivo. Furthermore, it is not certain whether cellular proteins of eukaryotes are broken down within lysosomes (3, 6-9) or in the soluble phase of the cytoplasm (4, 10, 11), or if different classes of proteins are degraded by independent mechanisms (12, 13). Particularly important questions about this process could be asked if it were possible to isolate and identify intermediates, defined as fragments of proteins, the degradation of which has been initiated but not yet completed at the time of their extraction. One could then ask where in cells do the intermediates accumulate, or whether the degradation of a protein in a given cell-free system proceeds through the same sequence of reactions as in vivo. Unfortunately, the demonstration of intermediates in the in vio degradation of proteins has been elusive. Small amounts of low molecular weight intermediates have been reported in Escherichia coli, after partial disruption of the cell structure with toluene treatment (14) or as a result of multiple mutations affecting proteolytic enzymes (15). Another E. coli mutation resulted in the accumulation of a large molecular weight protein which appeared to be an intermediate in the degradation of 3-galactosidase (16). Comparable genetic manipulations have not yet been accomplished in mammalian cells. Proteinase inhibitors could conceivably be used to study accumulation of degradation intermediates. A number of these inhibitors have been shown to affect the degradation of cellular proteins in mammalian cells (12, 13), but we know of no report in which treatment has led to significant accumulation of in-
EXPERIMENTAL PROCEDURE Materials. L-[1-_4C]Leucine, L-[4,5-3H]leucine, Protosol, Aquasol, Biofluor and Econofluor were obtained from New England Nuclear; hemin, puromycin, cycloheximide, blue dextran, and Dowex 50X8-400 (H+ form), from Sigma; Bactopeptone from Difco; Sephadex G-10, particle size 40-120,um, from Pharmacia; 1-acetyl-2-phenylhydrazine, from Eastman Kodak; L-threo-a-amino-13-chlorobutyric acid, from Calbiochem. Bestatin was kindly provided by Hamao Umezawa, Microbial Chemistry Research Foundation (Tokyo, Japan). It was dissolved in 0.9% NaCl at 5 mg/ml and stored frozen. Reticulocytes. Reticulocytosis was induced in adult male CD1 mice (Charles River Breeding Laboratories) by five daily intraperitoneal injections of 1-acetyl-2-phenylhydrazine (2.8 mg/100 g of body weight). Four days after the last injection the animals were bled by decapitation. The cells were collected in heparinized tubes, separated from the plasma, and washed by three centrifugations (800 X g, 5 min) in 140 mM NaCl/5 mM KC1/1.5 mM magnesium acetate. The buffy coat containing leukocytes was removed. Each mouse yielded 0.3 to 0.4 ml of packed cells, approximately 80% of which were reticulocytes, identified by staining with new methylene blue. Labeling of Puromycinyl-Peptides. Packed cells (0.3 ml) were suspended in 1.7 ml of ice-cold Krebs-Ringer bicarbonate, supplemented with glucose, albumin, and amino acids (24). Leucine was adjusted to 0.02 mM and freshly dissolved hemin was added at a concentration of 0.1 mM. The suspension was
The publication costs of this article were defrayed in part by page charge payments. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
710
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Botbol and Scornik shaken at 370C in a 95%. 02/5% CO2 atmosphere for 10 min. Then, 3 ,uCi (1 Ci = 3.7 X 1010 becquerels) of L-[1-14C]leucine (55 ,tCi/tumol) was added, followed 20 s later by 0.2 ml of 10 mM puromycin (in 0.9% NaCi). One minute after the addition of puromycin, the incubation was stopped by combining the suspension with 0.2 ml of cycloheximide (15 mg/ml in 0.9% NaCI) and 10 ml of ice-cold 0.9% NaCI containing 2 mM Ieucine. The cells were centrifuged at 0°C and washed six times with the saline/leucine solution. The final pellet was suspended in 4.7 ml of the supplemented Krebs-Ringer bicarbonate containing 2 mM leucine. Labeling of Analog-Containing Protein. The packed cells were suspended as in the previous protocol, except that valine was omitted and the valine analog L-threo-a-amino-f3chlorobutyric acid was added at a final concentration of 1 mM. The cells were incubated for 5 min, at which time 3s,uCi of L[1-14C]ieucine was added. One minute later, the incubation was stopped as in the previous protocol. Degradation of Abnormal Proteins. A suspension of cells containing labeled abnormal proteins was prepared by either of the protocols described above; 0.1-ml aliquots were incubated at 37°C in a 95% 02/5% CO2 atmosphere for various intervals; the incubation was stopped with the addition of 0.6 ml of icecold 10% trichloroacetic acid, and the resulting precipitate was centrifuged down. A 0.3-ml portion of the supernatant was mixed with 0.05 ml of ninhydrin (90 mg/ml in ethanol), incubated for 1 hr at 900C, and shaken vigorously with 25 Al of 0.15 M NaHCO3 to eliminate the resulting 04CO2. Another 0.3-ml portion was treated in the same way, except that ninhydrin was omitted. Each portion was mixed with 10 ml of Aquasol and its radioactivity was determined by liquid scintillation spectrometry. The radioactivity in the first portion is referred to as ninhydrin-resistant radioactivity; that of the second portion, as total acid-soluble radioactivity. Protein radioactivity was measured in unincubated 0.1-ml aliquots after they were mixed with carrier liver homogenate (10 mg of tissue wet weight), precipitated with hot trichloroacetic acid, and extracted with organic solvents (25). The counting efficiency in each condition was determined with internal standards. Bestatin, at a concentration in the sample of 0.5 mg/ml, did not interfere with the decarboxylation by ninhydrin of added L-[1-14C]leucine (not shown). Sephadex Chromatography of the Ninhydrin-Resistant Material. Cell suspensions containing abnormal protein were incubated in the presence of bestatin (as will be described under Results and Discussion); 0.2 ml of the suspension was mixed with 0.6 ml of ice-cold 10% trichloroacetic acid and the precipitate was removed by centrifugation. The supernatant, with 10 mg of Bactopeptone (as carrier) and 0.1 ml of ninhydrin (90
>
711
100-
0 60
5
4-'
I-
40
/0 OL0
A
Incubation time, min FIG. 1. Accumulation of ninhydrin-resistant acid-soluble radioactivity in the presence of bestatin. A suspension of cells containing either labeled puromycinyl-peptides (Left} or analog-containing globin (Right) was prepared. The cell suspensions were incubated at 37°C for the times indicated, in the presence or absence of bestatin (0.5 mg/ml). The following parameters were measured: total trichloroacetic acid-soluble radioactivity, no bestatin; 0, total trichloroacetic acid-soluble radioactivity, with bestatin; A, ninhydrin-resistant radioactivity, no bestatin; 0, ninhydrin-resistant radioactivity, with bestatin. Also shown on the Left is the total acid-soluble radioactivity (o) in a cell suspension labeled in the same manner but lacking puromycin or the valine analog. ,,
mg/ml in ethanol) was heated at 90°C for 1 hr. A light brown precipitate formed and was removed by centrifugation. The supernatant was diluted with water to 3 ml and extracted thrice with 3 ml of water-saturated diethyl ether. The aqueous phase was percolated through a column (3 X 0.6 cm) of Dowex 50X8 (H+ form), and the column was washed with 5 ml of water. The radioactivity was eluted with 2 ml of 1 M NH40H, followed by 1 ml of water, evaporated to dryness in vacuo, and redissolved in 0.1 ml of chromatography buffer (sodium phosphate, pH 7, 0.1 M/NaCI, 0.05 M/mercaptoethanol, 0.1%/sodium dodecyl sulfate, 0.01%/urea, 6 M/and thymol, 0.1%); 10 ,u of 7% blue dextran and 0.01 ,uCi of L-[4,5-3H]leucine (10 ,uCi/ gAmol) were also added. The sample was chromatographed in a column (21 X 0.8 cm) of Sephadex G-10 equilibrated in chromatography buffer. The eluate was collected in 0.43-ml fractions, which were mixed with Biofluor and assayed for radioactivity. Recovery of the radioactivity through this procedure in three experiments was 55-68%. In one of these experiments the losses were traced as follows: 5% in the ether extraction, 19% in the Dowex step (of which half was not retained and the rest failed to elute), and 15% in the Sephadex chromatography. The results of the chromatography were not corrected for this recovery.
Table 1. Appearance of ninhydrin-resistant, acid-soluble radioactivity depends on the prior incorporation of radioactivity into protein
Radioactivity, dpm/0.1 ml cell suspension Acid-soluble, after 15 min NinhydrinProtein resistant Total (initial)
Conditions 310 1071 2426 a. Puromycinyl-peptides degraded in the presence of bestatin 28 1346 2426 b. Same as a, but bestatin omitted 6 4 128 c. Same as a, but cycloheximide added before the tracer A suspension of cells containing labeled puromycinyl-peptides was prepared and aliquots were incubated in the presence (a) or absence (b) of bestatin (0.5 mg/ml). With another portion of the cells (c), cycloheximide was added 60 s before the tracer (rather than 80 s afterward); the cells were exposed to the labeled leucine for 80 s and then washed and suspended as in the regular procedure. Aliquots of this suspension were incubated in the presence of bestatin. All values represent the average of triplicate aliquots. Counting efficiency was 74%.
712
Biochemistry: Botbol and Scornik
RESULTS AND DISCUSSION Abnormal proteins that were produced in intact reticulocytes by the effect of puromycin (Fig. 1 left) or the valine analog L-threo-a-amino--1-chlorobutyric acid (Fig. 1 right) were pulse-labeled with carboxy-labeled leucine. The cells were washed free of the tracer and incubation was continued. A large portion of the radioactive protein was rapidly degraded to trichloroacetic acid-soluble products, largely the free amino acids. This we know because the radioactivity disappeared after treatment with ninhydrin at acid pH, which decarboxylates only free amino acids (26). Addition of bestatin (0.5 mg/ml) to the incubation mixture had little effect on the rate at which the abnormal proteins were degraded to acid-soluble products but changed the nature of these products, one-fourth of which became now ninhydrin resistant (Fig. 1). In the experiment presented in Fig. 2, we observed the effects of different concentrations of the peptidase inhibitor. When the results were examined in a double reciprocal plot, they fit a straight line extrapolating to a maximum possible accumulation of ninhydrin-resistant material equivalent to 55% of the total acid-soluble radioactivity. It is not surprising that a fraction of all small peptides (the other 45%) resulting from endoproteolytic attack of the globin molecules may be degraded to amino acids by bestatin-insensitive enzymes. Bestatin is known to inhibit aminopeptidase B and leucine aminopeptidase, but not aminopeptidase A or endopeptidases (21).
40r
Proc. Natl. Acad. Sci. USA 76 (1979) Table 2. Acid hydrolysis of the ninhydrin-resistant material
_ -
-
+
+
+
A suspension of cells containing radioactive puromycinyl-peptides was prepared as described under Experimental Procedure (except that the tracer was twice the usual specific radioactivity). Aliquots (0.1 ml) were incubated for 15 min in the presence of bestatin (0.5 mg/ml). Each aliquot then received 0.6 ml of 10% trichloroacetic acid. The total trichloroacetic acid-soluble radioactivity (first line) and the ninhydrin-resistant fraction (second line) were determined as explained under Experimental Procedure. The trichloroacetic acid extract of a second aliquot was combined with 1 vol of concentrated HCl and incubated under reduced pressure for 20 hr at 1061C. This hydrolyzed sample was then evaporated to dryness and redissolved in 0.6 ml of 10% trichloroacetic acid. The total radioactivity (third line) and ninhydrin-resistant fraction (fourth line) were determined as before. Counting efficiency was 74%.
We believe that the acid-soluble radioactivity resistant to ninhydrin represented peptide intermediates in the degradation of the abnormal proteins, on the basis of the following evidence. (i) There was a precursor-product relationship between the labeling of the protein and the appearance of the ninhydrinresistant radioactivity. When cycloheximide was added before the labeled leucine, so that incorporation of the tracer into protein was prevented, further incubation in the presence of V0 594 342
\4 4
A 20
30
10
20 *, C._0J
0.5
C, mg/ml
0.3-
131
'I
10
-0
OL
"
I,_ 1
4.'
I
0
0
1/A
2683 607 2432 36
+
30F
OL 0 0.05
Radioactivity, dpm/0.1 ml cell suspension
Treatment Acid hydrolysis Ninhydrin
20
I
-
%
0.2 -
-I
%
0.1I4
1/C, ml/mg
PIG. 2. Dependence of ninhydrin-resistant radioactivity on bestatin concentration. A suspension of cells containing labeled puromycinyl-peptides was incubated with bestatin at 1.0, 0.5, 0.05, or 0 mg/ml. After 15 min the ninhydrin-resistant and the total acidsoluble radioactivities were determined. The percentage A of the acid-soluble radioactivity that became ninhydrin-resistant is plotted as a function of the bestatin concentration C (Upper). Also, the value at 0 mg/ml was subtracted from the average of the duplicates, and the reciprocal of the resulting value was plotted against the reciprocal of the bestatin concentration (Lower). The values fit a straight line extrapolating on the ordinate to 0.018, the reciprocal of 55%.
5
6
7
8
Effluent volume, ml FIG. 3. Size distribution of the ninhydrin-resistant material. Cells containing labeled puromycinyl-peptides (Upper) or analog-containing protein (Lower) were incubated in the presence of bestatin, 0.5 mg/ml (20 min in first case, and 15 min in the second). The size distribution of the ninhydrin-resistant (0) material was examined by chromatography on a Sephadex G-10 column. The position of the blue dextran (V0) and the elution profile of the internal standard of [3H]leucine (Mr 131) (0) are also shown. The positions of raffinose (Mr 594) and sucrose (Mr 342) were determined separately; their peaks are indicated by arrows and identified by their molecular weights.
Biochemistry: Botbol and Scornik Table 3. Retention by the cells of the ninhydrin-resistant material
Ninhydrin-resistant radioactivity, dpm/0.1 ml cell suspension Incubations Total Cells Medium 370C, 15 min 310 256 54 370C, 15min;0C, 120min 226 167 58 A suspension of cells containing radioactive puromycinyl-peptides was prepared, and 0.1-ml aliquots were incubated in the presence of bestatin (0.5 mg/ml) at the indicated temperatures. At the end of the incubation, duplicate aliquots were diluted with 0.6 ml of ice-cold 0.9% NaCl and the cells were separated from the medium by centrifugation. The medium received 0.05 ml of 100% trichloroacetic acid. Other duplicate (undiluted) aliquots received 0.6 ml of 10% trichloroacetic acid. The acid precipitate was removed by centrifugation in all aliquots and the ninhydrin-resistant radioactivity was determined in the supernatant. The values are the average of both aliquots. The fraction contained in the cells is calculated as the difference between the other two values. Counting efficiency was 74%.
bestatin yielded very little ninhydrin-resistant radioactivity in the acid-soluble extract (Table 1). (ii) Treatment with ninhydrin at acid pH decarboxylates only free amino acids (26). If the radioactivity of the ninhydrin-resistant material represents carboxy-labeled leucine, of which either the carboxylic C or the a-amino N is engaged in peptide bonds, these bonds should be broken by intensive acid hydrolysis, releasing the radioactivity in a ninhydrin-sensitive form. This was indeed the case (Table 2). (iii) The ninhydrin-resistant material accumulated in the presence of bestatin during the degradation of puromycinylpeptides (Fig. 3 upper) or analog-containing proteins (Fig. 3 lower), eluted from a Sephadex G-10 column as an approximately symmetrical peak with an apparent size distribution averaging about 300 daltons. This molecular mass is consistent with leucine-containing di- and tripeptides, or both, and could be expected from the substrate specificity of the enzymes known to be inhibited by bestatin (21). The radioactivity must represent a heterogeneous mixture; the sequences of a and f chains of mouse hemoglobin (27) contain a total of 34 leucine residues, which could possibly integrate 28 different di- and 83 different tripeptide sequences. These intermediates are largely retained by the cells; this was shown in an experiment in which the reticulocytes were separated from the suspension by centrifugation. After incubation for 15 min at 37°C, the cells contained more than 80% of the ninhydrin-resistant material. The distribution was similar even after storage of the cell suspension at 0°C for 2 hr (Table 3). Thus a peptidase inhibitor has been successfully used in the production of low molecular weight peptide intermediates in the in vivo degradation of cellular proteins. Further characterization of the intermediates and studies of their subcellular localization should provide valuable insight into the pathway(s) of degradation of abnormal proteins.
Proc. Natl. Acad. Sci. USA 76 (1979)
713
We are grateful to Dr. Hamao Umezawa for a generous gift of bestatin and Dr. Mary Lee S. Ledbetter for critical revision of the manuscript. This research was supported by Grant AM 13336 of the National Institutes of Health. 1. Barrett, -A. J., ed. (1977) Proteinases in Mammalian Cells and Tissues (North-Holland, Amsterdam). 2. Hanson, H., Ansorge, S. & Bohley, P., eds. (1977) Acta Biol. Med. Ger. 36, 1503-1968. 3. Mortimore, G. E. & Ward, W. F. (1976) in Lysosomes in Biology and Pathology, eds. Dingle, J. T. & Dean, R. T. (North-Holland, Amsterdam), Vol. 5, pp. 157-184. 4. Etlinger, J. D. & Goldberg, A. L. (1977) Proc. Natl. Acad. Sci. USA 74, 54-58. 5. Holzer, H. (1978) in Symposium on Protein Turnover and Lysosomal Function, eds. Segal, H. & Doyle, D. (Academic, New York), pp. 305-314. 6. de Duve, C. & Wattiaux, R. (1966) Annu. Rev. Physiol. 28, 435-492. 7. Dean, R. T. (1977) Acta Biol. Med. Ger. 36, 1815-1820. 8. Khairallah, E. A., Airhart, J., Bruno, M. K., Puchalsky, D. & Khairallah, L. (1977) Acta Biol. Med. Ger. 36, 1735-1745. 9. Rudek, D. E., Dien, P. Y. & Schneider, D. L. (1978) Biochem. Biophys. Res. Commun. 82,342-347. 10. Ciehanover, A., Hod, Y. & Hershko, A. (1978) Biochem. Biophys. Res. Commun. 81, 1100-1105. 11. Daniels, R. S. & Hipkiss, A. R. (1978) Biochem. Soc. Trans. 6, 623-625. 12. Goldberg, A. L. & St. John, A. C. (1976) Annu. Rev. Biochem. 45, 747-803. 13. Ballard, F. J. (1977) Essays Biochem. 13, 1-37. 14. Pine, M. J. (1975) in Intracellular Protein Turnover, eds. Schimke, R. T. & Katunuma, N. (Academic, New York), pp. 65-76. 15. Miller, C. G. (1975) Annu. Rev. Microbiol. 29,485-504. 16. Kowit, J. D. & Goldberg, A. L. (1977) J. Biol. Chem. 252, 8350-8357. 17. Rabinovitz, M. & Fisher, J. M. (1961) Biochem. Biophys. Res. Commun. 6, 449-453. 18. Morris, A., Arlinghaus, R., Favelukes, S. & Schweet, R. (1963) Biochemistry 2, 1084-1090. 19. McIlhinney, A. & Hogan, B. L. M. (1974) FEBS Lett. 40,297301. 20. DeSimone, J., Kleve, L., Longley, M. A. & Schaeffer, J. (1974) Biochem. Biophys. Res. Commun. 57,248-254. 21. Umezawa, H. & Aoyagi, T. (1977) in Proteinases in Mammalian Cells and Tissues, ed. Barrett, A. J. (North-Holland, Amsterdam), pp. 637-662. 22. Umezawa, H. (1977) Acta Biol. Med. Ger. 36, 1899-1915. 23. Hopgood, M. F., Clark, M. G. & Ballard, F. J. (1977) Biochem. J. 164, 399-407. 24. Scornik, 0. A., Schadel, B. F., Franze-Fernandez, M. T. & Botbol, V. (1978) Fed. Proc. Fed. Am. Soc. Exp. Biol. 37, 1332
(abstr.). 25. Scornik, 0. A. (1974) J. Biol. Chem. 249,3876-3883. 26. Van Slyke, D. D., Dillon, R. T., MacFadyen, D. A. & Hamilton, P. (1941) J. Biol. Chem. 141, 627. 27. Dayhoff, M., ed. (1972) Atlas of Protein Sequence and Structure
(National Biomedical Research Foundation, Washington, DC), Vol. 5, pp. D-58, D-69.
