Biomedical subjects

V Turk

Publications and source records attributed to V Turk.

At least 91 records · Page 5Linked to original sources

PubMed1994

Structural characterisation of human stefin A in solution and implications for binding to cysteine proteinases.

Stefin A is a member of the cystatin superfamily of proteins which are tight and reversibly binding inhibitors of the papain-like cysteine proteinases. The 1H-NMR and 15N-NMR resonances of human stefin A have been sequentially assigned using two-dimensional homonuclear and heteronuclear NMR techniques in conjunction with three-dimensional heteronuclear methods. Characteristic sequential and medium range NOE contacts, J constants and hydrogen exchange data have been used to identify the secondary structural elements of the protein which consists of five anti-parallel beta-strands and a single alpha-helix. There is much similarity between the secondary structural features of stefin A and the homologous protein stefin B in its complex with papain [Stubbs, M. T., Laber, B., Bode, W., Huber, R., Jerala, R., Lenarcic, B. & Turk, V. (1990) EMBO. J. 9. 1939-1947] but also some important differences in regions which are fundamental to the binding event. The principal difference is the presence of two conformationally unrestricted regions in stefin A that form two of the components of the tripartite wedge which docks into the active site of the target proteinase. Specifically, these regions are the five N-terminal residues and the second binding loop, which form a turn and a short helix respectively, in the bound conformation of stefin B.

Amino Acid Sequence↗

PubMed1994

Elongation on the amino-terminal part of stefin B decreases inhibition of cathepsin H.

Two mutants of the cysteine proteinase inhibitor, stefin B, were prepared by ligating the amino-terminal region from cystatin C and kininogen, members of two other families of cystatin superfamily. The mutant proteins were expressed in Escherichia coli and purified to homogeneity. Inhibition and kinetic constants were determined for authentic and mutated stefins against the four different cysteine proteinases, papain and human cathepsins B, L and H. Inhibition of both amino-terminal elongated stefin B mutants was decreased particularly for cathepsin H. A model of the tertiary structure of cathepsin H and its complex with stefin B was constructed. The framework for the model of cathepsin H consisted of structurally conserved regions from tertiary structures of three cysteine proteinases. Variable regions were selected from fragments of other proteins from the protein data base. We suggest that reduced binding of stefins with elongated amino termini is caused by the mini chain of cathepsin H which is probably in close proximity to the amino termini in the complexes. This mini chain is bridged to Cys214 and has already been proposed to be responsible for the aminopeptidase activity of cathepsin H. We conclude that the amino-terminal region of stefin B plays an important role in determining the strength of inhibition of cathepsin H.

Amino Acid Sequence↗

PubMed1994

Sequence and structure similarities of cathepsin B from the parasite Schistosoma mansoni and human liver.

A three-dimensional structure of Schistosoma mansoni cathepsin B was modelled using the coordinates of the crystal structure of the human liver enzyme. Both enzymes appear to share remarkable structural similarity. However, an examination of the models complexed with two synthetic inhibitors revealed differences in inhibitor binding, as confirmed by differences in the 50% inhibitory concentration of the same inhibitor.

Amino Acid Sequence↗

PubMed1994

Stefin B, the major low molecular weight inhibitor in ovarian carcinoma.

Endogenous cysteine proteinase inhibitors (CPIs) presumably regulate lysosomal cysteine endopeptidases (CPs), such as cathepsins B and L, in vivo. An imbalance between CPs and CPIs in carcinomas, possibly due to impaired inhibition of proteinases, was reported. Ovarian carcinoma contain high levels of Stefin B and about twentyfold less Stefin A compared to normal epithelial tissue. Stefin B was isolated and characterized. We used alkaline treatment, affinity chromatography on Cm-papain Sepharose, followed by gel filtration and ion-exchange chromatography and anti-Stefin B-Sepharose 4B to isolate two major isoforms of Stefin B with pI values 5.9 and 6.5. M(r) of ovarian Stefin B was close to 14,000 as judged by SDS-PAGE and had a blocked N-terminus. It strongly inhibited papain (Ki = 0.11 nM) and cathepsin L (Ki = 0.035 nM), but only moderately cathepsin B (Ki = 130 nM). As these properties are similar to Stefin B from human and bovine origin, as well as to Stefin B from human histiosarcoma, we believe that tumor Stefin B does not differ from normal Stefin B.

Chromatography, Affinity↗

PubMed1994

The mechanisms and regulation of procathepsin L secretion from osteoclasts in bone resorption.

The secretion mechanisms of cathepsin L from osteoclasts in the process of bone resorption were investigated. The increases in bone pit numbers formed take place by PTH addition in parallel with the increases of cathepsin L and/or L-like proteinase activities in the culture medium of bone cells, and these were suppressed by the addition of calcitonin. The Z-Phe-Arg-MCA hydrolysing activity increased in the medium through the effect of PTH is considered to be a kind of procathepsin L by Western blotting analysis, and was suppressed by calcitonin addition. Furthermore, monensin inhibited not only the PTH-induced pit formation, but also cysteine proteinase activity in osteoclasts. Therefore, the procathepsin L excreted might be transferred from endothelial reticulum via Golgi and/or via lysosomes.

Animals↗

PubMed1994

Kinetics of inhibition of bovine cathepsin S by bovine stefin B.

The kinetics of the complex formation between bovine cathepsin S and bovine stefin B was studied by conventional and stopped-flow techniques. The inhibition at low inhibitor concentrations was tight and reversible (kass = 5.8 x 10(7) M-1.s-1, kdiss = 4.9 x 10(-4) s-1 at pH 6.0 and 25 degrees C), whereas at higher inhibitor concentrations it was pseudo-irreversible (kass = 6.14 x 10(7) M-1.s-1). The complex was formed directly lacking the fast pre-equilibrium step with the dissociation equilibrium constant of approximately 8 pM. The competitive nature of inhibition was confirmed. The kass was found to be pH-independent between pH 6.0 and 7.5 and decreased at lower or higher pH values in a way that strongly suggests involvement of two ionizable groups in the interaction (pKi = 5.2, pK2 = 8.3). The enzyme-substrate interaction seems to be influenced by different ionizable groups (pKi = 4.4, pK2 = 7.8).

Animals↗

PubMed1994

Improved expression and evaluation of polyethyleneimine precipitation in isolation of recombinant cysteine proteinase inhibitor stefin B.

Synthetic gene coding for human cysteine proteinase inhibitor stefin B was expressed in Escherichia coli by the use of pKP1500 plasmid-containing tac promotor and temperature-sensitive origin of replication, ensuring high plasmid copy number. Several parameters were varied in order to maximize the yield of inhibitory active protein: distance between RBS and initiator codon, temperature of fermentation, and conditions of fermentation. Production of stefin B was markedly improved by setting the RBS to ATG codon distance to 10 nt and with fermentation conditions that increased yield of biomass. The isolation procedure was modified by including precipitation with polyethyleneimine that removed contaminants such as nucleic acids and most bacterial (predominantly acidic) proteins. Precipitation itself produced more than 80% pure recombinant inhibitor, which was purified to homogeneity by a single chromatographic step. Isolated protein had the same inhibitory properties as authentic inhibitor.

Base Sequence↗

PubMed1994

Structural basis for the difference in thermodynamic properties between the two cysteine proteinase inhibitors human stefins A and B.

Homology modelling has been used to model stefin A based on the X-ray structure of stefin B. Several models have been produced by interactive modelling or positioning of the side chains by Monte Carlo procedure with simulated annealing. The quality of models was evaluated by calculation of the free energy of hydration, 3D-1D potential or buried area of surface accessibility. Stefin A is a thermostable protein, exhibiting a two-state denaturation, while stefin B denatures at a 40 degrees C lower temperature and forms a stable molten globule intermediate under mild denaturing conditions. From the tertiary structures, thermodynamic functions were predicted, conforming closely to the experimental calorimetric results. Polar and apolar buried areas of surface accessibility were obtained by structural deconvolution of the thermograms. It is suggested that the basic difference between the stefins is the domination of hydrophobic interaction in the stabilization of stefin B, which is due to its non-specific nature leading to the formation of a molten globule intermediate. Modelling of stefin A predicts increased numbers of hydrogen bonds which stabilize it and increase the cooperativity of its denaturation.

Amino Acid Sequence↗

PubMed1993

Purification of the complex of cathepsin L and the MHC class II-associated invariant chain fragment from human kidney.

The complex of cathepsin L and the fragment of the MHC class II-associated invariant chain was purified from human kidney. M(r) of the complex, as determined by gel filtration, is about 40,000. Both components were identified by amino acid and sequence analyses. The bound invariant chain fragment is almost identical to the additional segment found in p41, but not in the p31 form of the invariant chain. The complex has significantly enhanced stability at neutral and slightly alkaline pH, and reduced proteolytic activity against the synthetic substrate Z-Phe-Arg-MCA compared to free cathepsin L. The complex exhibits no enzymatic activity against the protein substrate azocasein. For the first time, the invariant chain was found in a complex with a protein, which was not an MHC molecule.

Amino Acid Sequence↗

PubMed1993

Molecular cloning of a putative homolog of proline/arginine-rich antibacterial peptides from porcine bone marrow.

Screening of a porcine bone marrow cDNA library with a PCR-derived probe from rabbit LPS-binding protein CAP18 led to the discovery of two closely related clones. The longer, full-length cDNA clone encodes a 228 amino acid residue protein similar to the family of antibacterial/LPS-binding cationic peptides. In contrast to other hitherto discovered precursors of Pro/Arg-rich peptides from this family, they have a novel, unique structure of the C-terminal region of 100 amino acid residues with a repeating sequence of ten residues (FPPPNXPGPR, where X = V or F). These precursors could represent a part of the antibacterial peptide repertoire of porcine bone marrow.

Amino Acid Sequence↗

PubMed1993

Pig leukocyte cysteine proteinase inhibitor (PLCPI), a new member of the stefin family.

A new stefin type low-M(r) cysteine proteinase inhibitor (PLCPI) was isolated from pig polymorphonuclear leukocytes as a contaminant of the cathelin sample. The inhibitor consists of 103 amino acids, and its M(r) was calculated to be 11,768. The inhibitor exhibits considerable sequence identity with inhibitors from the stefin family, particularly with human stefin A. The PLCPI is a fast acting inhibitor of papain and cathepsins L and S (k(ass) > or = 1 x 10(6) M-1 x s-1) and forms very tight complexes with these enzymes (Ki < or = 190 pM). The affinity for cathepsins B and H (Ki > or = 125 nM) was lower. These results also show that the inhibitory activity previously ascribed to cathelin was due to the presence of PLCPI.

Amino Acid Sequence↗

PubMed1993

The structures of native phosphorylated chicken cystatin and of a recombinant unphosphorylated variant in solution.

The solution structures of the phosphorylated form of native chicken cystatin and the recombinant variant AEF-S1M-M29I-M89L were determined by 2D, 3D and 4D-NMR. The structures turn out to be very similar, despite the substitutions and the phosphorylation of the wild-type. Their dominant feature is a five-stranded beta-sheet, which is wrapped around a five-turn alpha-helix, as shown by X-ray crystallographic studies of wild-type chicken cystatin. However, the NMR analysis shows that the second helix observed in the crystal is not present in solution. The phosphorylation occurs at S80, which is located in a flexible region. For this reason, very few effects on the structure are observed. Comparison of structures of the unphosphorylated variant and the wild-type shows small effects on H84 which is located in the supposed recognition site of the serine kinase. This recognition site appears to be well structured as a large loop-containing bulge of the beta-sheet. The N termini of both mutants, which contribute to a large extent to the binding to the proteinase, are very flexible. A loop structure involving the residues L7 to A10 as found in related inhibitors, such as in the kininogen domains 2 and 3, is not sufficiently populated to be observed.

Amino Acid Sequence↗

PubMed1993

Conformational variability of chicken cystatin. Comparison of structures determined by X-ray diffraction and NMR spectroscopy.

The structural model derived from X-ray crystallography for unphosphorylated wild-type chicken cystatin is compared with two chicken cystatin structures derived from NMR spectroscopy: the phosphorylated wild-type and the genetically engineered variant AEF-SIM-M29I-M89L. The comparison shows the same overall fold, but also significant differences in structurally variable segments of the polypeptide chain. The largest such segment, comprising residues 71 to 89, is a region characteristic of the family 2 cystatin inhibitors which contains a disulphide bridge (71-81) and the phosphorylation site (Ser80) discussed in the accompanying article. In the crystal structure, the segment 71 to 76 is found as a flexible loop, 77 to 85 as an alpha-helical segment, and 86 to 89 is completely undefined. The solution NMR structures on the other hand are disordered in the initial segment 72 to 80, have an extended conformation at 81 to 83 in contact with the beta-sheet, and clearly show a beta-turn at residues 87 to 90. The segment comprising residues 53 to 57, with smaller variability, is of particular interest as the hairpin loop conserved throughout the cystatin superfamily which binds to the cysteine proteinase. In most of the solution NMR structures, this segment adopts a conformation more like that of stefin B, a family 1 cystatin inhibitor, as was observed in the crystal structure of its inhibitory complex with papain. The differences between the structures are rationalized by an examination of the crystal contacts generated by hypothetical crystal packing of the NMR structures. Additionally, the X-ray refinement shows evidence of conformational disorder in the crystal. Joint refinement with NOE restraints and reflection data does not produce a structure to satisfy the restraints of both methods.

Animals↗

PubMed1993

The affinity-labelling of cathepsin S with peptidyl diazomethyl ketones. Comparison with the inhibition of cathepsin L and calpain.

Since peptidyl diazomethyl ketones are useful irreversible inhibitors for inactivating cysteinyl proteinases in vitro and in vivo and in order to reveal their role, we set out to obtain selective and effective reagents for cathepsin S. A number of such derivatives with hydrophobic amino acid residues, such as valine, leucine and tryptophane in positions adjacent to the primary specificity site were synthesized and these provided inhibitors rapidly acting at high dilution. For example, 1 nM Z-Leu-Leu-Nle-CHN2 inactivates cathepsin S with k2nd = 4.6 x 10(6) M-1 x s-1 at pH 6.5, 25 degrees C. Similarities to the specificities of cathepsin L and calpain were evident. However, Z-Val-Val-NleCHN2 is over 300 times more effective in inactivating S than L. On the other hand, Z-Phe-Tyr(t-Bu)CHN2 is about 10(4) more effective against L than S. Reagents are thus now available for a clear discrimination between these proteases.

Amino Acid Sequence↗

PubMed1993

The primary structure of inhibitor of cysteine proteinases from potato.

The complete amino acid sequence of the cysteine proteinase inhibitor from potato tubers was determined. The inhibitor is a single-chain protein having 180 amino acid residues. Its primary structure was elucidated by automatic degradation of the intact protein and sequence analysis of peptides generated by CNBr, trypsin and glycyl endopeptidase. A search through the protein sequence database showed homology to other plant proteinase inhibitors of different specificities and non-inhibitory proteins of M(r) around 20,000. On the basis of sequence homology, prediction of secondary structure and fold compatibility, based on a 3D-1D score to the three-dimensional profile of Erythrina caffra trypsin inhibitor, we suggest that the potato cysteine proteinase inhibitor belongs to the superfamily of proteins that have the same pattern of three-dimensional structure as soybean trypsin inhibitor. This superfamily would therefore include proteins that inhibit three different classes of proteinases-serine, cysteine and aspartic proteinases.

Amino Acid Sequence↗

PubMed1993

Monoclonal antibodies to human stefin B and determination of their epitopes.

Monoclonal antibodies (MAbs) to human stefin B were developed and three of them were characterised. Stefin B was cleaved into four peptides which were later subfragmented to smaller peptides. Only two peptides, of 24 and 30 amino acids, could bind to MAbs. In one instance, two peptides that were not consecutive in the sequence were recognised by the same antibody, proving that the epitope was discontinuous. Location of the epitopes was further narrowed by measuring the binding of MAbs to the complex of stefin B with papain. A sandwich ELISA (enzyme-linked immunosorbent assay), which measures the concentration of free inhibitor, was developed. It confirms that two out of three MAbs bind to different sites of stefin B. On the basis of the crystal structure of complex of stefin B with papain, the surface, accessible to a sphere with a radius of 5 A which simulates the accessibility of variable regions of antibody was determined. From the difference between accessibilities of free stefin B and stefin B in complex, the epitope location was determined more accurately.

Amino Acid Sequence↗

PubMed1993

Rapid affinity chromatographic method for the isolation of human cathepsin H.

Cathepsin H was purified by a single-step affinity chromatographic method from crude human kidney extract. The affinity medium consisted of low-molecular-mass cysteine proteinase inhibitors from potato tubers (PCPIs) coupled to cyanogen bromide-activated Sepharose. The yield of the method is comparable to that of the classical methods. Isoelectric focusing and sodium dodecyl sulphate polyacrylamide electrophoresis showed high purity of the isolated cathepsin H. N-Terminal sequence analysis revealed that intact single-chain cathepsin H was obtained. Binding of the enzyme to the PCPI-Sepharose showed that a free SH group in the cysteine proteinase is not required for complex formation.

Amino Acid Sequence↗

PubMed1993

Participation of cathepsin L on bone resorption.

The proteinase responsible for bone collagen degradation in osteo-resorption was examined. The bone pit formation induced by parathyroid hormone (PTH) was markedly suppressed by leupeptin, E-64 and cystatin A, while no inhibition was observed by CA-074, a specific inhibitor of cathepsin B. Pig leucocyte cysteine proteinase inhibitor (PLCPI), a specific inhibitor of cathepsin L, and chymostatin, a selective inhibitor of cathepsin L, completely inhibited the pit formation. Cathepsin L activity in osteoclasts was much higher than the other cathepsin activities. Serum calcium in rats placed on a low calcium diet was decreased by treatment of E-64 or cystatin A, but not by CA-074. These findings suggest that cathepsin L is the main proteinase responsible for bone collagen degradation.

Animals↗