Inhibition of cathepsin L-like proteases by cathepsin V propeptide

The N-terminal propeptide domains of several cathepsin L-like cysteine proteases have been shown to possess potent inhibitory activity. Here we report the first kinetic characterisation of the inhibition properties of the cathepsin V propeptide (CatV PP). Using a facile recombinant approach we demonstrate expression, purification and evaluation of the CatV PP. This propeptide was found to behave as a tight-binding inhibitor against CatV (K (i) 10.2 nm). It also functions as an inhibitor against other members of the CatL-like subclass (CatL, 9.8 nm; CatS, 10.7 nm; and CatK, 149 nm) and had no discernible effects upon the more distantly related CatB.     

An evolutionarily conserved tripartite tryptophan motif stabilizes the prodomains of cathepsin L-like cysteine proteases.

Cathepsin L-like cysteine proteinases contain an evolutionarily highly conserved alpha-helical motif in the proregion. This is called the ER(F/W)N(I/V)N motif according to the conserved amino acids along one side of the helix. We studied the function of this motif using site-directed mutagenesis experiments of human procathepsin S. We replaced each of these amino acids with alanine and constructed deletion mutants lacking parts of the helix. All mutants were expressed in HEK 293 cells, but only one, W52A, was not processed to mature cathepsin S, nor was it phosphorylated or secreted into the culture medium. W52 is part of the hydrophobic core in the propeptide region of cathepsin S comprising two additional tryptophan residues, W28 and W31, also conserved among cathepsin L-like cysteine peptidases. Replacement of the latter with alanine led to consequences similar to those with the W52A mutation. Recombinant propeptides containing mutations of one of the three tryptophan residues were three orders of magnitude less effective as inhibitors of mature cathepsin S than the wild-type propeptide. The results point to a dominant role of the respective hydrophobic stack in the proper folding, transport and maturation of procathepsin S and related cathepsin L-like cysteine proteinases.     

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.     

Folding incompetence of cathepsin L-like cysteine proteases may be compensated by the highly conserved, domain-building N-terminal extension of the proregion

Folding of cathepsins L and S depend upon their proregion which extends the enzyme part by about 100 amino acids. Only a minority of the prosequence follows the structural template provided by the enzyme part; the majority forms an autonomous minidomain fairly distant from the active site cleft. We suggest that this prodomain may be the structural correlate of a foldase function of the proregion within the cathepsin L-like subfamily of papain-type cysteine proteases and report on a functional approach supporting this hypothesis.     

Procongopain from Trypanosoma congolense is processed at basic pH: an unusual feature among cathepsin L-like cysteine proteases

Congopain, the major cysteine protease from Trypanosoma congolense, is synthesized as an inactive zymogen, and further converted into its active form after removal of the proregion, most probably via an autocatalytic mechanism. Processing of recombinant procongopain occurs via an apparent one-step or a multistep mechanism depending on the ionic strength. The auto-activation is pH-dependent, with an optimum at pH 4.0, and no activation observed at pH 6.0. After addition of dextran sulfate (10 microg/ml), an approx. 20-fold increase of processing (expressed as enzymatic activity) is observed. Furthermore, in the presence of dextran sulfate, procongopain can be processed at pH 8.0, an unusual feature among papain-like enzymes. Detection of procongopain and trypanosomal enzymatic activity in the plasma of T. congolense-infected cattle, together with the capacity of procongopain to be activated at weakly basic pH, suggest that procongopain may be extracellularly processed in the presence of blood vessel glycosaminoglycans, supporting the hypothesis that congopain acts as a pathogenic factor in host-parasite relationships.     

Inhibition of a cathepsin L-like cysteine protease by a chimeric propeptide-derived inhibitor

Like other papain-related cathepsins, congopain from Trypanosoma congolense is synthesized as a zymogen. We have previously identified a proregion-derived peptide (Pcp27), acting as a weak and reversible inhibitor of congopain. Pcp27 contains a 5-mer YHNGA motif, which is essential for selectivity in the inhibition of its mature form [Lalmanach, G., Lecaille, F., Chagas, J. R., Authié, E., Scharfstein, J., Juliano, M. A., and Gauthier, F. (1998) J. Biol. Chem. 273, 25112-25116]. In the work presented here, a homology model of procongopain was generated and subsequently used to model a chimeric 50-mer peptide (called H3-Pcp27) corresponding to the covalent linkage of an unrelated peptide (H3 helix from Antennapedia) to Pcp27. Molecular simulations suggested that H3-Pcp27 (pI = 9.99) maintains an N-terminal helical conformation, and establishes more complementary electrostatic interactions (E(coul) = -25.77 kcal/mol) than 16N-Pcp27, the 34-mer Pcp27 sequence plus the 16 native residues upstream from the proregion (E(coul) = 0.20 kcal/mol), with the acid catalytic domain (pI = 5.2) of the mature enzyme. In silico results correlated with the significant improvement of congopain inhibition by H3-Pcp27 (K(i) = 24 nM), compared to 16N-Pcp27 (K(i) = 1 microM). In addition, virtual alanine scanning of H3 and 16N identified the residues contributing most to binding affinity. Both peptides did not inhibit human cathepsins B and L. In conclusion, these data support the notion that the positively charged H3 helix favors binding, without modifying the selectivity of Pcp27 for congopain.     

Purification and characterization of cathepsin L-like proteinase from goat brain

Cathepsin L-like proteinase was purified approximately 1708-fold with 40% activity yield to an apparent electrophoretic homogeneity from goat brain by homogenization, acid-autolysis at pH 4.2, 30-80% (NH4)2SO4 fractionation, Sephadex G-100 column chromatography and ion-exchange chromatography on CM-Sephadex C-50 at pH 5.0 and 5.6. The molecular weight of proteinase was found to be approximately 65,000 Da, by gel-filtration chromatography. The pH optima were 5.9 and 4.5 for the hydrolysis of Z-Phe-Arg-4mbetaNA (benzyloxycarbonyl-L-phenylalanine-L-arginine-4-methoxy-beta-naphthylamide) and azocasein, respectively. Of the synthetic chromogenic substrates tested, Z-Phe-Arg-4mbetaNA was hydrolyzed maximally by the enzyme (Km value for hydrolysis was 0.06 mM), followed by Z-Val-Lys-Lys-Arg-4mbetaNA, Z-Phe-Val-Arg-4mbetaNA, Z-Arg-Arg-4mbetaNA and Z-Ala-Arg-Arg-4mbetaNA. The proteinase was activated maximally by glutathione in conjunction with EDTA, followed by cysteine, dithioerythritol, thioglycolic acid, dithiothreitol and beta-mercaptoethanol. It was strongly inhibited by p-hydroxymercuribenzenesulphonic acid, iodoacetic acid, iodoacetamide and microbial peptide inhibitors, leupeptin and antipain. Leupeptin inhibited the enzyme competitively with Ki value 44 x 10(-9) M. The enzyme was strongly inhibited by 4 M urea. Metal ions, Hg(2+), Ca(2+), Cu(2+), Li(2+), K(+), Cd(2+), Ni(2+), Ba(2+), Mn(2+), Co(2+) and Sn(2+) also inhibited the activity of the enzyme. The enzyme was stable between pH 4.0-6.0 and up to 40 degrees C. The optimum temperature for the hydrolysis of Z-Phe-Arg-4mbetaNA was approximately 50-55 degrees C with an activation energy Ea of approximately 6.34 KCal mole(-1).     

Fasciola hepatica cathepsin L-like proteases: biology,function, and potential in the development of first generation liver fluke vaccines

Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are members of a lineage that gave rise to the human cathepsin Ls, Ks and Ss, but while they exhibit similarities in their substrate specificities to these enzymes they differ in having a wider pH range for activity and an enhanced stability at neutral pH. There are presently 13 Fasciola cathepsin L cDNAs deposited in the public databases representing a gene family of at least seven distinct members, although the temporal and spatial expression of each of these members in the developmental stage of F. hepatica remains unclear. Immunolocalisation and in situ hybridisation studies, using antibody and DNA probes, respectively, show that the vast majority of cathepsin L gene expression is carried out in the epithelial cells lining the parasite gut. Within these cells the enzyme is packaged into secretory vesicles that release their contents into the gut lumen for the purpose of degrading ingested host tissue and blood. Liver flukes also express a novel multi-domain cystatin that may be involved in the regulation of cathepsin L activity. Vaccine trials in both sheep and cattle with purified native FheCL1 and FheCL2 have shown that these enzymes can induce protection, ranging from 33 to 79%, to experimental challenge with metacercariae of F. hepatica, and very potent anti-embryonation/hatch rate effects that would block parasite transmission. In this article we review the vaccine trials carried out over the past 8 years, the role of antibody and T cell responses in mediating protection and discuss the prospects of the cathepsin Ls in the development of first generation recombinant liver fluke vaccines.     

Cloning and functional expression of a Boophilus microplus cathepsin L-like enzyme

A cysteine proteinase gene homologous to cathepsins L genes was isolated from a B. microplus cDNA library. The precursor protein deduced from the nucleotide sequence contains 332 amino acid residues consisting of a signal sequence (pre-region), a pro-region and a mature proteinase. The DNA fragment coding for the proenzyme was cloned and expressed using the E. coli expression vector pMAL-p. The recombinant protein (MBP+PROCP) once activated is able to hydrolyze synthetic substrates as well as protein substrates like hemoglobin, vitellin and gelatin. Its optimal enzymatic activity on both fluorogenic and protein substrates was found to occur at an acidic pH. Expression of the proteinase gene was tested by RT-PCR with tick larvae RNA. Detection of amplified sequences indicates that the gene is expressed at this stage of the tick life cycle and the molecule is therefore potentially a target for chemotherapy or an immunogen in a vaccine.     

Inhibition of gastrulation in Xenopus embryos by an antibody against a cathepsin L-like protease

An antibody against cathepsin L-like protease (AACLP) was injected into one cell of 2-celled Xenopus embryos. The blastopores of AACLP-injected embryos either did not invaginate or failed to complete invagination. As a result of this failure to complete gastrulation, the body axes could not form normally and tail bud stage embryos were bent dorsally. Embryos injected with a control antibody (CA) developed normally through the tadpole stage. Mesodermal induction was not inhibited in embryos exhibiting this AACLP-induced gastrulation defect, but the mesodermal structure of these embryos was organized incorrectly due to the defective gastrulation during the early stages.     

Partial characterization of a novel cathepsin L-like protease from Fasciola hepatica

A 30-kDa protease, purified previously from Fasciola hepatica, was sequenced and the first 15 N-terminal residues were found to be 100% homologous to a region in the protein Fcp1c, which was cloned and expressed from F. hepatica. This terminal region was also 53 and 54% identical to two other cathepsin L-like proteases isolated from the same source. The 30-kDa protease demonstrated a specificity different from humancathepsin L when assayed with novel peptidyl enediones of the type Z-Phe-Ala-CH&dbond;CH(2)-CO(2)R (where R = Me/Et/Bu(t)). The ethyl ester peptide was a more efficient inhibitor of the protease than the corresponding methyl ester. This is in contrast to bovine cathepsin B and human cathepsin L where both are more readily inhibited by the methyl, rather than the ethyl ester peptide. These differences in the inhibition of the novel parasite protease may allow it to be exploited as a chemotherapeutic target.     

Expression and characterization of cathepsin L-like cysteine protease from Philasterides dicentrarchi

Philasterides dicentrarchi is a causative agent of scuticociliatosis in olive flounder Paralichthys olivaceus, aquaculture in Korea. In this study, a cDNA encoding a cathepsin L-like cysteine protease (PdCtL) of P. dicentrarchi (synonym Miamiensis avidus) was identified. To express the PdCtL recombinant protein in a heterologous system, 10 codons were redesigned to conform to the standard eukaryotic genetic code using polymerase chain reaction (PCR)-based site-directed mutagenesis. The recombinant P. dicentrarchi procathepsin L (proPdCtL) was expressed at high levels in E. coli Rosetta (DE3) pLysS with a pPET21a vector, and successfully refolded, purified, and activated into a functional and enzymatically active form. The optimal pH for protease activity was 5. Similar to other cysteine proteases, enzyme activity was inhibited by E64 and leupeptin. Immunogenicity of recombinant PdCtL was assessed by enzyme-linked immunosorbent assay, western blot, and specific anti-recombinant PdCtL antibodies were detected. Our results suggest that the biochemical characteristics of the recombinant ciliate proPdCtL protein are similar to those of the cathepsin L-like cysteine protease, that the PCR-based site-direct mutated ciliate gene was successfully expressed in a biochemically active form, and that the recombinant PdCtL acted as a specific epitope in olive flounder.     

Potent slow-binding inhibition of cathepsin B by its propeptide.

A peptide (PCB1) corresponding to the proregion of the rat cysteine protease cathepsin B was synthesized and its ability to inhibit cathepsin B activity investigated. PCB1 was found to be a potent inhibitor of mature cathepsin B at pH 6.0, yielding a Ki = 0.4 nM. This inhibition obeyed slow-binding kinetics and occurred as a one-step process with a k1 = 5.2 x 10(5) M-1 s-1 and a k2 = 2.2 x 10(-4) s-1. On dropping from pH 6.0 to 4.7, Ki increased markedly, and whereas k1 remained essentially unchanged, k2 increased to 4.5 x 10(-3) s-1. Thus, the increase in Ki at lower pH is due primarily to an increased dissociation rate for the cathepsin B/PCB1 complex. At pH 4.0, the inhibition was 160-fold weaker (Ki = 64 nM) than at pH 6.0, and the propeptide appeared to behave as a classical competitive inhibitor rather than a slow-binding inhibitor. Incubation of cathepsin B with a 10-fold excess of PCB1 overnight at pH 4.0 resulted in extensive cleavage of the propetide whereas no cleavage occurred at pH 6.0, consistent with the formation of a tight complex between cathepsin B and PCB1 at the higher pH. The synthetic propeptide of cathepsin B was found to be a much weaker inhibitor of papain, a structurally similar cysteine protease, and no pH dependence was observed. Inhibition constants of 2.8 and 5.6 microM were obtained for papain inhibition by PCB1 at pH 4.0 and 6.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leishmania aethiopica: identification and characterization of cathepsin L-like cysteine protease genes

There is limited information on the biology and pathogenesis of Leishmania aethiopica, causative agent of cutaneous leishmaniasis (CL) in Ethiopia. In this study we have identified and characterized two cathepsin L-like cysteine protease genes, Laecpa and Laecpb, from L. aethiopica. The predicted amino acid sequence of Laecpa and Laecpb is more than 75% identical with homologous cathepsin L-like cysteine protease genes of other Leishmania species and less than 50% identical with human cathepsin L. Laecpa is expressed predominantly in the stationary, and to a lower level, during the amastigote stage while Laecpb is specifically expressed in the stationary stage of L. aethiopica development. Phylogenetic analysis showed that the two genes are grouped into separate clades which are the result of gene duplication. The isolation of these genes will be useful in developing Leishmania species specific diagnostics for molecular epidemiological studies and serves as a first step to study the role of cysteine proteases in L. aethiopica pathogenesis.     

Structural requirements for cathepsin B and cathepsin H inhibition by kininogens

Domain 3 (D3) of human kininogens, the major cysteine proteinase inhibitors in plasma, has been shown to be the tightest binding inhibitory domain for cathepsins B and H. D3 was expressed in three fragments as its exon products as follows: exon 7 (Gly235-Gln292), exon 8 (Gln292-Gly328), and exon 9 (Gln329-Met357). Exon products 7, 8, and 9 alone as well as exon product 7 + 9 each exhibited an IC₅₀ value 5- to 30-fold higher (5–30M) than exon products 7 + 8 and 8 + 9 (0.9–1.3M) for cathepsins B and H, respectively. However, in turn, the exon products 7 + 8 and 8 + 9 seemed to be less potent inhibitors than the intact D3 (10, 200 nM) or HK (200, 500 nM) molecule. These results clearly indicate that an intact molecule of HK or its domain 3 as a whole is required for optimal inhibition of cathepsins B and H.     

Engineering of proteases and protease inhibition.

Proteases are unquestionably the single most studied class of enzymes and yet many questions still remain about their mechanisms and roles. Protein engineering offers the opportunity to provide some of the answers. In this review, recent advances towards the understanding of stability, mechanism, specificity and regulation of proteases and their inhibitors are outlined. In addition, the application of this increased understanding is also discussed.     

ATP-dependent ligases in trypanothione biosynthesis--kinetics of catalysis and inhibition by phosphinic acid pseudopeptides

Glutathionylspermidine is an intermediate formed in the biosynthesis of trypanothione, an essential metabolite in defence against chemical and oxidative stress in the Kinetoplastida. The kinetic mechanism for glutathionylspermidine synthetase (EC 6.3.1.8) from Crithidia fasciculata (CfGspS) obeys a rapid equilibrium random ter-ter model with kinetic constants K(GSH) = 609 microM, K(Spd) = 157 microM and K(ATP) = 215 microM. Phosphonate and phosphinate analogues of glutathionylspermidine, previously shown to be potent inhibitors of GspS from Escherichia coli, are equally potent against CfGspS. The tetrahedral phosphonate acts as a simple ground state analogue of glutathione (GSH) (K(i) approximately 156 microM), whereas the phosphinate behaves as a stable mimic of the postulated unstable tetrahedral intermediate. Kinetic studies showed that the phosphinate behaves as a slow-binding bisubstrate inhibitor [competitive with respect to GSH and spermidine (Spd)] with rate constants k(3) (on rate) = 6.98 x 10(4) M(-1) x s(-1) and k(4) (off rate) = 1.3 x 10(-3) s(-1), providing a dissociation constant K(i) = 18.6 nM. The phosphinate analogue also inhibited recombinant trypanothione synthetase (EC 6.3.1.9) from C. fasciculata, Leishmania major, Trypanosoma cruzi and Trypanosoma brucei with K(i)(app) values 20-40-fold greater than that of CfGspS. This phosphinate analogue remains the most potent enzyme inhibitor identified to date, and represents a good starting point for drug discovery for trypanosomiasis and leishmaniasis.     

Reversible inhibition of mitochondrial respiratory control by tetrabutylammonium bromide.

Influences of tetrabutylammonium bromide as a phosphorylation inhibitor and as an uncoupler of oxidative phosphorylation in rat liver mitochondria were reversed by washing the organelles. Uncoupling by 2,4-dinitrophenol was also reversible whereas gramicidin and the detergents, sodium dodecylsulfate and cetyltrimethylammonium bromide, were tightly bound uncouplers and they were not substantially removed by simple washing.     

Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease

A diagnostic ELISA with recombinant Fasciola hepatica cathepsin L-like protease as antigen was developed to detect antibodies against F. hepatica in sheep and cattle. The recombinant cathepsin L-like protease was generated by functional expression of the cDNA from adult stage F. hepatica flukes in Saccharomyces cerevisiae. Specificity and sensitivity of the cathepsin L enzyme-linked immunosorbent assay (ELISA) was assessed using sera from sheep and calves experimentally or naturally mono-infected with F. hepatica and six-seven other parasites. The sensitivity of the cathepsin L ELISA for sheep and cattle sera was 99.1 and 100%, respectively. In the experimental setting with established mono-infections, the specificity of the cathepsin L ELISA was 98.5% for cattle sera and 96.5% for sheep sera. In experimentally infected cattle and sheep, the first detection of F. hepatica-specific antibodies appeared first between 5 and 7 weeks post-infection, but depended on the infectious dose of F. hepatica. In ELISA the detection preceded first detection of the infection based on egg counts and remained detectable till at least 23 weeks after a primary F. hepatica infection. Detection of Fasciola gigantica infections was similar to detection of F. hepatica. The first detection occurred at week 5 and signals persisted for at least 20 weeks. All sera from naturally F. hepatica infected sheep were seropositive in the cathepsin L-like ELISA. The relevance of this ELISA format was also evaluated using sera from naturally infected cattle in the Netherlands, Ecuador and Vietnam and compared with results from egg-counts. For the latter two endemic areas with mixed parasitic infections the 'apparent' sensitivity of the cathepsin L ELISA was calculated for all serum samples together to be 90.2%. The 'apparent' specificity under these conditions was calculated to be 75.3%. In cattle, the cathepsin L ELISA was superior to the concurrently evaluated peptide ELISA format using a single epitope as the antigen both in controlled natural infections as well as in infections in endemic areas. The present ELISA-format contributes a relatively sensitive and reliable tool for the early serodiagnosis of bovine and ovine fasciolosis.     

Reversible inhibition of renal microsome calcium pump by furosemide.

Renal microsomes have been shown to have an energy-dependent calcium pump activity. We now demonstrate that three diuretic compounds inhibit this calcium pump $\text{in}$$\text{vitro}$. Characterization of furosemide inhibition demonstrates that this agent is a reversible, non-competitive inhibitor of the renal microsome calcium pump. Furosemide, under similar conditions does not inhibit Na/K-ATPase activity in the renal microsome fraction. Furosemide may be useful to define function of the microsomal calcium pump in non-muscle cells.