Biomedical subjects

Dieter Brömme

Publications and source records attributed to Dieter Brömme.

At least 19 recordsLinked to original sources

PubMed2006

Modulation of hypotensive effects of kinins by cathepsin K.

Kinins are pro-inflammatory peptides, which participate in the maintenance of cardiovascular homeostasis, and play a key role in numerous diseases, including lung fibrosis and hypertension. Evidence has been provided recently for the presence of alternative mechanisms of bradykinin generation and/or degradation. Here we showed that cathepsin K may act as a potent kinin-degrading enzyme in bloodstream. Contrary to cathepsin L, cathepsin K attenuates kallikrein-induced decrease of rat blood pressure, and reduces the hypotensive effect of bradykinin in a dose-dependent manner. Moreover, we identified, by engineering the S2 subsite of both recombinant enzymes, two critical residues involved respectively in the kininase activity of cathepsin K, i.e. Tyr67/Leu205, versus kininogenase activity of cathepsin L, i.e. Leu67/Ala205. In conclusion, according to its ability to modulate hypotensive effects of kinins, we propose that cathepsin K is a kininase of biological relevance, in complement of well-documented neutral endopeptidase or angiotensin-converting enzyme.

Animals↗

PubMed2006

Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities.

The substrate specificities of papain-like cysteine proteases (clan CA, family C1) papain, bromelain, and human cathepsins L, V, K, S, F, B, and five proteases of parasitic origin were studied using a completely diversified positional scanning synthetic combinatorial library. A bifunctional coumarin fluorophore was used that facilitated synthesis of the library and individual peptide substrates. The library has a total of 160,000 tetrapeptide substrate sequences completely randomizing each of the P1, P2, P3, and P4 positions with 20 amino acids. A microtiter plate assay format permitted a rapid determination of the specificity profile of each enzyme. Individual peptide substrates were then synthesized and tested for a quantitative determination of the specificity of the human cathepsins. Despite the conserved three-dimensional structure and similar substrate specificity of the enzymes studied, distinct amino acid preferences that differentiate each enzyme were identified. The specificities of cathepsins K and S partially match the cleavage site sequences in their physiological substrates. Capitalizing on its unique preference for proline and glycine at the P2 and P3 positions, respectively, selective substrates and a substrate-based inhibitor were developed for cathepsin K. A cluster analysis of the proteases based on the complete specificity profile provided a functional characterization distinct from standard sequence analysis. This approach provides useful information for developing selective chemical probes to study protease-related pathologies and physiologies.

Animals↗

PubMed2006

Degradation of apolipoprotein B-100 by lysosomal cysteine cathepsins.

Although the degradation of cellular or endocytosed proteins comprises the normal function of lysosomal proteinases, these enzymes were also detected extracellularly during diseases such as atherosclerosis. Since lysosomal cysteine cathepsins were demonstrated to transform native LDL particles into a proatherogenic type, the following study was undertaken to characterize the modification of LDL particles and the degradation of apolipoprotein B-100 in more detail. LDL was incubated with cathepsins B, F, K, L, S, and V at pH 5.5 and under physiological conditions (pH 7.4) for 2 h to mimic conditions of limited proteolysis. Gel electrophoretic analysis of the degradation products revealed that cathepsin-mediated proteolysis of apolipoprotein B-100 is a fast process carried out by all enzymes at pH 5.5, and by cathepsin S also at pH 7.4. Electron microscopic analysis showed that cathepsin-mediated degradation of apolipoprotein B-100 rendered LDL particles fusion-competent compared to controls. N-Terminal sequencing of cathepsin cleavage fragments from apolipoprotein B-100 revealed an abundance of enzyme-specific cleavage sites located in almost all structurally and functionally essential regions. Since the cleavage sites superimpose well with results from substrate specificity studies, they might be useful for the development of cathepsin-specific inhibitors and substrates.

Amino Acid Sequence↗

PubMed2005

The role of cathepsins in osteoporosis and arthritis: rationale for the design of new therapeutics.

Human cysteine proteases of the papain family have been recognized as potential drug targets for musculoskeletal diseases. Most of the interest is focused on cathepsins S and K, which display selective expression in cells of the immune system and cells capable to efficiently degrade extracellular matrix proteins, in particular collagens. The predominant expression of cathepsin K in osteoclasts has rendered the enzyme into a major target for the development of novel anti-resorptive drugs in osteoporosis whereas cathepsin S appears to be an attractive drug target candidate for various inflammatory diseases including rheumatoid arthritis. Since rheumatoid arthritis is at the same time an inflammatory and joint destructive disorder, the combined inhibition of both cathepsins S and K should be beneficial. This review will outline the rationale and recent progress for targeting cathepsins in arthritis and osteoporosis.

Arthritis, Rheumatoid↗

PubMed2005

Drug delivery strategies for cathepsin inhibitors in joint diseases.

Cathepsins play important roles in the development of joint and bone diseases such as osteoporosis, rheumatoid arthritis (RA) and osteoarthritis (OA). Cathepsin inhibitors are presently in development and clinical testing for use as novel disease-modifying drugs for the improved treatment of osteoporosis. They may also be applicable for the treatment of joint diseases. However, some barriers still hamper their clinical applications in these indications. Based on pathophysiological features of RA and OA, the authors discuss six potential drug delivery strategies for the effective delivery of cathepsin inhibitors or other antiarthritic drugs to the arthritic joint tissue. Successful application of these strategies may significantly contribute to a more effective and safe treatment of RA and OA.

Albumins↗

PubMed2004

Cathepsin K: a cysteine protease with unique kinin-degrading properties.

Taking into account a previous report of an unidentified enzyme from macrophages acting as a kininase, the ability of cysteine proteases to degrade kinins has been investigated. Wild-type fibroblast lysates from mice, by contrast with cathepsin K-deficient lysates, hydrolysed BK (bradykinin), and released two metabolites, BK-(1-4) and BK-(5-9). Cathepsin K, but not cathepsins B, H, L and S, cleaved kinins at the Gly4-Phe5 bond and the bradykinin-mimicking substrate Abz (o-aminobenzoic acid)-RPPGFSPFR-3-NO2-Tyr (3-nitrotyrosine) more efficiently (pH 6.0: kcat/K(m)=12500 mM(-1) x s(-1); pH 7.4: kcat/K(m)=6930 mM(-1) x s(-1)) than angiotensin-converting enzyme hydrolysed BK. Conversely Abz-RPPGFSPFR-3-NO2-Tyr was not cleaved by the Y67L (Tyr67-->Leu)/L205A (Leu205-->Ala) cathepsin K mutant, indicating that kinin degradation mostly depends on the S2 substrate specificity. Kininase activity was further evaluated on bronchial smooth muscles. BK, but not its metabolites BK(1-4) and BK(5-9), induced a dose-dependent contraction, which was abolished by Hoe140, a B2-type receptor antagonist. Cathepsin K impaired BK-dependent contraction of normal and chronic hypoxic rats, whereas cathepsins B and L did not. Taking together vasoactive properties of kinins and the potency of cathepsin K to modulate BK-dependent contraction of smooth muscles, the present data support the notion that cathepsin K may act as a kininase, a unique property among mammalian cysteine proteases.

Animals↗

PubMed2004

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.

Catalysis↗

PubMed2004

Identification and activity of a lower eukaryotic serine proteinase inhibitor (serpin) from Cyanea capillata: analysis of a jellyfish serpin, jellypin.

Delineating the phylogenetic relationships among members of a protein family can provide a high degree of insight into the evolution of domain structure and function relationships. To identify an early metazoan member of the high molecular weight serine proteinase inhibitor (serpin) superfamily, we initiated a cDNA library screen of the cnidarian, Cyanea capillata. We identified one serpin cDNA encoding for a full-length serpin, jellypin. Phylogenetic analysis using the deduced amino acid sequence showed that jellypin was most similar to the platyhelminthe Echinococcus multiocularis serpin and the clade P serpins, suggesting that this serpin evolved approximately 1000 million years ago (MYA). Modeling of jellypin showed that it contained all the functional elements of an inhibitory serpin. In vitro biochemical analysis confirmed that jellypin was an inhibitor of the S1 clan SA family of serine proteinases. Analysis of the interactions between the human serine proteinases, chymotrypsin, cathepsin G, and elastase, showed that jellypin inhibited these enzymes in the classical serpin manner, forming a SDS stable enzyme/inhibitor complex. These data suggest that the coevolution of serpin structure and inhibitory function date back to at least early metazoan evolution, approximately 1000 MYA.

Amino Acid Sequence↗

PubMed2004

Cathepsin V, a novel and potent elastolytic activity expressed in activated macrophages.

Atherosclerosis is characterized by a thickening and loss of elasticity of the arterial wall. Loss of elasticity has been attributed to the degradation of the arterial elastin matrix. Cathepsins K and S are papain-like cysteine proteases with known elastolytic activities, and both enzymes have been identified in macrophages present in plaque areas of diseased blood vessels. Here we demonstrate that macrophages express a third elastolytic cysteine protease, cathepsin V, which exhibits the most potent elastase activity yet described among human proteases and that cathepsin V is present in atherosclerotic plaque specimens. Approximately 60% of the total elastolytic activity of macrophages can be attributed to cysteine proteases with cathepsins V, K, and S contributing equally. From this 60%, two-thirds occur extracellularly and one-third intracellularly with the latter credited to cathepsin V. Ubiquitously expressed glycosaminoglycans (GAGs) such as chondroitin sulfate specifically inhibit the elastolytic activities of cathepsins V and K via the formation of specific cathepsin-GAG complexes. In contrast, cathepsin S, which does not form complexes with chondroitin sulfate is not inhibited; thus suggesting a specific regulation of elastolytic activities of cathepsins by GAGs. Because the GAG content is reduced in atherosclerotic plaques, an increase of cathepsins V and K activities may accelerate the destruction of the elastin matrix in diseased arteries.

Animals↗

PubMed2004

Cysteine protease cathepsin F is expressed in human atherosclerotic lesions, is secreted by cultured macrophages, and modifies low density lipoprotein particles in vitro.

During atherogenesis, low density lipoprotein (LDL) particles in the arterial intima become modified and fuse to form extracellular lipid droplets. Proteolytic modification of apolipoprotein (apo) B-100 may be one mechanism of droplet formation from LDL. Here we studied whether the newly described acid protease cathepsin F can generate LDL-derived lipid droplets in vitro. Treatment of LDL particles with human recombinant cathepsin F led to extensive degradation of apoB-100, which, as determined by rate zonal flotation, electron microscopy, and NMR spectroscopy, triggered both aggregation and fusion of the LDL particles. Two other acid cysteine proteases, cathepsins S and K, which have been shown to be present in the arterial intima, were also capable of degrading apoB-100, albeit less efficiently. Cathepsin F treatment resulted also in enhanced retention of LDL to human arterial proteoglycans in vitro. Cultured monocyte-derived macrophages were found to secrete active cathepsin F. In addition, similarly with cathepsins S and K, cathepsin F was found to be localized mainly within the macrophage-rich areas of the human coronary atherosclerotic plaques. These results suggest that proteolytic modification of LDL by cathepsin F may be one mechanism leading to the extracellular accumulation of LDL-derived lipid droplets within the proteoglycan-rich extracellular matrix of the arterial intima during atherogenesis.

Animals↗

PubMed2004

SRP-2 is a cross-class inhibitor that participates in postembryonic development of the nematode Caenorhabditis elegans: initial characterization of the clade L serpins.

High molecular weight serpins are members of a large superfamily of structurally conserved proteins that inactivate target proteinases by a suicide substrate-like mechanism. In vertebrates, different clades of serpins distribute predominantly to either the intracellular or extracellular space. Although much is known about the function, structure, and inhibitory mechanism of circulating serpins such as alpha(1)-antitrypsin (SERPINA1) and antithrombin III (SERPINC1), relatively little is known about the function of the vertebrate intracellular (clade B) serpins. To gain a better understanding of the biology of the intracellular serpins, we initiated a comparative genomics study using Caenorhabditis elegans as a model system. A screen of the C. elegans genomic and cDNA databases revealed nine serpin genes, tandemly arrayed on chromosome V. Although the C. elegans serpins represent a unique clade (L), they share significant functional homology with members of the clade B group of intracellular serpins, since they lack typical N-terminal signal peptides and reside intracellularly. To determine whether nematode serpins function as proteinase inhibitors, one family member, srp-2, was chosen for further characterization. Biochemical analysis of recombinant SRP-2 protein revealed SRP-2 to be a dual cross-class inhibitor of the apoptosis-related serine proteinase, granzyme B, and the lysosomal cysteine proteinases, cathepsins K, L, S, and V. Analysis of temporal and spatial expression indicated that SRP-2 was present during early embryonic development and highly expressed in the intestine and hypoderm of larval and adult worms. Transgenic animals engineered to overexpress SRP-2 were slow growing and/or arrested at the first, second, or third larval stages. These data suggest that perturbations of serpin-proteinase balance are critical for correct postembryonic development in C. elegans.

Amino Acid Sequence↗

PubMed2004

The amplified mouse squamous cell carcinoma antigen gene locus contains a serpin (Serpinb3b) that inhibits both papain-like cysteine and trypsin-like serine proteinases.

The clade B serpins occupy a unique niche among a larger superfamily by predominantly regulating intracellular proteolysis. In humans, there are 13 family members that map to serpin gene clusters at either 6p25 or 18q21. While most of these serpins display a unique inhibitory profile and appear to be well conserved in mammals, the clade B loci of several species show evidence of relatively recent genomic amplification events. However, it is not clear whether these serpin gene amplification events yield paralogs with functional redundancy or, through selective pressure, inhibitors with more diverse biochemical activities. A recent comparative genomic analysis of the mouse clade B cluster at 1D found nearly complete conservation of gene number, order, and orientation relative to those of 18q21 in humans. The only exception was the squamous cell carcinoma antigen (SCCA) locus. The human SCCA locus contains two genes, SERPINB3 (SCCA1) and SERPINB4 (SCCA2), whereas the mouse locus contains four serpins and three pseudogenes. At least two of these genes encoded functional, dual cross-class proteinase inhibitors. Mouse Serpinb3a was shown previously to inhibit both chymotrypsin-like serine and papain-like cysteine proteinases. We now report that mouse Serpinb3b extends the inhibitory repertoire of the mouse SCCA locus to include a second cross-class inhibitor with activity against both papain-like cysteine and trypsin-like serine proteinases. These findings confirmed that the genomic expansion of the clade B serpins in the mouse was associated with a functional diversification of inhibitory activity.

Amino Acid Sequence↗

PubMed2004

Pivotal role of cathepsin K in lung fibrosis.

The paramount importance of the homeostasis of the extracellular matrix for pulmonary function is exemplified by two opposing extremes: emphysema and pulmonary fibrosis. This study examined the putative role of cathepsin K (catK) in the pathology of lung fibrosis in mice and its relevance to the human disease activity. We compared the induction of lung fibrosis by administration of bleomycin. CTSK(-/-) mice deposited significantly more extracellular matrix than control mice. Primary lung fibroblasts derived from CTSK(-/-) mice showed a decreased collagenolytic activity indicating the role of catK in collagen degradation. Interestingly, CTSK(+/+) control mice revealed an increased expression of catK in fibrotic lung regions suggesting a protective role of catK to counter the excessive deposition of collagen matrix in the diseased lung. Similarly, in lung specimens obtained from patients with lung fibrosis fibroblasts expressed larger amounts of catK than those obtained from normal lungs. Activation of human pulmonary fibroblasts in primary cell cultures led to an increased activity of catK through enhanced gene transcription and protein expression and to increased intracellular collagenolytic activity. We believe that this is the first study to show that catK plays a pivotal role in lung matrix homeostasis under physiological and pathological conditions.

Animals↗

PubMed2004

Production and activation of recombinant papain-like cysteine proteases.

Papain-like cysteine proteases are the most numerous family of the cysteine protease class. They are expressed throughout the animal and plant kingdoms as well as in viruses and bacteria. More recently, this protease family has drawn attention as a potential pharmaceutical drug target in diseases characterized by excessive extracellular matrix degradation such as in osteoporosis, arthritis, vascular diseases, and cancer. Moreover, papain-like cysteine proteases have been identified as critical components of the life cycle and invasive potential of various human and live stock pathogens as well as major allergens. Therefore, this protease class is rigorously studied and requires sufficient amounts of protease protein to analyze structure-activity relationships, their 3-D structures as well as to screen for and optimize potent and selective inhibitors. This review summarizes approaches to generate active papain-like cysteine proteases by heterologous expression in a variety of expression systems.

Animals↗

PubMed2004

The human cysteine protease cathepsin V can compensate for murine cathepsin L in mouse epidermis and hair follicles.

Mice lacking the ubiquitously expressed lysosomal cysteine protease cathepsin L, show a complex skin phenotype consisting of periodic hair loss and epidermal hyperplasia with hyperproliferation of basal epidermal keratinocytes, acanthosis and hyperkeratosis. The recently identified human cathepsin L-like enzyme cathepsin V, which is also termed cathepsin L2, is specifically expressed in cornea, testis, thymus, and epidermis. To date, in mice no cathepsin V orthologue with this typical expression pattern has been identified. Since cathepsin V has about 75% protein sequence identity to murine cathepsin L, we hypothesized that transgenic, keratinocyte-specific expression of cathepsin V in cathepsin L knockout mice might rescue the skin and hair phenotype. Thus, we generated a transgenic mouse line expressing cathepsin V under the control of the human keratin 14 promoter, which mimics the genuine cathepsin V expression pattern in human skin, by directing it to basal epidermal keratinocytes and the outer root sheath of hair follicles. Subsequently, transgenic mice were crossed with congenic cathepsin L knockout animals. The resulting mice show normalization of epidermal proliferation and normal epidermal thickness as well as rescue of the hair phenotype. These findings provide evidence for keratinocyte-specific pivotal functions of cathepsin L-like proteolytic activities in maintenance of epidermis and hair follicles and suggest, that cathepsin V may perform similar functions in human skin.

Animals↗

PubMed2004

Human cathepsin F: expression in baculovirus system, characterization and inhibition by protein inhibitors.

Recombinant full-length human procathepsin F, produced in the baculovirus expression system, was partially processed during the purification procedure to a form lacking the N-terminal cystatin-like domain and activated with pepsin. Active cathepsin F efficiently hydrolyzed Z-FR-MCA (kcat/Km=106 mM(-1) s(-1)) and Bz-FVR-MCA (kcat/Km=8 mM(-1) s(-1)), whereas hydrolysis of Z-RR-MCA was very slow (kcat/Km<0.2 mM(-1) s(-1)). Cathepsin F was rapidly and tightly inhibited by cystatin C, chicken cystatin and equistatin with Ki values in the subnanomolar range (0.03-0.47 nM), whereas L-kininogen was a less strong inhibitor of the enzyme (Ki=4.7 nM). Stefin A inhibited cathepsin F slowly (kass=1.6 x 10(5) M(-1) s(-1)) and with a lower affinity (Ki=25 nM). These data suggest that cathepsin F differs from other related endopeptidases by considerably weaker inhibition by stefins.

Animals↗

PubMed2003

Cathepsins F and S block HDL3-induced cholesterol efflux from macrophage foam cells.

In atherosclerosis, accumulation of cholesterol in macrophages may partially depend on its defective removal by high-density lipoproteins (HDL). We studied the proteolytic effect of cathepsins F, S, and K on HDL(3) and on lipid-free apoA-I, and its consequence on their function as inductors of cholesterol efflux from cholesterol-filled mouse peritoneal macrophages in vitro. Incubation of HDL(3) with cathepsin F or S, but not with cathepsin K, led to rapid loss of prebeta-HDL, and reduced cholesterol efflux by 50% in only 1min. Cathepsins F or K partially degraded lipid-free apoA-I and reduced its ability to induce cholesterol efflux, whereas cathepsin S totally degraded apoA-I, leading to complete loss of apoA-I cholesterol acceptor function. These results suggest that cathepsin-secreting cells induce rapid depletion of lipid-poor (prebeta-HDL) and lipid-free apoA-I and inhibit cellular cholesterol efflux, so tending to promote the formation and maintenance of foam cells in atherosclerotic lesions.

Animals↗

PubMed2003

Regulation of collagenase activities of human cathepsins by glycosaminoglycans.

Cathepsin K, a lysosomal papain-like cysteine protease, forms collagenolytically highly active complexes with chondroitin sulfate and represents the most potent mammalian collagenase. Here we demonstrate that complex formation with glycosaminoglycans (GAGs) is unique for cathepsin K among human papain-like cysteine proteases and that different GAGs compete for the binding to cathepsin K. GAGs predominantly expressed in bone and cartilage, such as chondroitin and keratan sulfates, enhance the collagenolytic activity of cathepsin K, whereas dermatan, heparan sulfate, and heparin selectively inhibit this activity. Moreover, GAGs potently inhibit the collagenase activity of other cysteine proteases such as cathepsins L and S at 37 degrees C. Along this line MMP1-generated collagen fragments in the presence of GAGs are stable against further degradation at 28 degrees C by all cathepsins but cathepsin K, whereas thermal destabilization at 37 degrees C renders the fragments accessible to all cathepsins. These results suggest a novel mechanism for the regulation of matrix protein degradation by GAGs. It further implies that cathepsin K represents the only lysosomal collagenolytic activity under physiologically relevant conditions.

Animals↗