The effects of lactoyl-pepstatin and the pepsin inhibitor peptide on pig cathepsin D.
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ABSTRACT:
SUBMITTER: Kay J
PROVIDER: S-EPMC1158299 | biostudies-other | 1982 Jun
REPOSITORIES: biostudies-other
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Project description:There is evidence that pepsin can aggravate tonsil hypertrophy. Pepstatin is a potent inhibitor of pepsin activity and could protect patients against reflux tonsil hypertrophy by inhibiting pepsin. We examined the effects of pepstatin on the development of tonsil hypertrophy to investigate pepsin's role in the pathogenesis of tonsil lesions. We investigated whether pepstatin suppresses pepsin-mediated lymphocyte proliferation in tonsil hypertrophy. Forty-nine children with tonsil hypertrophy and twenty-two adults with tonsillitis were recruited to the study prior to surgery. Tonsil tissue from each patient was harvested and assessed for changes in the number of lymphocytes and macrophages in the presence of pepsin and pepstatin. We found that the proportions of CD4- and CD14-positive cells were significantly lower (p < 0.05), but that the proportions of CD19- and CD68-positive cells were significantly higher (p < 0.05), in children than in adults. There were significantly more CD4-positive cells after pepsin treatment, but these numbers were reduced by pepstatin. The levels of both interleukin-2 (IL-2) and interferon gamma (IFN-γ) increased significantly in response to pepsin, but were reduced when pepsin was inhibited by pepstatin. The level of IL-10 is reduced in pepsin-treated CD4 cells and the level is restored by pepstatin. IL-2 blocking reduced the increased CD4 cell number by pepsin. But, an additive or a synergic effect is not founded in combined with IL-2 blocking and pepstatin. Pepsin-positive cells did not co-localize with CD20 and CD45 cells, but they were found surrounding CD20- and CD45-positive hypertrophic tonsil cells. Pepsin-positive cells co-localized with CD68-positive cells. It is probable that pepsin from extraesophageal reflux aggravates tonsil hypertrophy and pepstatin exerts a protective effect by inhibiting pepsin activity.
| S-EPMC6224077 | biostudies-literature
Project description:1. Because of the proposed role of cathepsin D in a variety of biological and pathological processes, the characteristics of inhibition by the potentially useful agent, pepstatin, were determined. 2. The beta and gamma forms of human cathepsin D, separated by isoelectric focusing, have identical specific extinction coefficients and specific activity in the degradation of haemoglobin. 3. Cathepsin D showed tight binding of 1 mol of pepstatin per 43000 g of protein, indicating that titration with the inhibitor represents a useful method for determination of absolute concentrations of the enzyme. 4. The titration curves were used to determine apparent dissociation constants (KD) for the binding of pepstatin and pepstatin methyl ester at pH3.5; values of approx. 5 X 10(-10)M were obtained. 5. Pepstatinyl-[3H]glycine was synthesized and shown to have a KD similar to that of pepstatin. Gel-chromatographic experiments showed that the binding of pepstatin and its derivatives is strongly pH-dependent. 6. The effect of pH on the KD for pepstatinyl-glycine was determined by equilibrium dialysis. As the pH was raised from 5.0 to 6.4, KD rose from 5 X 10(-10)M to 2 X 10(-6)M. 7. The catalytic activity of cathepsin D declines essentially to zero on going from pH5.0 to pH7.0, and we suggest that the binding site for substrate and pepstatin is abolished by a conformational change in the enzyme molecule. 8. The data indicate that, in biological experiments near neutral pH, large molar excesses of pepstatin over cathepsin D will be required for efficient inhibition.
| S-EPMC1172808 | biostudies-other
Project description:A number of peptides were found to increase the activity of pig pepsin towards small synthetic substrates. The activators increase transpeptidation of both the acyl-transfer and the amino-transfer types by as much as 45-fold. The effect on hydrolysis varies from inhibition to modest activation, but is always less than the effect on transpeptidation. The kinetics of substrate cleavage are the converse of non-competitive inhibition and show an increase in kcat. and no effect on Km values. Lineweaver-Burk plots of results obtained in the presence of the activators indicate a substrate activation at high substrate concentration. This appears to be a co-operative effect, since it is not observed in the absence of the activators. The activation is greatest at pH 4.7, less at pH 3.4, and at pH 2.0 is observable only with some of the activator peptides. The results show directly the effect of secondary binding on the catalytic efficiency of pepsin. The most effective activators are those that are most hydrophobic. The results suggest that binding in the secondary binding sites causes an increase in hydrophobicity in the catalytic site which results in increased stability of the acyl and amino intermediates, and preferential reaction with acceptors other than water. The implication that the present results strengthen the case for a role of covalent intermediates in the hydrolysis of good substrates (high kcat. values) is discussed.
| S-EPMC1172639 | biostudies-other
Project description:Cathepsin D is an aspartic protease and one of the most abundant proteases. It is overexpressed in many cancers and plays an important role in tumor development, progression, and metastasis. While it is a physiologically intracellular protein, cathepsin D is secreted into the extracellular matrix under pathological conditions, making it an appealing target for drug delivery systems. Here, we present the development and evaluation of a new delivery system for tumor targeting based on immunoliposomes functionalized with pepstatin A-a natural peptide inhibitor of cathepsin D. A lipid tail was added to pepstatin A, enabling its incorporation into the liposomal lipid bilayer. The successful targeting of cathepsin D was confirmed using recombinant cathepsin D and in tumor cell lines, showing the feasibility of this targeting approach and its potential for in vivo use in theragnostic applications.
| S-EPMC10609775 | biostudies-literature
Project description: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.
| S-EPMC1163880 | biostudies-other
Project description:Numerous cathepsin B inhibitors have been developed and are under investigation as potential cancer treatments. They have been evaluated for their ability to inhibit cathepsin B activity and reduce tumor growth. However, they have shown critical limitations, including low anticancer efficacy and high toxicity, due to their low selectivity and delivery problems. In this study, we developed a novel peptide and drug conjugate (PDC)-based cathepsin B inhibitor using cathepsin-B-specific peptide (RR) and bile acid (BA). Interestingly, this RR and BA conjugate (RR-BA) was able to self-assemble in an aqueous solution, and as a result, it formed stable nanoparticles. The nano-sized RR-BA conjugate showed significant cathepsin B inhibitory effects and anticancer effects against mouse colorectal cancer (CT26) cells. Its therapeutic effect and low toxicity were also confirmed in CT26 tumor-bearing mice after intravenous injection. Therefore, based on these results, the RR-BA conjugate could be developed as an effective anticancer drug candidate for inhibiting cathepsin B in anticancer therapy.
| S-EPMC10141979 | biostudies-literature
Project description:The kinetic properties of two classical inhibitors of aspartic proteases (PRs), pepstatin A and acetyl-pepstatin, were compared in their interactions with HIV-1 and xenotropic murine leukemia virus related virus (XMRV) PRs. Both compounds are substantially weaker inhibitors of XMRV PR than of HIV-1 PR. Previous kinetic and structural studies characterized HIV-1 PR-acetyl-pepstatin and XMRV PR-pepstatin A complexes and suggested dramatically different binding modes. Interaction energies were calculated for the possible binding modes and suggested a strong preference for the one-inhibitor binding mode for HIV-1 PR-acetyl-pepstatin and the two-inhibitor binding mode for XMRV PR-pepstatin A interactions. Comparison of the molecular models suggested that in the case of XMRV PR the relatively unfavorable interactions at S3' and the favorable interactions at S4 and S4' sites with the statine residues may shift the ground state binding towards the two-inhibitor binding mode, whereas the single molecule ground state binding of statines to the HIV-1 PR appear to be more favorable. The preferred single molecular binding to HIV-1 PR allows the formation of the transition state complex, represented by substantially better binding constants. Intriguingly, the crystal structure of the complex of acetyl-pepstatin with XMRV PR has shown a mixed type of binding: the unusual binding mode of two molecules of the inhibitor to the enzyme, in a mode very similar to the previously determined complex with pepstatin A, together with the classical binding mode found for HIV-1 PR. The structure is thus in good agreement with the very similar interaction energies calculated for the two types of binding.
| S-EPMC6290463 | biostudies-literature
Project description:In this study, we gain insight into the extracellular proteolytic system of Sulfolobus solfataricus grown on proteinaceous substrates, providing further evidence that acidic proteases were specifically produced in response to peptide-rich media. The main proteolytic component was the previously isolated SsMTP (Sulfolobus solfataricus multi-domain thermopsin-like protease), while the less abundant (named SsMTP-1) one was purified, characterized and identified as the sso1175 gene-product. The protein revealed a multi-domain organization shared with the cognate SsMTP with a catalytic domain followed by several tandemly-repeated motifs. Moreover, both enzymes were found spread across the Crenarchaeota phylum and belonging to the thermopsin family, although segregated into diverse phylogenetic clusters. SsMTP-1 showed a 75-kDa molecular mass and was stable in the temperature range 50-90 °C, with optimal activity at 70 °C and pH 2.0. Serine, metallo and aspartic protease inhibitors did not affect the enzyme activity, designating SsMTP-1 as a new member of the pepstatin-insensitive aspartic protease family. The peptide-bond-specificity of SsMTP-1 in the cleavage of the oxidized insulin B chain was uncommon amongst thermopsins, suggesting that it could play a distinct, but cooperative role in the protein degradation machinery. Interestingly, predictions of the transmembrane protein topology of SsMTP and SsMTP-1 strongly suggest a possible contribution in signal-transduction pathways.
| S-EPMC3958906 | biostudies-literature
Project description:1. Several peptides containing either of the sequences -Phe(NO2)-Trp- and -Phe(NO2)-Phe- and an uncharged hydrophilic group were synthesized, and the steady-state kinetics of their hydrolysis by pig pepsin (EC 3.4.23.1) and chicken liver cathepsin D (EC 3.4.23.5) were determined. Despite the presence of a hydrophilic group to increase substrate solubility, it was not possible to achieve the condition [S]0 much greater than Km, and, in some cases, only values of kcat./Km could be determined by measuring the first-order rate constant when [S]0 much less than Km. 2. Occupancy of the P2 and P3 sites considerably enhanced the specificity constant, and alanine was more effective than glycine at site P2. 3. The specificity constants for the hydrolysis by pepsin of those substrates in the present series that contain an amino acid residue at site P3 are considerably lower than for comparable substrates containing a cationic group. This difference does not apply to cathepsin D. 4. Hydrolyses with cathepsin D commonly exhibited a lag phase, and a possible explanation for this is given.
| S-EPMC1154347 | biostudies-other
Project description:1. Pepstatinyl-cystamine was synthesized. The disulphide bond was cleaved and the pepstatin-bound thiol was made to react with monobromobimane. The fluorescent N-pepstatinyl-S-bimanyl-2-aminoethanethiol was purified. 2. Human cathepsin D showed tight binding of the bimane-labelled pepstatin at pH 3.5. The titration curves were used to determine the apparent dissociation constant, KD; values of approx. 1 x 10(-10) M were obtained. 3. Gel-chromatographic experiments showed that, like that of pepstatin, the binding of N-pepstatinyl-S-bimanyl-1-aminoethanethiol to cathepsin D was strongly pH-dependent. Binding was seen at pH 5.0, but could not be demonstrated at pH 7.4. 4. Cultured human synovial cells were fixed and incubated with the fluorescent inhibitor at pH 5.0 or pH 7.4. When examined by fluorescence microscopy the cells stained at pH 5.0 showed a punctate perinuclear distribution of bimane fluorescence. By contrast, the cells stained at pH 7.4 showed no fluorescence. 5. The distribution of cathepsin D, determined by indirect immunofluorescence at pH 7.4, closely resembled that of the fluorescent inhibitor seen at pH 5.0. 6. We conclude that N-pepstatinyl-S-bimanyl-2-aminoethanethiol is a fluorescent probe selective for the active conformation of cathepsin D.
| S-EPMC1163417 | biostudies-other