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.     

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.     

Evidence that catalytically-inactivated thrombin forms non-covalently linked dimers that bridge between fibrin/fibrinogen fibers and enhance fibrin polymerization

Phe-pro-arg-chloromethyl ketone-inhibited alpha-thrombin [FPR alpha-thr] retains its fibrinogen recognition site (exosite 1), augments fibrin/fibrinogen [fibrin(ogen)] polymerization, and increases the incorporation of fibrin into clots. There are two 'low-affinity' thrombin-binding sites in each central E domain of fibrin, plus a non-substrate 'high affinity' gamma' chain thrombin-binding site on heterodimeric 'fibrin(ogen) 2' molecules (gamma(A), gamma'). 'Fibrin(ogen) 1' (gamma(A), gamma(A)) containing only low-affinity thrombin-binding sites, showed concentration-dependent FPR alpha-thr enhancement of polymerization, thus indicating that low-affinity sites are sufficient for enhancing polymerization. FPR gamma-thr, whose exosite 1 is non-functional, did not enhance polymerization of either fibrin(ogen)s 1 or 2 and DNA aptamer HD-1, which binds specifically to exosite 1, blocked FPR alpha-thr enhanced polymerization of both types of fibrin(ogen) (1>2). These results showed that exosite 1 is the critical element in thrombin that mediates enhanced fibrin polymerization. Des B beta 1-42 fibrin(ogen) 1, containing defective 'low-affinity' binding sites, was subdued in its FPR alpha-thr-mediated reactivity, whereas des B beta 1-42 fibrin(ogen) 2 (gamma(A), gamma') was more reactive. Thus, the gamma' chain thrombin-binding site contributes to enhanced FPR alpha-thr mediated polymerization and acts through a site on thrombin that is different from exosite 1, possibly exosite 2. Overall, the results suggest that during fibrin clot formation, catalytically-inactivated FPR alpha-thr molecules form non-covalently linked thrombin dimers, which serve to enhance fibrin polymerization by bridging between fibrin(ogen) molecules, mainly through their low affinity sites.     

3-Acylamino-azetidin-2-one as a novel class of cysteine proteases inhibitors

A new class of inhibitors for cysteine proteases cathepsin B, L, K and S is described. These inhibitors are based on the beta-lactam ring designed to interact with the nucleophilic thiol of the cysteine in the active site of cysteine proteases. Some 3-acylamino-azetidin-2-one derivatives showed very potent inhibition activities for cathepsins L, K and S at the nanomolar or subnanomolar IC(50) values.     

Probing interactions between the coagulants thrombin, Factor XIII, and fibrin(ogen)

Thrombin cleaves fibrinopeptides A and B from fibrinogen leading to the formation of a fibrin network that is later covalently crosslinked by Factor XIII (FXIII). Thrombin helps activate FXIII by catalyzing hydrolysis of the FXIII activation peptides (AP). In the current work, the role of exosites in the ternary thrombin-FXIII-fibrin(ogen) complex was further explored. Hydrolysis studies indicate that thrombin predominantly utilizes its active site region to bind extended Factor XIII AP (FXIII AP 33-64 and 28-56) leaving the anion-binding exosites for fibrin(ogen) binding. The presence of fibrin-I leads to improvements in the K(m) for hydrolysis of FXIII AP (28-41), whereas peptides based on the cardioprotective FXIII V34L sequence exhibit less reliance on this cofactor. Surface plasmon resonance measurements reveal that d-Phe-Pro-Arg-chloromethylketone-thrombin binds to fibrinogen faster than to FXIII a(2) and dissociates from fibrinogen more slowly than from FXIII a(2). This system of thrombin exosite interactions with differing affinities promotes efficient clot formation.     

Localization of the domains of fibrin involved in binding to platelets

The molecular basis of platelet-fibrin interactions has been investigated by using synthetic peptides as potential inhibitors of fibrin protofibril and fibrinogen binding to ADP-stimulated platelets, adhesion of fibrin fibers to the platelet surface, and platelet-mediated clot retraction. Synthetic peptides of sequence RGDS and HHLGGAKQAGDV, corresponding to regions of the fibrinogen alpha- and gamma-chains previously identified as platelet recognition sites, inhibited the binding of radiolabelled soluble fibrin oligomers to ADP-stimulated platelets with IC50 values of 10 and 40 microM, respectively. Synthetic GPRP and GHRP, corresponding to the N-terminal tripeptide sequence of the fibrin alpha-chains and the tetrapeptide sequence of the beta-chains, respectively, were minimally effective in blocking soluble fibrin polymer binding to ADP-stimulated platelets. Platelet functions which are unique to the three-dimensional fibrin network were examined by measurements of the extent of adhesion of fluorophore-labelled fibrin to platelets with a microfluorimetric technique and by light scattering measurements of the time course of clot retraction. Inhibition of fibrin-platelet adhesion by RGDS, HHLGGAKQAGDV and GHRP exhibited a similar, linear dependence reaching 1/2 maximum at about 200 microM, suggesting nonspecific effects. GPRP inhibited fibrin assembly but did not appear to have specific effects on fibrin-platelet adhesion. Only RGDS effected clot retraction, causing a 4-6-fold decrease in rate at 230 microM. These results indicate that fibrinogen and fibrin protofibrils, which are obligatory intermediates on the fibrin assembly pathway, share a set of common platelet recognition sites located at specific regions of the alpha- and gamma-chains of the multinodular fibrin(ogen) molecules. The RGDS site is also involved in mediating interactions between the three-dimensional fibrin network and stimulated platelets.     

Human cathepsins K,L, and S: Related proteases,but unique fibrinolytic activity

Background

Fibrin formation and dissolution are attributed to cascades of protease activation concluding with thrombin activation, and plasmin proteolysis for fibrin breakdown. Cysteine cathepsins are powerful proteases secreted by endothelial cells and others during cardiovascular disease and diabetes. Their fibrinolytic activity and putative role in hemostasis has not been well described.

Cysteine cathepsins: from structure, function and regulation to new frontiers

It is more than 50 years since the lysosome was discovered. Since then its hydrolytic machinery, including proteases and other hydrolases, has been fairly well identified and characterized. Among these are the cysteine cathepsins, members of the family of papain-like cysteine proteases. They have unique reactive-site properties and an uneven tissue-specific expression pattern. In living organisms their activity is a delicate balance of expression, targeting, zymogen activation, inhibition by protein inhibitors and degradation. The specificity of their substrate binding sites, small-molecule inhibitor repertoire and crystal structures are providing new tools for research and development. Their unique reactive-site properties have made it possible to confine the targets simply by the use of appropriate reactive groups. The epoxysuccinyls still dominate the field, but now nitriles seem to be the most appropriate "warhead". The view of cysteine cathepsins as lysosomal proteases is changing as there is now clear evidence of their localization in other cellular compartments. Besides being involved in protein turnover, they build an important part of the endosomal antigen presentation. Together with the growing number of non-endosomal roles of cysteine cathepsins is growing also the knowledge of their involvement in diseases such as cancer and rheumatoid arthritis, among others. Finally, cysteine cathepsins are important regulators and signaling molecules of an unimaginable number of biological processes. The current challenge is to identify their endogenous substrates, in order to gain an insight into the mechanisms of substrate degradation and processing. In this review, some of the remarkable advances that have taken place in the past decade are presented. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

A single amino acid substitution affects substrate specificity in cysteine proteinases from Fasciola hepatica

The trematode Fasciola hepatica secretes a number of cathepsin L-like proteases that are proposed to be involved in feeding, migration, and immune evasion by the parasite. To date, six full cDNA sequences encoding cathepsin L preproproteins have been identified. Previous studies have demonstrated that one of these cathepsins (L2) is unusual in that it is able to cleave substrates with a proline in the P2 position, translating into an unusual ability (for a cysteine proteinase) to clot fibrinogen. In this study, we report the sequence of a novel cathepsin (L5) and compare the substrate specificity of a recombinant enzyme with that of recombinant cathepsin L2. Despite sharing 80% sequence identity with cathepsin L2, cathepsin L5 does not exhibit substantial catalytic activity against substrates containing proline in the P2 position. Molecular modeling studies suggested that a single amino acid change (L69Y) in the mature proteinases may account for the difference in specificity at the S2 subsite. Recombinant cathepsin L5/L69Y was expressed in yeast and a substantial increase in the ability of this variant to accommodate substrates with a proline residue in the P2 position was observed. Thus, we have identified a single amino acid substitution that can substantially influence the architecture of the S2 subsite of F. hepatica cathepsin L proteases.     

Incorporation of vitronectin into fibrin clots. Evidence for a binding interaction between vitronectin and gamma A/gamma' fibrinogen.

Vitronectin is an abundant plasma protein that regulates coagulation, fibrinolysis, complement activation, and cell adhesion. Recently, we demonstrated that plasma vitronectin inhibits fibrinolysis by mediating the interaction of type 1 plasminogen activator inhibitor with fibrin (Podor, T. J., Peterson, C. B., Lawrence, D. A., Stefansson, S., Shaughnessy, S. G., Foulon, D. M., Butcher, M., and Weitz, J. I. (2000) J. Biol. Chem. 275, 19788-19794). The current studies were undertaken to further examine the interactions between vitronectin and fibrin(ogen). Comparison of vitronectin levels in plasma with those in serum indicates that approximately 20% of plasma vitronectin is incorporated into the clot. When the time course of biotinylated-vitronectin incorporation into clots formed from (125)I-fibrinogen is monitored, vitronectin incorporation into the clot parallels that of fibrinogen in the absence or presence of activated factor XIII. Vitronectin binds specifically to fibrin matrices with an estimated K(d) of approximately 0.6 microm. Additional vitronectin subunits are assembled on fibrin-bound vitronectin multimers through self-association. Confocal microscopy of fibrin clots reveals the globular vitronectin aggregates anchored at intervals along the fibrin fibrils. This periodicity raised the possibility that vitronectin interacts with the gamma A/gamma' variant of fibrin(ogen) that represents about 10% of total fibrinogen. In support of this concept, the vitronectin which contaminates fibrinogen preparations co-purifies with the gamma A/gamma' fibrinogen fraction, and clots formed from gamma A/gamma' fibrinogen preferentially bind vitronectin. These studies reveal that vitronectin associates with fibrin during coagulation, and may thereby modulate hemostasis and inflammation.     

Human cathepsins F and W: A new subgroup of cathepsins.

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.     

Protein kinase C-alpha and -delta are required for NADPH oxidase activation in WKYMVm-stimulated IMR90 human fibroblasts

Gene duplications in rodents have given rise to a family of proteases that are expressed exclusively in placenta. To define the biological role of these enzymes specific inhibitors are needed to differentiate their activities from other more ubiquitously expressed proteases, such as cathepsins B and L. Libraries of peptidyl inhibitors based upon a 4-cyclohexanone pharmacophore were screened for inhibition of cathepsins P, L, and B. The tightest binding dipeptidyl inhibitor for cathepsin P contained Tyr in P(2) and Trp in P(2)('), consistent with the specificity of this enzyme for hydrophobic amino acids at these sites in synthetic substrates. An inhibitor containing Trp in both P(2) and P(2)(') provided better discrimination between cathepsin P and cathepsins B and L. Extension of the inhibitors to include P(3), and P(3)(') amino acids identified an inhibitor with Trp in P(2), P(2)('), and P(3), and Phe in P(3)(') that bound to cathepsin P with a K(i) of 32 nM. This specificity for inhibitors with hydrophobic aromatic amino acids in these four positions is unique among the lysosomal cysteine proteases. This inhibitor bound to cathepsin P an order of magnitude tighter than to mouse and human cathepsin L and two orders of magnitude tighter than to human cathepsin B. Cbz-Trp-Trp-4-cyclohexanone-Trp-Phe-OMe can discriminate cathepsin P from cathepsins B and L and consequently can be used to specifically inhibit and identify cathepsin P in cellular systems.     

Cathepsins L and Z are critical in degrading polyglutamine-containing proteins within lysosomes

In neurodegenerative diseases caused by extended polyglutamine (polyQ) sequences in proteins, aggregation-prone polyQ proteins accumulate in intraneuronal inclusions. PolyQ proteins can be degraded by lysosomes or proteasomes. Proteasomes are unable to hydrolyze polyQ repeat sequences, and during breakdown of polyQ proteins, they release polyQ repeat fragments for degradation by other cellular enzymes. This study was undertaken to identify the responsible proteases. Lysosomal extracts (unlike cytosolic enzymes) were found to rapidly hydrolyze polyQ sequences in peptides, proteins, or insoluble aggregates. Using specific inhibitors against lysosomal proteases, enzyme-deficient extracts, and pure cathepsins, we identified cathepsins L and Z as the lysosomal cysteine proteases that digest polyQ proteins and peptides. RNAi for cathepsins L and Z in different cell lines and adult mouse muscles confirmed that they are critical in degrading polyQ proteins (expanded huntingtin exon 1) but not other types of aggregation-prone proteins (e.g. mutant SOD1). Therefore, the activities of these two lysosomal cysteine proteases are important in host defense against toxic accumulation of polyQ proteins.     

Ovarian cysteine proteinases in the teleost Fundulus heteroclitus: molecular cloning and gene expression during vitellogenesis and oocyte maturation

The cysteine proteinases cathepsins B and L are members of the multigene family of lysosomal proteases that have been implicated in the processing of yolk proteins (YPs) in teleost oocytes. However, the full identification of the type of cathepsins expressed in fish ovarian follicles and embryos, as well as their regulatory mechanisms and specific function(s), are not yet elucidated. In this study, cDNAs encoding cathepsins B, L, F, K, S, Z, C, and H have been isolated from the teleost Fundulus heteroclitus, and the analysis of their deduced amino acid sequences revealed highly similar structural features to vertebrate orthologs, and confirmed in this species the existence of cathepsin L-like, cathepsin B-like, and cathepsin F-like subfamilies of cysteine proteinases. While all identified cathepsins were expressed in ovarian follicles, the corresponding mRNAs showed different temporal expression patterns. Thus, similar mRNA levels of cathepsins L, F, S, B, C, and Z were found throughout the oocyte growth or vitellogenesis period, whereas those for cathepsin H and K appeared to decrease as vitellogenesis advanced. During oocyte maturation, a transient accumulation of cathepsins L, S, H, and F mRNAs, approximately a 3-, 1.5-, 1.6-, and 6-fold increase, respectively, was detected in ovarian follicles within the 20-25 hr after hormone stimulation, coincident with the maximum proteolysis of the oocyte major YPs. The specific temporal pattern of expression of these genes may indicate a potential role of cathepsin L-like and cathepsin F proteases in the YP processing events occurring during fish oocyte maturation and/or early embryogenesis.     

Multiple sites on SARS‐CoV‐2 spike protein are susceptible to proteolysis by cathepsins B,K, L,S, and V

SARS‐CoV‐2 is the coronavirus responsible for the COVID‐19 pandemic. Proteases are central to the infection process of SARS‐CoV‐2. Cleavage of the spike protein on the virus''s capsid causes the conformational change that leads to membrane fusion and viral entry into the target cell. Since inhibition of one protease, even the dominant protease like TMPRSS2, may not be sufficient to block SARS‐CoV‐2 entry into cells, other proteases that may play an activating role and hydrolyze the spike protein must be identified. We identified amino acid sequences in all regions of spike protein, including the S1/S2 region critical for activation and viral entry, that are susceptible to cleavage by furin and cathepsins B, K, L, S, and V using PACMANS, a computational platform that identifies and ranks preferred sites of proteolytic cleavage on substrates, and verified with molecular docking analysis and immunoblotting to determine if binding of these proteases can occur on the spike protein that were identified as possible cleavage sites. Together, this study highlights cathepsins B, K, L, S, and V for consideration in SARS‐CoV‐2 infection and presents methodologies by which other proteases can be screened to determine a role in viral entry. This highlights additional proteases to be considered in COVID‐19 studies, particularly regarding exacerbated damage in inflammatory preconditions where these proteases are generally upregulated.     

Cysteine cathepsins: From structure, function and regulation to new frontiers

It is more than 50 years since the lysosome was discovered. Since then its hydrolytic machinery, including proteases and other hydrolases, has been fairly well identified and characterized. Among these are the cysteine cathepsins, members of the family of papain-like cysteine proteases. They have unique reactive-site properties and an uneven tissue-specific expression pattern. In living organisms their activity is a delicate balance of expression, targeting, zymogen activation, inhibition by protein inhibitors and degradation. The specificity of their substrate binding sites, small-molecule inhibitor repertoire and crystal structures are providing new tools for research and development. Their unique reactive-site properties have made it possible to confine the targets simply by the use of appropriate reactive groups. The epoxysuccinyls still dominate the field, but now nitriles seem to be the most appropriate “warhead”. The view of cysteine cathepsins as lysosomal proteases is changing as there is now clear evidence of their localization in other cellular compartments. Besides being involved in protein turnover, they build an important part of the endosomal antigen presentation. Together with the growing number of non-endosomal roles of cysteine cathepsins is growing also the knowledge of their involvement in diseases such as cancer and rheumatoid arthritis, among others. Finally, cysteine cathepsins are important regulators and signaling molecules of an unimaginable number of biological processes. The current challenge is to identify their endogenous substrates, in order to gain an insight into the mechanisms of substrate degradation and processing. In this review, some of the remarkable advances that have taken place in the past decade are presented. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Differential expression of cathepsins K,S and V between young and aged Caucasian women skin epidermis

Cutaneous aging translates drastic structural and functional alterations in the extracellular matrix (ECM). Multiple mechanisms are involved, including changes in protease levels. We investigated the age-related protein expression and activity of cysteine cathepsins and the expression of two endogenous protein inhibitors in young and aged Caucasian women skin epidermis. Immunofluorescence studies indicate that the expression of cathepsins K, S and V, as well as cystatins A and M/E within keratinocytes is reduced in photoprotected skin of aged women. Furthermore, the overall endopeptidase activity of cysteine cathepsins in epidermis lysates decreased with age. Albeit dermal elastic fiber and laminin expression is reduced in aged skin, staining of nidogen-1, a key protein in BM assembly that is sensitive to proteolysis by cysteine, metallo- and serine proteases, has a similar pattern in both young and aged skin. Since cathepsins contribute to the hydrolysis and turnover of ECM/basement membrane components, the abnormal protein degradation and deposition during aging process may be related in part to a decline of lysosomal/endosomal cathepsin K, S and V activity.     

Antifibrinolytic effect of single apo(a) kringle domains: relationship to fibrinogen binding

Elevated plasma concentrations of lipoprotein(a) [Lp(a)] are associated with an increased risk for the development of atherosclerotic disease which may be attributable to the ability of Lp(a) to attenuate fibrinolysis. A generally accepted mechanism for this effect involves direct competition of Lp(a) with plasminogen for fibrin(ogen) binding sites thus reducing the efficiency of plasminogen activation. Efforts to determine the domains of apolipoprotein(a) [apo(a)] which mediate fibrin(ogen) interactions have yielded conflicting results. Thus, the purpose of the present study was to determine the ability of single KIV domains of apo(a) to bind plasmin-treated fibrinogen surfaces as well to determine their effect on fibrinolysis using an in vitro clot lysis assay. A bacterial expression system was utilized to express and purify apo(a) KIV (2), KIV (7), KIV (9) DeltaCys (which lacks the seventh unpaired cysteine) and KIV (10) which contains a strong lysine binding site. We also expressed and examined three mutant derivatives of KIV (10) to determine the effect of changing critical residues in the lysine binding site of this kringle on both fibrin(ogen) binding and fibrin clot lysis. Our results demonstrate that the strong lysine binding site in apo(a) KIV (10) is capable of mediating interactions with plasmin-modified fibrinogen in a lysine-dependent manner, and that this kringle can increase in vitro fibrin clot lysis time by approximately 43% at a concentration of 10 microM KIV (10). The ability of the KIV (10) mutant derivatives to bind plasmin-modified fibrinogen correlated with their lysine binding capacity. Mutation of Trp (70) to Arg abolished binding to both lysine-Sepharose and plasmin-modified fibrinogen, while the Trp (70) -->Phe and Arg (35) -->Lys substitutions each resulted in decreased binding to these substrates. None of the KIV (10) mutant derivatives appeared to affect fibrinolysis. Apo(a) KIV (7) contains a lysine- and proline-sensitive site capable of mediating binding to plasmin-modified fibrinogen, albeit with a lower apparent affinity than apo(a) KIV (10). However, apo(a) KIV (7) had no effect on fibrinolysis in vitro. Apo(a) KIV (2) and KIV (9) DeltaCys did not bind measurably to plasmin-modified fibrinogen surfaces and did not affect fibrinolysis in vitro.     

6-Acylamino-penam derivatives: synthesis and inhibition of cathepsins B,L, K,and S

The synthesis of a new series of 6-acylamino penam derivatives and their inhibition of cysteine proteases cathepsins B, L, K, and S is described. The 6-acylamino-penam sulfone compounds showed excellent cathepsin L, K, and S inhibition activity with IC(50) values in the nanomolar and subnanomolar range.     

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.