Bestatin as an experimental tool in mammals.

Bestatin, an antibiotic of microbial origin, is a potent inhibitor of some, but not all aminopeptidases. It can be administered, with low toxicity, to cultured cells, intact animals and humans. It has become a useful tool in elucidating the physiological role of some mammalian exopeptidases in the regulation of the immune system, in the growth of tumors and their invasion of surrounding tissues, and in the degradation of cellular proteins. Bestatin-sensitive enzymes play important roles in the digestion and absorption of peptides in the brush border of the intestine and the kidney, in the reproductive system, and in the metabolism of opioid peptides and leukotrienes. Aminopeptidase N emerges as the major target for the effects of bestatin on the immune system and some of its effects on tumor growth and the endometrium. It is also the major bestatin-sensitive enzyme involved in the degradation of oligopeptides on the surface of intestine and kidney brush borders, and the inactivation of enkephalins in the brain. Bestatin-sensitive cytosolic exopeptidases are important in the degradation to amino acids of di- and tripeptides generated in most cells by cellular protein degradation, as well as those absorbed through the brush border of intestine and kidney. Inhibition of one of these exopeptidases, cytosol alanine aminopeptidase, results in apoptosis. Bestatin-sensitive cystinyl aminopeptidase is abundant in placenta. Two bestatin-sensitive enzymes, aminopeptidase B and nardilysin, are particularly abundant in late spermatids. Finally bestatin-sensitive LTA4 hydrolase generates the potent chemotactic agent, LTB4.

Cellular uptake of 3H-bestatin in tissues of mice after its intravenous injection.

Bestatin, a dipeptide analog, is a potent aminopeptidase inhibitor of bacterial origin. We have previously shown that bestatin inhibits cytosolic exopeptidases in mammalian cells, and results in the accumulation of di- and tripeptide intermediates in cellular protein degradation. Our primary interest is the uptake of bestatin in liver and muscle, 10 min after its intravenous injection into mice. In this short interval, peptide intermediates accumulate linearly in these tissues and permit an estimate of their rates of cellular protein breakdown. Male, CD-1 adult mice received the intravenous injection of 3H-bestatin and 14C-sucrose. The disappearance of 3H-bestatin from the plasma, when normalized by the injected radioactivity, was indistinguishable from that of 14C-sucrose. They both drop rapidly during the first 10 min after the injection, followed by a slower exponential disappearance of 3.4% per min, which extrapolates to an apparent volume of distribution of 25 ml/100 g body weight. In two mice, 3 hr after the injection, the urine contained 77.4% and 79.8% of the injected 14C-sucrose, and 70.9% and 73.9% of the injected 3H-bestatin. Other mice were killed 10 min after the injection of 5 mg of bestatin, and the concentration of 3H-bestatin and 14C-sucrose was determined in the plasma and various tissues. Using sucrose as a nonpermeant marker of the extracellular space, extracellular 3H-bestatin was calculated and subtracted from the total to estimate the cellular uptake of bestatin. Bestatin was taken up readily in the liver (383-452 microg/g), kidneys (175-191 microg/g), and intestine (137-179 microg/g), but much less in red cells (11 microg/g) or skeletal muscle (4.8 microg/g). Bestatin also entered slowly into erythrocytes in vitro (0.3%/min) by a nonsaturable process. It is suggested that bestatin is taken up through transporter-mediated processes in some cells but not others.

Measurement of muscle protein degradation in live mice by accumulation of bestatin-induced peptides.

Bestatin, an aminopeptidase inhibitor, permits the degradation of cellular proteins to di- and tripeptides but interferes with the further breakdown of these peptides to amino acids. We propose to measure instant rates of protein degradation in skeletal muscles of intact mice by the accumulation of bestatin-induced intermediates. Muscle protein was labeled by injection of L-[guanidino-14C]arginine; 3 days later, maximum accumulation of intermediates was measured in abdominal wall muscles 10 min after the intravenous injection of 5 mg of bestatin. The peptides were partially purified and hydrolyzed in 6 N HCl, and the radioactivity in peptide-derived arginine was determined, after conversion to 14CO2 by treatment with arginase and urease. The measurement of bestatin-induced intermediates provides a unique tool for studying acute changes in muscle protein turnover in live mice. We observed a 62% increase in muscle protein breakdown after a 16-h fast, which was reversed by refeeding for 3.5 h, and a 38% increase after 3 days of protein depletion.

Protein depletion and replenishment in mice: different roles of muscle and liver.

Fully grown male CD-1 mice, fed a protein-free diet for 3 days, received 1 g of starch with or without 300 mg casein by intragastric intubation. We surveyed the acute effects of these nutrients on protein synthesis in all tissues (by extrapolating to infinity the incorporation of radioactive leucine after its injection in massive doses) and protein degradation in skeletal muscle and liver (by the accumulation of bestatin-induced peptide intermediates). Muscle proteolysis was the major source of N during depletion. Compared with postabsorptive animals, starch suppressed muscle protein loss (synthesis +21%, degradation -24%, P < 0.01) and stimulated hepatic proteolysis (degradation +28%, P < 0.01). Addition of casein to the starch was anabolic in liver (synthesis +41%, degradation -33%, P < 0.01), gastrointestinal tract, pancreas, and skin (synthesis +38, +69 and +38%, respectively, P < 0.01) but had no effect on muscle. Protein turnover proved uniquely sensitive to the dietary supply of carbohydrates in muscle and to the endogenous or exogenous supply of amino acids in liver.

Measurement of instant rates of protein degradation in the livers of intact mice by the accumulation of bestatin-induced peptides.

Bestatin induces the accumulation of di- and tripeptide intermediates in cellular protein breakdown. In liver, a single set of bestatin-sensitive cytosolic peptidases are involved in the degradation to amino acids of the major classes of cellular proteins. Accumulation of bestatin-induced peptides, in isolated hepatocytes, is proportional to the rate of protein degradation (Botbol, V., and Scornik, O. A. (1989) J. Biol. Chem. 264, 13504-13509). Injection of 1 mg of bestatin into mice results in detectable amounts of hepatic intermediates in 15 min. We propose to use the accumulation of these peptides as a relative measurement of liver protein degradation. There is at present no other way to determine transient changes in protein breakdown in the tissues of intact animals. As an example of the applications of this procedure, we present the effects of a single meal on hepatic protein metabolism. Protein synthesis was estimated by the incorporation into liver protein of a massive dose of radioactive leucine (Scornik, O. A. (1974) J. Biol. Chem. 249, 3876-3883) and degradation of long-lived or short-lived proteins by the accumulation of bestatin-induced peptides, labeled in carboxy-C of their Leu or Arg moieties, 1 day or 1 h beforehand. A single meal resulted in an 18% increase in liver protein in 8 h, a 45% increase in the rate of hepatic protein synthesis, and a 3-fold decrease in the rate of breakdown of long-lived proteins. Short-lived proteins were not affected. To establish the efficiency with which bestatin-induced peptides accumulate in the livers of fasting mice, we compared them with the disappearance, in 1 day, of protein-bound 14C-guanidino-Arg residues, labeled by previous injection of 14C-bicarbonate (Swick, R. W., and Ip, M. M. (1974) J. Biol. Chem. 249, 6836-6841). From this comparison, we estimated that bestatin-induced Leu-labeled intermediates, accumulating in 15 min, represented 39% of the hepatic proteins degraded in that interval. For Arg-labeled intermediates the value was 55%. Correcting for these efficiencies, we estimate that in 4 h a meal decreased the rate of degradation of long-lived Arg-labeled proteins from 2.02 to 0.73%/h. For Leu-labeled proteins the estimated rates were 1.76 and 0.66%/h, respectively. Although a transient slowdown of liver protein degradation after a single meal had been suggested before, this is the first time that acute changes such as this can be determined directly in intact animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of bestatin-sensitive exopeptidases in the intracellular degradation of hepatic proteins.

Injection of bestatin into intact mice produces accumulation of di- and tripeptide intermediates in the degradation of short- and long-lived hepatic proteins, whereas lysosomal breakdown of endocytosed plasma asialoglycoproteins is not affected. The majority of the peptides are found in the liver cytosol, but a minor portion appears in a sedimentable fraction containing mitochondria and lysosomes (Botbol, V., and Scornik, O. A. (1983) J. Biol. Chem. 258, 1942-1949). We now report that (a) the primary location of the intermediates is the cytosol. The particulate fraction represents cytosolic peptides trapped within mitochondria, as evidenced by sedimentation equilibrium in sucrose gradients after loading lysosomes with Triton WR1339 and by the sensitivity of the particles to lysis by digitonin. (b) In isolated hepatocytes, where we can measure simultaneously protein breakdown and bestatin-induced peptides, the accumulation of intermediates parallels protein degradation of analog-containing, short- and long-lived proteins, even after stimulation of the latter by amino acid deprivation. These observations are consistent with the hypothesis that bestatin inhibits cytosolic exopeptidases that complete the intracellular breakdown to amino acids of the major classes of hepatic proteins. The role of cytosolic exopeptidases is expected in the rapid degradation of abnormal proteins, a demonstrated cytosolic process. In stimulated degradation of long-lived proteins, the importance of cytosolic exopeptidases implies either that this process is largely cytosolic or, more likely, that peptides escape from autophagic organelles.

Role of idle ribosomes in the response of Chinese hamster ovary cells to depletion of histidyl-tRNA.

In Chinese hamster ovary cells, histidine starvation and inactivation of histidyl-tRNA synthetase by mutations or histidinol result in stimulation of protein breakdown. We have previously shown that the regulatory mechanism recognizes the level of aminoacylation of tRNA(His). We now report that it is also sensitive to the functional state of the ribosomes. Cycloheximide, an inhibitor of peptidyl-tRNA translocation, decreases the sensitivity of the regulation. In the presence of 1.5 micrograms cycloheximide/ml, protein synthesis is inhibited to 6% of control; a full response can still be elicited by appropriate concentrations of histidinol, but it requires a more extensive depletion of histidyl-tRNA than in the absence of cycloheximide. The response is attained only when the depletion is sufficient to inhibit protein synthesis further and to increase the number of ribosomes idling in the histidine codon with an empty aminoacyl site, measured by their reactivity in vivo to low concentrations of puromycin. The results indicate that a simple depletion of his-tRNA is not sufficient to elicit the response and suggest that idle ribosomes are required for regulation.

Effects of inhibitors of protein degradation on the rate of protein synthesis in Chinese hamster ovary cells.

In the absence of serum and amino acids, cultured Chinese Hamster Ovary cells released to the medium two thirds of the leucine produced by protein degradation. Because protein synthesis requires all the amino acids, the loss of leucine implies incomplete reincorporation of the other amino acids as well. Leupeptin (0.45 mg/ml) and chloroquine (up to 40 microM) inhibited protein breakdown by 21 and up to 41%, respectively, and resulted in proportional decreases in protein synthesis. Chloroquine abolished the stimulation of protein breakdown by amino acid deprivation. From the values of protein synthesis and leucine output with and without chloroquine, it is estimated that the stimulation of protein degradation not only permitted continuing protein synthesis but also increased amino acid output. In the presence of serum or amino acids protein breakdown was slower than in their absence and less sensitive to inhibition by chloroquine, but proportional effects on synthesis and degradation were still observed. It is suggested that protein degradation may be necessary for the maintenance of optimum intracellular concentrations of amino acids even in the presence of extracellular amino acids.

Role of protein degradation in the regulation of cellular protein content and amino acid pools.

A decrease in the rate of protein degradation contributes to the accumulation of cellular protein during growth or storage of dietary amino acids. Examples of this process in the liver of live mice are reviewed. One aspect of this regulation is the direct effect of amino acids on the rate of protein breakdown. At least in the case of histidine starvation in Chinese hamster ovary cells, the regulatory mechanism recognizes the level of aminoacylation of tRNA.

Peptide intermediates in the degradation of cellular proteins. Bestatin permits their accumulation in mouse liver in vivo.

We have previously shown that bestatin (an aminopeptidase inhibitor) permits the accumulation of di- and tripeptide intermediates in the degradation of abnormal globin (Botbol, V., and Scornik, O. A. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 710-713; Botbol, V., and Scornik, O. A. (1979) J. Biol. Chem. 254, 11254-11257). We now report that this drug (1 mg, intravenously, 5-30 min) causes similar intermediates to appear during breakdown of normal cellular proteins in the livers of live mice labeled 1 or 20 h before with L-[1-14C]Leu or L-[1-14C]Arg. These intermediates represent an estimated 20% of all degraded cellular protein. Lysosomal degradation of labeled asialoglycoproteins taken up by endocytosis is less affected by bestatin. After homogenization and centrifugation of the livers, we find a major fraction of the intermediates in the cytosol. Another fraction sediments at 27,000 X g in 15 min. The fraction that sediments is larger for Arg-labeled (30%) than for Leu-labeled (10%) peptides. The particulate fraction does not represent soluble peptides trapped in the pellet during fractionation because it does not appear when soluble intermediates are added to nonradioactive homogenates. The presence of intermediates in particulate and soluble fractions could result from protein breakdown either in both compartments or in organelles from which the peptides escape. We favor the latter possibility because (a) the particulate fraction does not increase after stimulation of autophagy by injection of glucagon (40 micrograms, 75 min before killing), (b) the subcellular distribution is the same whether intermediates are produced during the degradation of short or long lived proteins, and (c) chromatographic fingerprints of the particulate and soluble components reveal the same seven bands. The presence of well defined intermediates of cellular protein degradation in the particulate fraction of liver homogenates provides a valuable marker in the isolation and characterization of autophagic organelles.

Faster protein degradation in response to decreases steady state levels of amino acylation of tRNAHis in Chinese hamster ovary cells.

The rate of protein degradation in cultured Chinese hamster ovary cells increases in response to histidine starvation. Using cell lines with defective histidyl-tRNA synthetase, or histidinol (a competitive inhibitor of the enzyme), we have previously demonstrated a functional connection between the increase in degradation and the amino acylation of this tRNA (Scornik, O. A., Ledbetter, M. L. S., and Malter, J. S. (1980) J. Biol. Chem. 255, 6322-6329). A correlation is shown here between the steady state level of histidyl-tRNA and the regulatory response. Cells were incubated for 15 min in the presence of L-[3H]histidine, at a concentration at which greater than 90% of histidine for protein synthesis derives from the medium. The level of histidyl-tRNA was measured by its radioactivity after purification by phenol extraction, ethanol precipitation, and mild alkaline hydrolysis. Protein degradation in each condition was determined by the release of acid-soluble radioactivity from cells labeled for 24 h with L-[1-14C]leucine. The steady state level of histidyl-tRNA was altered by either histidinol (which slows down its production) or cycloheximide (which interferes with its utilization). Cycloheximide counteracts the effects of histidinol both on the level of histidyl-tRNA and on the rate of protein degradation. Both effects can be obtained, however, even in the presence of cycloheximide, if higher concentrations of histidinol are used. The results indicate that this regulatory mechanism does not recognize the rate of amino acylation per se but rather, the steady state level of its product, amino acyl-tRNA.

Degradation of abnormal proteins in intact mouse reticulocytes: accumulation of intermediates in the presence of bestatin.

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-[1-14C]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 G-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.

Faster synthesis and slower degradation of liver protein during developmental growth.

A study is presented of the liver protein gain during the early stages of postnatal development. Fractional rates of protein synthesis and degradation were determined in vivo in livers of 4-day-old mice. At this age, liver protein accumulated at a rate of 18% per day. Synthesis was measured after the injection of massive amounts of radioactive leucine. Degradation was extimated as the balance between synthesis and accumulation of stable liver proteins, or from the disappearance of radioactivity from liver protein previously labelled by the administration of NaH14CO3. We found that the neonatal livers: (1) synthesize 139% as much protein per unit time and unit mass as adult tissue, which is accounted for by a higher ribosome concentration (synthesis per mg of RNA was the same); (2) retain 39% of the newly synthesized protein as stable liver components (compared with 48% in adult mice); (3) degrade protein at 56% of the rate in the adult liver. This lower rate of degradation is quantitatively the most significant difference between the growing and non-growing liver.