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PubMed2002

Novel inhibition of porcine pepsin by a substituted piperidine. Preference for one of the enzyme conformers.

Pepsin inhibition by 3-alkoxy-4-arylpiperidine (substituted piperidine; (3R,4R)-3-(4-bromobenzyloxy)-4-[4-(2-naphthalen-1-yl-2-oxo-ethoxy)phenyl]piperidine) has been studied using steady-state kinetic and pre-equilibrium binding methods. Data were compared with pepstatin A, a well known competitive inhibitor of pepsin. Steady-state analysis reveals that the substituted piperidine likewise behaves as a competitive inhibitor. Pre-equilibrium binding studies indicate that the substituted piperidine can displace a fluorescently labeled statine inhibitor from the enzyme active site. Simulation of the stopped-flow fluorescence transients provided estimates of the K(d) values of 1.4 +/- 0.2 microm and 39 +/- 2 nm for the piperidine and the fluorescently labeled statine, respectively. The effects of combinations of these two inhibitors resulted in a series of parallel lines when plotted by the method of Yonetani and Theorell (Yonetani, T., and Theorell, H. (1964) Arch. Biochem. Biophys. 106, 234-251), suggesting that the two inhibitors bind in a mutually exclusive fashion to pepsin. Fitting of the entire data set to the appropriate equation yielded an alpha factor of 8 +/- 1. The magnitude of this factor ( infinity > alpha > 1) can be explained by a conformational distinction between the enzyme species that bind each inhibitor. The effects of pH on the inhibition constants for pepstatin A and the substituted piperidine also suggest that the inhibitors bind to distinct conformational forms of the enzyme. No inhibition by the piperidine was observed at acidic pH, while pepstatin A inhibition is maximal at low pH values. Inhibition by the piperidine was maximal when a group with pK 4.8 +/- 0.2 was deprotonated and another group with pK 5.9 +/- 0.2 was protonated. Most likely these two groups are the catalytic aspartates with perturbed ionization properties as a result of a significant and unique conformational change. Taken together, these data suggest that the enzyme can readily interconvert between two conformers, one capable of binding substrate and pepstatin A and the other capable of binding the substituted piperidine.

Animals↗
PubMed2005

Cathepsin D and H2O2 stimulate degradation of thioredoxin-1: implication for endothelial cell apoptosis.

Cathepsin D (CatD) is a lysosomal aspartic proteinase and plays an important role in the degradation of proteins and in apoptotic processes induced by oxidative stress, cytokines, and aging. All of these stimuli are potent inducers of endothelial cell apoptosis. Therefore, we investigated the role of CatD in endothelial cell apoptosis and determined the underlying mechanisms. Incubation with 100-500 microm H2O2 for 12 h induced apoptosis in endothelial cells. To determine a role for CatD, we co-incubated endothelial cells with the CatD inhibitor pepstatin A. Pepstatin A as well as genetic knock down of CatD abolished H2O2-induced apoptosis. In contrast, overexpression of CatD wild type but not a catalytically inactive mutant of CatD (CatDD295N) induced apoptosis under basal conditions. To gain insights into the underlying mechanisms, we investigated the effect of CatD on reactive oxygen species (ROS) formation. Indeed, knocking down CatD expression reduced H2O2-induced ROS formation and apoptosis. The major redox regulator in endothelial cells is thioredoxin-1 (Trx), which plays a crucial role in apoptosis inhibition. Thus, we hypothesized that CatD may alter Trx protein levels and thereby promote formation of ROS and apoptosis. Incubation with 100 microm H2O2 for 6 h decreased Trx protein levels, whereas Trx mRNA was not altered. H2O2-induced Trx degradation was inhibited by pepstatin A and genetic knock down of CatD but not by other protease inhibitors. Incubation of unstimulated cell lysates with recombinant CatD significantly reduced Trx protein levels in vitro, which was completely blocked by pepstatin A pre-incubation. Overexpression of CatD reduced Trx protein in cells. Moreover, H2O2 incubation led to a translocation of Trx to the lysosomes prior to the induction of apoptosis. Taken together, CatD induces apoptosis via degradation of Trx protein, which is an essential anti-apoptotic and reactive oxygen species scavenging protein in endothelial cells.

Aging↗
PubMed1989

Renin inhibitor and converting enzyme inhibitors suppress vascular angiotensin II.

The direct effects of a renin inhibitor, N-acetyl-pepstatin and five angiotensin converting enzyme inhibitors, captopril and the active diacid forms of enalapril, ramipril, cilazapril, and CS-622, on the vascular renin-angiotensin system were examined in isolated perfused rat mesenteric arteries. Vascular renin activity and angiotensin II (Ang II) released into the perfusate were determined. Infusion of N-acetyl-pepstatin (5 X 10(-8)-5 X 10(-6) M) suppressed vascular renin activity and Ang II release dose dependently. Isoproterenol (10(-6) M) induced a 135 +/- 30% increase in Ang II release from the basal value. N-Acetyl-pepstatin (5 X 10(-6) M) suppressed isoproterenol-induced Ang II release. Infusions of 5 X 10(-6) M captopril and the diacid forms of enalapril, ramipril, cilazapril, and CS-622 by themselves had little effect on Ang II release, but concomitant infusion of isoproterenol with these angiotensin converting enzyme inhibitors significantly decreased Ang II release (71 +/- 21%, 51 +/- 40%, 8 +/- 21%, 69 +/- 24%, and 44 +/- 29% increase, respectively, from the basal values). These results indicate that N-acetyl-pepstatin suppresses the vascular renin-angiotensin system. This effect may in part contribute to the hypotensive actions of renin inhibitors. Although angiotensin converting enzyme inhibitors also suppress locally generated Ang II, the mechanism and physiological significance still remain to be clarified.

Angiotensin II↗
PubMedDate not supplied

Analysis of the endocytic pathway upon intracellular transport of IgG molecules through Fc receptors.

The uniformly distributed Fc receptors (FcRs) on the surface of many cell types are involved in a variety of immune reactions by non-specifically facilitating the entry of antigen-specific IgG molecules to the cell. Such reactions may be beneficial to the organism when foreign antigens are involved, or harmful in cases of self antigens and viruses. In order to avoid the IgG-mediated self antigen presentation or viral infection in autoimmunity and viral attack respectively, we attempt in this study to inhibit the intracellular transport of antibodies. This blockage, however, implies: efficacy of inhibition, inability of de novo exocytosis of the internalised antibody and finally maintenance of normal cell growth and morphology. We thus concentrate our interest on the endocytic pathway followed by a neutralising antibody in murine trophoblast cells where we try to inhibit antibody intracellular transport by various agents according to the criteria set above. In our model-system, IFN-gamma, upon induction of FcRs, facilitates endocytosis of the anti-p21ras antibody which blocks in turn the IFN-gamma-induced surface class II major histocompatibility complex (MHC) expression. Using various intracellular transport inhibitors, we study the required conditions by which these compounds cancel the inhibitory action of anti-p21ras and allow induction of class II MHC molecules by IFN-gamma. The effectiveness of the inhibitors in a ranking order is shown as following: monodansyl cadaverine > didansyl cadaverine > pepstatin A > leupeptin > NH4Cl > brefeldin A > ZPCK > TPCK. From these inhibitors, only brefeldin A, leupeptin, pepstatin and ZPCK do not allow exocytosis of the antibody in the culture medium and only didansyl cadaverine, pepstatin and leupeptin maintain cell viability and morphology. However, by sequential elimination based on this study's established criteria, only pepstatin A and leupeptin are shown to be effective inhibitors to specific antibody intracellular transport, protecting also the cell's viability and physiology.

Animals↗
PubMed1985

Macrophage endosomes contain proteases which degrade endocytosed protein ligands.

Rabbit alveolar macrophages rapidly internalize and degrade mannosylated bovine serum albumin (125I-mannose-BSA). Trichloroacetic acid-soluble degradation products appear in the cells as early as 6 min after uptake at 37 degrees C, and in the extracellular medium after 10 min. Incubation of endocytic vesicles containing this ligand in isotonic buffers at pH 7.4 + ATP resulted in intravesicular proteolysis, which was inhibited by monensin, nigericin, or ammonium chloride. At pH 5.0, degradation proceeded rapidly and was abolished by lysis of the vesicles with 0.1% Triton X-100. Readdition of lysosomes to the incubation mixture did not increase the rate of prelysosomal degradation. Proteolysis of 125I-mannose-BSA was optimal at pH 4.5, and inhibited by low concentrations of the cathepsin D inhibitor pepstatin A. After subcellular fractionation of the macrophages on Percoll gradients, 125I-mannose-BSA sedimented with prelysosomal vesicles and was not transported to secondary lysosomes. Addition of pepstatin A to extracellular medium during internalization of prebound 125I-mannose-BSA partially inhibited degradation of ligand, and resulted in transfer of undegraded 125I-mannose-BSA to lysosomes after 20 min. Using 125I-bovine serum albumin as a substrate for the protease in the presence of 0.1% Triton X-100, we have shown that as much as 36% of the total pepstatin A-sensitive activity sediments with nonlysosomal membranes. After intraendosomal iodination using lactoperoxidase, a labeled protease was isolated by affinity chromatography on pepstatin-agarose. The labeled protease, which had a subunit size of 46 kDa, was detected in endocytic vesicles after 5 min of internalization. These results suggest that a cathepsin D-like protease is responsible for the degradation of 125I-mannose-BSA in macrophages, and that this ligand is degraded in a prelysosomal vesicle.

Ammonium Chloride↗
PubMed1985

Comparative effects of cathepsin inhibitors on rat embryonic development in vitro. Evidence that cathepsin D is unimportant in the proteolytic function of yolk sac.

The effects of two proteinase inhibitors, leupeptin and pepstatin on the development of 9.5-day rat conceptuses in vitro has been studied. All cultures were of 48 h duration and the inhibitors were present throughout the entire period. When pepstatin was added to the culture medium (5-25 micrograms/ml) conceptuses developed and grew to an extent that did not differ from untreated controls. However, leupeptin (1-4 micrograms/ml) caused severe growth retardation and abnormal development of conceptuses. The effects of the two inhibitors on the hydrolysis of 125I-labelled BSA and haemoglobin by homogenates of 10.5-day yolk sac indicated the biochemical basis for the differential toxic effects of the two inhibitors on development. Leupeptin was highly inhibitory of the degration of both substrates whereas pepstatin caused no inhibition of 125I-labelled BSA hydrolysis, and only a slight inhibition of haemoglobin hydrolysis. These observations demonstrate that cathepsin D, a lysosomal aspartic proteinase that is specifically inhibited by pepstatin is not involved in yolk-sac-mediated protein utilization by early organogenesis-phase conceptuses and that lysosomal cysteine proteinases, specifically inhibited by leupeptin, are of paramount importance in this yolk sac function.

Animals↗
PubMed2000

Characterization of endopeptidase activity of tripeptidyl peptidase-I/CLN2 protein which is deficient in classical late infantile neuronal ceroid lipofuscinosis.

Endopeptidase activities of the CLN2 gene product (Cln2p)/tripeptidyl peptidase I (TPP-I), purified from rat spleen, were studied using the synthetic fluorogenic substrates. We designed and constructed decapeptides, based on the known sequence cleavage specificities of bacterial pepstatin-insensitive carboxyl proteases (BPICP). MOCAc-Gly-Lys-Pro-Ile-Pro-Phe-Phe-Arg-Leu-Lys(Dnp)r-NH(2) is readily hydrolyzed by Cln2p/TPP-I (K(cat)/K(m) = 7.8 s(-1) mM(-1)). The enzyme had a maximal activity at pH 3.0 for an endopeptidase substrate, but at pH 4.5 with respect to tripeptidyl peptidase activity. Both endopeptidase and tripeptidyl peptidase activities were strongly inhibited by Ala-Ala-Phe-CH(2)Cl, but not inhibited by tyrostatin, an inhibitor of bacterial pepstatin-insensitive carboxyl proteases, pepstatin, or inhibitors of serine proteases. Fibroblasts from classical late infantile neuronal ceroid lipofuscinosis patients have less than 5% of the normal tripeptidyl peptidase activity and pepstatin-insensitive endopeptidase activity. Cln2p/TPP-I is a unique enzyme with both tripeptidyl peptidase and endopeptidase activities for certain substrate specificity.

Amino Acid Sequence↗
PubMed2003

Roles of cathepsins in reperfusion-induced apoptosis in cultured astrocytes.

Astrocytic apoptosis may play a role in the central nervous system injury. We previously showed that reperfusion of cultured astrocytes with normal medium after exposure to hydrogen peroxide (H(2)O(2))-containing medium causes apoptosis. This study examines the involvement of the lysosomal enzymes cathepsins B and D in the astrocytic apoptosis. Reperfusion after exposure to H(2)O(2) caused a marked increase in caspase-3 and cathepsin D activities and a marked decrease in cathepsin B activity. Pepstatin A, an inhibitor of cathepsin D, and acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspart-1-aldehyde (Ac-DMQD-CHO), a specific inhibitor of caspase-3, blocked the H(2)O(2)-induced decrease in cell viability and DNA ladder formation in cultured rat astrocytes. The (L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl)-L-isoleucyl-L-proline methyl ester (CA074 Me), a specific inhibitor of cathepsin B, did not affect the H(2)O(2)-induced cell injury. On the other hand, CA074 Me decreased cell viability with DNA ladder formation when cultured in the presence of Ac-DMQD-CHO. This caspase-independent apoptosis was attenuated by the addition of the cathepsin D inhibitor pepstatin A. Caspase-3 like activity was markedly inhibited by Ac-DMQD-CHO and partially by pepstatin A. Pepstatin A and CA074 Me inhibited cathepsin B and cathepsin D activities, respectively, in the presence and absence of Ac-DMQD-CHO. These results suggest that cathepsins B and D are involved in astrocytic apoptosis: cathepsin D acts as a death-inducing factor upstream of caspase-3 and the caspase-independent apoptosis is regulated antagonistically by cathepsins B and D.

Animals↗
PubMed1999

A mechanism of TNFR type II (75 kDa) "shedding" in macrophages.

Adherent macrophage populations derived from monocytes isolated from peripheral blood were evaluated for their ability to "shed" the membrane-associated receptor for TNF-alpha (TNFR) following exposure to a calcium ionophor (A23187) and a synthetic chemotactic peptide (fMLP) reagent. A soluble fraction of TNFR was detected in "cell-free" supernatant produced by stimulated macrophage populations applying 125I-TNF-alpha and biotinylated TNF-alpha ligand-binding analysis (96-well format) in combination with conventional autoradiographic techniques. Approximate molecular weight of the shed TNFR glycoprotein fraction was estimated to be 75 kDa based on interpretation of nondenaturing PAGE gels transferred laterally onto sheets of nitrocellulose membrane subsequently probed by ligand-binding analysis applying 125I-TNF-alpha and biotinylated TNF-alpha as detection modalities. Immunorecognition techniques were also employed to detect TNFR fragments shed from macrophages using biotinylated anti-TNFR Type II (75 kDa) monoclonal antibody in combination with conjugated strepavidin:HRPO and a chemiluminescent substrate reagent. In an effort to identify the class of enzyme directly mediating TNFR Type II (75 kDa) shedding, a spectrum of carboxyl- (e.g., aspartate), hydroxyl- (e.g., serine), thiol (e.g., cysteine), and metalo- (e.g., Ca2+, Mg2+) protease-inhibiting agents were evaluated. Experimental findings implied that a carboxy (aspartate) peptidase, and possibly to a lesser extent, serine (hydroxyl), and thiol (cysteine) peptidases participate in macrophage TNFR Type II (75 kDa) shedding phenomena. Subsequent investigations demonstrated that the carboxy (aspartate) peptidase cathepsin-D promoted liberation of TNFR Type II (75 kDa) in unactivated populations of adherent macrophages. In an effort to complement these observations, a protein fraction with presumed carboxy (aspartate) protease activity was isolated from the cell-free supernatant generated by activated populations of adherent macrophages using immobilized pepstatin-A beaded agarose. Exposure of unstimulated populations of adherent macrophages to the partially purified pepstatin-A binding protein fractions resulted in the liberation of a soluble TNFR Type II (75 kDa) fragment based on interpretation of ligand-binding and immunorecognition analysis of samples developed by SDS-PAGE/PAGE format and transferred onto sheets of nitrocellulose membrane. The molecular weight of the macrophage pepstatin-A binding protein fraction was estimated to be 47-52 kDa with lesser bands also visible at approximately 26-32 kDa, and 100 kDa based on SDS-PAGE analysis. Nondenaturing hemoglobin-PAGE substrate gel analysis of protein fractions possessing pepstatin-A binding-avidity detected a protease with a molecular weight of approximately 47-52 kDa that proteolytically digested hemoglobin, in addition to a synthetic cathepsin-D specific peptide substrate. Collective interpretation of these experimental findings directly corresponds with many of the physical (molecular) and functional (biochemical) characteristics known to be associated with the leukocyte carboxy (aspartate) peptidase cathepsin-D, which is a non-metaloprotease known to exert relatively limited proteolytic activity.

Animals↗
PubMed1991

Proteolytic processing of thyroglobulin by extracts of thyroid lysosomes.

The release of T4 and T3 from the prohormone thyroglobulin (Tg) occurs in thyroid lysosomes. To examine the role of cathepsin-B, -D, and -L, the three major endopeptidases in this process, we incubated rabbit [125I]Tg, labeled in vivo, with lysosomal extracts from human thyroids. Iodopeptide formation was evaluated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after short term incubations (20-45 min), while iodoamino acid release was assessed by paper chromatography after long term incubations (8 and 24 h). Using pepstatin to inhibit cathepsin D, Z-Phe-Ala-CHN2 to inhibit both cathepsin B and L, and Z-Phe-Phe-CHN2 to selectively inhibit cathepsin L, we obtained the following results: 1) blocking of all three endopeptidases reduced both iodopeptide formation in short term experiments and iodoamino acid release in long term experiments by 80-90%; 2) iodopeptide formation was reduced by 85% with Z-Phe-Ala-CHN2, by 56% with Z-Phe-Phe-CHN2, and by 26% with pepstatin; 3) iodoamino acid release was reduced by 60-80% with Z-Phe-Ala-CHN2 and by 40-50% with either Z-Phe-Phe-CHN2 or pepstatin at 8 h, but by less than 20% at 24 h; pepstatin and Z-Phe-Phe-CHN2 together reduced iodoamino acid release by 80% and 60% at 8 and 24 h, respectively. Limited hydrolysis of Tg by lysosomal enzymes produced at least eight peptide fragments of less than 100,000 mol wt. Three of these, together representing 32% of the 125I released, resulted from cleavages in the C-terminal region of Tg corresponding to residues 2487, 2393, and 2390 of cDNA-derived human Tg. Several other peptides, together containing 38% of the 125I released, included the N-terminus of Tg. These C-terminal and N-terminal fragments contained three of Tg's four major hormonogenic sites, but none of the cleavage sites fell close to the hormone sites themselves. We conclude that 1) the formation of discrete iodopeptides precedes the release of iodothyronines and iodotyrosines from Tg; 2) the cysteine proteinases are more important than cathepsin D in this process; and 3) these endopeptidases selectively cleave Tg to favor the production of hormone-containing intermediates for subsequent processing by exopeptidases.

Chromatography, High Pressure Liquid↗
PubMed1998

Chemical modification studies of Rhizomucor miehei protease: evidence for the role of basic amino acids in enzyme catalysis.

The effect of chemical modification on milk clotting and proteolytic activities of aspartyl protease obtained from Rhizomucor miehei NRRL 3500 was examined in the absence and the presence of its specific inhibitor pepstatin A. The effect on the ratio of milk clotting activity (MC) to proteolytic activity (PA), an index of the quality of milk clotting proteases was also determined. Modification of the enzyme with trinitrobenzenesulfonic acid, diethylpyrocarbonate and phenylglyoxal produced an increase in the ratio of MC/PA, while modification with 2- hydroxy-5-nitrobenzyl bromide did not affect the ratio. Modification with N-acetylimidazole resulted in a marginal increase in MC/PA ratio. Protection using pepstatin A during modification with phenylglyoxal, N-acetylimidazole and 2-hydroxy-5-nitrobenzyl bromide, protected both MC and PA. In the case of modification by diethylpyrocarbonate, pepstatin A protected only MC. Pepstatin A did not protect both the activities on the modification of the enzyme by trinitrobenzene sulfonic acid. These observations indicate the presence of arginine, tyrosine and tryptophan at the catalytic site of the enzyme, for eliciting MC and PA of the enzyme. In general, modification of the positively charged residues increases the MC/PA ratio of the enzyme. In addition the modified lysine residues responsible for the inactivation of the enzyme were not involved in the active site of the enzyme. Thus the lysine residues might have a secondary role in enzyme catalysis. Further, histidine at the catalytic site was found to be exclusively involved in milk clotting activity. The enzyme with modified histidine residues were more susceptible to autocatalysis, indicating that histidine residues protect the enzyme against autolysis.

Aspartic Acid Endopeptidases↗
PubMed1979

The isolation of leukokinin-H and leukokininogen from human ascites fluid; their properties and role.

The leukokinin-leukokininogen system is a pathological kinin generating system which is catalyzed by acid proteases present in neoplastic cells, white cells and even normal tissues. The components of the human system including leukokinin-H and leukokininogen have now been isolated and characterized. Very specific protease inhibitors of the system such as pepstatin have been found and are now known to prevent "in vivo" the formation of pathological fluids such as neoplastic ascites. Strong evidence has been previously published and additional evidence has been presented here which indicates that pepstatin's actions are related to the inhibition of cathepsin-D in vivo and the inhibition of leukokinin formation. Both leukokinins and leukokininogens have been clearly defined and shown to differ from bradykinin and human bradykininogens. This clearly demonstrates the presence in pathological systems of a kinin-generating system which is separate and distinct from the bradykinin generating system. The importance of the leukokinin-leukokininogen system in disease would seem to be very great. The finding that pepstatin can inhibit the system in vivo opens the way for studies of pepstatin and related protease inhibitors as therapeutic agents in neoplastic disease and protease mediated inflammatory disorders.

Amino Acids↗
PubMed1977

Therapeutic trials in muscular dystrophy. III. Studies of microbial proteinase inhibitors in murine dystrophy.

The microbial antiproteinases--antipain, leupeptin, and pepstatin--have been reported to inhibit the degeneration of chicken dystrophic muscle in tissue culture. Trials of antipain and pepstatin, and of leupeptin and pepstatin administered subcutaneously in murine muscular dystrophy, failed to produce evidence of benefit. It is suggested that these antiproteinases cannot pass through an intact sarcolemma into muscle fibers. Further studies with liposomes may allow these agents to enter muscle fibers.

Animals↗
PubMed1999

The effect of inhibitor binding on the structural stability and cooperativity of the HIV-1 protease.

The effects of the peptide inhibitor acetyl pepstatin on the structural stability of the HIV-1 protease have been measured by high sensitivity calorimetric techniques. At 25 degrees C and pH 3.6, acetyl pepstatin binds to HIV-1 protease with an affinity of 1.6 x 10(7 )M-1 and an enthalpy of 7.3 +/- 0.5 kcal/mol, indicating that binding is not favored enthalpically and that the favorable Gibbs energy originates from a large positive entropy. Since the binding of acetyl pepstatin is associated with a negative change in heat capacity (-450 cal/K*mol) the association reaction becomes enthalpically favored at temperatures higher than 40 degrees C. The presence of the inhibitor stabilizes the dimeric structure of the protease in a fashion that can be quantitatively described by a set of thermodynamic linkage equations. The combination of titration and differential scanning calorimetry provides an accurate way of determining binding constants for high affinity inhibitors that cannot be determined by titration calorimetry alone. A structure-based thermodynamic analysis of the binding process indicates that the stabilization effect is not distributed uniformly throughout the protease molecule. The binding of the inhibitor selectively stabilizes those conformational states in which the binding site is formed, triggering a redistribution of the state probabilities in the ensemble of conformations populated under native conditions. As a result, the stability constants for individual residues do not exhibit the same change in magnitude upon inhibitor binding. Residues in certain areas of the protein are affected significantly whereas residues in other areas are not affected at all. In particular, inhibitor binding has a significant effect on those regions that define the binding site, especially the flap region which becomes structurally stable as a result of the additional binding free energy. The induced stabilization propagates to regions not in direct contact with the inhibitor, particularly to the strand between residues Pro9 and Ala22 and the helix between Arg87 and Gly94. On the other hand, the stability of the strand between Asp60 and Leu76 is not significantly affected by inhibitor binding. The structural distribution of binding effects define cooperative pathways within the protease molecule. Proteins 1999;36:147-156.

Algorithms↗
PubMed1995

Western blotting and isoform analysis of cathepsin D from normal and malignant human breast cell lines.

Cathepsin D from normal (Hs578Bst) and malignant (MCF7, MDA-MB-231) breast cell lines has been characterized with regard to its kinetic properties, activity levels, precursor and processed M(r) forms, and isoform composition. Normal cell cathepsin D appears to have a more neutral pH optimum (pH 3.5) than the cancer cell line (pH 3.0-3.2) and greater activity between pH values of 4.0 to 4.5. The two cancer cell lines have approximately 1.5 to 2.0-fold increased total acid protease activity and 2 to 3-fold increased pepstatin-inhibitable protease activity (i.e. cathepsin D) when compared to the normal breast cell line. Western blotting indicates that a major processed form of cathepsin D for all three cell lines occurs at 31 kDa. The cancer cell lines contain significant amounts of cathepsin D precursors of 47 and 42 kDa whereas the normal cell line contains little if any of these precursors. Isoelectric focusing indicates that the normal cell line contains approximately 50% of its total acid protease activity at pIs above 4 whereas the cancer cell lines contain 70-80% of their protease activity at such pIs. In addition, the cancer cell lines contain two to three major isoforms between pIs of 5.5 and 6.3 which were not present in the normal cell line. The isoforms from pI values of 5.5 to 7.3 for all three cell lines are 100% pepstatin-inhibitable. In addition, Western blot analysis indicates that these isoforms contain the processed 31 kDa form of cathepsin D. The combined results indicate that the two breast cancer cell lines are similar to biopsied malignant breast tissue in exhibiting altered acid protease isoform profiles with increased relative amounts of pepstatin-inhibitable and immunoreactive acid protease activity (cathepsin D) compared to normal breast tissue or cells.

Aspartic Acid Endopeptidases↗
PubMed1989

Evidence that aspartic proteinase is involved in the proteolytic processing event of procathepsin L in lysosomes.

Our recent studies have shown that cathepsin L is first synthesized as an enzymatically inactive proform in endoplasmic reticulum and is successively converted into an active form during intracellular transport and we postulated that aspartic proteinases would be responsible for the intracellular propeptide-processing step of procathepsin L accompanied by the activation of enzyme (Y. Nishimura, T. Kawabata, and K. Kato (1988) Arch. Biochem. Biophys. 261, 64-71). To better understand this proposed mechanism, we investigated the effect of pepstatin, a potent inhibitor of aspartic proteinases, on the intracellular processing kinetics of cathepsin L analyzed by pulse-chase experiments in vivo with [35S]methionine in the primary cultures of rat hepatocytes. In the pepstatin-treated cells, the proteolytic conversion of cellular procathepsin L of 39 kDa to the mature enzyme was significantly inhibited and considerable amounts of proenzyme were found in the cell after 5-h chase periods. Further, the subcellular fractionation experiments demonstrated that the intracellular processing of procathepsin L in the high density lysosomal fraction was significantly inhibited and that considerable amounts of the procathepsin L form were still observed in the light density microsomal fraction after 2 h of chase. These results suggest that pepstatin treatment caused a significant inhibitory effect on the intracellular processing and also on the intracellular movement of procathepsin L from the endoplasmic reticulum to the lysosomes. These findings provide the first evidence showing that aspartic proteinase may play an important role in the intracellular proteolytic processing and activation of lysosomal cathepsin L in vivo. Therefore, we suggest that cathepsin D, a major lysosomal aspartic proteinase, is more likely to be involved in this proposed model in the lysosomes.

Animals↗
PubMed1987

Specific proteolysis regulates fusion between endocytic compartments in Xenopus oocytes.

We examined the role of proteolytic ligand modification in endosomal targeting using vitellogenin (VTG) uptake by Xenopus oocytes as a model system. Non-cleavable VTG is internalized, but does not appear in yolk platelets. We identified two inhibitors of VTG processing into the yolk proteins: the ionophore monensin and pepstatin A, a specific inhibitor of cathepsin D. Pepstatin neither affected ligand binding and internalization, nor inhibited the degradation of nonspecifically incorporated proteins, whereas monensin inhibited all of these processes. Inhibiting VTG processing prevented its deposition into yolk platelets by strongly interfering with endosome-yolk platelet fusion. Monensin treatment resulted in morphologically abnormal endosomes, while pepstatin only inhibited VTG cleavage and the subsequent fusion of endosomes with yolk platelets. Since VTG cleavage is initiated prior to its deposition in platelets, we postulate that ligand proteolysis could be necessary for normal endosomal targeting.

Animals↗
PubMed1991

The use of protease inhibitors in experimental allergic neuritis.

In experimental allergic neuritis (EAN) break-down of myelin is attributed to macrophages, which among other factors contain and secrete proteases. In vitro studies have shown that cathepsin D, an acidic aspartyl endopeptidase, and plasmin can degrade myelin proteins. In order to elucidate a potential therapeutic effect of protease inhibitors we treated Lewis rats, immunized with bovine peripheral nervous system myelin, with epsilon-amino-caproic acid (EACA) or pepstatin. EACA or pepstatin was administered twice daily by intraperitoneal injection beginning on day 6 postimmunization or from the onset of disease (on day 12) through day 24. Compared to saline-treated controls, animals treated with either of the inhibitors showed delayed development of clinical signs and electrophysiological abnormalities. Maximal severity and the further course of disease, however, were not different in control and treated groups. Immunohistological evaluation of sciatic nerve specimens on day 24 postimmunization showed equal numbers of cells positive for ED1 (macrophages) and cathepsin D in all animal groups. There was also no difference in the spontaneous proteolytic activity of the sciatic nerve homogenates at pH 2.8, 5.0, and 7.4. Incubation of the homogenates with pepstatin, however, significantly reduced proteolytic activity at pH 2.8 and 5.0, while EACA had no effect at any pH tested. These results imply that treatment to limit the infiltration of cathepsin D-positive cells or to reduce the induction or activity of cathepsin D may provide a therapeutic avenue for treating inflammatory demyelination of the peripheral nervous system.

Aminocaproic Acid↗