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Antigen-presenting cells (APC) play a pivotal role in the initiation of the T cell-mediated and antigen-specific immune response. The suggested role of endogenous inhibitor cystatin C (CyC) is to modulate cysteine proteases (cathepsins) present in human APC. To test this hypothesis, dendritic cells (DC) were generated in vitro from isolated monocytes, and changes in content, localization, and secretion of CyC and cathepsins S, L, and H (CatS, -L, and -H, repsectively) were followed in response to interleukin-4, enabling monocyte differentiation, and to tumor necrosis factor α (TNF-α), enabling DC maturation. A large increase in intracellular CyC accompanied the differentiation of monocytes to immature DC, also shown by strong immunolabeling of Golgi in immature DC. On DC maturation, intracellular CyC levels decreased, and CyC was mostly absent from the Golgi. On prolonged incubation of mature DC with TNF-α, CyC was found located in the proximity of the plasma membrane, indicating that the transport of CyC from Golgi was not blocked as the result of the arrested exocytosis in mature DC. The secretion of CyC ceased, consistent with the peak of the surface expression of phenotypic markers (CD40, CD54, CD80, CD83, CD86, and major histocompatibility complex class II), characteristic for the mature DC stage, whereas the secretion of cathepsins did not correlate with the maturation stage. The difference in localization of CyC and of CatS, -L, and -H in immature and mature DC shows that the regulatory potential of CyC toward CatS, -L, and -H inside DC is limited. However, these interactions may occur extracellularly in lymph, as suggested by the large excess of CyC over secreted CatS, -L, and -H, and they may facilitate DC migration to lymph nodes.

Stefin B (cystatin B) is an inhibitor of lysosomal cysteine cathepsins and does not inhibit cathepsin D, E (aspartic) or cathepsin G (serine) proteinases. In this study, we have investigated apoptosis triggered by camptothecin, staurosporin (STS), and anti-CD95 monoclonal antibody in the thymocytes from the stefin B-deficient mice and wild-type mice. We have observed increased sensibility to STS-induced apoptosis in the thymocytes of stefin B-deficient mice. Pretreatment of cells with pan-caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone completely inhibited phosphatidylserine externalization and caspase activation, while treatment with inhibitor of calpains- and papain-like cathepsins (2S,3S)-trans-epoxysuccinyl-leucylamido-3-methyl-butane ethyl ester did not prevent caspase activation nor phosphatidylserine exposure. We conclude that sensitization to apoptosis induced by STS in thymocytes of stefin B-deficient and wild-type mice is not dependent on cathepsin inhibition by stefin B.

Cystatin F is a recently discovered type II cystatin expressed almost exclusively in immune cells. It is present intracellularly in lysosome-like vesicles, which suggests a potential role in regulating papain-like cathepsins involved in antigen presentation. Therefore, interactions of cystatin F with several of its potential targets, cathepsins F, K, V, S, H, X and C, were studied in vitro. Cystatin F tightly inhibited cathepsins F, K and V with Ki values ranging from 0.17 nM to 0.35 nM, whereas cathepsins S and H were inhibited with 100-fold lower affinities (Ki approximately 30 nM). The exopeptidases, cathepsins C and X were not inhibited by cystatin F. In order to investigate the biological significance of the inhibition data, the intracellular localization of cystatin F and its potential targets, cathepsins B, H, L, S, C and K, were studied by confocal microscopy in U937 promonocyte cells. Although vesicular staining was observed for all the enzymes, only cathepsins H and X were found to be colocalized with the inhibitor. This suggests that cystatin F in U937 cells may function as a regulatory inhibitor of proteolytic activity of cathepsin H or, more likely, as a protection against cathepsins misdirected to specific cystatin F containing endosomal/lysosomal vesicles. The finding that cystatin F was not colocalized with cystatin C suggests distinct functions for these two cysteine protease inhibitors in U937 cells.

Loss of function mutations in the gene (CSTB) encoding human cystatin B, a widely expressed cysteine protease inhibitor, are responsible for a severe neurological disorder known as an Unverricht-Lundborg disease (EPM1). EPM1 had been linked to chromosome 21q22.3 in Finnish families and it is an autosomal recessive inherited disorder with a homozygous minisatellite expansion in the cystatin B gene (stefin B gene). This disease is difficult to treat because it is refractory to most antiepileptic drugs and using multiple medications had been unsuccessful so far. To come a step closer to understanding of the nature of this disease, especially about the events on the molecular level, in vitro properties of missense EPM1 mutant G4R were determined. It was observed that the mutant has a prolonged lag phase of fibrillation at the same protein stability, which could indicate it were more toxic to the cells. Similar experiments with the N-terminal fragment of 67 aminoacid residues are ongoing, showing higher propensity to aggregate. Therefore, a hypothesis is launched that at least in a subset of Unverricht-Lundborg disease patients, cystatin B protein may aggregate in the cell. Protein aggregation can be secondary to external insults or aging, however, inherited forms of neurodegenerative diseases, such as familial Parkinson's, Huntington's or familial Alzheimer's disease, are directly linked to the mutant proteins aggregation. Protein aggregates in the form of amyloid plaques, neurofibrilary tangles, intra-cytoplasmic or intra-nuclear inclusions lead to increased production of the reactive oxygen species and dysfunction of the ubiquitine/proteasome system. Finally, mitochondrial dysfunction and cell death are observed. Certainly, it remains to be checked by experiments whether overexpression in cell culture of the missense mutants G4R and N-terminal fragment to residue 68 lead to cellular inclusions and the accompanying changes characteristic for the conformational disorders.

During larva to adult transition, the larval fat body of the Medfly (Ceratitis capitata) progressively disintegrates to be replaced by the adult one, after imago ecdysis. Here we show that a temporal correlation exists among the microscopy images of fat body progressive disintegration, the activation of fat body lysosomes (as judged by acid phosphatase activity), and the activity of a novel fat body aspartyl proteinase. The enzyme was purified and partially characterized. This proteinase exhibited a wide range of acid isoforms with isoelectric points from 5.6 to 7.3, an optimum pH of 3.0 for hemoglobin digestion, and was completely inhibited by pepstatin A. The apparent molecular weight was estimated (42 ± 1 kDa) and the protein was characterized as N-glycosylated, judging from affinity to Concanavalin A. From the biochemical characteristics, the enzyme that we called “Early Metamorphosis Aspartyl Proteinase” (EMAP) appears to be similar to mammalian Cathepsin D. However, the N-terminal sequence of EMAP showed no similarity with any known animal Cathepsins and exhibited an important instability to neutral and alkaline pH. This feature seems to be a peculiar characteristic of insect aspartyl proteinases. The temporal activity profile of EMAP during metamorphosis correlated well with the microscopy images of fat body cell autolytic death. Our data support the notion that EMAP is a metamorphosis-specific lysosomal proteinase, mostly expressed during larval fat body histolysis. Arch. Insect Biochem. Physiol. 57:51–67, 2004. © 2004 Wiley-Liss, Inc.

Increasing evidence suggests that lysosomal proteases are actively involved in apoptosis. Using HeLa cells as the model system, we show that selective lysosome disruption with L-leucyl-L-leucine methyl ester results in apoptosis, characterized by translocation of lysosomal proteases into the cytosol and by the cleavage of a proapoptotic Bcl-2-family member Bid. Apoptosis and Bid cleavage, but not translocation of lysosomal proteases to the cytosol, could be prevented by 15 microM L-trans-epoxysuccinyl(OEt)-Leu-3-methylbutylamide, an inhibitor of papain-like cysteine proteases. Incubation of cells with 15 microM N-benzoyloxycarbonyl-VAD-fluoromethyl ketone prevented apoptosis but not Bid cleavage, suggesting that cathepsin-mediated apoptosis in this system is caspase-dependent. In vitro experiments performed at neutral pH showed that papain-like cathepsins B, H, L, S, and K cleave Bid predominantly at Arg(65) or Arg(71). No Bid cleavage was observed with cathepsins C and X or the aspartic protease cathepsin D. Incubation of full-length Bid treated with cathepsins B, H, L, and S resulted in rapid cytochrome c release from isolated mitochondria. Thus, Bid may be an important mediator of apoptosis induced by lysosomal disruption.

Human procathepsin H was expressed in the form of inclusion bodies in Escherichia coli. Following refolding and autocatalytic activation, a recombinant cathepsin H form lacking the mini chain was produced. Removal of the mini chain completely abolished aminopeptidase activity of the enzyme and largely increased its endopeptidase activity (approximately 40-fold). Similarly to cathepsin S, Bz-FVR-AMC (k(cat)/K(m) value of 1070 mM(-1) s(-1)) was found to be the preferred substrate of recombinant cathepsin H. However, substrate inhibition was observed at a higher substrate (Z-FR-AMC, Bz-FVR-AMC) concentration. Endopeptidase activity of recombinant cathepsin H was seen also with the protein substrate insulin beta-chain with the major cleavage site between Glu13-Ala14. Recombinant human cathepsin H was inhibited by chicken cystatin, stefin A, and stefin B with the K(i) values in the range of 0.05-0.1 nM, which is slightly tighter than the inhibition of purified cathepsin H by the same inhibitors. These results thus indicate that the cathepsin H mini chain is essential for the aminopeptidase activity of the enzyme but has only a minor effect on the inhibition by cystatins.

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.

Lysosomal cysteine proteinase cathepsin B is implicated in remodeling the extracellular matrix, a crucial step in the process of tumor cell invasion. In this study the contributions of intracellular and extracellular cathepsin B activities in the invasion of ras-transformed human breast epithelial cells, MCF-10A neoT, were assessed using specific cathepsin B neutralizing monoclonal antibody (Mab) 2A2, together with other general and specific cysteine proteinase inhibitors. We showed that the degradation of extracellular matrix by living MCF-10A neoT cells was predominantly intracellular, as imaged by confocal assays using quenched fluorescent substrate DQ-collagen IV. CA-074, a membrane-impermeable cathepsin B-selective inhibitor and its membrane-permeable analogue CA-074Me showed similar inhibition of invasion at 10 microM, i.e., 24.9 and 27.0%, respectively. Neutralizing monoclonal antibody exhibited a significantly higher inhibitory effect, decreasing invasion at 0.5 microM by 42.7%. Tumor cells may internalize monoclonal antibody; therefore, 2A2 Mab could impair both the intracellular and the extracellular fractions of cathepsin B activity. However, both 2A2 Mab and cathepsin B-selective inhibitors were less potent than the general cysteine proteinase inhibitors chicken cystatin and E-64, indicating that other cysteine proteinases, presumably cathepsin L, are involved in invasion. Our results show that intracellular and extracellular cathepsin B activity contribute to in vitro invasion of MCF-10A neoT cells and suggest that inhibitors capable of impairing both fractions have a potential as new anticancer drugs.

Extracellular cysteine cathepsins are known to drive cancer progression, but besides degradation of extracellular matrix proteins little is known about their physiological substrates and thus the molecular mechanisms they deploy. One of the major mechanisms used by other extracellular proteases to facilitate cancer progression is proteolytic release of the extracellular domains of transmembrane proteins or ectodomain shedding. Here we show using a mass spectrometry-based approach that cathepsins L and S act as sheddases and cleave extracellular domains of CAM adhesion proteins and transmembrane receptors from the surface of cancer cells. In cathepsin S-deficient mouse pancreatic cancers, processing of these cathepsin substrates is highly reduced, pointing to an essential role of cathepsins in extracellular shedding. In addition to influencing cell migration and invasion, shedding of surface proteins by extracellular cathepsins impacts intracellular signaling as demonstrated for regulation of Ras GTPase activity, thereby providing a putative mechanistic link between extracellular cathepsin activity and cancer progression. The MS data is available via ProteomeXchange with identifier PXD002192.

Cathepsin B (CtsB) is a lysosomal cysteine proteinase that is specifically translocated to the extracellular milieu during cancer progression. The development of a lipidated CtsB inhibitor incorporated into the envelope of a liposomal nanocarrier (LNC-NS-629) is described. Ex vivo and in vivo studies confirmed selective targeting and internalization of LNC-NS-629 by tumor and stromal cells, thus validating CtsB targeting as a highly promising approach to cancer diagnosis and treatment.

Human cysteine protease cathepsin L was inactivated at acid pH by a first-order process. The inactivation rate decreased with increasing concentrations of a small synthetic substrate, suggesting that substrates stabilize the active conformation. The substrate-independent inactivation rate constant increased with organic solvent content of the buffer, consistent with internal hydrophobic interactions, disrupted by the organic solvent, also stabilizing the enzyme. Circular dichroism showed that the inactivation is accompanied by large structural changes, a decrease in α-helix content being especially pronounced. The high activation energy of the reaction at pH 3.0 (200 kJ·mol−1) supported such a major conformational change occurring. The acid inactivation of cathepsin L was irreversible, consistent with the propeptide being needed for proper folding of the enzyme. Aspartic protease cathepsin D was shown to cleave denatured, but not active cathepsin L, suggesting a potential mechanism for in-vivo regulation and turnover of cathepsin L inside lysosomes.

Dipeptidyl peptidase I (DPPI) or cathepsin C is the physiological activator of groups of serine proteases from immune and inflammatory cells vital for defense of an organism. The structure presented shows how an additional domain transforms the framework of a papain-like endopeptidase into a robust oligomeric protease-processing enzyme. The tetrahedral arrangement of the active sites exposed to solvent allows approach of proteins in their native state; the massive body of the exclusion domain fastened within the tetrahedral framework excludes approach of a polypeptide chain apart from its termini; and the carboxylic group of Asp1 positions the N-terminal amino group of the substrate. Based on a structural comparison and interactions within the active site cleft, it is suggested that the exclusion domain originates from a metallo-protease inhibitor. The location of missense mutations, characterized in people suffering from Haim–Munk and Papillon–Lefevre syndromes, suggests how they disrupt the fold and function of the enzyme.

From their discovery in the first half of the 20th century, lysosomal cysteine proteases have come a long way: from being the enzymes non-selectively degrading proteins in lysosomes to being those responsible for a number of important cellular processes. Some of the features and roles of their structures, specificity, regulation and physiology are discussed.

We measured and compared the activities of various kinds of proteinases, such as cysteine, serine, aspartic, and metalloproteinases, in synovial fluids of 16 patients with rheumatoid arthritis (RA) and 18 patients with osteoarthritis (OA). More than 19-fold higher activity of cathepsin B and about 6-fold higher activity of prolylendopeptidase, compared to those of OA, were accumulated in RA fluid. Moreover, levels of cathepsins B and S using the corresponding sandwich enzyme immunoassays were statistically higher in RA fluid than those in OA. Significant amounts of 41-kDa and 35-kDa procathepsin L were detected in RA fluid using gelatin zymography, while 41-kDa enzyme alone was detected in OA. Cathepsin B in RA fluid could degrade collagen, and this degradation was suppressed by the addition of CA-074, a specific inhibitor of cathepsin B. Therefore, cathepsin B may participate in joint destruction of RA, and its inhibitor may be effective for RA care.

We investigated the mechanism of lysosome-mediated cell death using purified recombinant pro-apoptotic proteins, and cell-free extracts from the human neuronal progenitor cell line NT2. Potential effectors were either isolated lysosomes or purified lysosomal proteases. Purified lysosomal cathepsins B, H, K, L, S, and X or an extract of mouse lysosomes did not directly activate either recombinant caspase zymogens or caspase zymogens present in an NT2 cytosolic extract to any significant extent. In contrast, a cathepsin L-related protease from the protozoan parasiteTrypanosoma cruzi, cruzipain, showed a measurable caspase activation rate. This demonstrated that members of the papain family can directly activate caspases but that mammalian lysosomal members of this family may have been negatively selected for caspase activation to prevent inappropriate induction of apoptosis. Given the lack of evidence for a direct role in caspase activation by lysosomal proteases, we hypothesized that an indirect mode of caspase activation may involve the Bcl-2 family member Bid. In support of this, Bid was cleaved in the presence of lysosomal extracts, at a site six residues downstream from that seen for pathways involving capase 8. Incubation of mitochondria with Bid that had been cleaved by lysosomal extracts resulted in cytochrome c release. Thus, cleavage of Bid may represent a mechanism by which proteases that have leaked from the lysosomes can precipitate cytochrome c release and subsequent caspase activation. This is supported by the finding that cytosolic extracts from mice ablated in the bid gene are impaired in the ability to release cytochrome c in response to lysosome extracts. Together these data suggest that Bid represents a sensor that allows cells to initiate apoptosis in response to widespread adventitious proteolysis.

Stefins A and B are cysteine proteinase inhibitors that have considerable sequence similarity but marked differences in their stability and folding properties. Two chimeric proteins were designed to shed light on these differences. The chimeric mutants have been expressed in Escherichia coli and have been isolated. The first, A37B, consists of 37 residues of stefin A, comprising the N-terminal and the alpha-helix, joined to 61 residues of stefin B; the second, A61B, consists of 61 N-terminal residues of stefin A, followed by 37 residues of stefin B. Spectroscopic properties of the chimeric proteins (absorption, CD, and NMR spectra), together with activity measurements, have confirmed that both have well-defined tertiary structure and are active as cysteine proteinase inhibitors. Characterization consisted of GuHCl denaturation, ANS binding as a function of pH, and monitoring of dimerization under partially denaturing conditions. The c(m) values are 1.3 M GuHCl for A61B as compared with 2.7 M GuHCl for stefin A, and 2.1 M GuHCl for A37B as compared with 1.4 M GuHCl for stefin B (all at pH 7.5, 25 degrees C). However (G degrees (N-U) is lower for both chimeric proteins (18 +/- 3 kJ/mol) than for the parent stefins (28 +/- 3 kJ/mol). In pH denaturation, unlike stefin B, neither chimeric mutant unfolds to I(N) below pH 5.4. At pH 3, where stefin B forms a molten globule and stefin A is native, both A37B and A61B show increased ANS fluorescence and aggregate visibly. Dimers at pre-denaturation conditions are observed in all the proteins under study, but they remain "trapped" only in stefin A.

Human dipeptidyl peptidase I was expressed in the insect cell/baculovirus system and purified in its active (rhDPPI) and precursor (pro-rhDPPI) forms. RhDPPI was very similar to the purified enzyme (hDPPI) with respect to glycosylation, enzymatic processing, oligomeric structure, CD spectra, and catalytic activity. The precursor, which was a dimer, could be activated approximately 2000-fold with papain. Cathepsin L efficiently activated pro-rhDPPI in vitro at pH 4.5 (k(app) approximately 2 x 10(3) min(-)(1) M(-)(1)), and two cleavage pathways were characterized. The initial cleavage was within the pro region between the residual pro part and the activation peptide. Subsequently, the activation peptide was cleaved from the catalytic region, and the latter was cleaved into the heavy and light chains. Alternatively, the pro region was first separated from the catalytic region. Cathepsin S was a less efficient activating enzyme. Cathepsin B and rhDPPI did not activate pro-rhDPPI, and the proenzyme was incapable of autoactivation. Incubation of both pro-rhDPPI and rhDPPI with cathepsin D resulted in degradation. Cystatin C and stefins A and B inhibited rhDPPI with K(i) values in the nanomolar range (K(i) = 0.5-1.1 nM). The results suggest that cathepsin L could be an important activator of DPPI in vivo and that cathepsin D and possibly the cystatins may contribute to DPPI downregulation.

Cathepsin X, purified to homogeneity from human liver, is a single chain glycoprotein with a molecular mass of < 33 kDa and pI 5.1‐5.3. Cathepsin X was inhibited by stefin A, cystatin C and chicken cystatin (Kia 1.7‐15.0 nm), but poorly or not at all by stefin B (Ki . 250 nm) and l-kininogen, respectively. The enzyme was also inhibited by two specific synthetic cathepsin B inhibitors, CA-074 and GFG-semicarbazone. Cathepsin X was similar to cathepsin B and found to be a carboxypeptidase with preference for a positively charged Arg in P1 position. Contrary to the preference of cathepsin B, cathepsin X normally acts as a carboxymonopeptidase. However, the preference for Arg in the P1 position is so strong that cathepsin X cleaves substrates with Arg in antepenultimate position, acting also as a carboxydipeptidase. A large hydrophobic residue such as Trp is preferred in the P1 0 position, although the enzyme cleaved all P1 0 residues investigated (Trp, Phe, Ala, Arg, Pro). Cathepsin X also cleaved substrates with amide-blocked C-terminal carboxyl group with rates similar to those of the unblocked substrates. In contrast, no endopeptidase activity of cathepsin X could be detected on a series of o-aminobenzoic acid-peptidyl-N-[2,-dinitrophenyl]ethylenediamine substrates. Furthermore, the standard cysteine protease methylcoumarine amide substrates (kcat /Km < 5.0 10 3 m 21 ·s 21 ) were degraded < 25-fold less efficiently than the carboxypeptidase substrates (kcat /Km < 120.0 10 3 m 21 ·s 21 ).

Lysosomal cysteine proteases were believed to be mainly involved in intracellular protein degradation. Under special conditions they have been found outside lysosomes resulting in pathological conditions. With the discovery of a series of new cathepsins with restricted tissue distributions, it has become evident that these enzymes must be involved in a range of specific cellular tasks much broader than as simple housekeeping enzymes. It is therefore timely to review and discuss the various physiological roles of mammalian lysosomal papain-like cysteine proteases as well as their mechanisms of action and the regulation of their activity.

Background: Cathepsin X is a widespread, abundantly expressed papain-like mammalian lysosomal cysteine protease. It exhibits carboxy-monopeptidase as well as carboxy-dipeptidase activity and shares a similar activity profile with cathepsin B. The latter has been implicated in normal physiological events as well as in various pathological states such as rheumatoid arthritis, Alzheimer's disease and cancer progression. Thus the question is raised as to which of the two enzyme activities has actually been monitored. Results: The crystal structure of human cathepsin X has been determined at 2.67 A resolution. The structure shares the common features of a papain-like enzyme fold, but with a unique active site. The most pronounced feature of the cathepsin X structure is the mini-loop that includes a short three-residue insertion protruding into the active site of the protease. The residue Tyr27 on one side of the loop forms the surface of the S1 substrate-binding site, and His23 on the other side modulates both carboxy-monopeptidase as well as carboxy-dipeptidase activity of the enzyme by binding the C-terminal carboxyl group of a substrate in two different sidechain conformations. Conclusions: The structure of cathepsin X exhibits a binding surface that will assist in the design of specific inhibitors of cathepsin X as well as of cathepsin B and thereby help to clarify the physiological roles of both proteases.

In order to evaluate the role of cysteine peptidase cathepsin H (Cath H) in human lung cancer its protein levels were determined in 148 pairs of lung tumour tissue and adjacent non-tumourous lung parenchyma using the enzyme-linked immunosorbent assay technique. Additionally, Cath H levels were determined in sera of 171 patients with malignant tumours, 34 patients with benign lung diseases and 47 healthy controls. The median level of Cath H in tumour tissue was 0.64 times that in the corresponding lung parenchyma. Relating tumour levels with histological type we found higher Cath H levels in small-cell and adenocarcinomas and lower levels in squamous cell carcinoma, large-cell carcinoma and secondary tumours. A significant difference in Cath H level between lung tumour tissue and non-tumourous lung parenchyma was associated with the group of cigarette smokers (156 vs 263 ng mg−1protein, P< 0.001). For this group of patients Cath H tumour levels correlated with the survival rate, while for the entire patient population this was not the case. Smokers with high tumour levels of Cath H experienced poor survival. Cath H was significantly higher in sera of patients with malignant and benign lung diseases than in control sera (P< 0.001). The increase was significant for all histological types, being the highest in small-cell and squamous cell carcinomas. Our study reveals that in lung tumours there is different behaviour of Cath H compared with other cysteine peptidases, e.g. cathepsin B and cathepsin L. Variations between tissue and serum levels of Cath H indicate either reduced expression or enhanced secretion of this enzyme in lung tumours. © 2000 Cancer Research Campaign

We found that pyridoxal phosphate shows considerable inhibition of cathepsins. CLIK-071, in which the phosphate ester of position 3 of pyridoxal phosphate was replaced by propionate, strongly inhibited cathepsin B. Three new types of synthetic pyridoxal propionate derivatives showing specific inhibition of cathepsin K were developed. New synthetic pyridoxal propionate derivatives, -162, -163, and -164, in which the methyl arm of position 6 of CLIK-071 was additionally modified, strongly inhibited cathepsin K and cathepsin S weakly, but other cathepsins were not inhibited. CLIK-166, in which the position 4 aldehyde of CLIK-071 is replaced by a vinyl radical and position 5 is additionally modified, showed cathepsin K-specific inhibition at 10−5 M. Pit formation due to bone collagen degradation by cathepsin K of rat osteoclasts was specifically suppressed by administration of CLIK-164, but not by inhibitors of cathepsin L or B.

Specific inhibitors for cathepsin L and cathepsin S have been developed with the help of computer-graphic modeling based on the stereo-structure. The common fragment, N -( L - trans -carbamoyloxyrane-2-carbonyl)-phenylalanine-dimethylamide, is required for specific inhibition of cathepsin L. Seven novel inhibitors of the cathepsin L inhibitor Katunuma (CLIK) specifically inhibited cathepsin L at a concentration of 10 −7 M in vitro, while almost no inhibition of cathepsins B, C, S and K was observed. Four of the CLIKs are stable, and showed highly selective inhibition for hepatic cathepsin L in vivo. One of the CLIK inhibitors contains an aldehyde group, and specifically inhibits cathepsin S at 10 −7 M in vitro.

The discovery of a fragment from the p41 form of invariant chain tightly bound to cathepsin L provided the first direct link between MHC class II molecules and the regulation of activity of lysosomal cysteine proteases. We recently determined the crystal structure of this p41 invariant chain fragment in complex with cathepsin L [EMBO J. 18, 793-803 (1999)]. This structure explains the specificity of the observed interactions and actually provides a tool, which can be utilized by means of molecular biology, to explore and understand the specificity of thyroglobulin type I domains and thus allow the design of specific inhibitors of papain-like cysteine proteases. The structure further supports the hypothesis that the thyroglobulin type I and II domains present in various proteins, sometimes in multiple repeats, are regulatory elements of the processing of these proteins by proteolytic cleavage.

Equistatin from sea anemone is a protein composed of three thyroglobulin-type 1 domains known to inhibit papain-like cysteine proteinases, papain, and cathepsins B and L. Limited proteolysis was used to dissect equistatin into a first domain, eq d-1, and a combined second and third domain, eq d-2,3. Only the N-terminal domain inhibits papain (K i = 0.61 nm). Remarkably, equistatin also strongly inhibits cathepsin D withK i = 0.3 nm but not other aspartic proteinases such as pepsin, chymosin, and HIV-PR. This activity resides on the eq d-2,3 domains (K i = 0.4 nm). Papain and cathepsin D can be bound and inhibited simultaneously by equistatin at pH 4.5, confirming the physical separation of the two binding sites. Equistatin is the first inhibitor of animal origin known to inhibit cathepsin D. The obtained results demonstrate that the widely distributed thyroglobulin type-1 domains can support a variety of functions.

It has been shown that hu- man stefin B exhibits molten globule intermedi- ates when denatured by acid or GuHCl. In the presence of TFE, it transforms into a highly helical state. In our first study on its folding mechanism (Zerovnik et al., Proteins 32:296- 303), the kinetics measured by circular dichro- ism (CD) and fluorescence were correlated. In the present work the kinetics of folding were moni- tored by tyrosine fluorescence, ANS fluores- cence, and, for certain reactions, far ultravio- let (UV) CD. The folding was started from the unfolded state in 3.45 M GuHCl, the acid dena- tured state at pH 1.8 6 0.2, an acid molten globule intermediate I1 (pH 3.3 6 0.1, low salt), a more structured acid molten globule interme- diate I2 (pH 3.3 6 0.1, 0.42 M NaCl), and the TFE state (pH 3.3 6 0.1, 42% TFE). It has been found that all denatured states, including GuHCl, TFE, acid denatured and acid molten globule intermediate I1, fold with the same kinetics, provided that the final conditions are identi- cal. This does not apply to the second acid molten globule intermediate I2, which demon- strates a higher rate of folding by a factor of 270. Different energy of activation and pH depen- dence were found for folding from states I1 or I2. Proteins 32:304-313, 1998. r 1998 Wiley-Liss, Inc.

Synthesis of proteases as inactive zymogens is a very important mechanism for the regulation of their activity. For lysosomal proteases proteolytic cleavage of the propeptide is triggered by the acidic pH. By using fluorescence, circular dichroism, and NMR spectroscopy, we show that upon decreasing the pH from 6.5 to 3 the propeptide of cathepsin L loses most of the tertiary structure, but almost none of the secondary structure is lost. Another partially structured intermediate, prone to aggregation, was identified between pH 6.5 and 4. The conformation, populated below pH 4, where the activation of cathepsin L occurs, is not completely unfolded and has the properties of molten globule, including characteristic binding of the 1-anilinonaphthalene-8-sulfonic acid. This pH unfolding of the propeptide parallels a decrease of its affinity for cathepsin L and suggests the mechanism for the acidic zymogen activation. Addition of anionic polysaccharides that activate cathepsin L already at pH 5.5 unfolds the tertiary structure of the propeptide at this pH. Propeptide of human cathepsin L which is able to fold independently represents an evolutionary intermediate in the emergence of novel inhibitors originating from the enzyme proregions.

Kinetic stopped-flow measurements of refolding of the recombinant sulphonated procathepsin S from 6 M urea are presented. The experiments were performed using intrinsic tryptophan fluorescence and fluorescence of the hydrophobic probe 1-anilino-naphthalene-8-sulfonate (ANS). Initially, (t1/2 = 3 +/- 1 ms) an intermediate with increased ANS fluorescence and protected tryptophan environment is formed. Much later, a slow increase in ANS fluorescence occurs with no accompanying changes in tryptophan fluorescence. The reaction of the slow ANS fluorescence increase correlates with the rate of aggregation as shown by the size exclusion chromatography (SEC). For comparison, the folding reactions of the reduced chicken cystatin were measured, both, by intrinsic tryptophan and extrinsic ANS fluorescence. An early intermediate forms very fast in the refolding of reduced chicken cystatin on 6-fold dilution from 5.7 M GuHCl (t1/2 = 5 +/- 2 ms), similarly to that observed for the sulphonated procathepsin S. ANS fluorescence and tryptophan fluorescence decrease further (t1/2 = 100 +/- 50 ms) leading to a late, 'more structured' intermediate which is prone to dimerization.

Proteolytic enzymes have been proposed as new biological prognostic indicators to facilitate decisions about treatment of breast cancer patients following surgery. We reported earlier that the activities of cysteine proteinases (CP), cathepsin (Cat) B and cathepsin (Cat) L and the expression of stefin A might be associated with breast tumor progression and prognosis. Here, the protein concentrations of Cats D, B and L and stefin A have been measured in a series of 60 matched pairs of breast tumours and control adjacent tissues, using ELIS As developed in our laboratory. Median tumor concentrations of Cat D (47 pm/mg), Cat B (222 ng/mg) and Cat L (88 ng/mg) were significantly (p

Cell lines derived from human squamous cell (EPCL), large cell (LCLC), and small cell lung cancer (SCLC) lines were investigated for the expression of cathepsin B (Cat B) and cysteine proteinase inhibitors (CPIs). The EPLC and LCLC lines expressed 5- to 50-fold more Cat B activity and contained more mature Cat B of M(r) 27-29 kDa (> 2.5 microg/mg total protein) than the SCLC lines ( or = 67 kDa). The LMM inhibitors of M(r) 10-15 kDa were cystatin C and stefins A and B, which were quantitated by ELISA: stefins A and B were present in cell extracts and medium in similar concentrations (5-200 ng/10(6) cells), while 80-99% of the cystatin C was released in the medium (10-195 ng/10(6) cells). Phorbol ester (PMA), which induces protein-kinase C mediated signal transduction and enhances cellular differentiation in many non-small cell lung cancer (NSCLC) cell lines, increased intracellular Cat B activity and Cat B protein as well as its secretion in some cell lines but not in others, regardless of their histological type. PMA significantly (P < 0.049) decreased intracellular stefin A concentrations in two EPLC lines and non-significantly in two LCLC lines. PMA decreased secretion of stefin A in all EPLC lines, but not in LCLC lines, while IGF-I significantly increased stefin B secretion in both SCLC lines. These data showed that lung tumor cells produce both cysteine proteinases and cystatins. As the antagonistic molecules are regulated differently in histologically different types of lung tumor cells, it is possible that an imbalance between the proteinases and their specific inhibitors plays a role in progression of certain types of lung tumors in vivo.

Trypanosoma cruzi, the parasitic protozoan which causes the American Trypanosomiasis, Chagas disease, contains a major cysteine proteinase (CP), cruzipain. The enzyme belongs to the papain family, but contains, as other CPs from Trypanosomatids, an unusual C-terminal extension. This C-terminal domain contains a number of post-translational modifications and is responsible for the immunodominant antigenic character of cruzipain in natural human infections. In addition, this domain is probably the cause of most of the microheterogeneities found in natural cruzipain. Irreversible inhibitors of CPs are able to block the parasite's life cycle at the differentiation steps, suggesting an essential role for CPs for parasite survival, and opening up possibilities of developing new chemotherapeutic agents against Chagas disease based on specific cruzipain inhibitors.

The potential role of cysteine cathepsins in tumor necrosis factor-related apoptosis-inducing ligand(TRAIL/Apo2L)- and CD95 (Fas/APO-1)-induced apoptosis was investigated using four different cell lines (HeLa, HuH-7, Jurkat, and U-937). All four cell lines exhibited different levels of cathepsins and responded differently to apoptosis triggering, with Jurkat cells being the most sensitive and the only ones that were sensitive to the agonistic anti-APO-1 antibody. Apoptosis was accompanied by caspase activation, loss of the mitochondria and lysosome integrity, and the release of cysteine cathepsins into the cytosol, as judged based on the hydrolysis of the cysteine cathepsin substrate benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin and by the immunological detection of cathepsin B. The inhibition of caspases by the broad-spectrum inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone prevented apoptosis,including the mitochondrial and lysosomal membrane permeabilization, as well as cathepsin release into the cytosol, consistent with caspases playing a crucial role in the process. Conversely, however, although the broad-spectrum cysteine cathepsin inhibitor (2 S ,3 S )-trans -epoxysuccinyl-leucyl amido-3-methyl-butane ethyl ester and the more cathepsin B-selective inhibitor[(2 S ,3 S )-3-propylcarbamoyloxirane-2-carbonyl]-l-isoleucyl-l-proline methyl ester completely blocked cathepsin activity, these inhibitors neither prevented apoptosis including the mitochondrial and lysosomal membrane permeabilization, as well as cathepsin release into the cytosol, consistent with caspases playing a crucial role in the process. Conversely, however, although the broad-spectrum cysteine cathepsin inhibitor (2 S ,3 S )-trans -epoxysuccinyl-leucylamido-3-methyl-butane ethyl ester and the more cathepsin B-selective inhibitor[(2 S ,3 S )-3-propylcarbamoyloxirane-2-carbonyl]-l-isoleucyl-l-proline methyl ester completely blocked cathepsin activity, these inhibitors neither prevented apoptosis and its progression nor the mitochondrial and lysosomal membrane permeabilization associated with this type of cell death. Consequently, cathepsin release into the cytosol was also not prevented. Together, these data indicate that cysteine cathepsins are not required for the TRAIL- and CD95-mediated apoptosis in various human cancer cell lines. This does not, however, rule out that lysosomes and cysteine cathepsins are involved in the amplification, but not in the initiation, of death receptor-mediated apoptosis in certain cell lines or under different stimulation conditions than the ones employed here.

Osteoarthritis and rheumatoid arthritis are destructive joint diseases that involve the loss of articular cartilage. Degradation of cartilage extracellular matrix is believed to occur due to imbalance between the catabolic and anabolic processes of resident chondrocytes. Previous work has suggested that various lysosomal cysteine cathepsins participate in cartilage degeneration; however, their exact roles in disease development and progression have not been elucidated. In order to study degradation processes under conditions resembling the in vivo milieu of the cartilage, we cultivated chondrocytes on a type II collagen-containing matrix. Stimulation of the cultivated chondrocytes with interleukin-1α and/or tumor necrosis factor α resulted in a time-dependent increase in cathepsin S expression and induced its secretion into the conditioned media. Using a novel bioluminescent activity-based probe, we were able to demonstrate a significant increase in proteolytic activity of cathepsin S in the conditioned media of proinflammatory cytokine-stimulated chondrocytes. For the first time, cathepsin S was demonstrated to be secreted from chondrocytes upon stimulation with the proinflammatory cytokines, and displayed proteolytic activity in culture supernatants. Its stability at neutral pH and potent proteolytic activity on extracellular matrix components mean that cathepsin S may contribute significantly to cartilage degradation and may thus be considered a potential drug target in joint diseases.

Fluorescence titrations showed that high-molecular-weight kininogen binds two molecules of papain, cruzipain and cathepsin S with high affinity. The 2:1 binding stoichiometry was confirmed by stopped-flow kinetic measurements of papain binding, which also revealed that the two sites bind the enzyme with different association rate constants (kass,1 = 23.0 x 10(6) M-1 s-1 and kass,2 = 3.4 x 10(6) M-1 s-1). As for low-molecular-weight kininogen, comparison of these kinetic constants with previous data for intact low- and high-molecular-weight kininogen and the separated domains indicated that the faster-binding site is also the tighter-binding site and is that of domain 3, whereas the slower-binding, lower-affinity site is on domain 2. The results further demonstrate that there is no appreciable steric interference between the two domains or by the kininogen light chain in the binding of proteinases. Similarly, the binding of kininogen via its light chain to a surface, as indicated by the binding to the model surface, heparin, did not affect the inhibitory properties of kininogen. The M(r) of high-molecular-weight kininogen was determined to be 83,500 by sedimentation equilibrium measurements, in agreement with the value calculated from amino acid sequence and carbohydrate analysis.

The invariant chain (Ii) is associated with major histocompatibility complex class II molecules during early stages of their intracellular transport. In an acidic endosomal/lysosomal compartment, it is proteolytically cleaved and removed from class II heterodimers. Participation of aspartic and cysteine proteases has been observed in in vitro degradation of Ii, but the specific enzymes responsible for its in vivo processing are as yet undefined. We have previously isolated a noncovalent complex of the lysosomal cysteine protease cathepsin L with a peptide fragment derived from the p41 form of Ii from human kidney. Here we show that this Ii fragment, which is identical to the alternatively spliced segment of p41, is a very potent competitive inhibitor of cathepsin L (equilibrium inhibition constant Ki = 1.7 X 10(-12) M). It inhibits two other cysteine proteases, cathepsin H and papain, but to much lesser extent. Cysteine proteases cathepsins B, C, and S, as well as representatives of serine, aspartic, and metalloproteases, are not inhibited at all. These findings suggest a novel role for p41 in the regulation of various proteolytic activities during antigen processing and presentation. The Ii inhibitory fragment shows no sequence homology with the known cysteine protease inhibitors, and may, therefore, represent a new class.

Cystatin C, a low Mr cysteine proteinase inhibitor was isolated from bovine parotid glands by a procedure which includes alkaline treatment of the homogenate, affinity chromatography, gel filtration and ion exchange chromatography. The purified inhibitor has a pl of 8.0 and Mr of 14500. The identity with bovine cystatin C from colostrum was confirmed by N-terminal sequence of the inhibitor and amino acid composition. Cystatin C rapidly (kass = 5.5 x 10(7) M-1s-1) and tightly inhibits papain (Ki = 0.02 nM), whereas its interaction with bovine cathepsin B is substantially weaker (Ki = 4.4 nM). Bovine cystatin C also shows a weak antiviral effect on poliovirus infected human Hela cells.

Human low-molecular-weight kininogen (LK) was shown by fluorescence titration to bind two molecules of cathepsins L and S and papain with high affinity. By contrast, binding of a second molecule of cathepsin H was much weaker. The 2:1 binding stoichiometry was confirmed by titration monitored by loss of enzyme activity and by sedimentation velocity experiments. The kinetics of binding of cathepsins L and S and papain showed the two proteinase binding sites to have association rate constants kass,1 = 10.7-24.5 x 10(6) M-1 s-1 and kass,2 = 0.83-1.4 x 10(6) M-1 s-1. Comparison of these kinetic constants with previous data for intact LK and its separated domains indicate that the faster-binding site is also the tighter-binding site and is present on domain 3, whereas the slower-binding, lower-affinity site is on domain 2. These results also indicate that there is no appreciable steric hindrance for the binding of proteinases between the two binding sites or from the kininogen light chain.

Cathepsin C has been purified from human kidney by a modified procedure. Human cathepsin C was isolated as pure protein with a pI close to 6.0. The enzyme was shown to have a molecular mass of 200 kDa and to consist of four identical subunits, each composed of three different polypeptide chains, two of them disulfide-bound. Their NH2-terminal amino acid sequences were determined. Two chains showed pronounced similarity with the heavy and light chains of other papain-like cysteine proteinases, whereas the third one corresponded to the prosequence of the enzyme, thus showing that a substantial part of the proregion remains bound in the mature enzyme. The kinetics of substrate hydrolysis deviated substantially from standard Michaelis-Menten kinetics, demonstrating substrate inhibition at higher substrate concentrations. These data are explained by a sequential cooperative interaction model, where an enzyme molecule can bind up to four substrate molecules but where only the binary enzyme-substrate complex is catalytically active. Substrate inhibition was observed over the whole range of pH activity. From the pH activity profile it can be concluded that at least three ionizable groups with pKa values 4.2, 6.8, and 7.7 are involved in substrate hydrolysis. Human cathepsin C thus appears to differ qualitatively from other cysteine proteinases of different origin.

Crystals of cysteine protease human cathepsin B inhibited with CA030 (ethyl ester of epoxysuccinyl-Ile-Pro-OH) [Murata, M., et al. (1991) FEBS Lett. 280, 307-310; Towatari, T., et al. (1991) FEBS Lett. 280, 311-315] were isomorphous to a previous published structure of cathepsin B [Musil, D., et al. (1991) EMBO J. 10, 2321-2330]. The crystal structure of the complex was refined at 2.0-A resolution to an R-value of 0.194. CA030 is well-defined in the electron density. The Ile-Pro-OH part of CA030 mimics a substrate P1' and P2' residues. The structure thus reveals for the first time a substratelike interaction in the S1' and S2' sites of a papain-like cysteine protease. The CA030 ethyl ester group occupies the S2 site. The structure confirms the role of residues His 110 and His 111 as the receptors of a peptidic substrate C-terminal carboxylic group. The structure suggests that an epoxysuccinyl fragment can be used to extend binding into primed and nonprimed substrate binding sites of a papain-like cysteine protease.

A cDNA clone encoding human procathepsin B was expressed at a high level in Escherichia coli using a T7 polymerase expression system, resulting in the formation of insoluble cytoplasmic protein aggregates (inclusion bodies). The recombinant product was solubilized and renatured by refolding and reoxidation. The proenzyme was subsequently processed with pepsin to produce an enzymically active enzyme. By systematic variation of the parameters influencing the folding, formation of disulphide bonds, and processing of procathepsin B to the catalytically active mature form, a simple renaturation procedure was designed, allowing the production of about 3 mg purified active cathepsin B/l E. coli culture broth. The enzyme obtained in this way consists of a single chain and, as a consequence of pepsin treatment, possesses a three-amino-acid extension at its N-terminus. The enzyme has similar kinetic and immunological properties to native human cathepsin B.

The three-dimensional solution structure of recombinant human stefin A has been determined by a simulated annealing protocol using a total of 1113 distance and angle constraints obtained from 1H and 15N HMR spectroscopy. The solution structure is represented by a family of 17 conformers with an average root-mean-square deviation relative to the mean structure of 0.44 A for backbone atoms and 0.94 A for all heavy atoms for the main body of the structure. The protein has a well-defined global fold consisting of five anti-parallel beta-strands wrapped around a central five-turn alpha-helix. There is considerable similarity between the structural features of free stefin A in solution and the X-ray structure of the homologous protein stefin B in its complex with papain, but there are also some important differences in the regions which are fundamental to proteinase binding. The differences consist primarily of two regions of high conformational heterogeneity in free stefin A which correspond in stefin B to two of the components of the tripartite wedge that docks into the active site of the target proteinase. These regions, which are shown to be mobile in solution, are the five N-terminal residues and the second binding loop. In the bound conformation of stefin B they form a turn and a short helix, respectively.

The kinetics of pH-induced inactivation of human cathepsins B and L was studied by conventional and stopped-flow methods. The inactivation of both enzymes was found to be an irreversible, first-order process. The inactivation rate constants increased exponentially with pH for both enzymes. From log kinac vs pH plots, 3.0 and 1.7 protons were calculated to be desorbed for pH-induced inactivation of cathepsins L and B. Cathepsin B was thus substantially more stable than cathepsin L (approximately 15-fold at pH 7.0 and 37 degrees C). Cathepsin B was efficiently inhibited by cystatin C at pH 7.4, whereas the inhibition by stefin B and high molecular weight kininogen was only moderate. In contrast, cathepsin L was efficiently inhibited by both chicken cystatin and stefin B at this pH kass approximately 3.3 x 10(7) m-1 s-1).

In cytosols of tumour and normal tissue of 53 patients suffering from head and neck carcinoma cathepsins D, B, H and L were measured using quantitative immunoreactive assays (ELISA). The values of cathepsins D, B and L were significantly higher in tumour tissue, whereas cathepsin H concentration was lower in tumour than in normal tissue. Median cathepsin D values were 27 pmol (tumour tissue) vs. 12 pmol (normal tissue) per mg of total protein, median cathepsin B values were 1.25 micrograms/mg (tumour tissue) vs. 0.23 micrograms/mg (normal tissue) and median cathepsin L values were 39.8 ng/mg (tumour tissue) vs. 20.0 ng/mg (normal tissue). Median cathepsin H values were 1.05 micrograms/mg and 2.20 micrograms/mg for tumour and normal tissue, respectively. Additionally, stefin A and stefin B were measured in tumour and normal tissue samples. In contrast to the cathepsins, the concentrations of these inhibitors of cysteine proteinases was not significantly different between tumour and normal samples. The concentrations of cathepsins D, B, H and L and stefins A and B measured in head and neck tumours, were independent of standard clinical and histological prognostic factors. Significant correlation of tumour tissue values was observed between cathepsins B and L and between both stefins.