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Biomedical subjects

V Turk

Publications and source records attributed to V Turk.

At least 37 records · Page 2Linked to original sources
PubMed1997

Thermal denaturation of human cystatin C and two of its variants; comparison to chicken cystatin.

Thermal denaturation of the recombinant human cystatin C, an 8-residue shorter variant (Leu-9 cystatin C), and the W106S mutant were measured using differential scanning calorimetry (DSC). The finding that Leu-9 cystatin C is of similar stability to the full length protein is in accordance with its nearly normal inhibitory activity. The variant W106S cystatin C exhibits a higher melting temperature by 4 degrees than the wild-type protein. This contrasts with its reduced inhibitory activity and represents an example where activity changes are due to local effects and are not correlated to stability. From the ratio between Van't Hoff and calorimetric enthalpies it is judged that recombinant human cystatin C and Leu-9 cystatin C are dimeric prior to thermal unfolding whereas W106S cystatin C is monomeric. Melting temperatures and estimated stabilities for some other members of the cystatin superfamily of the cysteine proteinase inhibitors are presented which have been recorded previously or were collected for this study (chicken cystatin). It is concluded that thermal stability of human cystatin C (Tm = 82 degrees C) is placed in between the more stable human stefin A (Tm = 95 degrees C) and the less stable human stefin B (Tm = 66 degrees C) whereas chicken cystatin behaves as a thermophilic protein, melting above 115 degrees C. To illustrate secondary structure changes, thermal denaturations of the recombinant human cystatin C and of W106S cystatin C were followed by circular dichroism in the far UV. It was found that the change in tertiary structure (revealed by DSC) precedes the major change in secondary structure.

Animals↗
PubMed1996

Differences in specificity for the interactions of stefins A, B and D with cysteine proteinases.

Four different stefin-type cysteine proteinase inhibitors have been isolated from porcine thymus and skin. Amino acid sequence determination revealed the presence of stefin A and stefin B type inhibitors and two new inhibitors, designated as porcine stefin D1 and stefin D2. Stefin D1 was identified as PLCPI, an inhibitor recently characterized from porcine polymorphonuclear leukocytes [Lenarcic et al. (1993) FEBS Lett. 336, 289-292]. Stefin A is composed of 101 amino acids and has an Mr of 11 391 while stefin B contains 98 amino acids, has an Mr of 11 174 and is N-terminally blocked. All inhibitors were found to be fast-acting inhibitors of papain, cathepsin L and cathepsin S (Ki = 0.009-0.161 nM). Stefins A and B also bind tightly and rapidly to cathepsin H (Ki = 0.027 and 0.069 nM, respectively), while stefins D1 and D2 have been shown to be very poor inhibitors of cathepsin H (Ki = 102-150 nM). The decreased affinity of these inhibitors toward cathepsin B (Ki = 2-1700 nM) was shown to be mainly due to the low second order association rate constants. The presence of a highly negatively charged N-terminus on stefin D1 constitutes a likely structural determinant of inhibitor specificity.

Amino Acid Sequence↗
PubMed1996

Interaction of human cathepsin C with chicken cystatin.

Cathepsin C was purified from human spleen by a rapid procedure, which included homogenization, ammonium sulfate precipitation, gel filtration on Sephacryl S-200 and finally affinity chromatography on chicken cystatin-Sepharose. The interaction between cathepsin C and chicken cystatin was further characterized. It was found to be accompanied by a maximum decrease in fluorescence emission intensity at 336 nm. Fluorescence titration showed that human cathepsin C can bind four chicken cystatin molecules. The 4:1 binding stoichiometry was confirmed by titration monitored by the loss of enzyme activity. A non-competitive-competitive type of inhibition was determined from a double-reciprocal Lineweaver-Burk plot with a Ki value of 0.22 nM for the non-competitive inhibition.

Animals↗
PubMed1996

High-molecular-weight kininogen binds two molecules of cysteine proteinases with different rate constants.

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

Animals↗
PubMed1996

Crystal structures of human procathepsin B at 3.2 and 3.3 Angstroms resolution reveal an interaction motif between a papain-like cysteine protease and its propeptide.

A wild-type human procathepsin B was expressed, crystallized in two crystal forms and its crystal structure determined at 3.2 and 3.3 Angstroms resolution. The structure reveals that the propeptide folds on the cathepsin B surface, shielding the enzyme active site from exposure to solvent. The structure of the enzymatically active domains is virtually identical to that of the native enzyme [Musil et al. (1991) EMBO J. 10, 2321-2330]: the main difference is that the occluding loop residues are lifted above the body of the mature enzyme, supporting the propeptide structure.

Binding Sites↗
PubMed1996

Major histocompatibility complex class II-associated p41 invariant chain fragment is a strong inhibitor of lysosomal cathepsin L.

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

Amino Acid Sequence↗
PubMed1996

Folding and activation of human procathepsin S from inclusion bodies produced in Escherichia coli.

Human procathepsin S was produced in the form of insoluble inclusion bodies in Escherichia coli using an inducible T7-based expression system. After cell disruption, the dissolved inclusion body proteins were S-sulphonated with 2-nitro-5-thiosulphobenzoate and purified by gel filtration. Recombinant procathepsin S was renatured at pH 7.6 by a two-step dilution which significantly increased the yield of production compared to single-step dilution. The proenzyme was autocatalytically processed to active cathepsin S at pH 4.5 in the presence of an excess of cysteine and catalytic amounts of dextran sulphate. Most of the loss of procathepsin S occurred during folding, probably because of aggregation. Concentrations lower than 20 microgram/ml of procathepsin S were necessary to minimise such aggregation. The recombinant cathepsin S was catalytically active on fluorogenic substrates and had kinetic properties similar to those of recombinant enzyme produced in yeast. The expression, renaturation, and activation procedures used enable the production of up to 2 mg of catalytically active recombinant human cathepsin S/l fermentation broth.

Base Sequence↗
PubMed1996

Characterization of cystatin C from bovine parotid glands: cysteine proteinase inhibition and antiviral properties.

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

Amino Acid Sequence↗
PubMed1996

Inhibition of bovine cathepsins L and S by stefins and cystatins.

Inhibition of bovine cathepsins L and S by bovine stefin B, human stefins A and B and cystatin C was studied under pseudo-first-order conditions by continuous fluorimetric assay. All inhibitors formed very tight complexes with the enzymes (Ki < or = 29 pM). The binding was reversible (kdiss = 0.52 - 16.7 x 10(-4) s-1) and very fast (kass = 2.8 - 6.2 x 10(7) M-1 S-1). Cystatin C was the strongest inhibitor of the enzymes, but the affinity was too tight to be measured accurately by this method. Consistently weaker inhibition of cathepsin S by all the stefins is apparent due mainly to the higher dissociation rate constants.

Animals↗
PubMed1995

Oligomeric structure and substrate induced inhibition of human cathepsin C.

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

Amino Acid Sequence↗
PubMed1995

Inhibition of cruzipain, the major cysteine proteinase of the protozoan parasite, Trypanosoma cruzi, by proteinase inhibitors of the cystatin superfamily.

Cruzipain, the major cysteine proteinase from Trypanosoma cruzi epimastigotes, purified to a sequentially pure form, exists in multiple forms with pI values between 3.7 and 5.1, and an apparent molecular mass of 41 kDa. The enzyme is stable between pH 4.5-9.5. Cruzipain was found to be rapidly and tightly inhibited by various protein inhibitors of the cystatin superfamily (kass = 1.7-79 x 10(6) M-1s-1, Kd = 1.4-72 pM). These results suggest a possible defensive role for the host's cystatins after parasite infection, and may be of use for the design of new therapeutic drugs.

Animals↗
PubMed1995

Molecular cloning and sequence analysis of human preprocathepsin C.

A cDNA clone (C1) coding for human preprocathepsin C was isolated from a human ileum cDNA library using a rat kidney-derived RT-PCR probe and its complete nucleotide sequence determined. The full-length 1857 bp sequence codes for a protein of 463 amino acid residues with a calculated molecular mass of 51848 Da. Comparison of the deduced amino acid sequence with that of rat preprocathepsin C indicates an 87.5% identity. A multiple alignment of the deduced cathepsin C sequence of 233 residues which, by analogy to other cystein proteinases, corresponds to the mature protein, confirms that human cathepsin C belongs to the papain superfamily.

Amino Acid Sequence↗
PubMed1995

The preparation of catalytically active human cathepsin B from its precursor expressed in Escherichia coli in the form of inclusion bodies.

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

Amino Acid Sequence↗
PubMed1995

Crystal structure of cathepsin B inhibited with CA030 at 2.0-A resolution: A basis for the design of specific epoxysuccinyl inhibitors.

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

Amino Acid Sequence↗
PubMed1995

Identification of bovine stefin A, a novel protein inhibitor of cysteine proteinases.

For the first time, three different stefins, A, B and C, have been isolated from a single species. The complete amino acid sequence of bovine stefin A was determined. The inhibitor, with a calculated M(r) of 11,123, consists of 98 amino acid residues. Although it exhibits considerable similarity to human and rat stefin A, some significant differences in inhibition kinetics were found. Bovine stefin A bound tightly and rapidly to cathepsin L (kass = 9.6 x 10(6) M-1.s-1, Ki = 29 pM). The binding to cathepsin H was also rapid (kass = 2.1 x 10(6) M-1.s-1), but weaker (Ki = 0.4 nM) due to a higher dissociation rate constant. In contrast, the binding to cathepsin B was much slower (kass = 1.4 x 10(5) M-1.s-1), but still tight (Ki = 1.9 nM).

Amino Acid Sequence↗
PubMed1995

The three-dimensional solution structure of human stefin A.

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

Amino Acid Sequence↗
PubMed1995

High-affinity binding of two molecules of cysteine proteinases to low-molecular-weight kininogen.

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

Amino Acid Sequence↗