Immunochemical localisation of cathepsin S, cathepsin L and MHC class II-associated p41 isoform of invariant chain in human lymph node tissue

Antigen presentation by MHC class II molecules requires cysteine proteases (CP) for two convergent proteolytic processes: stepwise degradation of the invariant chain (Ii) and generation of immunogenic peptides. Their activity is controlled by intracellular CP inhibitors, including presumably the p41 isoform of invariant chain (p41 Ii), which is in vitro a potent inhibitor of cathepsin L but not of cathepsin S. In order to evaluate the inhibitory potential of p41 Ii in antigen-presenting cells (APC), these three proteins were stained in lymph node tissue using specific monoclonal and polyclonal antibodies. The most abundant labelling was observed in subcapsular (cortical) and trabecular sinuses of the lymph node. In this area the most frequent APC were macrophages, as confirmed by the CD68 cell marker. Using confocal fluorescence microscopy, co-localisation of p41 Ii with cathepsin S, but not with cathepsin L was found in these cells. Our results are consistent with the hypothesis that cathepsin S participates in degradation of the invariant chain, but they do not support the association between cathepsin L and p41 Ii in APC.     

Identification of cathepsin B,D and H in the larval midgut of colorado potato beetle,Leptinotarsa decemlineata say (Coleoptera: Chrysomelidae)

The larval midgut of the Colorado beetle, Leptinotarsa decemlineata contains cathepsin B, D and H activity detected by use of haemoglobin, synthetic substrates specific for each enzyme, pH at which the substrate was maximally hydrolysed and effects of potential activators and inhibitors on proteolytic activity. Cysteine proteases cathepsin B, and H were activated by thiol compounds and inhibited by iodoacetamide, TLCK and epoxysuccinyl-leucyl-amido(guanidino)butane (E-64) a cysteine specific proteinase inhibitor. Cathepsin B was distinguished from H by hydrolysis of benzoyloxycarbonyl-Ala-Arg-Arg-methoxynaphthylamide, a cathepsin B specific substrate and inhibition of substrate hydrolysis by leupeptin. Cathepsin H activity, detected using the specific substrate arginine-naphthylamide, was insensitive to leupeptin. Cathepsin D had maximal activity at pH 4.5 and was inhibited by pepstatin, an aspartic proteinase inhibitor.     

Detection of procathepsin D in rat milk

The presence of procathepsin D, a zymogen of the soluble lysosomal aspartic proteinase cathepsin D, was detected in rat milk using Western blot analysis and assay of proteolytic activity in acidic buffers. No other forms of cathepsin D were found. Two different polyclonal anti-procathepsin D antibodies were used for immunochemical detection of procathepsin D. Both antibodies we found to recognize rat procathepsin D. Proteolytic activity in acidic buffers was detected using a fluorogenic substrate specific for cathepsin D and was abolished by pepstatin A, a specific inhibitor of aspartic proteinases. This study represents third demonstration of presence of procathepsin D in mammal breast milk. Potential sources and physiological functions are discussed.     

Identification and discrimination of extracellularly active cathepsins B and L in high-invasive melanoma cells

We established a novel protocol for lithium dodecyl sulfate (LDS) gelatin zymography, which operates under reducing conditions and at a slightly acidic pH value (6.5). This zymographic assay is based on polyacrylamide gel electrophoresis and facilitates the electrophoretic separation of human cathepsins in an active state. By this technique, activity of purified human liver cathepsin B was detected at a concentration as low as 50 ng and was blocked only in the presence of the cysteine protease inhibitor E-64 and the specific cathepsin B inhibitor CA-074 but not by aspartate, serine, or matrix metalloprotease inhibitors. The method was applied to analyze cathepsin activities in cell culture supernatants of the high-invasive melanoma cell line MV3. Interestingly, LDS zymography of MV3 cell supernatants in combination with specific inhibitors of cathepsins B and L identified three forms of extracellularly active cathepsin B and two forms of proteolytically active cathepsin L. We herein describe the generation and biochemical significance of acidic LDS zymography. This novel method permits not only the enzymatic analysis of purified cysteine proteases but also the identification and discrimination of different cathepsin activities in biological fluids, cell lysates, or supernatants, especially of cathepsins B and L, which are closely linked to major inflammatory and malignant processes.     

Role of the prosegment of Fasciola hepatica cathepsin L1 in folding of the catalytic domain

The N-terminal propeptides of cysteine proteinases play regulatory roles in the folding and stability of their catalytic domains, as well as being potent and highly specific inhibitors of their parental mature enzymes. Cysteine proteinases play a major role in the biology of the parasitic trematode Fasciola hepatica; in particular, this organism secretes significant amounts of cathepsin L enzymes. The isolated propeptide of F. hepatica cathepsin L1 functioned as a chaperone for the mature enzyme in renaturation experiments. A double point mutation (N701/F721) within the GxNxFxD motif of the propeptide affected its conformation and markedly decreased its affinity for the mature enzyme. When this mutation was introduced into preprocathepsin L1 expressed in yeast, the secretion of active enzyme dropped dramatically. However, significant enzyme activity was recovered from the culture supernatants after denaturation and renaturation in the presence of native propeptide. Thus, the variant prosegment gave rise to an enzyme with altered conformation, which could be refolded to the active form with the assistance of the native propeptide.     

Intracellular proteolysis of pancreatic zymogens.

Activation of pancreatic digestive zymogens within the pancreatic acinar cell may be an early event in the development of pancreatitis. To detect such activation, an immunoblot assay has been developed that measures the relative amounts of inactive zymogens and their respective active enzyme forms. Using this assay, high doses of cholecystokinin or carbachol were found to stimulate the intracellular conversion of at least three zymogens (procarboxypeptidase A1, procarboxypeptidase B, and chymotrypsinogen 2) to their active forms. Thus, this conversion may be a generalized phenomenon of pancreatic zymogens. The conversion is detected within ten minutes of treatment and is not associated with changes in acinar cell morphology; it has been predicted that the lysosomal thiol protease, cathepsin B, may initiate this conversion. Small amounts of cathepsin B are found in the secretory pathway, and cathepsin B can activate trypsinogen in vitro; however, exposure of acini to a thiol protease inhibitor (E64) did not block this conversion. Conversion was inhibited by the serine protease inhibitor, benzamidine, and by raising the intracellular pH, using chloroquine or monensin. This limited proteolytic conversion appears to require a low pH compartment and a serine protease activity. After long periods of treatment (60 minutes), the amounts of the active enzyme forms began to decrease; this observation suggested that the active enzyme forms were being degraded. Treatment of acini with E64 reduced this late decrease in active enzyme forms, suggesting that thiol proteases, including lysosomal hydrolases, may be involved in the degradation of the active enzyme forms. These findings indicate that pathways for zymogen activation as well as degradation of active enzyme forms are present within the pancreatic acinar cell.     

Proteolytic activation of internalized cholera toxin within hepatic endosomes by cathepsin D

We have defined the in vivo and in vitro metabolic fate of internalized cholera toxin (CT) in the endosomal apparatus of rat liver. In vivo, CT was internalized and accumulated in endosomes where it underwent degradation in a pH-dependent manner. In vitro proteolysis of CT using an endosomal lysate required an acidic pH and was sensitive to pepstatin A, an inhibitor of aspartic acid proteases. By nondenaturating immunoprecipitation, the acidic CT-degrading activity was attributed to the luminal form of endosomal cathepsin D. The rate of toxin hydrolysis using an endosomal lysate or pure cathepsin D was found to be high for native CT and free CT-B subunit, and low for free CT-A subunit. On the basis of IC(50) values, competition studies revealed that CT-A and CT-B subunits share a common binding site on the cathepsin D enzyme, with native CT and free CT-B subunit displaying the highest affinity for the protease. By immunofluorescence, partial colocalization of internalized CT with cathepsin D was confirmed at early times of endocytosis in both hepatoma HepG2 and intestinal Caco-2 cells. Hydrolysates of CT generated at low pH by bovine cathepsin D displayed ADP-ribosyltransferase activity towards exogenous Gsalpha protein suggesting that CT cytotoxicity, at least in part, may be related to proteolytic events within endocytic vesicles. Together, these data identify the endocytic apparatus as a critical subcellular site for the accumulation and proteolytic degradation of endocytosed CT, and define endosomal cathepsin D an enzyme potentially responsible for CT cytotoxic activation.     

Autolytic degradation of chicken intestinal proteins

Data on the exhaustive degradation of chicken intestinal proteins by endogenous proteases, which could be utilized as a means to prepare protein hydrolysate, is reported in the present paper. Chicken intestine possesses proteolytic activities (cathepsin B, D, H, L, aminopeptidases and alkaline proteases) comparable to that in organ tissues like liver and spleen, which could degrade the tissue proteins extensively. The autolytic degradation was found to be optimum at pH 2.5 and 60 degrees C. Analysis by SDS-PAGE showed a time dependent degradation of proteins to low molecular weight (<10 kDa) products. Kinetic studies employing specific inhibitors indicated that the degradation (90-94%) of proteins at acidic pH is governed largely by pepstatin sensitive proteases. The acidic extract of the tissue was found to hydrolyse albumin, casein and soybean proteins efficiently. Results point to the possible application of tissue autolysis for obtaining protein hydrolysates from chicken intestine. Chicken intestine could also serve as a potential source of much needed proteolytic enzymes for food and pharmaceutical applications.     

Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors

Proteolytic activities in alfalfa weevil (Hypera postica) larval midguts have been characterized. Effects of pH, thiol activators, low-molecular weight inhibitors, and proteinase inhibitors (PIs) on general substrate hydrolysis by midgut extracts were determined. Hemoglobinolytic activity was highest in the acidic to mildly acidic pH range, but was maximal at pH 3.5. Addition of thiol-activators dithiothreitol (DTT), 2-mercaptoethanol (2-ME), or L-cysteine had little effect on hemoglobin hydrolysis at pH 3.5, but enhanced azocaseinolytic activity two to three-fold at pH 5.0. The broad cysteine PI E-64 reduced azocaseinolytic activity by 64% or 42% at pH 5 in the presence or absence of 5 mM L-cysteine, respectively. Inhibition by diazomethyl ketones, Z-Phe-Phe-CHN(2) and Z-Phe-Ala-CHN(2), suggest that cathepsins L and B are present and comprise approximately 70% and 30% of the cysteine proteolytic activity, respectively. An aspartyl proteinase component was identified using pepstatin A, which inhibited 32% (pH 3.5, hemoglobin) and 50% (pH 5, azocasein) of total proteolytic activity. This activity was completely inhibited by an aspartyl proteinase inhibitor from potato (API), and is consistent with the action of a cathepsin D-like enzyme. Hence, genes encoding PIs with specificity toward cathepsins L, B and D could potentially be effective for control of alfalfa weevil using transgenic plants.     

Design of FRET-based GFP probes for detection of protease inhibitors

In this study, tandem Green fluorescent protein (GFP) fusion proteins were designed to detect proteolytic activity of thrombin based on the principle of fluorescence resonance energy transfer (FRET). The thrombin-specific recognition sequence, LVPR, was strategically placed in between a cyan-emitting mutant of the green fluorescent protein and an enhanced yellow-emitting fluorescent protein to allow thrombin-specific cleavage with detectable changes of FRET signal. A 4.6-fold increase of fluorescence emission ratio was observed upon addition of thrombin. This FRET-based probe was further tested for dose-dependent effects of thrombin specific inhibitor, hirudin. Our result showed a nice correlation between fluorescence emission ratios and concentrations of hirudin with subnanomolar sensitivity. We propose that FRET-based GFP probes can be used for high-throughput screening of protease inhibitors.     

Development and validation of a simple cell-based fluorescence assay for dipeptidyl peptidase 1 (DPP1) activity

Dipeptidyl peptidase 1 (DPP1) (EC 3.4.14.1; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.     

Intracellular proteolytic activity of cathepsin B is associated with capillary-like tube formation by endothelial cells in vitro

The lysosomal cysteine protease cathepsin B is implicated in degradation of extracellular matrix (ECM), a crucial step in a variety of physiological and pathological processes, including tumor dissemination and angiogenesis. In this study, we analyzed the contribution of extracellular and intracellular cathepsin B activity on the formation of capillary-like tubular structures by human umbilical vein endothelial cells (HUVECs) grown on Matrigel matrix, using general and specific cysteine protease inhibitors. We demonstrated, by confocal assay using quenched fluorescent protein substrate DQ-collagen IV, that endothelial cells degrade ECM both intracellularly and pericellularly. Intracellular cathepsin B activity detected by degradation of Z-Arg-Arg cresyl violet substrate was co-localized with the products of DQ-collagen IV degradation in the perinuclear region and in the capillary-like tubular structures. Treatment of cells with membrane-permeable CA-074 Me effectively abolished intracellular cathepsin B activity, and resulted in reduced tube length (32.3+/-9.4% at 10 microM), total tubule area (49.6+/-12.4% at 10 microM), and the number of branch points of tubules (47.5+/-7.7% at 10 microM) in a dose-dependent manner. In contrast, CA-074 (0.1-10 microM), a membrane-impermeable cathepsin B specific inhibitor, general cysteine protease inhibitors chicken cystatin (5 microM) and E-64 (10 microM), and the metalloprotease inhibitor Minocycline (10 microM) showed no significant inhibitory effect in our angiogenesis model. These results show that, besides multiple regulatory molecules, intracellular cathepsin B also contributes to the neovascularization process and should be considered as a potential therapeutic target.     

Activation mechanism of erythrocyte cathepsin E. evidence for the occurrence of the membrane-associated active enzyme

Activation of the erythrocyte cathepsin E located on the cytoplasmic surface of the membrane in a latent form was studied in stripped inside-out membrane vesicles prepared from human erythrocyte membranes. Incubation of the vesicles at 40 degrees C at pH 4 resulted in increased degradation of the membrane proteins, especially band 3. This proteolysis was selectively inhibited by the inclusion of pepstatin (isovaleryl-Val-Val-statyl-Ala-statine) or H 297 [Pro-Thr-Glu-Phe(CH2-NH)Nle-Arg-Leu] in the incubation mixtures, indicating that cathepsin E, as the only aspartic proteinase in erythrocytes, is responsible for the proteolysis. Two potential active-site-directed inhibitors of aspartic proteinases, pepstatin and H 297, were used to prove the occurrence of the membrane-associated active enzyme. To minimize potential errors arising from non-specific binding, the concentrations of the inhibitors used in the binding assay (pepstatin, 5 x 10(-8) M; H 297, 1 x 10(-5) M) were determined by calibration for purified and membrane-associated cathepsin E. The inhibition of the membrane-associated cathepsin E by each inhibitor, which showed the binding of the inhibitor to the activated enzyme, was temperature- and time-dependent. The binding of each inhibitor to the enzyme on the exposed surface of the membrane at pH 4 was highly specific, saturable, and reversible. The present study thus provides the first evidence that cathepsin E tightly bound to the membrane is converted to the active enzyme in the membrane-associated form, and suggests that this enzyme may be responsible for the degradation of band 3.     

A principal role for the proteasome in endoplasmic reticulum-associated degradation of misfolded intracellular cystic fibrosis transmembrane conductance regulator.

Endoplasmic reticulum-associated degradation of misfolded cystic fibrosis transmembrane conductance regulator (CFTR) protein is known to involve the ubiquitin-proteasome system. In addition, an ATP-independent proteolytic system has been suggested to operate in parallel with this pathway and become up-regulated when proteasomes are inhibited (Jensen, T. J., Loo, M. A., Pind, S., Williams, D. B., Goldberg, A. L., and Riordan, J. R. (1995) Cell 83, 129-135). In this study, we use two independent techniques, pulse-chase labeling and a noninvasive fluorescence cell-based assay, to investigate the proteolytic pathways underlying the degradation of misfolded CFTR. Here we report that only inhibitors of the proteasome have a significant effect on preventing the degradation of CFTR, whereas cell-permeable inhibitors of lysosomal degradation, autophagy, and several classes of protease had no measurable effect on CFTR degradation, when used either alone or in combination with the specific proteasome inhibitor carbobenzoxy-l-leucyl-leucyl-l-leucinal (MG132). Our results suggest that ubiquitin-proteasome-mediated degradation is the dominant pathway for disposal of misfolded CFTR in mammalian cells and provide new mechanistic insight into endoplasmic reticulum-associated degradation.     

A homologue of cathepsin L identified in conditioned medium from Sf9 insect cells

Gelatin zymography revealed the presence of proteolytic activity in conditioned medium (CM) from a serum-free, non-infected Spodoptera frugiperda, Sf9 insect cell culture. Two peptidase bands at about 49 and 39 kDa were detected and found to be proform and active form of the same enzyme. The 49-kDa form was visible on zymogram gels in samples of CM taken on days 4 and 5 of an Sf9 culture, while the 39-kDa form was seen on days 6 and 7. On basis of the inhibitor profile and substrate range, the enzyme was identified as an Sf9 homologue of cathepsin L, a papain-like cysteine peptidase. After lowering the pH of Sf9 CM to 3.5, an additional peptidase band at 22 kDa appeared. This peptidase showed the same inhibitor profile, substrate range and optimum pH (5.0) as the 39-kDa form, indicating that Sf9 cathepsin L has two active forms, at 39 and 22 kDa. Addition of the cysteine peptidase inhibitor E-64c to an Sf9 culture inhibited all proteolytic activities of Sf9 cathepsin L but did not influence the proliferation of Sf9 cells.     

Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants

Green fluorescent protein (GFP) makes it possible for organelles and protein transport pathways to be visualized in living cells. However, GFP fluorescence has not yet been observed in the vacuoles of any organs of higher plants. We found that the fluorescence of a vacuole-targeted GFP was stably observed in the vacuoles of transgenic Arabidopsis plants under dark conditions, and that the fluorescence rapidly disappeared under light conditions. The vacuolar GFP was rapidly degraded within 1 h in the light, especially blue light. An inhibitor of vacuolar type H+-ATPase, concanamycin A, and an inhibitor of papain-type cysteine proteinase, E-64d, abolished both the light-dependent disappearance of GFP fluorescence and GFP degradation in the vacuoles. An in vitro assay showed that bacterially expressed GFP was degraded by extracts of Arabidopsis cultured-cell protoplasts at an acidic pH in the light. These results suggest that blue light induced a conformational change in GFP, and the resulting GFP in the vacuole was easily degraded by vacuolar papain-type cysteine proteinase(s) under the acidic pH. The light-dependent degradation accounts for the failure to observe GFP fluorescence in the vacuoles of plant organs. Our results show that stable GFP-fluoresced vacuoles are achieved by transferring the plants from the light into the dark before inspection with a fluorescent microscope. This might eliminate a large hurdle in studies of the vacuolar-targeting machinery and the organ- and stage-specific differentiation of endomembrane systems in plants.     

Progesterone-dependent cathepsin L proteolytic activity in cat uterine flushings

Sequence analysis of a cDNA clone for the progesterone-dependent protein (PDP) of the cat uterus revealed that PDP may be cathepsin L. This study was undertaken to directly measure the cathepsin L activity in uterine flushings from pregnant and ovariectomized steroid-treated animals in order to confirm that PDP is cathepsin L. Optimum activity toward the substrate Z-Phe-Arg-NMec was observed at a pH of 5-6. Z-Phe-Phe-CHN2, a specific inhibitor of cathepsin L, significantly inhibited the proteolytic activity present in uterine flushings. Immunoabsorption of PDP from uterine flushings obtained from progesterone (P)-treated cats reduced cathepsin L proteolytic activity to levels observed in ovariectomized and estradiol (E2)-treated animals. In E2-primed and E2 + P-treated animals, proteolytic activity in uterine flushings was detectable after 7 days and peaked after 11-13 days of E2 + P treatment. This proteolytic activity was also dramatically increased before implantation (10-12 days after coitus) in pregnant cats. Thus, our data indicate that changes in cathepsin L activity in uterine flushings are correlated with changes in PDP, the uterine protein synthesized and released from the epithelial cells of the deep uterine glands. PDP, via its cathepsin L proteolytic activity, may play a role in the implantation process.     

A homogeneous caspase-3 activity assay using HTRF technology

Caspases are cysteine proteases presenting a conserved active site that cleaves protein substrates at a highly specific position. They are involved in different aspects of the active cell death pathway. Most of them act through proteolytic degradations of cellular components. This paper describes the assay development, assay validation, and screening for inhibitors of this enzyme, which could be potential drug candidates. The assay uses homogeneous time-resolved fluorescence based on energy transfer from europium cryptate as donor to cross-linked allophycocyanin as acceptor (XL665). A double-tagged substrate, biotinyl-epsilon-aminocaproyl-L-aspartyl-L-glutamyl-L-valyl-Laspartyl-L-alanyl-L-propyl-N(epsilon)-(2,4-dinitrophenyl)-L-lysine-amide (biotin-X-DEVDAPK(dnp)-NH(2)), is conjugated with streptavidin cryptate and anti-dnp-XL665 monoclonal antibody. The close proximity between donor and acceptor induces a specific time-resolved fluorescence signal. In the presence of enzyme activity, the substrate cleavage induces an unlinking of the two fluorescent probes and, subsequently, the disappearance of the specific signal as a result of loss of proximity. Experiments to optimize the reagent concentration, incubation times, precision, reproducibility, and robustness are discussed in comparison with a fluorometric method.     

Depolarization after resonance energy transfer (DARET): a sensitive fluorescence-based assay for botulinum neurotoxin protease activity

The DARET (depolarization after resonance energy transfer) assay is a coupled Förster resonance energy transfer (FRET)–fluorescence polarization assay for botulinum neurotoxin type A or E (BoNT/A or BoNT/E) proteolytic activity that relies on a fully recombinant substrate. The substrate consists of blue fluorescent protein (BFP) and green fluorescent protein (GFP) flanking SNAP-25 (synaptosome-associated protein of 25 kDa) residues 134–206. In this assay, the substrate is excited with polarized light at 387 nm, which primarily excites the BFP, whereas emission from the GFP is monitored at 509 nm. Energy transfer from the BFP to the GFP in the intact substrate results in a substantial depolarization of the GFP emission. The energy transfer is eliminated when the fluorescent domains separate on cleavage by the endopeptidase, and emission from the directly excited GFP product fragment is then highly polarized, resulting in an overall increase in polarization. This increase in polarization can be monitored to assay the proteolytic activity of BoNT/A and BoNT/E in real time. It allows determination of the turnover rate of the substrate and the kinetic constants (V_max and k_cat) based on the concentration of cleaved substrate determined directly from the measurements using the additivity properties of polarization. The assay is amenable to high-throughput applications.