Biomedical subjects

V Turk

Publications and source records attributed to V Turk.

At least 19 recordsLinked to original sources

PubMed1999

Crystal structure of MHC class II-associated p41 Ii fragment bound to cathepsin L reveals the structural basis for differentiation between cathepsins L and S.

The lysosomal cysteine proteases cathepsins S and L play crucial roles in the degradation of the invariant chain during maturation of MHC class II molecules and antigen processing. The p41 form of the invariant chain includes a fragment which specifically inhibits cathepsin L but not S. The crystal structure of the p41 fragment, a homologue of the thyroglobulin type-1 domains, has been determined at 2.0 A resolution in complex with cathepsin L. The structure of the p41 fragment demonstrates a novel fold, consisting of two subdomains, each stabilized by disulfide bridges. The first subdomain is an alpha-helix-beta-strand arrangement, whereas the second subdomain has a predominantly beta-strand arrangement. The wedge shape and three-loop arrangement of the p41 fragment bound to the active site cleft of cathepsin L are reminiscent of the inhibitory edge of cystatins, thus demonstrating the first example of convergent evolution observed in cysteine protease inhibitors. However, the different fold of the p41 fragment results in additional contacts with the top of the R-domain of the enzymes, which defines the specificity-determining S2 and S1' substrate-binding sites. This enables inhibitors based on the thyroglobulin type-1 domain fold, in contrast to the rather non-selective cystatins, to exhibit specificity for their target enzymes.

Amino Acid Sequence↗

PubMed1999

Thyroglobulin type-1 domains in equistatin inhibit both papain-like cysteine proteinases and cathepsin D.

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 (Ki = 0.61 nM). Remarkably, equistatin also strongly inhibits cathepsin D with Ki = 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 (Ki = 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.

Amino Acid Sequence↗

PubMed1999

Acidic pH as a physiological regulator of human cathepsin L activity.

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 alpha-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.

Amino Acid Sequence↗

PubMed1998

On the mechanism of human stefin B folding: II. Folding from GuHCl unfolded, TFE denatured, acid denatured, and acid intermediate states.

It has been shown that human stefin B exhibits molten globule intermediates 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 dichroism (CD) and fluorescence were correlated. In the present work the kinetics of folding were monitored by tyrosine fluorescence, ANS fluorescence, and, for certain reactions, far ultraviolet (UV) CD. The folding was started from the unfolded state in 3.45 M GuHCl, the acid denatured state at pH 1.8+/-0.2, an acid molten globule intermediate I1 (pH 3.3+/-0.1, low salt), a more structured acid molten globule intermediate I2 (pH 3.3+/-0.1, 0.42 M NaCl), and the TFE state (pH 3.3+/-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 identical. This does not apply to the second acid molten globule intermediate I2, which demonstrates a higher rate of folding by a factor of 270. Different energy of activation and pH dependence were found for folding from states I1 or I2.

Anilino Naphthalenesulfonates↗

PubMed1998

On the mechanism of human stefin B folding: I. Comparison to homologous stefin A. Influence of pH and trifluoroethanol on the fast and slow folding phases.

The folding of human stefin B has been studied by several spectroscopic probes. Stopped-flow traces obtained by circular dichroism in the near and far UV, by tyrosine fluorescence, and by extrinsic probe ANS fluorescence are compared. Most (60+/-5%) of the native signal in the far UV circular dichroism (CD) appeared within 10 ms in an unresolved "burst" phase, which was followed by a fast phase (t = 83 ms) and a slow phase (t = 25s) with amplitudes of 30% and 10%, respectively. Similar fast and slow phases were also evident in the near UV CD, ANS fluorescence, and tyrosine fluorescence. By contrast, human stefin A, which has a very similar structure, exhibited only one kinetic phase of folding (t = 6s) detected by all the spectroscopic probes, which occurred subsequent to an initial "burst" phase observed by far UV CD. It is interesting that despite close structural similarity of both homologues they fold differently, and that the less stable human stefin B folds faster by an order of magnitude (comparing the non-proline limited phase). To gain more information on the stefin B folding mechanism, effects of pH and trifluoroethanol (TFE) on the fast and slow phases were investigated by several spectroscopic probes. If folding was performed in the presence of 7% of TFE, rate acceleration and difference in the mechanism were observed.

Cystatin A↗

PubMed1998

Substrate inhibition of cruzipain is not affected by the C-terminal domain.

Endogenous and recombinant cruzipain, the major cysteine proteinase from the protozoan parasite Trypanosoma cruzi, exhibit differences in the protein and circular dichroism spectra probably attributed to the absence of the C-terminal domain in the recombinant enzyme. Substrate hydrolysis of both molecules at 25 degrees C and neutral pH obeyed Michaelis-Menten kinetics whereas significant substrate inhibition was observed above neutral pH. The results suggest that substrate inhibition of cruzipain is pH-dependent, and that the C-terminal domain does not play an essential role in this process.

Animals↗

PubMed1998

pH-induced conformational transitions of the propeptide of human cathepsin L. A role for a molten globule state in zymogen activation.

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.

Cathepsin L↗

PubMed1998

Refolding of recombinant sulphonated procathepsin S and of reduced chicken cystatin; implications for renaturation experiments.

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.

Animals↗

PubMed1998

Stoichiometry and heterogeneity of the pro-region chain in tetrameric human cathepsin C.

The subunit structure and composition of mature human cathepsin C, an oligomeric cysteine proteinase, has been characterised in detail. The heavy chain, light chain and pro-region peptides are shown to be held together solely by non-covalent interactions, and to be present in equimolar ratio, suggesting an important structural role for the residual pro-region chain which is strongly bound to the enzyme. The mass of the light chain, as determined by mass spectrometry, combined with its N-terminal sequence, determines the position of cleavage from the heavy chain. Amino-acid sequencing has led to definition of the 13.5 kDa N-terminal part of the pro-region which remains in the mature enzyme, the C-terminal moiety of 10 kDa being cleaved out and lost from the pro-peptide on activation. The residual pro-region is heterogeneous, a proportion being intact and the remainder being cleaved at alternative positions 58 or 61, yielding two smaller peptides joined by disulphide bond. The proportion of cleaved form was found to vary with tissue and enzyme preparation but did not affect enzyme activity. The molecular masses of the constituent chains after deglycosylation lead to a protein mass of 158 kDa. All four potential glycosylation sites are glycosylated.

Alkylation↗

PubMed1998

Crystal structure of porcine cathepsin H determined at 2.1 A resolution: location of the mini-chain C-terminal carboxyl group defines cathepsin H aminopeptidase function.

BACKGROUND: Cathepsin H is a lysosomal cysteine protease, involved in intracellular protein degradation. It is the only known mono-aminopeptidase in the papain-like family and is reported to be involved in tumor metastasis. The cathepsin H structure was determined in order to investigate the structural basis for its aminopeptidase activity and thus to provide the basis for structure-based design of synthetic inhibitors. RESULTS: The crystal structure of native porcine cathepsin H was determined at 2.1 A resolution. The structure has the typical papain-family fold. The so-called mini-chain, the octapeptide EPQNCSAT, is attached via a disulfide bond to the body of the enzyme and bound in a narrowed active-site cleft, in the substrate-binding direction. The mini-chain fills the region that in related enzymes comprises the non-primed substrate-binding sites from S2 backwards. CONCLUSIONS: The crystal structure of cathepsin H reveals that the mini-chain has a definitive role in substrate recognition and that carbohydrate residues attached to the body of the enzyme are involved in positioning the mini-chain in the active-site cleft. Modeling of a substrate into the active-site cleft suggests that the negatively charged carboxyl group of the C terminus of the mini-chain acts as an anchor for the positively charged N-terminal amino group of a substrate. The observed displacements of the residues within the active-site cleft from their equivalent positions in the papain-like endopeptidases suggest that they form the structural basis for the positioning of both the mini-chain and the substrate, resulting in exopeptidase activity.

Amino Acid Sequence↗

PubMed1998

The influence of Ala205 on the specificity of cathepsin L produced by dextran sulfate assisted activation of the recombinant proenzyme.

Human procathepsin L has been expressed in E. coli in the form of inclusion bodies. The recombinant protein was isolated, refolded and processed at pH 5.5 by the addition of dextran sulfate which increased the overall yield of cathepsin L almost 10-fold. After the auto-activation of the 38 kDa procathepsin L at least three processing sites were determined by N-terminal amino acid sequencing. After replacing the Ala205 residue by glutamic acid, cathepsin B-like specificity was introduced into cathepsin L. This mutation resulted in a 15-fold increased activity toward the substrate Z-Arg-Arg-AMC and in a 29-fold decreased activity toward Z-Phe-Arg-AMC. Residue 205 is thereby confirmed experimentally to be critical for the specificity of cathepsins B and L.

Alanine↗

PubMed1997

Crystal structure of the wild-type human procathepsin B at 2.5 A resolution reveals the native active site of a papain-like cysteine protease zymogen.

The structure of the wild-type human procathepsin B has been refined to a crystallographic R-value of 0.18 and R-free of 0.23 exploiting the data obtained from new crystals that diffract beyond 2.5 A resolution. The structure confirms two previously presented, lower-resolution structures. The structure of the propeptide chain folds on the surface of the enzyme domains and blocks access of substrate to the already formed active site. Abundant solvent molecules fill the cavities between the propeptide and the enzyme part of the molecule. The propeptide structure is compared with a substrate model in the S2, S1, S1' and S2' binding sites. In this crystal form the cathepsin B occluding loop residues adopt yet another conformation. The structures show that the occluding loop region between the residues Cys108 and Cys119 behaves quite independently from the rest of the structure and easily adapts to changes in environment. The variety of the observed conformations of the occluding loop is in agreement with other data showing that the loop is responsible for limiting cathepsin B activity to that of a carboxydipeptidase. The region before Cys108 is essentially the same as in the mature structure, whereas the region from Cys119 to Thr125 is raised compared to the mature form by the propeptide squeezed between it and the enzyme domains, surface. The structure strongly suggests that processing of procathepsin B during its autoactivation is not unimolecular.

Amino Acid Sequence↗

PubMed1997

Equistatin, a new inhibitor of cysteine proteinases from Actinia equina, is structurally related to thyroglobulin type-1 domain.

It is well known that the activities of the lysosomal cysteine proteinases are tightly regulated by their endogenous inhibitors, cystatins. Here we report a new inhibitor of cysteine proteinases isolated from sea anemone Actinia equina. The inhibitor, equistatin, is an acidic protein with pI 4.7 and molecular weight of 14,129. It binds tightly and rapidly to cathepsin L (ka = 5.7 x 10(7) M-1 s-1, Ki = 0.051 nM) and papain (ka = 1.2 x 10(7) M-1 s-1, Ki = 0.57 nM). The lower affinity for cathepsin B (Ki = 1.4 nM) was shown to be due mainly to a lower second order association rate constant (ka = 0.04 x 10(6) M-1 s-1). The inhibitor is composed of 128 amino acids forming two repeated domains with 48% identity. Neither of the domains shows any sequence homology to cystatins, but they do show a significant homology to thyroglobulin type-1 domains. A highly conserved consensus sequence motif of Cys-Trp-Cys-Val together with conserved Cys, Pro, and Gly residues is present in major histocompatibility complex class II-associated p41 invariant chain, nidogen, insulin-like growth factor proteins, saxiphilin domain a, pancreatic carcinoma marker proteins (GA733), and chum salmon egg cysteine proteinase inhibitor. In each of the domains of the equistatin, the three residues are similarly conserved, and the sequences Val-Trp-Cys-Val and Cys-Trp-Cys-Val are present in domains a and b, respectively. We suggest that equistatin belongs to a new superfamily of protein inhibitors of cysteine proteinases named thyroglobulin type-1 domain inhibitors. This superfamily currently includes equistatin, major histocompatibility complex class II- associated p41 invariant chain fragment, and chum salmon egg cysteine proteinase inhibitor.

Amino Acid Sequence↗

PubMed1997

Characterization of the equilibrium intermediates in acid denaturation of human stefin B.

Acid-induced denaturation of recombinant human stefin B was followed using circular dichroism (CD) and fluorimetry. By comparing different spectroscopic probes, a number of equilibrium intermediates were detected. In pH denaturation at very low salt concentration (0.03 M NaCl) four states can be distinguished: N - I(N) - I1 - U, where N is the native state, I(N) is a native-like intermediate, I1 is an acid intermediate state with properties of a molten globule and U is the unfolded state. State 1, exhibits no near-ultraviolet CD but has some residual far-ultraviolet CD. It differs from U in its ability to increase fluorescence of 1-anilino-naphthalene 8-sulfonate (ANS). In 0.42 M salt, the pH denaturation is three-state between the dimeric native state N2 and intermediates I(N2) and I2, which are also dimeric according to size-exclusion chromatography. The acid intermediate I2 is more structured than I1: it binds ANS to a lower extent an I1, its Tyr residues are protected from the solvent, it shows some near-ultraviolet CD and its far-ultraviolet CD is even more intense than that for the native state. 1H-NMR spectra confirmed the overall structural features of the acid intermediates. To obtain the enthalpies of unfolding, microcalorimetric measurements were performed under conditions where the acid intermediates are maximally populated (18 degrees C): state I(N) from pH 5.0 to 4.6, 0.03 M salt: state I1 below pH 3.8, 0.42 M salt; and state I1 in equilibrium with I(N) at pH 4.05, 0.03 M salt. Enthalpies of unfolding for states I(N) and I1 were comparable to those of the native state. The enthalpy of unfolding for state I1 could not be determined.

Anilino Naphthalenesulfonates↗

PubMed1997

Enzyme-linked immunosorbent assay for the detection of total cathepsin H in human tissue cytosols and sera.

An enzyme-linked immunosorbent assay (ELISA) was constructed for the determination of total human cathepsin H concentration in clinical samples. Utilising monoclonal and polyclonal antibodies, raised to human liver cathepsin H, the assay is able to detect a mature protein, a precursor molecule and enzyme-inhibitor complexes. The test system permits sensitive and reliable detection of analyte either in tissue cytosols or in sera. The detection limit is 2 ng/ml (n = 10, mean of zero standard +/- 3 SD). The average recovery of cathepsin H, added to the low content samples, was 95.3% +/- 1.8%. The within-run and between-run coefficient of variance (CV) varied from 2.3% to 8.9% and 12.7% to 16.4%, respectively, indicating satisfactory reproducibility of the method. The level of cathepsin H was defined in tissue cytosols of human heart, muscle and kidney and in sera from 30 healthy individuals. Additionally, cathepsin H was measured in sera from 55 patients with primary skin melanoma and from 42 patients with metastatic melanoma. The mean cathepsin H level was significantly higher for both groups of patients compared to normal sera level, being highest for metastatic melanoma patients.

Animals↗

PubMed1997

A fragment of the major histocompatibility complex class II-associated p41 invariant chain inhibits cruzipain, the major cysteine proteinase from Trypanosoma cruzi.

A peptide fragment derived from the p41 form of the invariant chain (Ii) associated with the major histocompatibility complex (MHC) class II molecule has been shown to inhibit the mammalian lysosomal cysteine proteinase, cathepsin L, and to be a novel cysteine proteinase inhibitor, distinct from cystatins. Here we report that this same fragment also binds to and inhibits cruzipain, the cathepsin L-like enzyme from the protozoan parasite Trypanosoma cruzi. The binding of the Ii fragment to cruzipain is fast (k(ass) = 2.4 x 10(7) M(-1) s(-1) and tight (Ki = 5.8 x 10(-11) M). The inhibition is competitive. These results suggest the possibility of using the invariant chain as a model for the specific inhibition of cruzipain in vivo, i.e. as a potential drug to combat Chagas' disease.

Alternative Splicing↗

PubMed1997

Cathepsin B and cysteine proteinase inhibitors in human lung cancer cell lines.

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 (< 1.0 microg/mg total protein). The LPLC lines also secreted the highest amounts of Cat B precursor of M(r) about 46 kDa. Inhibitory activities against Cat B and papain were associated with high molecular mass (HMM) and low molecular mass (LMM) inhibitory proteins, both in cell extracts and in media. About 75% of the inhibitory activity was associated with HMM inhibitors, the majority of which were kininogens (M(r) > 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.

Carcinogens↗

PubMed1997

Cruzipain, the major cysteine proteinase from the protozoan parasite Trypanosoma cruzi.

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.

Amino Acid Sequence↗