ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 203, No. 2, September, pp. 676-680, 1980

Inhibition of intracellular Protein Degradation by Pepstatin, Poly(L-Lysine), and Pepstatinyl-Poly(L-Lysine)

PATRICIA CAMPBELL,’ GEORGE I. GLOVER, AND J. MARTYN GUNN2

Departments of Chemistry and Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77X&’

Received February 22, 1980

Coupling the cathepsin D inhibitor pepstatin to poly(~-Lys) (M, 13,000) is shown to enhance its inhibition of protein breakdown in whole cell systems. Rates of intracellular protein breakdown for prelabeled proteins of Balb/c 3T3 fibroblasts were measured in the presence and absence of amino acids and insulin to generate basal and enhanced rates of protein breakdown. Pepstatin and poly(~-Lys) inhibited rates of degradation 5-7% and 16-23%, respectively, under each condition. Pepstatinyl-poly(L-Lys), containing 9 mol pep- statinimol polymer, inhibited enhanced rates of degradation a further 24-33% compared to poly(~-Lys), but this extra increment was not seen under basal conditions. Although the mechanism of inhibition of intracellular protein breakdown by poly(~-Lys) presently is unknown, the data obtained with free and conjugated pepstatin indicate the lysosomal system degrades proteins under both basal and enhanced conditions.

Umezawa and co-workers (1) have isolated several low molecular weight peptides from Streptomycetes that are very effective in- hibitors of intracellular, particularly lyso- somal, proteases. As well as inhibiting puri- fied enzymes, these inhibitors also inhibit intracellular protein degradation in cultured cells (2,3), in perfused liver (4,5), in skeletal muscle in vitro (6, 7), and have been shown to delay the degeneration of muscle tissue in genetically dystrophic chickens (8). How- ever, in some cases, certain of these inhibi- tors do not appear to be too effective in whole cell systems. Presumably this reflects failure of the inhibitors to penetrate cell membranes to the site of intracellular proteolysis.

Enhanced cellular penetration of a variety of different molecules can be achieved using positively charged proteins or polyamino acids as carriers (9, 10). In this paper we describe procedures for covalently attach- ing the specific cathepsin D inhibitor pep- statin to poly(~-Lys). We show that, com- pared to free pepstatin and poly(~-Lys),

’ Present address: Beckman Instruments, Inc., Somerset, N. J. 08873.

2 To whom reprint requests should be sent, % De- partment of Biochemistry.

pepstatinyl-poly(L-Lys) gives enhanced in- hibition of that component of intracellular pro- tein degradation attributable to autophagy (5), and provide evidence supporting the idea (5) that the lysosomal system degrades pro- teins even in the absence of overt autophagy.

MATERIALS AND METHODS

Pepstatin was purchased from the Peptide Insti- tute, Inc., Osaka, Japan. Poly(L-lysine) hydrobromide (Sigma Chemical Co.) had an average molecular weight of 13,000. Tissue culture supplies were from Flow Lab- oratories, Inc., L-[4,5-3H]leucine (40-60 Ci/mmol) from the Amersham Corporation, and monocomponent porcine insulin (Actrapid) from Novo Laboratories, Inc. All other reagents were of the highest grade available.

Pepstatin (73.4 mg) was activated with N-hydroxy- succinimide (29.2 mg) and dicyclohexylcarbodiimide (47.5 mg) in dry dimethylformamide (10 ml) at room temperature for 24 h. After filtration to remove the dicyclohexylurea, the solution was added to an equal volume of poly(~-Lys) (201 mg) in water at pH 9.6. The mixture was vigorously stirred at room tempera- ture for 2 days. Water (160 ml) was added, the solution acidified to pH 4.7 by addition of aqueous HBr, con- centrated to 10 ml, and centrifuged to remove unreacted pepstatin. The supernatant was chromatographed on Sephadex G-15, eluting with water, to separate the pepstatinyl-poly(L-Lys) from other reaction compo- nents. The pooled fractions from the first eluting peak

0003-9861/80/100676-05$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

676

INHIBITION OF PROTEIN DEGRADATION BY PEPSTATINYL-POLY(L-LYSINE) 677

were lyophilized, yielding 10.6 mg of product. Amino acid analysis of this material indicated that 5 of 33 lysine residues were covalently modified with pepstatin, i.e., the pepstatin concentration was approximately 9 mol/mol polymer.

Rates of intracellular protein degradation in Balb/c 3T3 fibroblasts and their simian virus 40-transformed variant were measured by a pulse-chase technique as previously described (ll), but with slight modifica- tions for use with 24-place multiwell dishes. Briefly, confluent monolayers were labeled for 16 h in Eagle’s minimum essential medium (MEM)3 containing 1% fetal calf serum and with 1 @J/ml [3H]leucine as the only source of leucine. This was removed, the monolayers were washed once with Earle’s salts, and MEM was added for 2 h. From this point all degradation media were supplemented with 0.1% bovine serum albumin, 2 mM leucine, and 20 mM N-tris (hydroxymethyl)- methyl-2-aminoethanesulfonate (Tes), pH 7.3. The deg- radation period was begun by replacing the medium with either Earle’s salts, MEM, or MEM plus insulin (lo-” M), each containing additions as designated in the tables. This provided a means for comparing the efficacy of the inhibitors against three different rates of protein degradation: the basal rate (MEM plus in- sulin), a partially enhanced rate (MEM), and a fully enhanced rate (Earle’s salts).

After 4 h the medium was transferred to centrifuge tubes, trichloroacetic acid was added to give a final concentration of 0.8 M, and the mixtures were kept at 0°C overnight. One milliliter of 0.1 M NaOH con- taining 0.1% Triton X-100 was added to each mono- layer, and the digested cells were transferred to tubes, thoroughly mixed, and 50-~1 portions sampled for ra- dioactivity determination using an NCS-based scintil- lation fluid (12). Radioactivity in these digests was assumed to be cell protein since other measurements showed less than 0.2% present as trichloroacetic acid- soluble material. The medium with trichloroacetic acid was centrifuged and 50-J portions of supernatant were taken for radioactivity measurements of medium amino acids. The remaining supernatant was discarded and the pellet (medium protein) dissolved in 1 ml of 0.5 N NaOH and 50 ~1 taken for radioactivity measurements. Protein breakdown was expressed as the percentage of labeled protein degraded over the 4-h period and was equal to loo-times the radioactivity in medium amino acids divided by the total radioactivity in amino acids plus protein. A comparable measurement at 0 h was subtracted. This was less than 0.5%. Although the measurements of medium protein were not essential to the procedure, they indicated the viability of mono- layers under different conditions since such radioactiv- ity increases either if proteins leak from cells or if cells detach from the monolayer.

3 Abbreviations used: MEM, minimum essential medium; Tes, N-tris(hydroxymethyl)methyl-2-amino- ethanesulfonate.

RESULTS

A number of methods for coupling pep- statin to poly(~-Lys) were attempted. Mix- ing the two components in an aqueous buffer with a water-soluble carbodiimide as the coupling catalyst resulted in the formation of a gel in which no reaction occurred. If the N-hydroxysuccinimide-activated pep- statin was mixed with poly(~-Lys) in dry dimethylformamide, no coupling was de- tected even after long reaction times. The 1:l dimethylformamide-H,O solvent system described was found to be the most success- ful, with all components being soluble at the end of the reaction.

Addition of poly(L-Lys) to the culture me- dium at 0.1 to 10 PM was found to progres- sively inhibit intracellular protein degrada- tion in Balb/c 3T3 cells (Table I). Adsorptive endocytosis of exogenous proteins is known to decrease rates of endogenous protein breakdown in other systems (13). Presum- ably the poly(~-Lys) accumulates in second- ary lysosomes and is preferentially degraded by proteinases with trypsin-like specificity. Since there are several proteinases with other specificities, this process would ac- count for part, but not all, of the inhibition of endogenous protein degradation observed in Table I. At high concentrations (10 PM), poly(~-Lys) causes cells to detach from the monolayer. This we attribute to its effect as a polycation acting on cell membranes (see Ref. (14) and references therein), and it is possible that this contributed also to the inhibition observed.

The pentapeptide pepstatin is a specific inhibitor of cathepsin D activity in vitro, giving half-maximal inhibition at approxi- mately lo-* M (1). In cultured 3T3 and SV40- 3T3 cells approximately 15 FM pepstatin (10 pg/ml) has little effect on intracellu- lar protein degradation under partially en- hanced conditions (Table II), presumably because the inhibitor does not penetrate cell membranes readily. However, addition of 1 PM pepstatinyl-poly(L-Lys) conjugate (ap- proximately 9 @M with respect to pepstatin) to 3T3 cells produced a marked inhibition (32%) over and above that due to the poly- cation alone. Similar results were obtained with SV40-transformed 3T3 cells (Table II).

678 CAMPBELL, GLOVER, AND GUNN

However, it is interesting to note, first, that, as observed previously (ll), rates of proteolysis are less in transformed compared to normal cells; second, that poly(~-Lys) has iess of an effect on rates of intracellular protein breakdown in transformed cells; and third, that the effect of conjugated pep- statin also is considerably reduced in trans- formed cells. That these effects are due to the conjugate and not to an association, or cotransportation of the inhibitor with the polycation, is shown by the fact that poly(~- Lys) plus pepstatin has no extra inhibitory effect compared to either agent alone (Table II). Increasing the concentration of conju- gated pepstatin 7.5-fold increased the inhibition of protein degradation to 50% in 3T3 and 20% in SV40-3T3 cells, but at the same time caused large-scale detachment of cells from the monolayer, making any dis- tinction of specific versus nonspecific and toxic effects dubious.

Protease inhibitors such as pepstatin are reported not to inhibit basal rates of protein degradation in cultured cells (2, 15). There- fore we compared the effects of free and conjugated pepstatin at inhibiting rates of intracellular proteolysis in 3T3 cells under the extremes of basal and twofold depriva- tion-enhanced conditions (Table III). As might be expected, under fully enhanced conditions (in Earle’s salts), the results were essentially the same as those reported in Table II, namely: a 7% inhibition of intracel- lular protein degradation with 15 PM pep- statin; 16% with 1 PM poly(~-Lys); and an

TABLE I

INHIBITION OF DEPRIVATION-ENHANCED ENDOGENOUSPROTEIN BREAKDOWN BY

POLY(L-LYS)IN 3T3 CELLS

POlY(L-Lys) Protein degradation (PM) (% in 4 h)

0 8.99 f 0.15 0.1 8.11 2 0.06 1.0 6.89 r 0.02

10.0 3.69 k 0.02

Note. Rates of protein degradation were determined in MEM as described under Materials and Methods. Values are means k SEM of six determinations.

TABLE II

INHIWFIONOF ENHANCEDPI~OTBIN DEGRADATION BY PEPSTATINYL-WLY(L-LYS)~N 3T3

ANaSV40-3T3CELLS

Protein degradation (% in 4 h)

Addition 3T3 SV40-3T3

0 9.05 2 0.06 6.7’7 + 0.10 Pepstatin 8.57 -t 0.12” 6.56 + 0.17 POlY(L-Lys) 7.05 ” 0.26” 5.68 k 0.15” POlY(L-Lys) plus

pepstatin Pepstatinyl-

7.11 f 0.17” 5.40 ? 0.17”

POW-LYSY 4.79 2 0.16a,” 4.94 2 O.ll”,b

Note. Rates of protein degradation were determined in MEM as described under Materials and Methods. Pepstatin was added to a final concentration of 15 PM; poly(L-Lys), 1 PM; and pepstatinyl-poly(L-Lys) at 9 PM with respect to pepstatin. All incubations contained 0.1% dimethylsulfoxide. Values are the means 2 SEM of lo-12 determinations.

” P < 0.05 compared to control. * P < 0.05 compared to poly(~-Lys).

extra 24% with pepstatinyl-poly(L-Lys) con- taining 9 j.&M pepstatin. In the presence of amino acids and insulin, which give basal conditions for proteolysis (Gunn, Hedges, and Walker, unpublished observations), pep- statin inhibited intracellular protein deg- radation by 9% and poly(~-Lys) by 23%, whereas pepstatinyl-poly(L-Lys) had no further effect over and above that of poly(~- Lys) alone (Table III).

DISCUSSION

The enhanced penetration of molecules not ordinarily transported across cell mem- branes, and the restoration of normal trans- port in transport-deficient mutants, may be achieved by covalently linking the compound to a positively charged protein or polyamino acid. For example, the transport of human serum albumin and horseradish peroxidase into mouse fibroblasts is enhanced if the pro- teins are attached to poly(~-Lys) (9). More- over, the conjugation of methotrexate with poly(~-Lys) increases the cellular uptake of the drug in transport-deficient cells and overcomes the drug resistance of mutants deficient in methotrexate transport (10).

INHIBITION OF PROTEIN DEGRADATION BY PEPSTATINYL-POLY&-LYSINE) 679

TABLE III

EFFECT OF PEPSTATIN, POLY(L-LYS), AND PEP- STATINYL-POLY(L-LYS) ON ENHANCED AND BASAL RATES OF PROTEIN DEGRADATION IN 3T3 CELLS

Protein degradation (% in 4 h)

Addition Earle’s salts MEM

+ insulin

0 Pepstatin POlY(L-Lys) Pepstatinyl-

12.13 + 0.12 6.16 + 0.09 11.23 k 0.14” 5.61 2 0.15” 10.20 k 0.12” 4.72 f 0.04”

POiY(L-LYS) 7.77 + 0.11aa* 4.61 ‘- 0.07”,”

Note. Rates of protein degradation were determined in either Earle’s salts or MEM plus insulin (10-O M) as described under Materials and Methods and the legend of Table II. Values are means + SEM of 12 determinations.

fl P < 0.05 compared to control. h P < 0.05 compared to poly(~-Lys). ’ P < 0.186 (two-tailed test) or 0.093 (one-tailed

test) compared to poly(~-Lys).

Although pepstatin is an excellent inhibi- tor of cathepsin D in vitro, inhibition of in- tracellular protein degradation in cultured cells is poor even at concentrations two or- ders of magnitude greater than that required for half-maximum inhibition of the enzyme. However, as reported here, poly(~Lys) con- jugated with pepstatin causes a significant inhibition of deprivation-enhanced intracel- lular protein breakdown in both normal and transformed cells. Such conjugates are taken up by adsorptive endocytosis into vesicles which eventually fuse with primary lyso- somes (9). In the case of pepstatinyl-poly- (L-Lys) this provides for the delivery of the inhibitor directly to the organelle containing cathepsin D. During this process, the con- jugate is degraded, presumably by trypsin- like enzymes, to free Lys and pepstatin or pepstatinyl-Lys. Presumptive evidence for this is twofold. First, a mixture of poly(~- Lys) and pepstatin had no additional effect on degradation over that of either agent alone, indicating that the active species trans- ported is the conjugate. Second, pepstatinyl- poly(~-Lys) had no effect on cathepsin D activity in vitro as measured by release of A,,,-absorbing, trichloroacetic acid-soluble material from hemoglobin (data not reported),

indicating that release of pepstatin is a nec- essary prerequisite for inhibition to occur.

We know of two other systems used for the specific direction of protease inhibitors. The first involves the use of albumin micro- spheres as carriers of the specific elastase inhibitor succinoyl-Ala- Ala-Pro-valyl-chlo- romethyl ketone (16). However, although this conjugate is a good inhibitor of leuko- cyte elastase and can be targeted to specific tissues, it has yet to be tested as an inhibitor of protein breakdown in vivo. The second involves the use of phospholipid vesicles. In the perfused liver, Dean (4) used this system to facilitate transport of pepstatin (50 pg/ml) resulting in a 50% inhibition of endogenous intracellular protein breakdown. This com- pares with 24-32% inhibition obtained here with pepstatin at 9 pM (approximately 6 pgl ml) in conjugated form. Because pepstatin acts on cathepsin D, both studies provide direct evidence for the involvement of lyso- somes in the degradation of intracellular pro- teins under deprivation-enhanced conditions, i.e., inhibition of that component of degra- dation attributable to autophagy (5). More- over, because the route of penetration is that used in the uptake and degradation of exogenous proteins, these studies also sug- gest the involvement of the same cell com- partment in the degradation of both extra- cellular and intracellular proteins.

More recently, Ward et al. (5), using a pepstatin-liposome complex similar to that of Dean (4), obtained a 20% inhibition of enhanced proteolysis in the perfused liver, a result in good agreement with the data reported here for 3T3 cells. In addition they found that combinations of pepstatin and leupeptin inhibit both basal and enhanced rates of protein degradation, implicating a role for lysosomes under both conditions. Previous work in cultured cells (2, 15) failed to show additivity for combinations of lyso- somotropic agents such as insulin plus leupeptin or pepstatin, indicating that lysosomes may not be involved in basal proteolysis to any great extent. Although pepstatin solubilized in dimethylsulfoxide does not readily inhibit intracellular protein degradation in 3T3 cells compared to the per- fused liver (5), or primary cultures of hepa- tocytes (3), nevertheless, we consistently ob-

680 CAMPBELL, GLOVER, AND GUNN

tamed inhibition of both enhanced and basal proteolysis. Since pepstatin is thought to inhibit only lysosomal cathepsin D, this im- plicates the involvement of lysosomes in pro- tein degradation even under physiologically basal conditions.

If poly(~-Lys), through its effects on pro- tein degradation, can be considered either a lysosomotropic agent or an inhibitor of au- tophagocytosis, then it too inhibits under all conditions. Although the mechanism of poly(~-Lys) inhibition is presently unknown, this may be ascribed either to its effect as a competitive inhibitor of trypsin-like protein- ases, or to its effect as a polycation on cellu- lar membranes (14), which might, through the insulin-like action of polyamines (17), inhibit autophagy, or to a combination of both. The failure of pepstatinyl-poly(L-Lys) to further inhibit basal rates of degradation may then be attributed to the totality of the effects of amino acids, insulin, and poly- (L-Lys) on bulk proteolysis (i.e., autophagy), and to the possibility that lysosomal cathep- sin D no longer plays a role in protein deg- radation when this system is completely inhibited. Such an interpretation requires that the autophagyl-lysosomal system be re- sponsible for part of the intracellular pro- tein breakdown measured under physiologi- cally basal conditions.

In this regard it is interesting to note that the physiological basal rate of protein degradation for 3T3 cells, obtained with MEM plus insulin, was 6.2% per 4 h, whereas the lowest rate was 4.7% per 4 h found for MEM plus pepstatinyl-poly(L-Lys) and for MEM plus insulin plus poly(~-Lys) or pepstatinyl-poly(cLys). These combinations resulted in a 60% inhibition of the maximum rate of protein breakdown observed under deprivation-enhanced conditions, and a 24% inhibition of the basal rate. Ward et al. (5) report a basal rate of 1.6% per hour for the perfused liver, and, using combinations of amino acids, insulin, pepstatin, and leupep- tin, obtained 80 and 56% inhibition of en- hanced and basal rates, respectively. In view of the differences in cell type and experi- mental procedures, the two reports may be considered to be in substantial agreement. Both provide evidence indicating a role for lysosomal cathepsin D and the autophagic

process in the degradation of intracellular proteins even under physiologically basal conditions, that is, even in the absence of overt autophagy.

ACKNOWLEDGMENTS

This work was supported by USPHS Grants AM- 19891, AM 26132, NSF Grant PCM76-15688, the Robert A. Welch Foundation, and the Texas Agricultural Ex- periment Station. Ms. Grace Walker provided excellent technical assistance.

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