IGF-I receptor phosphorylation is impaired in cathepsin X-deficient prostate cancer cells.

The cysteine-type peptidase cathepsin X is highly upregulated in several cancers and presumably promotes tumor invasion through bypassing cellular senescence. Here, we present first evidence that the underlying mechanism may involve the regulation of the insulin-like growth factor (IGF) system, a well-known activator of proliferating tumor cells. Cathepsin X deficiency leads to a reduced phosphorylation of the IGF-I receptor in response to IGF-I stimulation. In addition, downstream signaling through focal adhesion kinase was also affected. Taken together, our results indicate that cathepsin X is able to assist in IGF signaling, which may be an important progress toward understanding cathepsin X-dependent tumorigenesis.

Proteomic identification of protease cleavage sites characterizes prime and non-prime specificity of cysteine cathepsins B, L, and S.

Cysteine cathepsins mediate proteome homeostasis and have pivotal functions in diseases such as cancer. To better understand substrate recognition by cathepsins B, L, and S, we applied proteomic identification of protease cleavage sites (PICS) for simultaneous profiling of prime and non-prime specificity. PICS profiling of cathepsin B endopeptidase specificity highlights strong selectivity for glycine in P3' due to an occluding loop blocking access to the primed subsites. In P1', cathepsin B has a partial preference for phenylalanine, which is not found for cathepsins L and S. Occurrence of P1' phenylalanine often coincides with aromatic residues in P2. For cathepsin L, PICS identifies 845 cleavage sites, representing the most comprehensive PICS profile to date. Cathepsin L specificity is dominated by the canonical preference for aromatic residues in P2 with limited contribution of prime-site selectivity determinants. Profiling of cathepsins B and L with a shorter incubation time (4 h instead of 16 h) did not reveal time-dependency of individual specificity determinants. Cathepsin S specificity was profiled at pH 6.0 and 7.5. The PICS profiles at both pH values display a high degree of similarity. Cathepsin S specificity is primarily guided by aliphatic residues in P2 with limited importance of prime-site residues.

Cellular senescence induced by cathepsin X downregulation.

Cellular senescence represents a powerful tumor suppressor mechanism to prevent proliferation and invasion of malignant cells. Since tumor cells as well as primary fibroblasts lacking the lysosomal cysteine-type carboxypeptidase cathepsin X exhibit a reduced invasive capacity, we hypothesized that the underlying reason may be the induction of cellular senescence. To investigate the cellular and molecular mechanisms leading to diminished migration/invasion of cathepsin X-deficient cells, we have analyzed murine embryonic fibroblasts (MEF) derived from cathepsin X-deficient mice and neonatal human dermal fibroblasts (NHDF) transfected with siRNAs targeting cathepsin X. Remarkably, both cell types exhibited a flattened and enlarged cell body, a characteristic phenotype of senescent cells. Additional evidence for accelerated senescence was obtained by detection of the common senescence marker ß-galactosidase. Further examination revealed increased expression levels of senescence-associated genes such as p16, p21, p53, and caveolin in these cells along with a reduced proliferation rate. The accelerated cellular senescence induced by cathepsin X deficiency was rescued by simultaneous expression of exogenous cathepsin X. Finally, cell cycle analysis confirmed a marked reduction of the synthesis rate and prolongation of the S-phase, while susceptibility to apoptosis of cathepsin X-deficient cells remained unchanged. In conclusion, cathepsin X deficiency leads to accelerated cellular senescence and consequently to diminished cellular proliferation and migration/invasion implying a potential role of cathepsin X in bypassing cellular senescence.

A cysteine-type carboxypeptidase, cathepsin X, generates peptide receptor agonists.

The kallikrein-kinin system and the renin-angiotensin system interact at different levels and are linked by various molecules such as angiotensin-converting enzyme which degrades bradykinin into inactive peptides. Here we report that a cysteine-type carboxypeptidase, cathepsin X, is able to modulate the kallikrein-kinin system through carboxyterminal processing of the small peptide hormones bradykinin and kallidin. Both peptides are thereby converted from bradykinin B(2) receptor ligands to bradykinin B(1) receptor specific ligands. Cathepsin X, which has previously been recognized as an inflammatory marker may therefore act as a type I kininase. In addition, we have identified cathepsin X as an alternative possible link between the kallikrein-kinin system and the renin-angiotensin system in that it not only cleaves kinins C-terminally, but also converts angiotensin I to angiotensin II.

T-SP1: a novel serine protease-like protein predominantly expressed in testis.

Here, we describe a novel member in the group of membrane-anchored chymotrypsin (S1)-like serine proteases, namely testis serine protease 1 (T-SP1), as it is principally expressed in testis tissue. The human T-SP1 gene encompasses 28.7 kb on the short arm of chromosome 8 and consists of seven exons. Rapid amplification of cDNA ends (RACE) experiments revealed that due to alternative splicing three different variants (T-SP1/1, -2, -3) are detectable in testis tissue displaying pronounced heterogeneity at their 3'-end. T-SP1/1 consists of an 18 amino acid signal peptide and of a 49 amino acid propeptide. The following domain with the catalytic triad of His(108), Asp(156), and Ser(250) shares sequence identities of 42% and 40% with the blood coagulation factor XI and plasma kallikrein, respectively. Only T-SP1/1 contains a hydrophobic part at the C-terminus, which provides the basis for cell membrane anchoring. Using a newly generated polyclonal anti-T-SP1 antibody, expression of the T-SP1 protein was found in the Leydig and Sertoli cells of the testis and in the epithelial cells of the ductuli efferentes. Notably, T-SP1 protein was also detectable in prostate cancer and in some ovarian cancer tissues, indicating tumor-related synthesis of T-SP1 beyond testis tissue.

Kinin receptors in stimulated and characterized decidua tissue-derived cells.

Bradykinin and its kinin B(2) receptor are autocrine and paracrine mediators in foetal membranes and decidua. As a first step we characterized the intracellular morphology of decidual cells. Cultured decidua tissue-derived cells immunolabel for vimentin fibrils, and are considered to be of mesenchymal origin. They show characteristics of macrophages and can be distinguished from endothelial cells and cells of the trophoblast lineage. These cellular features were determined by means of immunocytochemistry. Furthermore cultured decidua tissue-derived cells express kinin B(2) receptors and in this context we demonstrated its expression at mRNA level by in situ reverse transcriptase polymerase chain reaction. Following stimulation with bacterial lipopolysaccharide, we have observed a marginal upregulation of the expression of kinin B(1) receptors and carboxypeptidase M by quantitative RT-PCR. Equilibrium binding experiments with [(3)H]des-Arg(10)-kallidin, the kinin B(1) receptor agonist, did not result in detectable binding sites.

The contact system--a novel branch of innate immunity generating antibacterial peptides.

Activation of the contact system has two classical consequences: initiation of the intrinsic pathway of coagulation, and cleavage of high molecular weight kininogen (HK) leading to the release of bradykinin, a potent proinflammatory peptide. In human plasma, activation of the contact system at the surface of significant bacterial pathogens was found to result in further HK processing and bacterial killing. A fragment comprising the D3 domain of HK is generated, and within this fragment a sequence of 26 amino acids is mainly responsible for the antibacterial activity. A synthetic peptide covering this sequence kills several bacterial species, also at physiological salt concentration, as effectively as the classical human antibacterial peptide LL-37. Moreover, in an animal model of infection, inhibition of the contact system promotes bacterial dissemination and growth. These data identify a novel and important role for the contact system in the defence against invasive bacterial infection.

RGD-dependent binding of procathepsin X to integrin alphavbeta3 mediates cell-adhesive properties.

Secreted lysosomal cysteine proteases (cathepsins) are involved in degradation and remodeling of the extracellular matrix, thus contributing to cell adhesion and migration. Among the eleven human lysosomal cysteine proteases, only procathepsin X contains an RGD motif located in a highly exposed region of the propeptide, which may allow binding of the proenzyme to RGD-recognizing integrins. Here, we have tested procathepsin X for cell-adhesive properties and found that it supports integrin alpha(v)beta(3)-dependent attachment and spreading of human umbilical vein endothelial cells. Using site-directed mutants of procathepsin X, we proved that this effect is mediated by the RGD sequence within the proregion of the protease. Endogenous procathepsin X is transported to the plasma membrane, accumulates in vesicles at lamellipodia of the human umbilical vein endothelial cell, and is partly associated with the cell surface, as shown by immunofluorescence. In addition, procathepsin X is partly co-localized with integrin beta(3), as detected by immunogold electron microscopy. A direct interaction between endogenous procathepsin X and alpha(v)beta(3) was demonstrated by co-immunoprecipitation. Moreover, surface plasmon resonance analysis revealed significant and RGD-dependent binding of procathepsin X to integrin alpha(v)beta(3). Our results provide for the first time evidence that the extracellular function of cathepsin X may include binding to integrins thereby modulating the attachment of migrating cells to ECM components.

An enzyme-linked immunosorbent assay for human cathepsin X, a potential new inflammatory marker.

The human lysosomal cysteine-type carboxypeptidase cathepsin X is mainly present in monocytes and macrophages and may be released into the circulation due to constitutive and/or regulated secretion by (activated) immune cells. To define its potential diagnostic value as an inflammatory marker, we have developed a highly sensitive and specific sandwich-type immunoassay (ELISA) for cathepsin X permitting both intra- and extracellular detection and quantification. The dynamic range of the cathepsin X ELISA was determined to be 100 (detection limit) to 8000 pg/ml. Reproducibility of both within and between runs yielded coefficients of variation (CVs) of 2.7-3.5% and 6.3-7.3%, respectively. Cross-reactivity with other members (cathepsin B, L) of the thiol-dependent cathepsin family was not observed. The ELISA was used to quantify cathepsin X in leukocytes as well as in plasma of healthy volunteers and patients with multiple trauma. During the first 72 h after trauma, plasma levels of cathepsin X increased significantly, particularly in patients who died during the posttraumatic period. In comparison to the well-known inflammation marker neutrophil elastase, cathepsin X levels predicted survival with a higher significance in the later posttraumatic phase. In conclusion, this report provides the first evidence of cathepsin X immunoreactivity not only in cell lysates but also in plasma samples. We suggest that the newly developed highly reproducible ELISA will be of great value for further evaluation of this protease as a diagnostic and/or prognostic marker in inflammatory diseases.

Up-regulation of cathepsin X in Helicobacter pylori gastritis and gastric cancer.

Recently, we identified increased cathepsin X expression in H. pylori-infected gastric mucosa. Here, we describe further up-regulation in gastric cancer and report on the role of inflammatory cytokines required for cathepsin X up-regulation in H. pylori-infected gastric mucosa, as well as on consequences for cellular invasion. Biopsy specimens were taken from the antrum, corpus and cardia of H. pylori-infected and non-infected patients. Gastric cancer samples were obtained from patients undergoing gastric surgery. Cathepsin X was detected in gastric mucosa by quantitative real-time RT-PCR, western blotting and immunohistochemistry. Induction of cathepsin X expression in epithelial and inflammatory cells caused by H. pylori infection was tested in in vitro contact and non-contact co-cultures of AGS cells and monocytic cells. Patients with H. pylori gastritis showed significantly higher cathepsin X mRNA (2.5-fold) and protein (1.6-fold) expression than H. pylori-negative patients. Cathepsin X was also up-regulated in gastric cancer (3-12-fold) compared to non-neoplastic mucosa. Cathepsin X was predominantly expressed by macrophages in the mucosal stroma and in glands of the antral mucosa. In addition, tumour cells stained for cathepsin X in 26 (68%) patients with gastric carcinoma. In general, staining was significantly more common (20 vs. 6 patients) and more intense (3.55 vs. 0.83) in intestinal type gastric cancer than in the diffuse type. In vitro cell culture experiments revealed that intercellular signalling between pathogenicity island (PAI)-positive H. pylori-infected epithelial cells and macrophages via soluble factors in the culture medium seems to be responsible for increased expression of cathepsin X in monocytes. Using antisense oligonucleotides, cathepsin X up-regulation was directly associated with higher invasiveness in vitro. Although no correlation of cathepsin X expression and TNM stage was found, our study demonstrates that cathepsin X plays a role not only in the chronic inflammation of gastric mucosa but also in the tumourigenesis of gastric cancer.

Identification of Chlamydia pneumoniae in intracranial and extracranial arteries in patients with stroke and in controls: combined immunohistochemical and polymerase chain reaction analyses.

An association of the obligatory intracellular gram-negative pathogen Chlamydia pneumoniae with coronary artery disease, myocardial infarction, and atherosclerosis was suggested. The presence of C pneumoniae was determined in different arteries (n = 165) from 23 control cases and 10 patients with stroke including coronary arteries, carotid arteries, basilar artery, and middle cerebral arteries of normal controls and patients with stroke using nested polymerase chain reaction (PCR) and immunohistochemistry (IHC). Atherosclerosis was detected in 51.5% of all investigated arteries. No significant differences were detected between controls (59.1% by IHC, 45.5% by nested PCR) and patients with stroke (40% by IHC, 40% by nested PCR). This is the first investigation demonstrating C pneumoniae by IHC and nested PCR in different intracerebral arteries in control persons and patients with stroke. No significant correlation between the presence of chlamydial DNA or antigens in arteries and stroke could be demonstrated. The presence of the C pneumoniae is indicative of a correlation between infection and atherosclerosis, but not of a specific vascular neuropathology such as stroke.

The SEP domain of p47 acts as a reversible competitive inhibitor of cathepsin L.

The solution structure of the human p47 SEP domain in a construct comprising residues G1-S2-p47(171-270) was determined by NMR spectroscopy. A structure-derived hypothesis about the domains' function was formulated and pursued in binding experiments with cysteine proteases. The SEP domain was found to be a reversible competitive inhibitor of cathepsin L with a Ki of 1.5 microM. The binding of G1-S2-p47(171-270) to cathepsin L was mapped by biochemical assays and the binding interface was investigated by NMR chemical shift perturbation experiments.

Up-regulation of cathepsin X in prostate cancer and prostatic intraepithelial neoplasia.

BACKGROUND: Evidence is accumulating that several proteases are involved in prostate cancer progression. A locus which is often amplified in prostate cancer is the chromosomal region 20q13. Interestingly, one of the genes encoding the cysteine protease cathepsin X maps to this region. The aim of this study was to assess the expression pattern of cathepsin X in malignant and non-malignant prostatic tissue samples. METHODS: Matched malignant and non-malignant tissue specimens were obtained from 56 men after radical prostatectomy. Cathepsin X was quantified at both protein and mRNA levels using several detection methods: Western blotting, immunohistochemistry, quantitative RT-PCR, and in situ hybridization. Furthermore, genomic DNA was analyzed by PCR for possible gene amplification. RESULTS: Immunohistochemical analysis of formalin-fixed, paraffin-embedded sections of radical prostatectomy specimens was performed utilizing a polyclonal antibody against human procathepsin X and revealed staining of acinar basal cells in normal prostate glands. Prostatic intraepithelial neoplasias (PINs) and prostate carcinomas stained highly positive for cathepsin X, showing a significant difference to the staining of normal prostate glands. In contrast, relatively weak and heterogeneous staining was observed for cathepsins F, B, and L. Up-regulation of cathepsin X at the protein level was confirmed by Western blotting. No statistically significant difference was observed at the mRNA level. PCR of genomic DNA revealed that cathepsin X up-regulation most likely occurs in the absence of genomic amplification. CONCLUSIONS: The high expression levels of cathepsin X both in PIN and invasive adenocarcinomas of the prostate suggest that cathepsin X may play a role in the early tumorigenesis of prostate cancer. Further studies are needed to define the utility of this cysteine protease as a diagnostic marker for the early detection of prostate cancer.

Family C1 cysteine proteases: biological diversity or redundancy?

Recent progress in the identification and partial characterization of novel genes encoding cysteine proteases of the papain family has considerably increased our knowledge of this family of enzymes. Kinetic data available to date for this large family indicate relatively broad, overlapping specificities for most enzymes, thus inspiring a growing conviction that they may exhibit functional redundancy. This is also supported in part by phenotypes of cathepsin knockout mice and suggests that several proteases can substitute for each other to degrade or process a given substrate. On the other hand, specific functions of one particular protease have also been documented. In addition, differences in cellular distribution and intracellular localization may contribute to defining specific functional roles for some of these proteases.

A noninvasive cell-based assay for monitoring proteolytic activity within a specific subcellular compartment.

A noninvasive cell-based assay has been developed to monitor the proteolytic activity of cathepsin L within a specific subcellular compartment, the lysosome. The green fluorescent protein (GFP) of Aequorea victoria was selected as a substrate. Targeting to lysosomes was achieved by fusing GFP to preprocathepsin L, which also ensures colocalization of the enzyme and the substrate. Stably transfected HeLa-rtTA (reverse tetracycline-controlled transactivator) cells were induced with doxycycline and cultured in the presence of various concentrations of cysteine protease inhibitors for 48 h. In the absence of inhibitor, proteolytic degradation of GFP leads to loss of fluorescence, which is due almost exclusively to the action of recombinant cathepsin L. However, a dose-dependent increase of GFP fluorescence is observed for cells treated with the potent cathepsin L inhibitor benzyloxycarbonyl-LeuLeuTyr-CHN(2). Fluorescence is also observed when GFP is fused to an inactive preprocathepsin L (C25A mutant). Targeting of GFP to an acidic cellular compartment can destabilize the protein and render it susceptible to proteolytic degradation. The approach should be generally applicable for proteases localized in acidic environments. Such an assay can be of great value in validating the participation of a specific enzyme in a given process or in testing the ability of putative inhibitors to reach their intracellular target.

S2' substrate specificity and the role of His110 and His111 in the exopeptidase activity of human cathepsin B.

The ability of the lysosomal cysteine protease cathepsin B to function as a peptidyldipeptidase (removing C-terminal dipeptides) has been attributed to the presence of two histidine residues (His(110) and His(111)) present in the occluding loop, an extra peptide segment located in the primed side of the active-site cleft. Whereas His(111) is unpaired, His(110) is present as an ion pair with Asp(22) on the main body of the protease. This ion pair appears to act as a latch to hold the loop in a closed position. The exopeptidase activity of cathepsin B, examined using quenched fluorescence substrates, was shown to have a 20-fold preference for aromatic side chains in the P2' position relative to glutamic acid as the least favourable residue. Site-directed mutagenesis demonstrated that His(111) makes a positive 10-fold contribution to the exopeptidase activity, whereas His(110) is critical for this action with the Asp(22)-His(110) ion pair stabilizing the electrostatic interaction by a maximum of 13.9 kJ/mol (3.3 kcal/mol). These studies showed that cathepsin B is optimized to act as an exopeptidase, cleaving dipeptides from protein substrates in a successive manner, because of its relaxed specificity in P2' and its other subsites.