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PubMed2000

Localization of pepstatin's inhibitory action during Fc-mediated antibody internalization: possible implications for antibody-mediated viral transmission.

Antibody internalization via Fc receptors is an important cellular mechanism, possibly facilitating the entry of antigenic peptides or viral particles into cells when specific antibodies are present at the periphery. Using an experimental model of trophoblast cells, we have shown that anti-p21(ras) monoclonal antibodies can use IFN-gamma-induced surface Fcgamma receptors to enter the cell. This entry of anti-p21(ras) antibodies ultimately inhibits IFN-gamma-mediated class II antigen induction. Since there may be obvious and inevitable harmful aspects of this mechanism, during which Fc-mediated viral particle or autoantigen transport may occur, we concentrated efforts on defining a potent inhibitor able to eliminate such uptake. The results presented here show that the protease inhibitor pepstatin A efficiently inhibits Fcgamma receptor induction by IFN-gamma and also blocks the endocytic pathway followed by an antibody when it enters the cell at the level of early endosomal compartments. We thus postulate that the use of pepstatin A, because of its inhibition of autoantigen presentation or viral transmission, including that of HIV, may find important applications in therapeutic protocols.

Animals↗
PubMed1978

Treatment of experimental allergic encephalomyelitis with an inhibitor of cathepsin D (pepstatin).

Intraperitoneal administration of pepstatin (2 mg/day for 5 weeks) to Lewis rats subjected to experimental allergic encephalomyelitis (EAE) (induced by guinea pig spinal cord and pertussis vaccine) suppressed the appearance of clinical signs of disease, and reduced the severity and incidence of CNS lesions normally associated with this disease. Administration of pepstatin for shorter periods to Lewis rats, or BSVS mice, or guinea pigs challenged with myelin basic protein delayed, but did not prevent clinical signs of EAE, but was accompanied in all cases by a less severe histopathology.

Animals↗
PubMed1990

Evidence for pepstatin-sensitive conversion of porcine big endothelin-1 to endothelin-1 by the endothelial cell extract.

We designed a radioimmunoassay (RIA) specific for the C-terminal fragment (CTF, 22-39) of porcine big endothelin-1 (big ET-1, 1-39), and investigated whether the generation of ET-1 (1-21) is concomitant with that of the CTF during incubation of big ET-1 with the endothelial cell (EC)-extract. When the incubation mixture was applied to reverse-phase high performance liquid chromatography coupled with the RIAs for CTF and ET, immunoreactive (IR)-CTF eluted as one major and one minor peak, and IR-ET as one major peak. The retention times of each peak corresponded to those of synthetic CTF, big ET-1 and ET-1, respectively. An aspartic protease inhibitor pepstatin-A completely inhibited the generation of CTF- and ET-1-like materials. Both materials were also detected in culture medium of porcine aortic ECs. These findings strongly suggest that ET-1 is generated from big ET-1 via a single cleavage between Trp21 and Val22 in vascular ECs and that a pepstatin-sensitive aspartic protease is a possible candidate for big ET-1 converting enzyme.

Animals↗
PubMed1985

The rennet-induced clotting of para-kappa-casein revisited: inhibition experiments with pepstatin A.

The proteolysis of micellar kappa-casein by rennet was followed by SDS-polyacrylamide gel-electrophoresis and the clotting of the para-kappa-casein formed by absorbance measurements. Up to a degree of proteolysis of about 0.4 the enzyme-inhibitor pepstatin A proved able to instantaneously stop the clotting. This effect is explained by the rapid condensation of monofunctional, monomeric and polymeric particles of para-kappa-casein. At higher degrees of proteolysis pepstatin was no longer able to completely block the polymerization. This is explained by the retardation of the condensation of the monofunctionals as their size grows larger. A kinetic analysis of the enzyme-controlled stage of the clotting process predicts that the system should gel at an early degree of proteolysis of about 0.07. The actual gel points occur at considerably higher degrees of proteolysis. This suggests that the enzymic attack of the polymeric inert kappa-casein particles is not completely at random. Primary micelles of kappa-casein, however, are degraded by random attack rather than by a 'catch-and-razor' mechanism.

Caseins↗
PubMed1987

Purification and properties of a pepstatin-insensitive carboxyl proteinase from a gram-negative bacterium.

A carboxyl proteinase was found in the culture filtrate of a Gram-negative bacterium. The optimum for the action of the purified enzyme was approx. pH 3 and its caseinolytic activity was not inhibited by carboxyl proteinase inhibitors, such as pepstatin, Streptomyces pepsin inhibitor and diazoacetyl-DL-norleucine methyl ester. 1,2-epoxy-3-(p-nitrophenoxy)propane modified the enzyme with concomitant loss of its enzyme activity. The enzymatic and physicochemical properties of the enzyme were compared with those of known pepstatin- and diazoacetyl-DL-norleucine methyl ester-insensitive carboxyl proteinases previously reported. To our knowledge, this is the first carboxyl proteinase isolated from bacteria.

Amino Acids↗
PubMed2000

pH-dependent reversible inhibition of violaxanthin de-epoxidase by pepstatin related to protonation-induced structural change of the enzyme.

The redox enzyme violaxanthin de-epoxidase (VDE) was found to be sensitive to pepstatin, a specific inhibitor of aspartic protease. The inhibition was similar to that of aspartic protease in that it was reversible and accompanied by the protonation of the enzyme. Of the two peaks of VDE appearing on anion exchange chromatography, VDE-I predominated at pH 7.2. On lowering the pH of the chromatography, VDE-I decreased and VDE-II increased. Furthermore, re-chromatography of either peak yielded both peaks. These results suggest that VDE-I and VDE-II are interconvertible depending on pH, and thus, they represent the de-protonated and protonated forms of the enzyme, respectively. Presumably the protonation-induced structural change of the enzyme is responsible for the interaction with pepstatin, and also with substrate.

Chromatography, Ion Exchange↗
PubMed1994

Solvent isotope effects on the onset of inhibition of porcine pepsin by pepstatin.

Pepstatin is a slow and tight-binding inhibitor of pepsin. Preincubating enzyme and inhibitor in H2O and in D2O in the absence of substrate generates an inverse solvent isotope effect of Dk = 0.69 +/- 0.06 on the apparent first-order rate constant for the decay in enzymatic activity. Proton inventory analysis of the inverse isotope effect suggests a single transition-state proton with a fractionation factor of 1.41 +/- 0.05. In contrast, combining enzyme with inhibitor and substrate (Leu-Ser-p-nitro-Phe-Nle-Ala-Leu-OMe) simultaneously along with observing the decay in enzymatic activity during catalytic turnovers generates a normal solvent isotope effect of Dk = 1.25 +/- 0.09. Proton inventory analysis of the normal isotope effect suggests a single reactant-state proton with a fractionation factor of 1.46 +/- 0.03. These two experimental designs are often considered equivalent, but the differences in isotopic data require that the pathway for onset of pepstatin inhibition in the absence of substrate must be different from the pathway in the presence of substrate. In the former, the inhibitor can only bind to free enzyme; in the latter, the inhibitor is hindered from binding to free enzyme because of competition with substrate but can bind to intermediate forms of enzyme generated during catalytic turnovers, downstream from enzyme-product complexes.

Animals↗
PubMed1982

Three-dimensional structure of the complex of the Rhizopus chinensis carboxyl proteinase and pepstatin at 2.5-A resolution.

An X-ray diffraction analysis has been carried out at 2.5-A resolution of the three-dimensional structure of the Rhizopus chinensis carboxyl proteinase complexed with pepstatin. The resulting model of the complex supports the hypothesis [Marciniszyn, J., Hartsuck, J.A., & Tang, J. (1976) J. Biol. Chem. 251, 7088-7094] that statine (3-hydroxy-4-amino-6-methylheptanoic acid) approaches an analogue of the transition state for catalysis. The way in which pepstatin binds to the enzyme can be extended to provide a model of substrate binding and a model of the transition-state complex. This in turn has led to a proposed mechanism of action based on general acid-base catalysis with no covalent intermediates. These predictions are in general agreement with kinetic studies using several carboxyl proteinases, which together with their sequence homology and their common three-dimensional structures suggest that this mechanism can be extrapolated to all carboxyl proteinases.

Aspartic Acid Endopeptidases↗
PubMed1982

Synthesis of analogues of the carboxyl protease inhibitor pepstatin. Effect of structure in subsite P3 on inhibition of pepsin.

A series of pepstatin analogues having minimum structural requirements for tight-binding inhibition has been synthesized and tested on porcine pepsin. Subtle changes in the geometry and size of side chains at the valine-1 position of pepstatin were found to dramatically affect inhibitor potency as well a the type of kinetic behavior observed. The inhibitors reported here can be grouped into two categories: the more potent inhibitors are slow-binding inhibitors, i.e., exhibit slow, time-dependent inhibition: the weaker inhibitors, with Ki values greater than 10(-8) M, are not time-dependent inhibitors. A minimum kinetic mechanism is proposed to account for the observed kinetic behavior.

Binding Sites↗
PubMed1983

Synthesis of analogues of pepstatin. Effect of structure in subsites P1', P2', and P2 on inhibition of porcine pepsin.

A series of pepstatin analogues having structural variations in the P2', P1', and P2 positions have been synthesized and tested for inhibition of porcine pepsin. The standard peptide for this study was Iva-Sta-Val-Ala-Iaa. Structural variations in the P2' and P1' positions have relatively little effect on Ki; however, small variations in the P2 position have a more dramatic effect on Ki and time-dependent inhibition. A series of pepstatin fragments were also synthesized and tested for inhibition of porcine pepsin.

Animals↗
PubMed1998

Auxiliary agents for the peroral administration of peptide and protein drugs: synthesis and evaluation of novel pepstatin analogues.

The peroral administration of (poly)peptide drugs requires the development of delivery systems, which provide a protective effect toward a gastrointestinal enzymatic attack. A promising strategy for such systems represents polymer-enzyme inhibitor conjugates in which the embedded therapeutic agent is protected. However, the practical use of polymer-inhibitor conjugates has so far been limited by high production costs of these auxiliary agents. To solve this problem for delivery systems shielding from pepsinic degradation, structurally simplified analogues of the pepsin inhibitor pepstatin A have been synthesized. The synthesis of tripeptide analogues, described by McConnell et al., led us to pursue further modifications varying the C-terminus. Our target to attach a spacer moiety-enabling the free access of pepsin to the inhibitor-should be combined with an attractive synthetic approach providing low production costs in large-scale preparation. Structure modifications comprised either the side chain of the third amino acid which served as starting compound designing the C-terminus (L-leucine, L-isoleucine, L-norvaline) as the length of the spacer link, simulated by a linear alkyl group (n-butyl, n-hexyl, and n-octyl). The inhibitory activities which have been evaluated by an enzyme assay were significantly dependent on the nature of the side chain, whereas the length of the spacer had no influence on the inhibitory effect. Analogues bearing the isobutyl or n-propyl moiety as side chain displayed a strong inhibitory effect which was comparable to that pepstatin A. These congeners represent promising auxiliary agents for the peroral administration of (poly)peptide drugs.

Administration, Oral↗
PubMed1976

Interaction of pepstatin with pig pepsinogen.

In contrast with pepsin, pepsinogen does not bind pepstatin at pH values between 5.3 and 2.5. Pepsinogen is not retarded by pepstatin immobilized on to aminohexyl-Sepharose at pH5.3 or 4.1, whereas at pH3.0 activation takes place during the chromatography, with retardation of the resultant pepsin.

Amino Acids↗
PubMed1980

Interaction of dinitrophenyl-pepstatins with human cathepsin D and with anti-dinitrophenyl antibody. Development of potential reagents for the localization in vivo of active proteinases at sites of tissue injury.

Extracellular cathepsin D has been observed by various cytochemical methods at sites of tissue injury. However, the role of this enzyme in connective tissue matrix degradation is uncertain because there are no histochemical methods for determining whether or not the cathepsin D is active at such sites in living tissues. We considered that the combined use of a labelled tight-binding inhibitor with immunoprecipitation of the enzymes might overcome this problem. We have explored the application of derivatives of the inhibitor pepstatin, as only active cathepsin D binds pepstatin tightly. A series of N-pepstatinyl-N'-dinitrophenyl-alpha, omega-diaminoalkanes were synthesized with alkyl-chain lengths of two, four and six carbon atoms. These compounds were tight-binding inhibitors of human cathepsin D. In fluorescence-quenching titrations the dinitrophenyl groups were also fully available to bind high-affinity anti-dinitrophenyl antibody. It was shown by immunodiffusion in gels and by gel permeation chromatography that N-pepstatinyl-N'-dinitrophenyl-1,6-diaminohexane was a bifunction inhibitor able to bind cathepsin D and anti-dinitrophenyl antibody at the same time.

Antibody Affinity↗
PubMed2001

Effects of pressure on the activity and spectroscopic properties of carboxyl proteinases. Apparent correlation of pepstatin-insensitivity and pressure response.

The pressure dependence of the activity and spectroscopic properties of four carboxyl proteinases were investigated. Two were pepstatin-sensitive carboxyl proteinases (porcine pepsin and proteinase A from baker's yeast) and two were pepstatin-insensitive carboxyl proteinases (from Pseudomonas sp. 101 (pseudomonapepsin; PCP) and Xanthomonas sp. T-22 (xanthomonapepsin; XCP)). The specificity constant [k(cat)/K(m(app))] of PCP and XCP for a synthetic peptide substrate showed only a slight decrease with increasing pressure, whereas pepsin and proteinase A showed substantial disactivation at higher pressures. The calculated apparent activation volume (Delta V((k(cat)/(K(m)) was about 1, 3, 13, and 14 mL.mol(-1) for PCP, XCP, pepsin, and proteinase A, respectively. The hydrolysis of acid-denatured myoglobin by the four carboxyl proteinases was only slightly affected by high pressure (except for proteinase A at 400 MPa), in contrast to the results for the peptide hydrolysis. In fact, PCP, XCP, and proteinase A actually showed slightly higher degradations of acid-denatured myoglobin at higher pressures. The residual activities of these enzymes after the incubation at high pressures implied a pressure-induced stabilization towards autolysis. The changes in the fourth derivative near-UV absorbance spectrum of the four enzymes in aqueous solution were measured at various pressures from 0.1 to 400 MPa. Upon an increase in pressure, the peaks from PCP and XCP red-shifted slightly, whereas pepsin and proteinase A blue-shifted substantially, thus indicating a more polar environment. The intrinsic fluorescence also decreased upon increasing pressure. However, the change for XCP was rather small, but the change for the other three was very large. The changes in the peak wavelength for pepsin and proteinase A were characteristic, and also indicated a more polar environment under high pressure. An analysis by the center of spectra mass (CSM) gave the Delta G and Delta V of transition as 9.8 kJ x mol(-1) and -24 mL x mol(-1) (pepsin) and 11.7 kJ x mol(-1) and -43 mL x mol(-1) (proteinase A), respectively, by assuming a simple two-state transition. The circular dichroism (CD) showed relatively small changes after 1-h incubations at 400 MPa, indicating that the secondary structures were largely maintained.

Animals↗
PubMed1982

Conformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin.

Crystals of the molecular complex between the esterified tripeptide fragment of pepstatin and the aspartyl proteinase penicillopepsin are isomorphous with crystals of native penicillopepsin. The difference electron-density map at 1.8-A resolution, computed by using the amplitude differences and refined phases of reflections from the crystal of native penicillopepsin, unambiguously showed the binding mode of isovaleryl-Val-Val-StaOEt, where StaOEt is the ethyl ester of statine [(4S,3S)-4-amino-3-hydroxyl-6-methylheptanoic acid]. In addition, a major conformational change in penicillopepsin involving the large beta loop of residues from Trp-71 to Gly-83 (the so-called "flap" region) occurs as a result of this inhibitor binding. This structural movement provides the first confirmation of the importance of enzyme flexibility in the aspartyl proteinase mechanism. The 3-hydroxyl group of the Statine residue and the carbonyl oxygen atom of the ethyl ester are situated on either side of the approximate plane containing the hydrogen-bonded carboxyl groups of Asp-33 and Asp-213. The observed binding mode of the pepstatin tripeptide fragment is similar to that predicted for the binding of good substrates with penicillopepsin [James, M. N. G. (1980) Can. J. Biochem. 58, 251-271].

Aspartic Acid Endopeptidases↗
PubMed1988

Human immunodeficiency virus has an aspartic-type protease that can be inhibited by pepstatin A.

The protease encoded by the human immunodeficiency virus (HIV) processes the viral gag and gag-pol protein precursor by posttranslational cleavage. In this study we have demonstrated by site-specific mutagenesis (Asp----Thr) and by pepstatin A inhibition that the recombinant HIV protease is an aspartic-type protease. Furthermore, incubation of HIV-infected H9 cells with pepstatin A inhibited part of the intracellular processing of the HIV gag protein yet had no apparent toxicity on HIV-infected cells during 48 hr of incubation.

Animals↗
PubMed1997

Solution structure of the HIV protease inhibitor acetyl-pepstatin as determined by NMR and molecular modeling.

The structure of acetyl-pepstatin has been investigated in solution by two-dimensional NMR spectroscopy and molecular modeling. The analysis of DQFCOSY, TOCSY and NOESY spectra lead to a full assignment of the -NMR signals both in DMSO-d6 and in TFE-d3:H2O 1:1. Interproton distances, dihedral angles and exchanger regimes of NH or OH protons were derived from ROESY connectivities, coupling constants and temperature dependences of the chemical shifts, respectively. Molecular modeling using the NMR distance and dihedral angle constraints obtained in DMSO-d6 yielded a model showing a well-defined structure for the N-terminal segment Ac-1 to Sta-4, but a flexible structure for the C-terminal segment. The structure was less defined in TFE-d3:H2O 1:1 and 13C T1 measurements are indicative of higher mobility. Comparison of the NMR-determined solution structure of acetyl-pepstatin with its crystal structure when bound to HIV-1 protease shows that the conformation is more extended in the complex as a result of intermolecular interactions.

Carbon Isotopes↗
PubMed1995

The primary structure of pepstatin-insensitive carboxyl proteinase produced by Pseudomonas sp. No. 101.

A unique carboxyl proteinase [EC 3.4.23.33] from Pseudomonas sp. No. 101 is the first example of a prokaryotic enzyme which is insensitive to the classical inhibitor, pepstatin. The primary structure of the proteinase was determined by conventional methods. Pseudomonas carboxyl proteinase consists of 370 amino acid residues with one disulfide bond. This enzyme has no homologous sequence with any other known carboxyl proteinase, including carboxyl proteinase B from Scytalidium lignicolum, which is a pepstatin-insensitive carboxyl proteinase. In addition, Pseudomonas carboxyl proteinase lacks the Asp*-Thr-Gly, Glu*-Thr-Gly, and Asp*-Thr-Ser-Gly (*indicates the catalytic residue) sequences which are known as the motif sequences around a pair of catalytic residues in carboxyl proteinases reported so far. The results strongly indicate that Pseudomonas carboxyl proteinase is a new type of carboxyl proteinase.

Amino Acid Sequence↗