Cathepsin L, but not cathepsin B, is a potential kininogenase

Although papain-like enzymes are strongly inhibited by their natural tight-binding inhibitors of the cystatin superfamily, cathepsins B and L may still retain some residual proteolytic activity toward Z-Phe-Arg-AMC in the presence of an excess of kininogen. This activity is abolished by adding E-64 or chicken cystatin. Cathepsins B and L show a single band of gelatinolytic activity when subjected to gelatin-SDS-PAGE. Adding high Mr kininogen, low Mr kininogen, T-kininogen, or chicken cystatin to cathepsin L results in additional intense bands of enzyme activity corresponding to the protease-inhibitor complexes. Cathepsin B does not produce these additional bands. This gelatinolytic activity was inhibited by E-64, but not by EDTA, PMSF or Pefabloc. Cathepsin L also specifically generated kinins from high and low molecular weight kininogens in vitro, but cathepsin B did not. T-kininogen did not release any immunoreactive kinins when complexed with cathepsin L, as previously observed using tissue kallikreins. The ability of cathepsin L to generate vasoactive peptides raises the question of the physiological significance of this mechanism during inflammation.     

Decreased activity of cathepsins L+B and decreased invasive ability of PC3 prostate cancer cells

Cancer metastasis involves multiple factors, one of which is the production and secretion of matrix degrading proteases by the cancer cells. Many metastasizing cancer cells secrete the lysosomal proteases, cathepsins L and B, which implicates them in the metastatic process. Cathepsins L and B are regulated by endogenous cysteine proteinase inhibitors (CPI) known as cystatins. An imbalance between cathepsin L and/or B and cystatin expression/activity may be a characteristic of the metastatic phenotype. To determine whether cystatins can attenuate the invasive ability of PC3 prostate cancer cells, cells were transfected with a cDNA coding for chicken cystatin. Expression of chicken cystatin mRNA was determined by PCR analysis. Total cysteine proteinase inhibitory activity, cathepsins L+B activity, and invasion through a Matrigel® matrix were assessed. Stably transfected cells expressed the chicken cystatin mRNA and exhibited a significant decrease in secreted cathepsin L+B activity and a small increase in secreted cysteine proteinase inhibitor activity. The ability of cystatin transfected cells to invade the reconstituted basement membrane, Matrigel®, was attenuated compared to nontransfected cells or cells transfected with vector alone. We have demonstrated that the cysteine proteinases cathepsins L and B participate in the invasive ability of the PC3 prostate cancer cell line, and we discuss here the potential of using cysteine proteinase inhibitors such as the cystatins as anti-metastatic agents.     

Decreased activity of cathepsins L+B and decreased invasive ability of PC3 prostate cancer cells

Cancer metastasis involves multiple factors, one of which is the production and secretion of matrix degrading proteases by the cancer cells. Many metastasizing cancer cells secrete the lysosomal proteases, cathepsins L and B, which implicates them in the metastatic process. Cathepsins L and B are regulated by endogenous cysteine proteinase inhibitors (CPI) known as cystatins. An imbalance between cathepsin L and/or B and cystatin expression/activity may be a characteristic of the metastatic phenotype. To determine whether cystatins can attenuate the invasive ability of PC3 prostate cancer cells, cells were transfected with a cDNA coding for chicken cystatin. Expression of chicken cystatin mRNA was determined by PCR analysis. Total cysteine proteinase inhibitory activity, cathepsins L+B activity, and invasion through a Matrigel® matrix were assessed. Stably transfected cells expressed the chicken cystatin mRNA and exhibited a significant decrease in secreted cathepsin L+B activity and a small increase in secreted cysteine proteinase inhibitor activity. The ability of cystatin transfected cells to invade the reconstituted basement membrane, Matrigel®, was attenuated compared to nontransfected cells or cells transfected with vector alone. We have demonstrated that the cysteine proteinases cathepsins L and B participate in the invasive ability of the PC3 prostate cancer cell line, and we discuss here the potential of using cysteine proteinase inhibitors such as the cystatins as anti-metastatic agents.     

Invasion of ras-transformed breast epithelial cells depends on the proteolytic activity of cysteine and aspartic proteinases

It has been suggested that the lysosomal proteinases cathepsin B, L and D participate in tumour invasion and metastasis. Whereas for cathepsins B and L the role of active enzyme in invasion processes has been confirmed, cathepsin D was suggested to support tumour progression via its pro-peptide, rather than by its proteolytic activity. In this study we have compared the presence of active cathepsins B, L and D in ras-transformed human breast epithelial cells (MCF-10A neoT) with their ability to invade matrigel. In this cell line high expression of all three cathepsins was detected by immunofluorescence microscopy. The effect of proteolytic activity on cell invasion was studied by adding various natural and synthetic cysteine and aspartic proteinase inhibitors. The most effective compound was chicken cystatin, a general natural inhibitor of cysteine proteinases, (82.8+/-1.6% inhibition of cell invasion), followed by the synthetic inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64). CLIK-148, a specific inhibitor of cathepsin L, showed a lower effect than chicken cystatin and E-64. Pepstatin A weakly inhibited invasion, whereas the same molar concentrations of squash aspartic proteinase (SQAPI)-like inhibitor, isolated from squash Cucurbita pepo, showed significant inhibition (65.7+/-1.8%). We conclude that both cysteine and aspartic proteinase activities are needed for invasion by MCF-10A neoT cells in vitro.     

Cysteine proteinases and their inhibitors in extracellular fluids: markers for diagnosis and prognosis in cancer

Cathepsins B, H and L have been shown to participate in processes of tumor growth, vascularization, invasion and metastasis. Their levels in tumor tissue extracts can provide useful clinical information to predict disease-free and overall survival in breast, lung, colorectal, brain and head and neck cancer patients. Recently we have found that both cysteine cathepsins and their endogenous protein inhibitors stefins and cystatin C can also predict prognosis when measured extracellularly. In melanoma and colorectal cancer patients high serum levels of cathepsins B and H correlated with shorter survival. Similarly, increased extracellular levels of stefins A and B and cystatin C correlated significantly with high risk of adverse outcome in cancer patients. However, the cathepsin B/cystatin C complex was found to be less abundant in sera of patients with malignant tumors than in those with benign diseases or in healthy controls, suggesting an imbalance between the enzyme and its inhibitor in cancer patients.     

Potent and selective cathepsin L inhibitors do not inhibit human osteoclast resorption in vitro

Cathepsins K and L are related cysteine proteases that have been proposed to play important roles in osteoclast-mediated bone resorption. To further examine the putative role of cathepsin L in bone resorption, we have evaluated selective and potent inhibitors of human cathepsin L and cathepsin K in an in vitro assay of human osteoclastic resorption and an in situ assay of osteoclast cathepsin activity. The potent selective cathepsin L inhibitors (K(i) = 0.0099, 0.034, and 0.27 nm) were inactive in both the in situ cytochemical assay (IC(50) > 1 micrometer) and the osteoclast-mediated bone resorption assay (IC(50) > 300 nm). Conversely, the cathepsin K selective inhibitor was potently active in both the cytochemical (IC(50) = 63 nm) and resorption (IC(50) = 71 nm) assays. A recently reported dipeptide aldehyde with activity against cathepsins L (K(i) = 0.052 nm) and K (K(i) = 1.57 nm) was also active in both assays (IC(50) = 110 and 115 nm, respectively) These data confirm that cathepsin K and not cathepsin L is the major protease responsible for human osteoclastic bone resorption.     

An activity-based probe for the determination of cysteine cathepsin protease activities in whole cells

We describe a novel diazomethylketone-containing irreversible inhibitor (BIL-DMK) which is specific for a subset of pharmaceutically important cysteine cathepsin proteases. BIL-DMK rapidly inactivates cathepsins B, F, K, L, S, and V in isolated enzyme assays and labels cathepsins in whole cells. The presence of catalytically active cathepsins B, L, and K or S was demonstrated using radioiodinated BIL-DMK in HepG2 (hepatoma), HIG82 (rabbit synoviocyte), and Ramos (B lymphoma) cell lines, respectively. The identity of each protein labeled was confirmed from the isoelectric point and molecular mass of the radioactive spots on two-dimensional gel and by comigration with each cathepsin as identified by immunoblotting. These cell lines were used to establish whole-cell enzyme occupancy assays to determine the potency of both irreversible and reversible inhibitors against each cathepsin in their native cellular lysosomal or endosomal environment. These whole-cell enzyme occupancy assays are useful to determine the cellular permeability of competing inhibitors and have the advantage of not requiring specific substrates for each cathepsin of interest.     

Manipulating substrate and pH in zymography protocols selectively distinguishes cathepsins K, L, S, and V activity in cells and tissues

Cathepsins K, L, S, and V are cysteine proteases that have been implicated in tissue-destructive diseases such as atherosclerosis, tumor metastasis, and osteoporosis. Among these four cathepsins are the most powerful human collagenases and elastases, and they share 60% sequence homology. Proper quantification of mature, active cathepsins has been confounded by inhibitor and reporter substrate cross-reactivity, but is necessary to develop properly dosed therapeutic applications. Here, we detail a method of multiplex cathepsin zymography to detect and distinguish the activity of mature cathepsins K, L, S, and V by exploiting differences in individual cathepsin substrate preferences, pH effects, and electrophoretic mobility under non-reducing conditions. Specific identification of cathepsins K, L, S, and V in one cell/tissue extract was obtained with cathepsin K (37 kDa), V (35 kDa), S (25 kDa), and L (20 kDa) under non-reducing conditions. Cathepsin K activity disappeared and V remained when incubated at pH 4 instead of 6. Application of this antibody free, species independent, and medium-throughput method was demonstrated with primary human monocyte-derived macrophages and osteoclasts, endothelial cells stimulated with inflammatory cytokines, and normal and cancer lung tissues, which identified elevated cathepsin V in lung cancer.     

Insights into the roles of cathepsins in antigen processing and presentation revealed by specific inhibitors

Eleven human cathepsins have been identified, however, the in vivo roles of individual cathepsins are still largely unknown. In this brief review we will summarize the functions of individual cathepsins in antigen processing and presentation, which are the initial steps of the immune response. Two general inhibitors of papain-like cysteine proteases, E-64 and pyridoxal phosphate, can completely suppress antigen presentation in vivo. To evaluate the contribution of individual cathepsins, specific inhibitors have been developed based on cathepsin tertiary structures: CA-074 for cathepsin B, CLIK-148 and -195 for cathepsin L, CLIK-60 for cathepsin S. Administration of CA-074, a cathepsin B inhibitor, suppresses the response to exogenous antigens, such as hepatitis B virus antigen, ovalbumin and Leishmania major antigen, and induces switching of the helper T cell responses from Th-2 to Th-1 of CD4+ T cells, thereby downregulating the production of IgE and IgG1. Administration of the cathepsin S inhibitor CLIK-60 impairs presentation of an autoantigen, alpha-fodrin, in Sjogren's syndrome and suppresses the Th-1 response and autoantibody production.     

Rat liver thiol proteinases: cathepsin B, cathepsin H and cathepsin L

Data on following points of lysosomal thiol proteinases (cathepsins B, H and L) from rat liver are described in this paper: Partial amino acid sequence of cathepsin B, substrate specificity of cathepsin L, immunological studies of cathepsin B and H and effectiveness of E-64, specific thiol proteinase inhibitor in vivo.     

Cathepsin B, cathepsin H, cathepsin X and cystatin C in sera of patients with early-stage and inflammatory breast cancer

Numerous studies have linked cathepsins and their inhibitor cystatin C to tumor invasion and metastasis. We examined whether cathepsin B, cathepsin H, cathepsin X and cystatin C could be detected in sera from women with early stage or inflammatory breast cancer and whether they correlated with clinicopathological characteristics. Preoperative serum was obtained from 176 patients with early-stage breast cancer (tumor size 相似文献   

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.     

Active cathepsins B, H, K, L and S in human inflammatory bronchoalveolar lavage fluids

BACKGROUND INFORMATION: Chronic inflammation and tissue remodelling result from an imbalance between proteolytic enzymes and their inhibitors in the lungs in favour of proteolysis. While many studies have examined serine proteases (e.g. cathepsin G and neutrophil elastase) and matrix metalloproteases, little is known about the role of papain-like CPs (cysteine proteases). The present study focuses on the thiol-dependent cathepsins (CPs) and their specific cystatin-like inhibitors [CPIs (CP inhibitors)] in human inflammatory BALFs (BAL fluids, where BAL stands for broncho-alveolar lavage). RESULTS: Cathepsins B, K and S found were mostly zymogens, whereas cathepsins H and L were predominantly in their mature forms. Little immunoreactive cystatin C was found and the high- and low-molecular-mass ('weight') kininogens were extensively degraded. The BALF procathepsins B and L could be activated autocatalytically, indicating that alveolar fluid pro-CPs are reservoirs of mature enzymes. Hydrolysis patterns of 7-amino-4-methylcoumarin-derived peptide substrates showed that extracellular alveolar CPs remain proteolytically active, and that cathepsins B and L are the most abundant thiol-dependent endoproteases. The CP/CPI balance was significantly tipped in favour of cathepsins (3- or 5-fold), as confirmed by the extensive CP-dependent degradation of exogenous kininogens by BALFs. CONCLUSIONS: Although their importance for inflammation remains to be clarified, the presence of active cathepsins L, K and S suggests that they contribute to the extracellular breakdown of the extracellular matrix.     

Biochemical properties and regulation of cathepsin K activity

Cysteine cathepsins (11 in humans) are mostly located in the acidic compartments of cells. They have been known for decades to be involved in intracellular protein degradation as housekeeping proteases. However, the discovery of new cathepsins, including cathepsins K, V and F, has provided strong evidence that they also participate in specific biological events. This review focuses on the current knowledge of cathepsin K, the major bone cysteine protease, which is a drug target of clinical interest. Nevertheless, we will not discuss recent developments in cathepsin K inhibitor design since they have been extensively detailed elsewhere. We will cover features of cathepsin K structure, cellular and tissue distribution, substrate specificity, and regulation (pH, propeptide, glycosaminoglycans, oxidants), and its putative roles in physiological or pathophysiological processes. Finally, we will review the kinetic data of its inhibition by natural endogenous inhibitors (stefin B, cystatin C, H- and L-kininogens).     

SAR studies of some acetophenone phenylhydrazone based pyrazole derivatives as anticathepsin agents

Cathepsins have emerged as promising molecular targets in a number of diseases such as Alzeimer’s, inflammation and cancer. Elevated cathepsin’s levels and decreased cellular inhibitor concentrations have emphasized the search for novel inhibitors of cathepsins. The present work is focused on the design and synthesis of some acetophenone phenylhydrazone based pyrazole derivatives as novel non peptidyl inhibitors of cathepsins B, H and L. The synthesized compounds after characterization have been explored for their inhibitory potency against cathepsins B, H and L. The results show that some of the synthesized compounds exhibit anti-catheptic activity with K_i value of the order of 10⁻¹⁰ M. Differential inhibitory effects have been observed for cathepsins B, H and L. Cathepsin L is inhibited more pronounced than cathepsin B and cathepsin H in that order.     

Matrigel® invasion by the prostate cancer cell lines, PC3 and DU145, and cathepsin L+B activity

Cathepsins L and B are lysosomal cysteine proteinases whose activities and cellular location are altered in many types of cancers and cancer cell lines. Cathepsins L and B play an unspecified role in cancer invasion and metastasis. The purpose of our study was to determine whether cathepsins L and B are important for the ability of two prostate cancer cell lines, PC3 and DU 145, to invade the basement membrane-like preparation, Matrigel®. Exposure of PC3 and DU145 to the irreversible cysteine proteinase inhibitor, E64, decreases the invasive ability of DU145, but not PC3. PC3 and DU145 were treated with the phorbol ester analogue, phorbol 12-myristate 13-acetate (PMA), a known tumor promoter that activates protein kinase C and contributes to the metastatic phenotype. PMA increased secreted cathepsin L+B activity and the invasive ability of PC3 and DU145; co-exposure to E64 and PMA decreased both cathepsin L+B activity and invasion. We conclude that DU145 requires cathepsin L+B activity more than PC3 for the invasion of the Matrigel®. When the amount of secreted cathepsin L+B activity is increased by PMA treatment, however, PC3 becomes dependent on cathepsin L+B for invasion. Our study demonstrates that modulation of the amount of secreted cathepsin L+B activity influences the invasive phenotype of PC3 and DU145.     

Comparative substrate specificity analysis of recombinant human cathepsin V and cathepsin L

Cathepsins V and L have high identity and few structural differences. In this paper, we reported a comparative study of the hydrolytic activities of recombinant human cathepsins V and L using fluorescence resonance energy transfer peptides derived from Abz-KLRSSKQ-EDDnp (Abz = ortho-aminobenzoic acid and EDDnp = N-(2,4-dinitrophenyl)ethylenediamine). Five series of peptides were synthesized to map the S3 to S2' subsites. The cathepsin V subsites S1 and S3 present a broad specificity while cathepsin L has preference for positively charged residues. The S2 subsites of both enzymes require hydrophobic residues with preference for Phe and Leu. The S1' and S2' subsites of cathepsins V and L are less specific. Based on these data we designed substrates to explore the electrostatic potential differences of them. Finally, the kininogenase activities of these cathepsins were compared using synthetic human kininogen fragments. Cathepsin V preferentially released Lys-bradykinin while cathepsin L released bradykinin. This kininogenase activity by cathepsins V and L was also observed from human high and low molecular weight kininogens.     

Matrigel invasion by the prostate cancer cell lines, PC3 and DU145, and cathepsin L+B activity.

Cathepsins L and B are lysosomal cysteine proteinases whose activities and cellular location are altered in many types of cancers and cancer cell lines. Cathepsins L and B play an unspecified role in cancer invasion and metastasis. The purpose of our study was to determine whether cathepsins L and B are important for the ability of two prostate cancer cell lines, PC3 and DU 145, to invade the basement membrane-like preparation, Matrigel. Exposure of PC3 and DU145 to the irreversible cysteine proteinase inhibitor, E64, decreases the invasive ability of DU145, but not PC3. PC3 and DU145 were treated with the phorbol ester analogue, phorbol 12-myristate 13-acetate (PMA), a known tumor promoter that activates protein kinase C and contributes to the metastatic phenotype. PMA increased secreted cathepsin L+B activity and the invasive ability of PC3 and DU145; co-exposure to E64 and PMA decreased both cathepsin L+B activity and invasion. We conclude that DU145 requires cathepsin L+B activity more than PC3 for the invasion of the Matrigel. When the amount of secreted cathepsin L+B activity is increased by PMA treatment, however, PC3 becomes dependent on cathepsin L+B for invasion. Our study demonstrates that modulation of the amount of secreted cathepsin L+B activity influences the invasive phenotype of PC3 and DU145.     

Toward very potent,non-covalent organophosphonate inhibitors of cathepsin C and related enzymes by 2-amino-1-hydroxy-alkanephosphonates dipeptides

Cathepsins play an important role in several human disorders and therefore the design and synthesis of their inhibitors attracts considerable interest in current medicinal chemistry approaches. Due to the presence of a strong sulphydryl nucleophile in the active center of the cysteine type cathepsins, most strategies to date have yielded covalent inhibitors. Here we present a series of non-covalent β-amino-α-hydroxyalkanephosphonate dipeptidic inhibitors of cathepsin C, ranking amongst the best low-molecular weight inhibitors of this enzyme. Their binding modes determined by molecular modelling indicate that the hydroxymethyl fragment of the molecule, not the phosphonate moiety, acts as a transition state analogue of peptide bond hydrolysis. These dipeptide mimetics appear also to be potent inhibitors of other cysteine proteases such as papain, cathepsin B and cathepsin K, thus providing new leading structures for these medicinally important enzymes.     

Functional in vivo imaging of cysteine cathepsin activity in murine model of inflammation

Near-infrared fluorophore (NIRF)-labeled imaging probes are becoming increasingly important in bio-molecular imaging applications, that is, in animal models for tumor imaging or inflammation studies. In this study we showed that the previously introduced chemical concept of ‘Reverse Design’ represents an efficient strategy for the generation of selective probes for cysteine proteases from chemically optimized protease inhibitors for investigations in proteomic lysates as well as for in vivo molecular imaging studies. The newly developed activity-based probe AW-091 was demonstrated to be highly selective for cathepsin S in vitro and proved useful in monitoring cysteine cathepsin activity in vivo, that is, in zymosan-induced mouse model of inflammation. AW-091 showed higher signal-to-background ratios at earlier time points than the commercially available polymer-based ProSense680 (VisEn Medical) and thus represents an efficient new tool for studying early proteolytic processes leading to various diseases, including inflammation, cancer, and rheumatoid arthritis. In addition, the fluorescent signal originating from the cleaved AW-091 was shown to be reduced by the administration of an anti-inflammatory drug, dexamethasone and by the cathepsin inhibitor E-64, providing a valuable system for the evaluation of small-molecule inhibitors of cathepsins.