Inhibition of cathepsin L-like proteases by cathepsin V propeptide

The N-terminal propeptide domains of several cathepsin L-like cysteine proteases have been shown to possess potent inhibitory activity. Here we report the first kinetic characterisation of the inhibition properties of the cathepsin V propeptide (CatV PP). Using a facile recombinant approach we demonstrate expression, purification and evaluation of the CatV PP. This propeptide was found to behave as a tight-binding inhibitor against CatV (K (i) 10.2 nm). It also functions as an inhibitor against other members of the CatL-like subclass (CatL, 9.8 nm; CatS, 10.7 nm; and CatK, 149 nm) and had no discernible effects upon the more distantly related CatB.

Fasciola hepatica cathepsin L-like proteases: biology, function and potential in the development of first generation liver fluke vaccines.

Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are members of a lineage that gave rise to the human cathepsin Ls, Ks and Ss, but while they exhibit similarities in their substrate specificities to these enzymes they differ in having a wider pH range for activity and an enhanced stability at neutral pH. There are presently 13 Fasciola cathepsin L cDNAs deposited in the public databases representing a gene family of at least seven distinct members, although the temporal and spatial expression of each of these members in the developmental stage of F. hepatica remains unclear. Immunolocalisation and in situ hybridisation studies, using antibody and DNA probes, respectively, show that the vast majority of cathepsin L gene expression is carried out in the epithelial cells lining the parasite gut. Within these cells the enzyme is packaged into secretory vesicles that release their contents into the gut lumen for the purpose of degrading ingested host tissue and blood. Liver flukes also express a novel multi-domain cystatin that may be involved in the regulation of cathepsin L activity. Vaccine trials in both sheep and cattle with purified native FheCL1 and FheCL2 have shown that these enzymes can induce protection, ranging from 33 to 79%, to experimental challenge with metacercariae of F. hepatica, and very potent anti-embryonation/hatch rate effects that would block parasite transmission. In this article we review the vaccine trials carried out over the past 8 years, the role of antibody and T cell responses in mediating protection and discuss the prospects of the cathepsin Ls in the development of first generation recombinant liver fluke vaccines. Author Keywords: Helminths; Trematodes; Parasites; Cathepsins; Proteases; Vaccines; Immunology; Biochemistry

Recombinant cathepsin S propeptide attenuates cell invasion by inhibition of cathepsin L-like proteases in tumor microenvironment

Human cathepsin L along with cathepsin S, K, and V are collectively known as cathepsin L-like proteases due to their high homology. The overexpression and aberrant activity of each of these proteases has been implicated in tumorigenesis. These proteases contain propeptide domains that can potently inhibit both their cognate protease and other proteases within the cathepsin L-like subfamily. In this investigation, we have produced the cathepsin S propeptide recombinantly and have shown that it is a potent inhibitor of the peptidolytic, elastinolytic, and gelatinolytic activities of the cathepsin L-like proteases. In addition, we show that this peptide is capable of significantly attenuating tumor cell invasion in a panel of human cancer cell lines. Furthermore, fusion of an IgG Fc-domain to the COOH terminus of the propeptide resulted in a chimeric protein with significantly enhanced ability to block tumor cell invasion. This Fc fusion protein exhibited enhanced stability in cell-based assays in comparison with the unmodified propeptide species. This approach for the combined inhibition of the cathepsin L-like proteases may prove useful for the further study in cancer and other conditions where their aberrant activity has been implicated. Furthermore, this strategy for simultaneous inhibition of multiple cysteine cathepsins may represent the basis for novel therapeutics to attenuate tumorigenesis.

Cathepsin F cysteine protease of the human liver fluke, Opisthorchis viverrini

The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities.

A bioavailable cathepsin S nitrile inhibitor abrogates tumor development

BACKGROUND: Cathepsin S has been implicated in a variety of malignancies with genetic ablation studies demonstrating a key role in tumor invasion and neo-angiogenesis. Thus, the application of cathepsin S inhibitors may have clinical utility in the treatment of cancer. In this investigation, we applied a cell-permeable dipeptidyl nitrile inhibitor of cathepsin S, originally developed to target cathepsin S in inflammatory diseases, in both in vitro and in vivo tumor models.

METHODS: Validation of cathepsin S selectivity was carried out by assaying fluorogenic substrate turnover using recombinant cathepsin protease. Complete kinetic analysis was carried out and true K i values calculated. Abrogation of tumour invasion using murine MC38 and human MCF7 cell lines were carried out in vitro using a transwell migration assay. Effect on endothelial tube formation was evaluated using primary HUVEC cells. The effect of inhibitor in vivo on MC38 and MCF7 tumor progression was evaluated using cells propagated in C57BL/6 and BALB/c mice respectively. Subsequent immunohistochemical staining of proliferation (Ki67) and apoptosis (TUNEL) was carried out on MCF7 tumors.

RESULTS: We confirmed that this inhibitor was able to selectively target cathepsin S over family members K, V, L and B. The inhibitor also significantly reduced MC38 and MCF7 cell invasion and furthermore, significantly reduced HUVEC endothelial tubule formation in vitro. In vivo analysis revealed that the compound could significantly reduce tumor volume in murine MC38 syngeneic and MCF7 xenograft models. Immunohistochemical analysis of MCF7 tumors revealed cathepsin S inhibitor treatment significantly reduced proliferation and increased apoptosis.

CONCLUSIONS: In summary, these results highlight the characterisation of this nitrile cathepsin S inhibitor using in vitro and in vivo tumor models, presenting a compound which may be used to further dissect the role of cathepsin S in cancer progression and may hold therapeutic potential.

Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases

The cysteine protease cathepsin C (CatC) activates granule-associated proinflammatory serine proteases in hematopoietic precursor cells. Its early inhibition in the bone marrow is regarded as a new therapeutic strategy for treating proteolysis-driven chronic inflammatory diseases, but its complete inhibition is elusive in vivo Controlling the activity of CatC may be achieved by directly inhibiting its activity with a specific inhibitor or/and by preventing its maturation. We have investigated immunochemically and kinetically the occurrence of CatC and its proform in human hematopoietic precursor cells and in differentiated mature immune cells in lung secretions. The maturation of proCatC obeys a multistep mechanism that can be entirely managed by CatS in neutrophilic precursor cells. CatS inhibition by a cell-permeable inhibitor abrogated the release of the heavy and light chains from proCatC and blocked ∼80% of CatC activity. Under these conditions the activity of neutrophil serine proteases, however, was not abolished in precursor cell cultures. In patients with neutrophilic lung inflammation, mature CatC is found in large amounts in sputa. It is secreted by activated neutrophils as confirmed through lipopolysaccharide administration in a nonhuman primate model. CatS inhibitors currently in clinical trials are expected to decrease the activity of neutrophilic CatC without affecting those of elastase-like serine proteases.

The role of cathepsin C in Papillon-Lefèvre syndrome, prepubertal periodontitis, and aggressive periodontitis

We have previously reported that loss-of-function mutations in the cathepsin C gene (CTSC) result in Papillon Lefevre syndrome, an autosomal recessive condition characterized by palmoplantar keratosis and early,onset, severe periodontitis. Others have also reported CTSC mutations in patients with severe prepubertal periodontitis, but without any skin manifestations. The possible role of CTSC variants in more common types of non-mendelian, early-onset, severe periodontitis ("aggressive periodontitis") has not been investigated. In this study, we have investigated the role of CTSC in all three conditions. We demonstrate that PLS is genetically homogeneous and the mutation spectrum that includes three novel mutations (c.386T>A/p. V129E, c.935A>G/p.Q312R, and c.1235A>G/p.Y412C) in 21 PLS families (including eight from our previous study) provides an insight into structure-function relationships of CTSC. Our data also suggest that a complete loss-of-function appears to be necessary for the manifestation of the phenotype, making it unlikely that weak CTSC mutations are a cause of aggressive periodontitis. This was confirmed by analyses of the CTSC activity in 30 subjects with aggressive periodontitis and age-sex matched controls, which demonstrated that there was no significant difference between these two groups (1,728.7 +/- SD 576.8 mu moles/mg/min vs. 1,678.7 +/- SD 527.2 mu moles/mg/min, respectively, p = 0.73). CTSC mutations were detected in only one of two families with prepubertal periodontitis; these did not form a separate functional class with respect to those observed in classical PLS. The affected individuals in the other prepubertal periodontitis family not only lacked CTSC mutations, but in addition did not share the haplotypes at the CTSC locus. These data suggest that prepubertal periodontitis is a genetically heterogeneous disease that, in some families, just represents a partially penetrant PLS. (C) 2004 Wiley-Liss, Inc.

The clinical significance of cathepsin S expression in human astrocytomas

Early local invasion by astrocytoma. cells results in tumor recurrence even after apparent total surgical resection, leading to the poor prognosis associated with malignant astrocytomas. Proteolytic enzymes have been implicated in facilitating tumor cell invasion and the current study was designed to characterize the expression of the cysteine proteinase cathepsin S (CatS) in astrocytomas and examine its potential role in invasion. Immunohistochemical analysis of biopsies demonstrated that CatS was expressed in astrocytoma cells but absent from normal astrocytes, oligodendrocytes, neurones and endothelial cells. Microglial cells and macrophages were also positive. Assays of specific activity in 59 astrocytoma biopsies confirmed CatS expression and in addition demonstrated that the highest levels of activity were expressed in grade IV tumors. CatS activity was also present in astrocytoma cells in vitro and the extracellular levels of activity were highest in cultures derived from grade IV tumors. In vitro invasion assays were carried out using the U251MG cell line and the invasion rate was reduced by up to 61% in the presence of the selective CatS inhibitor 4-Morpholineurea-LeuHomoPhe-vinylsulphone. We conclude that CatS expression is up-regulated in astrocytoma. cells and provide evidence for a potential role for CatS in invasion.

English Trade Mark Law in the Eighteenth Century: Blanchard v Hill Revisited - Another 'Case of Monopolies'?

The decision of Lord Hardwicke LC in Blanchard v Hill in 1742 is the earliest reported case on the equitable jurisdiction to grant injunctive relief against trade mark piracy. The ambiguous manner in which the case was reported led to the decision being interpreted as either the basis of equitable jurisdiction or a denial of jurisdiction. This article seeks to establish the background to the case, what actually happened, and the immediate impact of the decision. The scene is set, however, in a parallel symbolic universe – heraldry – because in 1740, the officers of arms were confronted with a trade mark case.