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Publications and source records attributed to D Brömme.
The role of the newly discovered cysteine protease, cathepsin K, in osteoporosis and rheumatoid arthritis is reviewed. The current development of cathepsin K inhibitors and their targeted delivery using synthetic polymer carriers are discussed. Future challenges and possible strategies to improve these delivery systems are addressed.
The rare osteosclerotic disease, pycnodysostosis, is characterized by decreased osteoclastic bone collagen degradation due to the absence of active cathepsin K. Although this enzyme is primarily expressed by osteoclasts, there is increasing evidence that it may also be present in other cells, including fibroblasts. Since fibroblasts are known to degrade collagen intracellularly following phagocytosis, we analyzed various soft connective tissues (periosteum, perichondrium, tendon, and synovial membrane) from a 13-week-old human fetus with pycnodysostosis for changes in this collagen digestion pathway. In addition, the same tissues from cathepsin K-deficient and control mice were analyzed. Microscopic examination of the human fetal tissues showed that cross-banded collagen fibrils had accumulated in lysosomal vacuoles of fibroblasts. Morphometric analysis of periosteal fibroblasts revealed that the volume density of collagen-containing vacuoles was 18 times higher than in fibroblasts of control patients. A similar accumulation was seen in periosteal fibroblasts of three children with pycnodysostosis. In contrast to the findings in humans, an accumulation of internalized collagen was not apparent in fibroblasts of mice with cathepsin K deficiency. Our observations indicate that the intracellular digestion of phagocytosed collagen by fibroblasts is inhibited in humans with pycnodysostosis, but probably not in the mouse model mimicking this disease. The data strongly suggest that cathepsin K is a crucial protease for this process in human fibroblasts. Murine fibroblasts may have other proteolytic activities that are expressed constitutively or up regulated in response to a deficiency of cathepsin K. This may explain why cathepsin K-deficient mice lack the dysostotic features that are prominent in patients with pycnodysostosis.
We report here that a number of commonly used small peptide caspase inhibitors consisting of a caspase recognition sequence linked to chloromethylketone, fluoromethylketone or aldehyde reactive group efficiently inhibit other cysteine proteases than caspases. The in vitro studies included cathepsins B, H, L, S, K, F, V, X and C, papain and legumain. Z-DEVD-cmk was shown to be the preferred irreversible inhibitor of most of the cathepsins in vitro, followed by Z-DEVD-fmk, Ac-YVAD-cmk, Z-YVAD-fmk and Z-VAD-fmk. Inactivation of legumain by all the inhibitors investigated was moderate, whereas cathepsins H and C were poorly inhibited or not inhibited at all. Inhibition by aldehydes was not very potent. All the three fluoromethylketones efficiently inhibited cathepsins in Jurkat and human embryonic kidney 293 cells at concentrations of 100 microM. Furthermore, they completely inhibited cathepsins B and X activity in tissue extracts at concentrations as low as 1 microM. These results suggest that data based on the use of these inhibitors should be taken with caution and that other proteases may be implicated in the processes previously ascribed solely to caspases.
Human cathepsin W (also called lymphopain) is a recently described papain-like cysteine protease of unknown function whose gene expression was found to be restricted to cytotoxic cells. Here we demonstrate that cathepsin W is expressed predominantly in NK cells and, to a lesser extent, in CTLs. Quantitative RT-PCR revealed that NK cells contained approximately 21 times more cathepsin W transcript than CTLs. The predominant expression of cathepsin W in NK cells was further confirmed by Western blot analysis and immunohistochemistry. IL-2-mediated stimulation of NK cells and CTLs revealed a stronger up-regulation of the cathepsin W gene and protein expression in NK cells (7-fold) than in CTLs (2-fold). Transfection experiments of HeLa cells and biochemical analyses revealed that cathepsin W is exclusively "high mannose-type" glycosylated and is mainly targeted to the endoplasmic reticulum (ER). Interestingly, the ER localization of cathepsin W was also found in NK cells, in which colocalization studies revealed an overlapping staining of cathepsin W and Con A, an ER-specific lectin. Furthermore, subcellular fractionation of cathepsin W-expressing cells confirmed the ER localization and showed that cathepsin W is membrane associated. Based on the results of this study, cathepsin W might represent a putative component of the ER-resident proteolytic machinery. The constitutive expression in NK cells and the stronger up-regulation of cathepsin W by IL-2 in NK cells than CTLs suggest that cathepsin W is not just a marker of cytotoxic cells but is, rather, specifically expressed in NK cells.
A human cDNA of 2166 bp encoding a novel cathepsin B-related protein was isolated and characterized. The amino acid sequence of the predicted protein of 467 aa was 46% identical with that of human tubulointerstitial nephritis antigen (TIN-ag), and therefore, the protein was tentatively designated as the TIN-ag-related protein (TIN-ag-RP). The amino acid sequence of TIN-ag-RP is composed of a 21 aa long signal sequence, a 181 aa long N-terminal domain containing two epidermal growth factor-like domains, a follistatin motif, and a 265 aa long cathepsin B-like domain. Interestingly, a serine residue has replaced the active site cysteine residue in the cathepsin B-like domain, resulting in a proteolytically inactive protein. Evolutionary analysis revealed that a distinct family of "TIN-ag-like" proteins had evolved in vertebrates. Northern blot analysis revealed a single TIN-ag-RP transcript of 2.4 kb in various tissues with the highest transcript levels detected in aorta, heart, placenta, skeletal muscle, kidney, and a colorectal adenocarcinoma cell line. Using a polyclonal anti-TIN-ag-RP antibody, TIN-ag-RP expression was predominantly seen in vascular smooth muscle (VSM) cells, but also in cardiac and skeletal muscle cells as well as in kidney. Interestingly, uterine smooth muscle cells completely lacked TIN-ag-RP expression, implying a regulated gene expression. Localization studies in HeLa cells stably transfected with TIN-ag-RP cDNA showed that TIN-ag-RP is glycosylated and actively secreted, a finding in line with its proposed function as a structural or regulatory protein similar to TIN-ag.
Cathepsin K is a cysteine protease with high matrix-degrading activity. Initially, cathepsin K was described as being expressed exclusively by osteoclasts. It was suggested that cathepsin K expression is a specific feature of cells involved in bone remodelling. The aim of this study was to investigate the hypothesis that cathepsin K is expressed not only in bone-resorbing macrophages, but also more generally in specifically differentiated macrophages, such as epithelioid cells and multinucleated giant cells in soft tissues. Specimens obtained from different organs and anatomical locations of patients suffering from sarcoidosis, tuberculosis, granulomas caused by foreign materials, and sarcoid-like lesions were investigated for the expression of cathepsins B, K, and L. Immunohistochemistry and in situ hybridization showed cathepsin K in epithelioid cells and multinucleated giant cells irrespective of the pathological condition and anatomical location, but not in normal resident macrophages. By immunoelectron microscopy, cathepsin K was discovered in cytoplasmic granules of multinucleated giant cells. In contrast, cathepsin B and cathepsin L were expressed ubiquitously in CD68-positive tissue macrophages, epithelioid cells, and multinucleated giant cells. The results demonstrate that cathepsin K, but not cathepsin B or cathepsin L, differentiates specific phenotypes of macrophages independently of the anatomical site. Its enzymatic characteristics, particularly its high matrix-degrading activity, suggest that cathepsin K-positive epithelioid cells and multinucleated giant cells are characterized by an enhanced specific proteolytic capability.
Synovial fibroblasts (SFs) play a critical role in the pathogenesis of rheumatoid arthritis (RA) and are directly involved in joint destruction. Both SF-resident matrix metalloproteases and cathepsins have been implicated in cartilage degradation although their identities and individual contributions remain unclear. The aims of this study were to investigate the expression of cathepsin K in SFs, the correlation between cathepsin K expression and disease severity, and the contribution of cathepsin K to fibroblast-mediated collagen degradation. Immunostaining of joint specimens of 21 patients revealed high expression of cathepsin K in SFs in the synovial lining and the stroma of synovial villi, and to a lesser extent in CD68-positive cells of the synovial lining. Cathepsin K-positive SFs were consistently observed at sites of cartilage and bone degradation. Expression levels of cathepsin K in the sublining and vascularized areas of inflamed synovia showed a highly significant negative correlation with results derived from the Hannover Functional Capacity Questionnaire (r = 0.78, P = 0.003; and r = 0.70, P = 0.012, respectively) as a measure of the severity of RA in individual patients. For comparison, there was no correlation between Hannover Functional Capacity Questionnaire and cathepsin S whose expression is limited to CD-68-positive macrophage-like synoviocytes. The expression of cathepsin K was also demonstrated in primary cell cultures of RA-SFs. Co-cultures of SFs on cartilage disks revealed the ability of fibroblast-like cells to phagocytose collagen fibrils whose intralysosomal hydrolysis was prevented in the presence of a potent cathepsin K inhibitor but not by an inhibitor effective against cathepsins L, B, and S. The selective and critical role of cathepsin K in articular cartilage and subchondral bone erosion was further corroborated by the finding that cathepsin K has a potent aggrecan-degrading activity and that cathepsin K-generated aggrecan cleavage products specifically potentiate the collagenolytic activity of cathepsin K toward type I and II collagens. This study demonstrates for the first time a critical role of cathepsin K in cartilage degradation by SFs in RA that is comparable to its well-known activity in osteoclasts.
The aims of this study were to investigate the role of cathepsin K in the pathology of amyloidosis by demonstrating its presence in multinucleated giant cells (MGCs) adjacent to amyloid deposits, and determining its ability to degrade amyloid fibril proteins in vitro. The study was performed using autopsy and biopsy specimens from patients with AA or AL amyloidosis. In six (55%) patients with AA amyloidosis and seven (58%) patients with AL amyloidosis, variable numbers of CD68-immunoreactive MGCs were found adjacent to amyloid deposits. In each case strong cytoplasmic immunostaining for cathepsin K was found in MGCs; immunostaining of amyloid deposits was present in five (45%) patients with AA amyloidosis and three (25%) patients with AL amyloidosis. In vitro degradation experiments showed that recombinant cathepsin K completely degraded AA amyloid fibril proteins at pH 5.5 as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Less effective degradation took place at pH 7.4 and there was no degradation in the presence of a general cysteine protease inhibitor (E64) or in the absence of cathepsin K. This is the first study to show that cathepsin K is expressed in MGCs adjacent to amyloid deposits and to demonstrate its ability to degrade amyloid fibril proteins.
A series of dipeptidyl alpha-keto-beta-aldehydes (glyoxals), prepared by solid-/solution-phase chemistries, were assessed for their inhibitory activity against cathepsin S, a lysosomal cysteine protease implicated in a number of important pathophysiological processes. The inhibitor Cbz-Phe-Leu-COCHO, which exhibits slow-binding kinetic characteristics, was found to be almost 400-fold more selective for cathepsin S (K(i) = 0.185 nM) than for cathepsin B (76 nM) and is, to our knowledge, the most potent, reversible, synthetic cathepsin S inhibitor reported to date.
The human squamous cell carcinoma antigens (SCCA) 1 and 2 are members of the serpin family that are 92% identical in their amino acid sequence. Despite this similarity, they inhibit distinct classes of proteinases. SCCA1 neutralizes the papain-like cysteine proteinases, cathepsins (cat) S, L, and K; and SCCA2 inhibits the chymotrypsin-like serine proteinases, catG and human mast cell chymase. SCCA2 also can inhibit catS, as well as other papain-like cysteine proteinases, albeit at a rate 50-fold less than that of SCCA1. Analysis of the mechanism of inhibition by SCCA1 revealed that the reactive site loop (RSL) is important for cysteine proteinase inhibition. The inhibition of catS by a mutant SCCA2 containing the RSL of SCCA1 is comparable to that of wild-type SCCA1. This finding suggested that there were no motifs outside and only eight residues within the RSL that were directing catS-specific inhibition. The purpose of this study was to determine which of these residues might account for the marked difference in the ability of SCCA1 and SCCA2 to inhibit papain-like cysteine proteinases. SCCA2 molecules containing different RSL mutations showed that no single amino acid substitution could convert SCCA2 into a more potent cysteine proteinase inhibitor. Rather, different combinations of mutations led to incremental increases in catS inhibitory activity with residues in four positions (P1, P3', P4', and P11') accounting for 80% of the difference in activity between SCCA1 and SCCA2. Interestingly, the RSL cleavage site differed between wild-type SCCA2 and this mutant. Moreover, these data established the importance of a Pro residue in the P3' position for efficient inhibition of catS by both wild-type SCCA1 and mutated SCCA2. Molecular modeling studies suggested that this residue might facilitate positioning of the RSL within the active site of the cysteine proteinase.
The human gene encoding a novel tubulointerstitial nephritis antigen (TIN-ag)-related protein (TIN-ag-RP) was isolated, and its genomic organization was determined. BLAST searches revealed the highest degree of homology to several mammalian TIN-ag orthologues, and a weak homology to cathepsin B-like proteases. The 12 kb gene was mapped by fluorescence in situ hybridization to chromosome 1p34.2-3, a locus neither related to that of the human TIN-ag (6p11.2-12) nor to that of cathepsin B (8p22-23.1). The TIN-ag-RP is encoded in ten exons with introns ranging from 83 bp to 4 kb. In addition, the gene contained one exon in the 5'UTR, but none in the 3'UTR. Five of the 10 splice sites of the TIN-ag-RP gene were fully conserved when compared to a related gene of C. elegans, whereas only one splice site was identical to those found in cathepsin B genes. Furthermore, human TIN-ag-RP tagged with the T7-epitope, was expressed in HeLa cells, and was found to be localized in vesicular compartments as well as secreted into the medium suggesting the involvement of the endosomal trafficking pathway. Based on the high degree of homology of the amino acid sequences and genomic organization between TIN-ag-RP and TIN-ag, we suggest that both molecules may form a distinct group or family of TIN-ag-like proteins.
Cathepsin K is the predominant cysteine protease in osteoclast-mediated bone remodeling, and the protease is thought to be involved in the pathogenesis of diseases with excessive bone and cartilage resorption. Osteoclastic matrix degradation occurs in the extracellular resorption lacuna and upon phagocytosis within the cell's lysosomal-endosomal compartment. Since glycosaminoglycans (GAGs) are abundant in extracellular matrixes of cartilage and growing bone, we have analyzed the effect of GAGs on the activity of bone and cartilage-resident cathepsins K and L and MMP-1. GAGs, in particular chondroitin sulfates, specifically and selectively increased the stability of cathepsin K but had no effect on cathepsin L and MMP-1. GAGs strongly enhanced the stability and, to a lesser extent, the catalytic activity of cathepsin K. To combine the activity and stability parameters, we defined a novel kinetic term, named cumulative activity (CA), which reflects the total substrate turnover during the life span of the enzyme. In the presence of chondroitin-4-sulfate (C-4S), the CA value increased 200-fold for cathepsin K but only 25-fold with chondroitin-6-sulfate (C-6S). C-4S dramatically increased the hydrolysis of soluble as well insoluble type I and II collagens, whereas the effects of C-6S and hyaluronic acid were less pronounced. C-4S acts in a concentration-dependent manner but reaches saturation at approximately 0.1%, a concentration similar to that found in the synovial fluid of arthritis patients. C-4S increased the cathepsin K-mediated release of hydroxyproline from insoluble type I collagen 10-fold but had only a less than 2-fold enhancing effect on the hydrolysis of intact cartilage. The relatively small increase in the hydrolysis of cartilage by C-4S was attributed to the endogenous chondroitin sulfate content present in the cartilage. Although C-4S increased the pH stability at neutral pH, a significant increase in the collagenolytic activity of cathepsin K at this pH was not observed, thus suggesting that the unique collagenolytic activity of cathepsin K at acidic pH is mechanistically determined and not by the enzyme's instability at neutral pH. The selective and significant stabilization and activation of cathepsin K activity by C-4S may provide a rationale for a novel mechanism to regulate the enzyme's activity during bone growth and aging, two processes known for significant changes in the GAG content.
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Tissue remodeling is crucial in different lung diseases, in the embryonal development as well as in bronchial carcinoma. Cathepsins were proposed to be involved in the degradation of matrix proteins. Cathepsin K is one of the most potent matrix-degrading cysteine proteinases known as yet. The elastinolytic and collagenolytic activity of this papain-like protease is comparable with that of neutrophil elastase. We have investigated the cathepsin K expression in normal adult lung tissues, in embryonal lung tissue and in bronchial carcinoma. With help of specific anti-cathepsin K antibodies it could be shown that cathepsin K was expressed in bronchial epithelial cells. These data could be confirmed at mRNA level using a quantitative RT-PCR as well as by visualisation of the specific enzymatic activity in epithelial cell lines. During the embryonal development cathepsin K was expressed in the epithelial cells of the developing bronchi. The expression seemed to be upregulated in parallel with the development of the bronchial and alveolar lumen. In the later phase of lung development the cathepsin K expression was restricted to bronchial epithelial cells. Furthermore, using quantitative RT-PCR it could be shown that cathepsin K-mRNA was upregulated in lung tumor tissues in comparison to normal tissues from the same patients. These data suggest that cathepsin K may play an important role in matrix remodeling of the lung under physiological and pathological conditions.
Several tumour-forming cell lines are known to overproduce the lysosomal cysteine peptidase cathepsin L. We have used an antisense approach to investigate whether inhibition of cathepsin L overexpression in two malignant cell lines (myeloma SP cells and L cells) reduces their tumorigenic potential. Two different cDNA fragments of murine cathepsin L were inserted in the antisense direction into the pcDNA3 vector, and SP and L cells were stably transfected with these plasmid constructs. Several of the selected clones expressing the antisense transcript showed specific reduction of the mRNA level and the intracellular activity of cathepsin L, and a greatly diminished amount of secreted procathepsin L. When tested in Balb/c nu/nu mice, the cell lines with low cathepsin L activity exhibited a significantly decreased potential for tumour growth when compared with control cells expressing wild-type levels of cathepsin L activity. This observation suggests that cathepsin L is a critical factor in tumour growth.
Extracellular proteolysis of thyroglobulin at the apical surface of thyroid epithelial cells results in liberation of thyroxine, and is mediated by lysosomal cysteine proteases such as cathepsins B and L. Here, we report on the expression of the cysteine protease cathepsin K in thyroid epithelial cells. The cDNA for porcine thyroid cathepsin K showed homologies ranging from 71% to 94% to the cDNA of cathepsin K from various species and cell types. The deduced amino acid sequence of porcine thyroid cathepsin K predicted a 37 kDa preproenzyme, with the active site residues Cys-140, His-277 and Asn-297, and one potential N-glycosylation site. The localization of cathepsin K was not restricted to lysosomes. Rather, secreted cathepsin K was predominantly found within the follicular lumen and in association with the apical plasma membrane of thyroid epithelial cells. Enzyme cytochemistry showed that cell-surface associated cathepsin K was proteolytically active at neutral pH. In vitro, recombinant cathepsin K liberated thyroxine from thyroglobulin by limited proteolysis at neutral pH. We postulate that its localization enables cathepsin K to contribute to the extracellular proteolysis of thyroglobulin, i.e. thyroid hormone liberation, at the apical surface of thyroid epithelial cells in situ.
Human cathepsin F is a recently described papain-like cysteine protease of unknown function. To investigate the evolutionary relatedness to other human cathepsins, we determined the genomic organization and the chromosomal localization of cathepsin F and isolated its putative promoter region. The gene of human cathepsin F (CTSF) is composed of twelve exons and eleven introns and was found to be similar to that of cathepsin W but different from the cathepsins K, S, L, O, B, and C. The splice sites of nine out of the eleven introns were identical to those determined in the cathepsin W gene (CTSW), whereas introns one and ten were unique for CTSF. The 4. 7 kb gene was mapped to the long arm of chromosome 11 at position q13.1-3, a locus shared with CTSW. Phylogenetic analysis of human cathepsin protein sequences demonstrated that (i) cathepsins F and W are evolutionarily separated from other human cathepsins, and (ii) cysteine proteases closely related to human cathepsin W and F are also expressed in parasites and mammals. Based on these phylogenetic findings, on the presence of a particular protein motif ("ERFNAQ") in the propeptides of cathepsins F and W as well as the genomic organization and chromosomal localization of their genes, we concluded that F and W form a novel subgroup of cathepsin proteases. We suggest the naming "cathepsin F-like" proteases distinct from the previously described cathepsins "L- and B-like" subgroups.
Cathepsin V, a thymus and testis-specific human cysteine protease, was expressed in Pichia pastoris, and its physicokinetic properties were determined. Recombinant procathepsin V is autocatalytically activated at acidic pH and is effectively inhibited by various cysteine protease class-specific inhibitors. The S2P2 subsite specificity of cathepsin V was found to be intermediate between those of cathepsins S and L. The substrate binding pocket, S2, accepted both aromatic and nonaromatic hydrophobic residues, whereas cathepsins L and S preferred either an aromatic or nonaromatic hydrophobic residue, respectively. In contrast to cathepsin L, but similar to cathepsin S, cathepsin V exhibited only a very weak collagenolytic activity. Furthermore, cathepsin V was determined to be significantly more stable at mildly acidic and neutral pH than cathepsin L, but distinctly less stable than cathepsin S. A homology structure model of cathepsin V revealed completely different electrostatic potentials on the molecular surface when compared with human cathepsin L. The model-based electrostatic potential of human cathepsin V was neutral to weakly positive at and in the vicinity of the active site cleft, whereas that of cathepsin L was negative over extended regions of the surface. Surprisingly, the electrostatic potential of the human cathepsin V model structure resembled that of the model structure of mouse cathepsin L. These differences in the electrostatic potential at the molecular surfaces provide a reactivity determinant that may be the source of differences in substrate selectivity and pH stability. Cathepsin V was mapped to the chromosomal region 9q22.2, a site adjacent to the cathepsin L locus. The high sequence identity and the overlapping chromosomal gene loci suggest that both proteases evolved from an ancestral cathepsin L-like precursor by gene duplication.