[Alteration of pulmonary epithelial permeability by cathepsin S in chronic obstructive pulmonary disease]. / Altération de la perméabilité épithéliale pulmonaire par la cathepsine S au cours de la bronchopneumopathie chronique obstructive.

Smoking is accountable for most of the chronic obstructive pulmonary disease (COPD) cases. COPD, which is characterized by the development of chronic bronchitis, could be associated with emphysema. In active smokers, there is an overexpression of cathepsin S, a cysteine protease, which participates in the development of emphysema via its elastinolytic activity. Likewise, we demonstrated that cathepsin S could degrade one or more protein constituents of cell junctions. This deleterious proteolytic activity leads to an alteration of the integrity of the lung epithelial barrier, which in turn could aggravate chronic inflammation and promote the exacerbation phases associated with infections.

Modulation of the expression and activity of cathepsin S in reconstructed human skin by neohesperidin dihydrochalcone.

Dysregulation of cathepsin S (Cat S), a cysteine protease involved in extracellular-matrix and basement membrane (BM) degradation, is a concomitant feature of several inflammatory skin diseases. Therefore, Cat S has been suggested as a potential therapeutic target. Flavonoids, which were identified as regulatory molecules of various proteolytic enzymes, exert beneficial effects on skin epidermis. Herein, thirteen flavonoid compounds were screened in vitro and in silico and neohesperidin dihydrochalcone (NHDC) was identified as a potent, competitive, and selective inhibitor (Ki=8±1 µM) of Cat S. Furthermore, Cat S-dependent hydrolysis of nidogen-1, a keystone protein of BM architecture, as well elastin, collagens I and IV was impaired by NHDC, while both expression and activity of Cat S were significantly reduced in NHDC-treated human keratinocytes. Moreover, a reconstructed human skin model showed a significant decrease of both mRNA and protein levels of Cat S after NHDC treatment. Conversely, the expression of nidogen-1 was significantly increased. NHDC raised IL-10 expression, an anti-inflammatory cytokine, and mediated STAT3 signaling pathway, which in turn dampened Cat S expression. Our findings support that NHDC may represent a valuable scaffold for structural improvement and development of Cat S inhibitors to preserve the matrix integrity and favor skin homeostasis during inflammatory events.

In silico and in vitro mapping of specificity patterns of glycosaminoglycans towards cysteine cathepsins B, L, K, S and V.

Human cysteine cathepsins are lysosomal proteases, which are involved in different biological processes. Their enzymatic activity can be regulated by glycosaminoglycans (GAGs): long linear periodic negatively charged polysaccharides, which dimeric building blocks consist of uronic acid and hexosamine monosaccharide units. In this study, molecular docking simulations of chondroitin 4-sulfate, chondroitin 6-sulfate, heparin, heparan sulfate, dermatan sulfate and hyaluronic acid of various chain lengths were performed with cathepsins B, L, K, S and V and followed by molecular dynamics-based refinement and binding free energy analysis. We concluded that electrostatics might be a driving force for cathepsin-GAG interactions; indeed as in most of characterised systems, the increase of GAG chain length consequently leads to a more pronounced effect on the strength of cathepsin-GAG interactions. Results also suggest that binding of GAGs at different regions on cathepsins surface affect differently their enzymatic activity and could is dependent on cathepsin and GAG type. Present data contribute to systematic description of cathepsin-GAG interactions, which is helpful in understanding the subtle molecular mechanisms of protease regulation behind their biological functions.

The occluding loop of cathepsin B prevents its effective inhibition by human kininogens.

Kininogens, the major plasma cystatin-like inhibitors of cysteine cathepsins, are degraded at sites of inflammation, and cathepsin B has been identified as a prominent mediator of this process. Cathepsin B, in contrast to cathepsins L and S, is poorly inhibited by kininogens. This led us to delineate the molecular interactions between this protease and kininogens (high molecular weight kininogen and low molecular weight kininogen) and to elucidate the dual role of the occluding loop in this weak inhibition. Cathepsin B cleaves high molecular weight kininogen within the N-terminal region of the D2 and D3 cystatin-like domains and close to the consensus QVVAG inhibitory pentapeptide of the D3 domain. The His110Ala mutant, unlike His111Ala cathepsin B, fails to hydrolyze kininogens, but rather forms a tight-binding complex as observed by gel-filtration analysis. K(i) values (picomolar range) as well as association rate constants for the His110Ala cathepsin B variant compare to those reported for cathepsin L for both kininogens. Homology modeling of isolated inhibitory (D2 and D3) domains and molecular dynamics simulations of the D2 domain complexed with wild-type cathepsin B and its mutants indicate that additional weak interactions, due to the lack of the salt bridge (Asp22-His110) and the subsequent open position of the occluding loop, increase the inhibitory potential of kininogens on His110Ala cathepsin B.

Evaluation of a peptide ELISA for the detection of rituximab in serum.

Pharmacokinetic studies of therapeutic monoclonal antibodies necessitate the measurement of their biologically active fraction. The aim of this work was to develop an enzyme-linked immunosorbent assay (ELISA) for rituximab, a chimeric anti-CD20 monoclonal antibody, based on its binding to a 20-mer peptide (P20) derived from the extracellular loop of human CD20 (residues 165-184). Derivatives of P20 were prepared by conjugation to bovine serum albumin (BSA-P20ACM) or biotin (Biot-P20ACM). Interactions of P20 and its derived peptides with rituximab were analyzed by surface plasmon resonance (SPR) and by ELISA. A monoclonal anti-idiotype antibody (MB2A4) was used as the reference in each case. SPR analysis showed that P20 (conjugated or unconjugated) had a lower affinity for rituximab than MB2A4. ELISA methods based on P20 or MB2A4 were both highly accurate and reproducible for rituximab measurement in spiked samples, but the MB2A4-based assay had a lower limit of quantification. Interestingly, discrepant results were obtained with the two ELISA methods when analyzing pharmacokinetic samples, with the rituximab concentrations obtained with the MB2A4-based method being systematically higher than those determined by the P20-based method. Possible interference of circulating CD20 with the P20-based method was supported by competition experiments. Rituximab aggregation in the bloodstream may also account for the bias observed in samples from healthy mice. The P20-based ELISA is far less sensitive than the MB2A-based ELISA, thus limiting its utility for pharmacokinetic studies. However, the discrepancy observed between two different approaches for rituximab measurement indicates that data from different studies should be interpreted with care.

A virus essential for insect host-parasite interactions encodes cystatins.

Cotesia congregata is a parasitoid wasp that injects its eggs in the host caterpillar Manduca sexta. In this host-parasite interaction, successful parasitism is ensured by a third partner: a bracovirus. The relationship between parasitic wasps and bracoviruses constitutes one of the few known mutualisms between viruses and eukaryotes. The C. congregata bracovirus (CcBV) is injected at the same time as the wasp eggs in the host hemolymph. Expression of viral genes alters the caterpillar's immune defense responses and developmental program, resulting in the creation of a favorable environment for the survival and emergence of adult parasitoid wasps. Here, we describe the characterization of a CcBV multigene family which is highly expressed during parasitism and which encodes three proteins with homology to members of the cystatin superfamily. Cystatins are tightly binding, reversible inhibitors of cysteine proteases. Other cysteine protease inhibitors have been described for lepidopteran viruses; however, this is the first description of the presence of cystatins in a viral genome. The expression and purification of a recombinant form of one of the CcBV cystatins, cystatin 1, revealed that this viral cystatin is functional having potent inhibitory activity towards the cysteine proteases papain, human cathepsins L and B and Sarcophaga cathepsin B in assays in vitro. CcBV cystatins are, therefore, likely to play a role in host caterpillar physiological deregulation by inhibiting host target proteases in the course of the host-parasite interaction.

Reversible inhibition of cathepsin L-like proteases by 4-mer pseudopeptides.

A library of 121 pseudopeptides was designed to develop reversible inhibitors of trypanosomal enzymes (cruzain from Trypanosoma cruzi and congopain from Trypanosoma congolense). The peptides share the framework: Cha-X1-X2-Pro (Cha=cyclohexyl-alanine, X1 and X2 were phenylalanyl analogs), based on a previous report [Lecaille, F., Authié, E., Moreau, T., Serveau, C., Gauthier, F. and Lalmanach, G. (2001) Eur. J. Biochem. 268, 2733-2741]. Five peptides containing a nitro-substituted aromatic residue (Tyr/Phe) and one a 4-chloro-phenylalanine at the X1 position, and 3-(2-naphthyl)-alanine, homocyclohexylalanine or 3-nitro-tyrosine (3-NO(2)-Tyr) at the X2 position, were selected. They inhibited congopain more effectively than cruzain, except Cha-4-NO(2)-Phe-3-NO(2)-Tyr-Pro which bound the two parasitic enzymes similarly. Among this series, Cha-3-NO(2)-Tyr-HoCha-Pro and Cha-4-NO(2)-Phe-3-NO(2)-Tyr-Pro are the most selective for congopain relative to host cathepsins. No hydrolysis occurred upon prolonged incubation time with purified enzymes. In addition introduction of non-proteogenic residues in the peptidyl backbone greatly enhanced resistance to proteolysis by mammalian sera.

Immunisation of cattle with cysteine proteinases of Trypanosoma congolense: targetting the disease rather than the parasite.

In order to test the hypothesis that trypanosome cysteine proteinases (CPs) contribute to pathology of trypanosomosis, cattle were immunised with CP1 and/or CP2, the major CPs of Trypanosoma congolense, and subsequently challenged with T. congolense. Immunisation had no effect on the establishment of infection and the development of acute anaemia. However, immunised cattle, unlike control cattle, maintained or gained weight during infection. Their haematocrit and leukocyte counts showed a tendency to recovery after 2-3 months of infection. Cattle immunised with CP2 mounted early and prominent IgG responses to CPs and to the variable surface glycoprotein following challenge. Thus trypanosome CPs may play a role in anaemia and immunosuppression; conversely, anti-CP antibody may modulate the trypanosome-induced pathology.

Functional expression of the catalytic domains of two cysteine proteinases from Trypanosoma congolense.

The catalytic domains of two closely related cysteine proteinases (CP1 and CP2) from Trypanosoma congolense, referred to as C1 and C2, were expressed as proforms in Escherichia coli (C1) and in the baculovirus system (C1 and C2). While the bacterial expression system did not allow recovery of active C1, the baculovirus system led to secretion of inactive zymogens which could be processed at acidic pH into mature enzymes. Active C1 and C2 were purified from serum-free culture supernatants by anion-exchange chromatography and characterised. Their kinetic parameters and pH activity profiles confirmed the relatedness between C2 and native CP2 (congopain). These properties also underline major functional differences between C1 and C2, that appear to relate to discrete but essential sequence differences. It is likely that these two enzymes perform distinct roles in vivo, in the parasite and/or in the host-parasite relationships.

Cathepsin L, but not cathepsin B, is a potential kininogenase.

Although papain-like enzymes are strongly inhibited by their natural tight-binding inhibitors of the cystatin superfamily, cathepsins B and L may still retain some residual proteolytic activity toward Z-Phe-Arg-AMC in the presence of an excess of kininogen. This activity is abolished by adding E-64 or chicken cystatin. Cathepsins B and L show a single band of gelatinolytic activity when subjected to gelatin-SDS-PAGE. Adding high Mr kininogen, low Mr kininogen, T-kininogen, or chicken cystatin to cathepsin L results in additional intense bands of enzyme activity corresponding to the protease-inhibitor complexes. Cathepsin B does not produce these additional bands. This gelatinolytic activity was inhibited by E-64, but not by EDTA, PMSF or Pefabloc. Cathepsin L also specifically generated kinins from high and low molecular weight kininogens in vitro, but cathepsin B did not. T-kininogen did not release any immunoreactive kinins when complexed with cathepsin L, as previously observed using tissue kallikreins. The ability of cathepsin L to generate vasoactive peptides raises the question of the physiological significance of this mechanism during inflammation.

Cysteine protease isoforms from Trypanosoma cruzi, cruzipain 2 and cruzain, present different substrate preference and susceptibility to inhibitors.

Cysteine-proteinases from parasitic protozoa have been recently characterized as factors of virulence and pathogenicity in several human and veterinary diseases. In Chagas' disease, the chronic infection caused by Trypanosoma cruzi, structure-functional studies on cysteine proteases were thus far limited to the parasite's major isoform, a cathepsin L-like lysosomal protease designated as cruzipain, cruzain or GP57/51. Encoded by a large gene family, cruzipain is efficiently targeted by synthetic inhibitors, which prevent parasite intracellular growth and differentiation. We have previously demonstrated that the multicopy cruzipain gene family includes polymorphic sequences, which could encode functionally different isoforms. We report here a comparative kinetic study between cruzain, the archetype of the cruzipain family, and an isoform, termed cruzipain 2, which is expressed preferentially by the mammalian stages of T. cruzi. Heterologous expression of the catalytic domain of cruzipain 2 in Saccharomyces cerevisae yielded an enzyme that differs markedly from cruzain with respect to pH stability, substrate specificity and sensitivity to inhibition by natural and synthetic inhibitors of cysteine proteases. We suggest that the structural-functional diversification imparted by genetic polymorphism of cruzipain genes may have contributed to T. cruzi adaptation to vertebrate hosts.

Subsite specificity of trypanosomal cathepsin L-like cysteine proteases. Probing the S2 pocket with phenylalanine-derived amino acids.

The S2 subsite of mammalian cysteine proteinases of the papain family is essential for specificity. Among natural amino acids, all these enzymes prefer bulky hydrophobic residues such as phenylalanine at P2. This holds true for their trypanosomal counterparts: cruzain from Trypanosoma cruzi and congopain from T. congolense. A detailed analysis of the S2 specificity of parasitic proteases was performed to gain information that might be of interest for the design of more selective pseudopeptidyl inhibitors. Nonproteogenic phenylalanyl analogs (Xaa) have been introduced into position P2 of fluorogenic substrates dansyl-Xaa-Arg-Ala-Pro-Trp, and their kinetic constants (Km, kcat/Km) have been determined with congopain and cruzain, and related host cathepsins B and L. Trypanosomal cysteine proteases are poorly stereoselective towards D/L-Phe, the inversion of chirality modifying the efficiency of the reaction but not the Km. Congopain binds cyclohexylalanine better than aromatic Phe derivatives. Another characteristic feature of congopain compared to cruzain and cathepsins B and L was that it could accomodate a phenylglycyl residue (kcat/Km = 1300 mM-1.s-1), while lengthening of the side chain by a methylene group only slightly impaired the specificity constant towards trypanosomal cysteine proteases. Mono- and di-halogenation or nitration of Phe did not affect Km for cathepsin L-like enzymes, but the presence of constrained Phe derivatives prevented a correct fitting into the S2 subsite. A model of congopain has been built to study the fit of Phe analogs within the S2 pocket. Phe analogs adopted a positioning within the S2 pocket similar to that of the Tyr of the cruzain/Z-Tyr-Ala-fluoromethylketone complex. However, cyclohexylalanine has an energetically favorable chair-like conformation and can penetrate deeper into the subsite. Fitting of modeled Phe analogs were in good agreement with kinetic parameters. Furthermore, a linear relationship could be established with logP, supporting the suggestion that fitting into the S2 pocket of trypanosomal cysteine proteases depends on the hydrophobicity of Phe analogs.

Altered expression of cruzipain and a cathepsin B-like target in a Trypanosoma cruzi cell line displaying resistance to synthetic inhibitors of cysteine-proteinases.

The therapeutic potential of synthetic inhibitors to the major cysteine-proteinase from Trypanosoma cruzi (cruzain or cruzipain) was recently demonstrated in animal models of Chagas' disease. A possible limitation of this strategy would be the emergence of parasite populations developing resistance to cysteine-proteinase inhibitors. Here, we describe the properties of a phenotypically stable T. cruzi cell line (R-Dm28) that displays increased resistance to Z-(SBz)Cys-Phe-CHN2, an irreversible cysteine-proteinase inhibitor which preferentially inactivates cathepsin L-like enzymes. Isolated from axenic cultures of the parental cells (IC50 1.5 microM), R-Dm28 epimastigotes exhibited 13-fold (IC50) 20 microM) higher resistance to this inhibitor and did not display cross-resistance to unrelated trypanocidal drugs, such as benznidazol and nifurtimox. Western blotting (with mAb), affinity labeling (with biotin-LVG-CHN2) and FACS analysis of R-Dm28 log-phase epimastigotes revealed that the cruzipain target was expressed at lower levels, as compared with Dm28c. Interestingly, this deficit was paralleled by increased expression of an unrelated Mr 30 000 cysteine-proteinase whose activity was somewhat refractory to inhibition by Z-(SBz)Cys-Phe-CHN,. N-terminal sequencing of the affinity-purified biotin-LVG-proteinase complex allowed its identification as a cathepsin B-like enzyme. Increased antigenic deposits of this proteinase were found in the grossly enlarged and electron dense reservosomes from R-Dm28 epimastigotes. Our data suggest that R-Dm28 resistance to toxic effects induced by the synthetic inhibitor may result from decreased availability of the most sensitive cysteine-proteinase target, cruzipain. The deficit in metabolic functions otherwise mediated by this cathepsin L-like proteinase is likely compensated by increased expression/accumulation of a cathepsin B-like target.

Chicken cystatin stimulates nitric oxide release from interferon-gamma-activated mouse peritoneal macrophages via cytokine synthesis.

Cystatins are natural tight-binding, reversible inhibitors of cysteine proteases. We have shown that cystatins also stimulate nitric oxide (NO) production by interferon-gamma-activated mouse peritoneal macrophages [Verdot, L., Lalmanach, G., Vercruysse, V., Hartman, S., Lucius, R., Hoebeke, J., Gauthier F. & Vray, B. (1996) J. Biol. Chem. 271, 28077-28081]. The present study was undertaken to further document this new function. Macrophages activated with interferon-gamma and then stimulated with interferon-gamma plus chicken cystatin generated increased amounts of NO in comparison with macrophages only activated with interferon-gamma. Interferon-gamma-activated macrophages must be incubated with chicken cystatin for at least 8 h to upregulate NO production. NO induction was due to increased inducible nitric oxide synthase protein synthesis. Macrophages incubated with chicken cystatin alone or with interferon-gamma plus chicken cystatin produced increased amounts of both tumor necrosis factor alpha and interleukin 10. The addition of recombinant murine tumor necrosis factor alpha alone or in combination with recombinant murine interleukin-10 mimicked the effect of chicken cystatin. The addition of neutralizing anti-(tumor necrosis factor alpha) antibodies reduced sharply NO production by chicken cystatin/interferon-gamma-activated mouse peritoneal macrophages. Taken together, these data suggest that chicken cystatin induces the synthesis of tumor necrosis factor alpha and interleukin 10. In turn, these two cytokines stimulate the production of NO by interferon-gamma-activated macrophages. The findings point to a new relationship between cystatins, cytokines, inflammation and the immune response.

Discrimination of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, and mammalian cathepsins B and L, by a pH-inducible fluorogenic substrate of trypanosomal cysteine proteinases.

The substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, was investigated using a series of dansyl-peptides based on the putative autoproteolytic sequence of the proteinase (VVG-GP) located at the hinge region between the catalytic domain and the C-terminal extension. Replacing Val with Pro at P2 in this sequence greatly improved the rate of cleavage by cruzipain. Tyr and Val residues are preferred at P3 by all cysteine proteinases whatever their origin, whereas only cruzipain and cathepsin L cleaved substrate with a His at that position. The combination of a Pro at P2 and His at P3 abolished cleavage by cathepsin L, so that only cruzipain was able to cleave the HPGGP peptide at the GG bond. A substrate with intramolecularly quenched fluorescence was raised on this sequence (Abz-HPGGPQ-EDDnp) which was also specifically cleaved by cruzipain (kcat/Km of 157 000 m-1. s-1) and by a homologous proteinase from Trypanosoma congolense. The pH activity profile of cruzipain on Abz-HPGGPQ-EDDnp showed a narrow peak with a maximum at pH 5.5 and no cleavage above pH 6.8, although trypanosomal cysteine proteinases remain active at basic pH. The lack of activity at neutral and basic pH was due to a decrease in kcat, while the Km remained essentially unchanged, demonstrating that the substrate still binds to the enzyme and therefore behaves as an inhibitor. Changing the substrate into an inhibitor depended on the deprotonation of the His residue in the substrate, as deduced from a comparison of the pH activity profile with that of a related, but uncharged, substrate. Abz-HPGGPQ-EDDnp also inhibited mammalian cathepsins B and L but was not cleaved by these proteinases at any pH. The importance of the His residue at P3 for cleavage by cruzipain was confirmed by substituting Lys for His at that position. The resulting peptide was not cleaved by cruzipain in spite of the presence of a positively charged group at P3, but still interacted with the enzyme. It was concluded that the presence of an imidazolium group at P3 was essential to endow the HPGGPQ sequence with the properties of a cruzipain substrate.

Inhibition of trypanosomal cysteine proteinases by their propeptides.

The ability of the prodomains of trypanosomal cysteine proteinases to inhibit their active form was studied using a set of 23 overlapping 15-mer peptides covering the whole prosequence of congopain, the major cysteine proteinase of Trypanosoma congolense. Three consecutive peptides with a common 5-mer sequence YHNGA were competitive inhibitors of congopain. A shorter synthetic peptide consisting of this 5-mer sequence flanked by two Ala residues (AYHNGAA) also inhibited purified congopain. No residue critical for inhibition was identified in this sequence, but a significant improvement in Ki value was obtained upon N-terminal elongation. Procongopain-derived peptides did not inhibit lysosomal cathepsins B and L but did inhibit native cruzipain (from Dm28c clone epimastigotes), the major cysteine proteinase of Trypanosoma cruzi, the proregion of which also contains the sequence YHNGA. The positioning of the YHNGA inhibitory sequence within the prosegment of trypanosomal proteinases is similar to that covering the active site in the prosegment of cysteine proteinases, the three-dimensional structure of which has been resolved. This strongly suggests that trypanosomal proteinases, despite their long C-terminal extension, have a prosegment that folds similarly to that in related mammal and plant cysteine proteinases, resulting in reverse binding within the active site. Such reverse binding could also occur for short procongopain-derived inhibitory peptides, based on their resistance to proteolysis and their ability to retain inhibitory activity after prolonged incubation. In contrast, homologous peptides in related cysteine proteinases did not inhibit trypanosomal proteinases and were rapidly cleaved by these enzymes.

A comparison of the enzymatic properties of the major cysteine proteinases from Trypanosoma congolense and Trypanosoma cruzi.

Congopain and cruzipain, the major cysteine proteinases from Trypanosoma congolense and Trypanosoma cruzi, were compared for their activities towards a series of new, sensitive fluorogenic substrates of the papain family of cysteine proteinases and for their sensitivity to inhibition by cystatins and related biotinylated peptidyl diazomethanes. Low Ki values, in the 10 pM range, were found for the interaction of both proteinases with natural cystatin inhibitors. The kinetic constants for the hydrolysis of cystatin-derived substrates, and the inhibition by related diazomethanes were essentially identical. Unlike cathepsins B and L, the related mammal papain family proteinases, congopain and cruzipain accomodate a prolyl residue in P2'. Substrates having the sequence VGGP from P2 to P2' were hydrolysed by both congopain and cruzipain with a k(cat)/Km greater than 4.10(3) mM(-1) s(-1). Irreversible diazomethane inhibitors, deduced from the unprime sequence of cystatin-derived substrates, inhibited the two parasite proteinases. N-terminal labelling of diazomethanes with a biotin group did not alter the rate of inhibition significantly, which provides a useful tool for examining the distribution of these enzymes in the parasite and in the host. Despite their similar activities on cystatin-derived substrates, congopain and cruzipain had significantly different pH-activity profiles when assayed with a cystatin-derived substrate. They were correlated with structural differences, especially at the presumed S2 subsites.

Cystatins up-regulate nitric oxide release from interferon-gamma-activated mouse peritoneal macrophages.

Up-regulation of nitric oxide (NO) production by activated murine macrophages was observed during infection by Trypanosoma cruzi, the etiological agent of Chagas' disease. Cell infection by T. cruzi depends at least in part on cruzipain, a membrane-associated papain-related proteinase which is sensitive to inhibition by synthetic inhibitors of cysteine proteinases. Using the natural cysteine proteinase inhibitor chicken cystatin, a representative member of cystatin family 2, to investigate the effect of cruzipain on macrophage infection and NO release, we found that the inhibitor alone up-regulated NO release from interferon-gamma-activated macrophages. A 12-fold increase in NO production was observed in the presence of 1 microM chicken cystatin. This overproduction was concentration-dependent and could be detected at concentrations as low as 10 nM and remained in the presence of polymyxin B. Representative members of the other cystatin families, i.e. stefin B (family 1), T-kininogen, and its inhibitory domains (family 3), were also able to enhance NO production from interferon-gamma-activated macrophages. Neither E64, an irreversible inhibitor of cysteine proteinases, nor inhibitors of aspartyl and serine proteinases (aprotinin, pepstatin, and soybean trypsin inhibitor) enhanced NO production. Upon complexation with saturating amounts of reduced-alkylated papain, cystatins still remained active in increasing NO production, suggesting that the cystatin inhibitory site was not involved in the mechanism. The results demonstrate that members of all 3 cystatin families share another common property unrelated to their function of cysteine proteinase inhibitors, i.e. up-regulation of NO production, which biological significance remains to be elucidated.

Biotin-labelled peptidyl diazomethane inhibitors derived from the substrate-like sequence of cystatin: targeting of the active site of cruzipain, the major cysteine proteinase of Trypanosoma cruzi.

Biotin-labelled peptidyl diazomethane inhibitors of cysteine proteinases, based on the N-terminal substrate-like segment of human cystatin C, a natural inhibitor of cysteine proteinases, were synthesized. These synthetic derivatives were tested as irreversible inhibitors of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, to compare the kinetics of the inhibition of the parasite proteinase with that of the mammalian cathepsins B and L. The accessibility of the active sites of these proteinases to these probes was also investigated. The inhibition of cruzipain by Biot-LVG-CHN2 (where Biot represents biotinyl and L,V and G are single-letter amino acid residue abbreviations) and Biot-Ahx-LVG-CHN2 (where Ahx represents 6-aminohexanoic acid) was similar to that of unlabelled inhibitor. Biotin labelling of the inhibitor slowed the inhibition of both cathepsin B and cathepsin L. Adding a spacer arm (Ahx) between the biotin and the peptide moiety of the derivative increased the inhibition of cathepsin B but not that of cathepsin L. The discrimination provided by this spacer is probably due to differences in the topologies of the binding sites of proteinases, a feature that can be exploited to improve targeting of individual cysteine proteinases. Analysis of the blotted proteinases revealed marked differences in the accessibility of extravidin-peroxidase conjugate to the proteinase-bound biotinylated inhibitor. Cruzipain molecules exposed to Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 were readily identified, but the reaction was much stronger when the enzyme was treated with the spacer-containing inhibitor. In contrast with the parasite enzyme, rat cathepsin B and cathepsin L treated with either Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 produced no detectable bands. Papain, the archetype of this family of proteinases, was poorly labelled with Biot-LVG-CHN2, but strong staining was obtained with Biot-Ahx-LVG-CHN2. These findings suggest that optimized biotinylated diazomethanes might considerably improve their selectivity for the T. cruzi target enzyme.

Inhibition of cathepsin B by its propeptide: use of overlapping peptides to identify a critical segment.

Ten overlapping 15-mer peptides (peptidyl amides) spanning the proregion of rat cathepsin B (residues 1p-60p) were constructed to identify minimal segments having inhibitory activity towards the mature enzyme, that could be used to develop a new generation of peptide-derived inhibitors specifically targeting the active site of the corresponding proteinase. Three synthetic peptides, containing the pentapeptide Leu-Cys-Gly-Thr-Val (residues 41p-45p) in their sequence, inhibited cathepsin B with Ki values in the micromolar range. Alkylation of the thiol group of Cys-42p of peptide PB8 (36p-50p) resulted in its rapid proteolytic degradation, suggesting that this residue is essential for inhibition. The inhibition constant was slightly improved (Ki = 2 microM) using a longer peptide (26p-50p) which was completely resistant to cleavage even after a prolonged incubation. Alkylation of its cysteinyl residue also resulted in rapid cleavage of the peptide chain. Peptides derived from the rat cathepsin B prosequence also inhibited human cathepsin B with similar Ki values. Unlike rat cathepsin B, which cleaves peptide PB8 at the G47p-G48p bond after prolonged incubation, the human enzyme cleaved both PB8 and PB11 at the Lys-40p-Leu-41p bond, in agreement with the different kinetic properties of these two proteinases. New probes with improved specificity for cysteine proteinases may therefore be designed based on the sequences of their propeptides.