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PubMed2006

Function of liver activation-regulated chemokine/CC chemokine ligand 20 is differently affected by cathepsin B and cathepsin D processing.

Chemokine processing by proteases is emerging as an important regulatory mechanism of leukocyte functions and possibly also of cancer progression. We screened a large panel of chemokines for degradation by cathepsins B and D, two proteases involved in tumor progression. Among the few substrates processed by both proteases, we focused on CCL20, the unique chemokine ligand of CCR6 that is expressed on immature dendritic cells and subtypes of memory lymphocytes. Analysis of the cleavage sites demonstrate that cathepsin B specifically cleaves off four C-terminally located amino acids and generates a CCL20(1-66) isoform with full functional activity. By contrast, cathepsin D totally inactivates the chemotactic potency of CCL20 by generating CCL20(1-55), CCL20(1-52), and a 12-aa C-terminal peptide CCL20(59-70). Proteolytic cleavage of CCL20 occurs also with chemokine bound to glycosaminoglycans. In addition, we characterized human melanoma cells as a novel CCL20 source and as cathepsin producers. CCL20 production was up-regulated by IL-1alpha and TNF-alpha in all cell lines tested, and in human metastatic melanoma cells. Whereas cathepsin D is secreted in the extracellular milieu, cathepsin B activity is confined to cytosol and cellular membranes. Our studies suggest that CCL20 processing in the extracellular environment of melanoma cells is exclusively mediated by cathepsin D. Thus, we propose a model where cathepsin D inactivates CCL20 and possibly prevents the establishment of an effective antitumoral immune response in melanomas.

Cathepsin B↗
PubMed1991

Primary structure of bovine cathepsin S. Comparison to cathepsins L, H, B and papain.

The primary structure of bovine cathepsin S was determined by combining results of protein and peptide sequencing with the sequence deduced from nucleic acid sequencing. Using polymerase chain reaction (PCR) technology, cDNA clones commencing at amino acid 22 of the mature enzyme and continuing through the 3' untranslated region of bovine cathepsin S mRNA were isolated and sequenced. The open reading frame in these overlapping clones correctly predicts the determined amino acid sequence of 13 tryptic peptides derived from purified bovine spleen cathepsin S. The deduced amino acid sequence shows that mature bovine cathepsin S consists of 217 amino acids corresponding to a molecular weight of 23.7 kDa. Cathepsin S belongs to the papain superfamily of lysosomal cysteine proteinases and shares 41% identity with papain. Amino acid sequence identities of bovine cathepsin S to human cathepsins L, H, and B are 56%, 47% and 31% respectively.

Amino Acid Sequence↗
PubMed2005

Lysosomotropism of basic cathepsin K inhibitors contributes to increased cellular potencies against off-target cathepsins and reduced functional selectivity.

The lysosomal cysteine protease cathepsin K is a target for osteoporosis therapy. The aryl-piperazine-containing cathepsin K inhibitor CRA-013783/L-006235 (1) displays greater than 4000-fold selectivity against the lysosomal/endosomal antitargets cathepsin B, L, and S. However, 1 and other aryl-piperazine-containing analogues, including balicatib (10), are approximately 10-100-fold more potent in cell-based enzyme occupancy assays than against each purified enzyme. This phenomenon arises from their basic, lipophilic nature, which results in lysosomal trapping. Consistent with its lysosomotropic nature, 1 accumulates in cells and in rat tissues of high lysosome content. In contrast, nonbasic aryl-morpholino-containing analogues do not exhibit lysosomotropic properties. Increased off-target activities of basic cathepsin K inhibitors were observed in a cell-based cathepsin S antigen presentation assay. No potency increases of basic inhibitors in a functional cathepsin K bone resorption whole cell assay were detected. Therefore, basic cathepsin K inhibitors, such as 1, suffer from reduced functional selectivities compared to those predicted using purified enzyme assays.

Animals↗
PubMed1996

Cytosolic proteolysis of tau by cathepsin D in hippocampus following suppression of cathepsins B and L.

Incubation of cultured hippocampal slices with an inhibitor [N-CBZ-L-phenylalanyl-L-alanine-diazomethyl ketone (ZPAD)] of cathepsins B and L resulted in the degradation of high molecular weight isoforms of tau protein and the production of a 29-kDa tau fragment (tau 29). A tau antibody that is sensitive to the phosphorylated state of its epitopes did not recognize tau proteins or the tau 29 fragment in slices that had been treated with a protein phosphatase inhibitor. This strongly suggests that the tau fragment was located in an extralysosomal compartment accessible to kinases and phosphatases. tau 29 exhibited a significant capacity for binding to microtubules and thus has the potential for interfering with normal tau-tubulin interactions. Three lines of evidence indicated that ZPAD-induced tau proteolysis was mediated by cathepsin D: (a) slices treated with the inhibitor had markedly elevated levels of cathepsin D in both lysosomal and extralysosomal compartments; (b) co-incubation of cathepsin D and tau at neutral pH resulted in a loss of intact tau proteins and production of a 28-kDa fragment; and (c) the lysosomotropic drug chloroquine blocked ZPAD-induced increases in mature cathepsin D, and this was accompanied by a suppression of ZPAD-induced tau proteolysis. Changes in lysosomal hydrolases and cytoskeletal perturbations occur during brain aging. The present results suggest that the enzymatic and structural effects are related and, more specifically, are linked by alterations in the concentration and localization of cathepsin D. The tau fragments with microtubule binding capacity generated by cathepsin D could also be a source for the small polypeptides found in association with age-related pathological features.

Animals↗
PubMed2005

Recombinant human cathepsin X is a carboxymonopeptidase only: a comparison with cathepsins B and L.

The S1 and S2 subsite specificity of recombinant human cathepsins X was studied using fluorescence resonance energy transfer (FRET) peptides with the general sequences Abz-Phe-Xaa-Lys(Dnp)-OH and Abz-Xaa-Arg-Lys(Dnp)-OH, respectively (Abz=ortho-aminobenzoic acid and Dnp=2,4-dinitrophenyl; Xaa=various amino acids). Cathepsin X cleaved all substrates exclusively as a carboxymonopeptidase and exhibited broad specificity. For comparison, these peptides were also assayed with cathepsins B and L. Cathepsin L hydrolyzed the majority of them with similar or higher catalytic efficiency than cathepsin X, acting as an endopeptidase mimicking a carboxymonopeptidase (pseudo-carboxymonopeptidase). In contrast, cathepsin B exhibited poor catalytic efficiency with these substrates, acting as a carboxydipeptidase or an endopeptidase. The S1' subsite of cathepsin X was mapped with the peptide series Abz-Phe-Arg-Xaa-OH and the enzyme preferentially hydrolyzed substrates with hydrophobic residues in the P1' position.

Carboxypeptidases↗
PubMed1995

Proteolysis of IGFBPs by cathepsin D in vitro and in cathepsin D-deficient mice.

Affinity-purified lysosomal protease cathepsin D cleaved recombinant human IGFBP-1 to -5 in fragments of defined sizes, while IGFBP-6 was not degraded. To assess the role of cathepsin D for proteolytic processing of IGFBP in vivo, serum from cathepsin D-deficient mice and conditioned media from cathepsin D-deficient fibroblasts and organ explants were analyzed. No differences for the pattern and level of IGFBPs were detected. When conditioned media from fibroblasts were incubated at acid pH, proteolysis of IGFBP-1 and -4 was observed only in media derived from cathepsin D-expressing cells. Additional experiments showed that the proteolysis of IGFBP-4 is mediated by cathepsin D and not by a protease activated by cathepsin D. The IGFBP-4 degrading activities in media from organ explants from cathepsin D-deficient mice were found to be sensitive to inhibitors of aspartyl and cysteine proteases. The data indicate that different classes of acid pH-dependent proteases can contribute to the regulation of IGFBP-4 abundance.

Animals↗
PubMedDate not supplied

Cathepsin D content in colorectal cancer. Correlation with cathepsin D activity and other biological parameters: a preliminary report.

Cathepsin D content and activity were determined in matched paired sets of colorectal tumor tissue and normal mucosa and correlated with a number of biological and clinical parameters. Significantly higher cathepsin D activity was measured in tumor cytosol compared to paired normal mucosa (p < 0.02), in Dukes' stage A tumors compared to Dukes' B and C (p < 0.05), in tumors < 5 cm compared to those > 5 cm, or in tumors with a low proliferation rate compared to those with a high proliferation rate (p < 0.05). Moreover, significant differences in enzyme activity between tumor tissue and paired normal mucosa were observed in node-positive and G2 tumors (p < 0.05). No significant correlation between cathepsin D activity and other biological parameters was found. Further, no differences in cathepsin D content between tumor tissue and paired normal mucosa were observed except in Dukes' stage A tumors (p < 0.02). A significantly increased cathepsin D content was also observed in tumors > 5 cm compared to tumors < 5 cm (p < 0.01). No relationship between tumor cathepsin D content and clinical stage was detected. However, a significant correlation (p < 0.05) was observed between the tumor-specific content of this enzyme and tumor grade. Finally, there was no relationship between tumor-specific cathepsin D activity and content (r = -0.27, p = 0.23). These data suggest that cathepsin D activity rather than content correlates with the malignant progression of colorectal cancer. This phenomenon should be taken into consideration when clinical studies are undertaken to assess the potential prognostic value of proteolytic enzymes involved in tumor progression.

Adult↗
PubMed1992

Characterization of recombinant rat cathepsin B and nonglycosylated mutants expressed in yeast. New insights into the pH dependence of cathepsin B-catalyzed hydrolyses.

The cysteine proteinase rat cathepsin B was expressed in yeast in an active form and was found to be heterogeneously glycosylated at the consensus sequence for N-linked oligosaccharide substitution. Purified enzyme fractions containing the highest levels of glycosylation were shown to have reduced activity. A glycosylation minus mutant constructed by site-directed mutagenesis (by changing the Ser to Ala in the consensus sequence) was still secreted by the yeast and was shown to be functionally identical with purified rat liver cathepsin B. Recombinant cathepsin B was used to further characterize the pH dependence of cathepsin B-catalyzed hydrolyses using 7-amido-4-methylcoumarin (AMC) and p-nitroaniline (pNA) substrates with arginine as the P1, and either arginine or phenylalanine as the P2 residue. The AMC and pNA groups give insights into the leaving group binding site (P') of cathepsin B. These studies show for the first time that at least seven dissociable groups are involved in substrate binding and hydrolysis in cathepsin B activity. Two of these groups, with pKa values of 6.9 and 7.7 in the recombinant enzyme, are in the leaving group binding site and are most likely His110 and His111. The same groups in rat liver cathepsin B have higher pKa values than in recombinant cathepsin B, but have identical function in the two enzymes. Two other groups are probably the active site Cys29 and His199 with pKa values of 3.6 and 8.6, respectively. A group with a pKa of 5.1 interacts with substrates containing Arg at P2, and the group is most likely Glu245. The remaining two groups, one with a pKa of about 4.9 and the other about 5.3, are most likely carboxyl residues possibly interacting with Arg at P1 in the substrate. The possible candidates on the basis of the x-ray structure are Asp22, Asp69, Glu171, and Glu122, all found within a 13 A radius from the active site thiol of Cys29.

Animals↗
PubMed1995

Comparative analysis of cathepsin L, cathepsin D, and collagenase messenger RNA expression in synovial tissues of patients with rheumatoid arthritis and osteoarthritis, by in situ hybridization.

OBJECTIVE: To compare the expression of cathepsin L, cathepsin D, and collagenase messenger RNA (mRNA) in synovial specimens from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: The expression of cathepsins L and D as well as collagenase mRNA in synovial tissues from 8 patients with RA, 6 patients with OA, and 2 patients with noninflamed joints was evaluated using in situ hybridization with digoxigenin-labeled RNA probes. RESULTS: Both RA and OA synovial tissue expressed cathepsins L and D as well as collagenase mRNA. The expression of the cathepsins was markedly higher in interstitial regions and, to some extent, in perivascular infiltrates of RA synovial tissue compared with OA specimens. CONCLUSION: Cathepsins L and D mRNA are expressed differently in RA and OA synovial tissues, supporting the concept that these enzymes may contribute to the influx of mononuclear cells into RA synovium. Moreover, the data reveal that the expression of collagenase and cathepsins in RA and OA synovial lining is otherwise largely similar, and suggest that the adhesion of synovial cells to cartilage mediates the invasive destructive process in RA.

Arthritis, Rheumatoid↗
PubMed1999

The contribution of N-terminal region residues of cystatin A (stefin A) to the affinity and kinetics of inhibition of papain, cathepsin B, and cathepsin L.

The affinity and kinetics of binding of three N-terminally truncated variants of the cysteine proteinase inhibitor cystatin A to cysteine proteinases were characterized. Deletion of Met-1 only minimally altered the inhibitory properties of the protein. However, deletion also of Ile-2 resulted in reduced affinities of 900-, >/=3-, and 200-fold for papain and cathepsins L and B, respectively. Further truncation of Pro-3 substantially increased the inhibition constants to approximately 0.5 microM for papain and cathepsin L and to 60 microM for cathepsin B, reflecting additionally 2 x 10(3)-, 2 x 10(4)-, and 400-fold decreased affinities, respectively. The reductions in affinity shown by the latter mutant indicate that the N-terminal region contributes about 40% of the total free energy of binding of cystatin A to cysteine proteinases. Moreover, Pro-3 and to a lesser extent Ile-2 are the residues responsible for this binding energy. The reduced affinities for papain and cathepsin L were due only to higher dissociation rate constants, whereas both lower association and higher dissociation rate constants contributed to the decreased affinity for cathepsin B. These differential effects indicate that the N-terminal portion of cystatin A primarily functions by stabilizing the complexes with enzymes having easily accessible active-site clefts, e.g., papain and cathepsin L. In contrast, the N-terminal region is required also for an initial binding of cystatin A to cathepsin B, presumably by promoting the displacement of the occluding loop and allowing facile interaction of the rest of the inhibiting wedge with the active-site cleft of the enzyme.

Animals↗
PubMed1998

Cathepsin D and cathepsin L activities in aortic aneurysm wall and parietal thrombus.

Deterioration of the aortic wall resulting in formation of aneurysms may be caused by increased activity of metalloproteases and lysosomal proteases. The aim of this work was the evaluation of cathepsin D and cathepsin L activities, and activities of inhibitors of cysteine cathepsins in the wall of aortic aneurysms and in parietal thrombus. Aortic aneurysms were obtained during operation. Aortas taken from organ donors and blood clots were used as control material. Activities of cathepsin D and cathepsin L in the aortic aneurysm wall and parietal thrombus were higher than in the control groups. The aneurysm wall showed lower activity of inhibitors of cysteine proteases than the normal aorta. Parietal thrombus had a higher level of cysteine protease inhibitor activity than blood clot. Cathepsin D and cathepsin L present in the aneurysm wall and in the parietal thrombus filling the aneurysm may act on proteins determining elasticity and mechanical resistance of arteries.

Aorta↗
PubMed2002

[Enzymatic activity of cathepsin B, cathepsin B and L, plasmin, trypsin and collagenase in hepatocellular carcinoma].

UNLABELLED: Hepatocellular carcinoma is one of the most common malignancies worldwide. Invasiveness of this tumour seems to be related to degradation of extracellular matrix. Such proteolytic enzymes as: cathepsin B and L, plasmin, collagenase and trypsin are thought to play a pivotal role in this process. Enzymatic activity depends on balance between enzymes and their inhibitors and--moreover--on interactions among these enzymes. The purpose of our study was to evaluate enzymatic activity of cathepsin B, cathepsin B and L, plasmin, collagenase and trypsin in patients with hepatocellular carcinoma in liver tissue and in peripheral blood. Then correlations between activity of enzymes (mentioned above) and clinical status, pathological findings and laboratory tests were assessed. Our study was conducted on 14 patients who underwent surgery because of hepatocellular carcinoma. Tissue samples were obtained during surgery from neoplastic area and from non-neoplastic area. Peripheral blood was withdrawn before surgery and within early post-operative period. Proteolytic activity of these enzymes was determined with use of fluorometric assay. Enzymatic activity in tissue samples was referred to protein concentration (BCA assay) and to DNA concentration (fluorometric assay). RESULTS: Proteolytic activity of plasmin and trypsin in neoplastic tissue were significantly lower as compared to non-neoplastic area of these patients (p = 0.0356; p = 0.0412, respectively). Activity of the remaining enzymes: cathepsin B, cathepsin B and L and collagenase did not differ significantly. No difference was demonstrated between activity of enzymes in peripheral blood withdrawn before surgery and in postoperative period. There was a statistically significant inverse correlation between serum AFP level and enzymatic activity of cathepsin B, cathepsin B and L and collagenase in tumor tissue. Lower activity of all investigated enzymes was observed in tumor tissue of HBV related hepatocellular carcinoma in comparison with the remaining tissue samples. Correlation between patients age and activity of enzymes was not statistically significant. CONCLUSION: Although the evaluation of presented enzymatic profile did not allow for the assessment of associations between investigated enzymes, our results demonstrated correlations between proteolytic activity of enzymes and serum AFP level, viral status, but it requires further investigations.

Adult↗
PubMed1995

[Role and behavior of cathepsin B and cathepsin L in gastric cancer].

Cathepsin B and cathepsin L--cysteine proteinases--may play an important role in cancer invasion and metastasis. The authors determined tissue antigen concentrations of cathepsins, using the ELISA method, in 25 patients with gastric cancer (17 males, 8 females, mean age 62, range 31-84). They evaluated the possible relationship that these proteinases may have with the presence of metastases, differentiation and histotype. Significantly higher cathepsin B and cathepsin L antigen levels were found: 1. in gastric cancer tissues vs. normal tissues distant from tumors (CATB: p < 0.05, CATL: p < 0.005); 2. in diffuse vs. intestinal type cancers (p < 0.05); 3. in patients with poorly vs. well-differentiated cancers (p < 0.05); in gastric cancers with vs. without metastasis (p < 0.05). Their results confirm that cathepsin B and L play an important role in gastric cancer invasion and metastasis. Considering the significantly higher cathepsins detected in cancers with metastasis, a poor differentiation and of diffuse histotype, these proteinases could be useful for identification gastric cancer patients with a poor prognosis.

Adult↗
PubMed2001

Human recombinant pro-dipeptidyl peptidase I (cathepsin C) can be activated by cathepsins L and S but not by autocatalytic processing.

Human dipeptidyl peptidase I was expressed in the insect cell/baculovirus system and purified in its active (rhDPPI) and precursor (pro-rhDPPI) forms. RhDPPI was very similar to the purified enzyme (hDPPI) with respect to glycosylation, enzymatic processing, oligomeric structure, CD spectra, and catalytic activity. The precursor, which was a dimer, could be activated approximately 2000-fold with papain. Cathepsin L efficiently activated pro-rhDPPI in vitro at pH 4.5 (k(app) approximately 2 x 10(3) min(-)(1) M(-)(1)), and two cleavage pathways were characterized. The initial cleavage was within the pro region between the residual pro part and the activation peptide. Subsequently, the activation peptide was cleaved from the catalytic region, and the latter was cleaved into the heavy and light chains. Alternatively, the pro region was first separated from the catalytic region. Cathepsin S was a less efficient activating enzyme. Cathepsin B and rhDPPI did not activate pro-rhDPPI, and the proenzyme was incapable of autoactivation. Incubation of both pro-rhDPPI and rhDPPI with cathepsin D resulted in degradation. Cystatin C and stefins A and B inhibited rhDPPI with K(i) values in the nanomolar range (K(i) = 0.5-1.1 nM). The results suggest that cathepsin L could be an important activator of DPPI in vivo and that cathepsin D and possibly the cystatins may contribute to DPPI downregulation.

Amino Acid Sequence↗
PubMed2000

Functions of cathepsin K in bone resorption. Lessons from cathepsin K deficient mice.

Cathepsin K is a cysteine proteinase expressed predominantly in osteoclasts. Cathepsin K cleaves key bone matrix proteins and is believed to play an important role in degrading the organic phase of bone during bone resorption. Pycnodysostosis, an autosomal recessive osteosclerosing skeletal disorder has recently been shown to result from mutations in the cathepsin K gene. Cathepsin K deficient mice generated by targeted disruption of this proteinase phenocopy many aspects of pycnodysostosis. They display an osteopetrotic phenotype with excessive trabeculation of the bone-marrow space accompanied by an altered ultrastructural appearance of the cathepsin K deficient osteoclasts. These cells also demonstrate an impaired resorptive activity in vitro. In contrast to other forms of osteopetrosis, which are due to disrupted osteoclastogenesis, cathepsin K deficiency is associated with an inhibition of osteoclast activity. Taken together the phenotype of cathepsin K knockout mice underlines the importance of this proteinase in bone remodelling.

Animals↗
PubMed1990

Molecular cloning of rat precursor cathepsin H and the expression of five lysosomal cathepsins in normal tissues and in a rat carcinosarcoma.

1. A rat cathepsin H cDNA was isolated from a rat liver cDNA library with synthetic oligonucleotide probes. 2. DNA sequence analysis indicated that it codes for rat preprocathepsin H. 3. Using this clone together with the cDNA for cathepsins B, D, L and S as probes, the expression of five major lysosomal proteinases was investigated in ten different normal rat tissues and in a rat carcinoma. 4. The common feature of their expression is that the five cathepsins have relatively high mRNA levels in lung and kidney, suggesting that they all play important roles in organs engaged in active protein metabolism. 5. In other tissues, the concentrations of the five cathepsin mRNAs are significantly different. This may indicate that their expressions are differentially regulated and that they may have specialized functions in specific tissues. 6. The cathepsin B mRNA level is at least 2.5-fold higher in the rat W256-carcinoma than in any of the normal rat tissues surveyed. 7. In contrast, the mRNA levels for the other four cathepsins show no comparable elevations. 8. This finding is consistent with previous observations reporting a correlation between cathepsins B expression and malignant tumors.

Amino Acid Sequence↗
PubMed1994

Neutralization of the activity of a Fasciola hepatica cathepsin L proteinase by anti-cathepsin L antibodies.

Fasciola hepatica secretes a cathepsin L proteinase that is suggested to play an in vivo role in immunoprotection since the enzyme can cleave host immunoglobulin. In the present report, rabbit anti-cathepsin L IgG was shown to bind to the cathepsin L enzyme and inhibit its ability to cleave IgG molecules. Cathepsin L can prevent the antibody-mediated attachment of eosinophils to newly excysted juveniles in in vitro assays; however, if anti-cathepsin L IgG are mixed with the cathepsin L prior to the addition of the enzyme to the assay, eosinophils attach to the newly excysted juveniles. Thus it is possible to prepare antibodies that can bind and disrupt the biological activity of the F. hepatica cathepsin L.

Animals↗
PubMed1995

Cathepsin D in primary breast carcinoma: adverse prognosis is associated with expression of cathepsin D in stromal cells.

The immunohistochemical expression of the aspartyl protease enzyme cathepsin D was examined in a consecutive series of 103 primary operable breast carcinomas with the polyclonal antibody NCL-CDp. Expression of cathepsin D was identified within the epithelial and stromal components of all tumours examined. No significant associations of increased cathepsin D expression in the epithelial tumour component with conventional prognostic indices such as tumour size, grade, lymph node stage, or patient survival were identified. However, significant associations of increased stromal cathepsin D expression and high tumour grade, chi 2 = 11.40 (df = 2), p = 0.003; increased tendency to local recurrence, chi 2 = 6.87 (df = 1), p = 0.009; regional recurrence, chi 2 = 7.44 (df = 1), p = 0.006; poorer disease free survival, chi 2 = 14.9 (df = 1), p = 0.0001; and poorer overall patient survival, chi 2 = 6.90 (df = 1), p = 0.0086, were identified. Cathepsin D expression is present in all breast tumours. Stromal cathepsin D expression is a neglected immunohistochemical prognostic parameter which could explain some of the previous apparently conflicting reports concerning the effect on patient prognosis of biochemical (i.e. total) and immunohistochemical estimations of cathepsin D in breast cancers.

Breast Neoplasms↗