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1

Squash inhibitor family of serine proteinases

100%

Otlewski J. , Krowarsch D.

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tom 43

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nr 3

EN

Squash inhibitors of serine proteinases form an uniform family of small proteins. They are built of 27-33 amino-acid residues and cross-linked with three disulfide bridges. The reactive site peptide bond (Pl-Pl') is between residue 5 (Lys, Arg or Leu) and 6 (always lie). High resolution X-ray structures are available for two squash inhibitors complexed with trypsin. NMR solution structures have also been determined for free inhibitors. The major structural motif is a distorted, triple-strai:ded antiparallel p-sheet. A similar folding motif has been recently found in a number of proteins, including: conotoxins from fish-hunting snails, carbo- xypeptidase inhibitor from potato, kalata B1 polypeptide, and in some growth factors (e.g. nerve growth factor, transforming growth factor P2, platelet-derived growth factor). Squash inhibitors are highly stable and rigid proteins. They inhibit a number of serine proteinases: trypsin, plasmin, kallikrein, blood clotting factors: Xa and XII*, cathepsin G. The inhibition spectrum can be much broadened if specific amino-acid substitutions are introduced, especially at residues which contact proteinase. Squash inhibitors inhibit proteinases via the standard mechanism. According to the mechanism, inhibitors are substrates which exibit at neutral pH a high fccWKm index for hydrolysis and resynthesis of the reactive site, and a low value of the hydrolysis constant.

2

Serine proteinase inhibitors from insect hemolymph

88%

Polanowski A. , Wilusz T.

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tom 43

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nr 3

EN

Insect hemolymph, like vertebrate serum, contains several different types of polypeptides that are able to inhibit the catalytic function of proteolytic enzymes, however studies on proteins possessing this capability have been limited to a relatively few species. A comparative examination of the inhibition of trypsin, chymo- trypsin, neutrophil elastase and cathepsin G and pancreatic elastase by the hemolymph of 14 insect species belonging to six orders showed great diversity in terms of both total proteinase inhibitory capacity and specificity. Most of the inhibitors examined fall into two groups: low molecular mass proteins (below 10 kDa) related to Kunitz type inhibitors, and proteins of about 45 kDa which belong to the serpin superfamily of serine proteinase inhibitors. This minireview describes the properties, characteristics and possible biological significance of selected inhibitors.

3

The interaction between some serine proteinases and horse leucocyte inhibitor

75%

Dubin A. , Potempa J. , Turyna B.

Folia Histochemica et Cytobiologica

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1986

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tom 24

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nr 2

4

Protein inhibitors of serine proteinases

75%

Otlewski J. , Krowarsch D. , Apostoluk W.

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1999

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tom 46

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nr 3

EN

Serine proteinases and their natural protein inhibitors belong to the most intensively studied models of protein-protein recognition. Protein inhibitors do not form a single group but can be divided into about 20 different families. Global structures of proteins representing different inhibitor families are completely different and comprise α-helical proteins, β-sheet proteins,α/β-proteins and different folds of small disulfide-rich proteins. Three different types of inhibitors can be distinguished: canonical (standard mechanism) inhibitors, non-canonical inhibitors, and serpins. The canonical inhibitor binds to the enzyme through the exposed and convex binding loop, which is complementary to the active site of the enzyme. The mechanism of inhibition in this group is consistently very similar and resembles that of an ideal substrate. Non-canonical inhibitors, originating from blood sucking organisms, specifically block enzymes of the blood clotting cascade. The interaction is mediated through inhibitor N-terminus which binds to the proteinase forming a parallel β-sheet. There are also extensive secondary interactions which provide an additional buried area and contribute significantly to the strength and specificity of recognition. Serpins are major proteinase inhibitors occurring in plasma. Similarly to canonical inhibitors, serpins interact with their target proteinases in a substrate-like manner. However, in the case of serpins, cleavage of a single peptide bond in a flexible and exposed binding loop leads to dramatic structural changes.

5

The mutual influence of proteins from Varroa destructor extracts and from honeybee haemolymph on their proteolytic activity – in vitro study

63%

Fraczek R. J. , Zoltowska K. , Lipinski Z. , Dmitryjuk M.

Acta Parasitologica

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2013

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tom 58

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nr 3

EN

The influence of extracts from Varroa destructor, a parasitic mite of the honeybee Apis mellifera, on the proteinase activity of worker bee haemolymph was analysed in vitro, along with the influence of bee haemolymph on the proteolytic activity of V. destructor extract. The study was conducted in three different environments: pH 7.5 (high activity of bee enzymes and very low activity of parasite enzymes), pH 5 (moderate activity of enzymes from both sources) and pH 3.5 (limited activity of bee proteinases and high activity of mite proteinases). Based on electrophoretic studies, the inhibition of the activity of bee haemolymph proteinases by V. destructor extracts was observed at each pH. The study at pH 7.5 with commercial inhibitors of the 4 main classes of proteinases (pepstatin A, ethylenediaminetetraacetic acid (EDTA), E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane), soybean trypsin inhibitor and Kunitz inhibitor) suggested that parasite extracts mainly inhibited serine proteinases and, to a lower degree, cysteine and aspartyl proteinases. At pH 3.5 and pH 5, a decrease of approximately 40% in parasite proteinase activity was also observed in the presence of bee haemolymph. The result points to the presence of aspartyl proteinase inhibitors in bee haemolymph, which may be an important defence element for bees during food intake by a mite. It was demonstrated that trypsin and trypsin inhibitors are active in the excretion/secretion products of V. destructor, the proteinases of which may assist the parasite in food suckling by preventing haemolymph coagulation, among other things.

6

Isolation and amino-acid sequence of two inhibitors of serine proteinase, members of the squash inhibitor family, from Echinocystis lobata seeds

63%

Stachowiak D. , Polanowski A. , Bieniarz G. , Wilusz T.

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tom 43

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nr 3

EN

Two serine proteinase inhibitors (ELTII and ELT1II) have been isolated from mature seeds of Echinocystis lobata by ammonium sulfate fractionation, methanol precipitation, ion exchange chromatography, affinity chromatography on immobilized anhydrotrypsin and HPLC. ELTI I and ELTI II consist of 33 and 29 amino-acid residues, respectively. The primary structures of these inhibitors are as follows: ELTI I KEEQRVCPRILMRCKRDSDCLAQCTCQQSGFCG ELTI II RVCPRILMRCKRDSDCLAQCTCQQSGFCG The inhibitors show sequence similarity with the squash inhibitor family. ELTI I differs from ELTI II only by the presence of the NH2-terminal tetrapeptide Lys- -Glu-Glu-Gln. The association constants (Ka) of F.LTI I and ELTI II with bovine-trypsin were determined to be 6.6 x 1010 M -1 and 3.1 x 1011 M -1, whereas the association constants of these inhibitors with cathepsin G were 1.2 x 107 M -1 and 1.1 x 107 M -1, respectively.

7

The effect of the Glu342Lys mutation in alpha1-antitrypsin on its structure, studied by molecular modelling medhods

63%

Jezierski G. , Pasenkiewicz-Gierula M.

Acta Biochimica Polonica

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2001

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tom 48

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nr 1

EN

The structure of native α1-antitrypsin, the most abundant protease inhibitor in human plasma, is characterised primarily by a reactive loop containing the centre of proteinase inhibition, and aβ-sheet composed of five strands. Mobility of the reactive loop is confined as a result of electrostatic interactions between side chains of Glu342 and Lys290, both located at the junction of the reactive loop and the β structure. The most common mutation in the protein, resulting in its inactivation, is Glu342->Lys, named the Z mutation. The main goal of this work was to investigate the influence of the Z mutation on the structure of α1-antitrypsin. Commonly used molecular modelling methods have been applied in a comparative study of two protein models: the wild type and the Z mutant. The results indicate that the Z mutation introduces local instabilities in the region of the reactive loop. Moreover, even parts of the protein located far apart from the mutation region are affected. The Z mutation causes a relative change in the total energy of about 3%. Relatively small root mean square differences between the optimised structures of the wild type and the Z mutant, together with detailed analysis of 'conformational searching' process, lead to the hypothesis that the Z mutation principally induces a change in the dynamics of α1-antitrypsin.

8

Non-conventional affinity chromatography of serine proteinases and their inhibitors

63%

Polanowski A. , Wilimowska-Pelc A. , Kowalska J. , Grybel J. , Zelazko M. , Wilusz T.

Acta Biochimica Polonica

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2003

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tom 50

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nr 3

EN

From among a wide variety of protein purification techniques affinity chromatography has proved to be particularly effective for separation of proteolytic enzymes and their inhibitors. In this article, following a general description of affinity adsorbents used for purification of proteinases, we overview a simple separation procedure for some serine proteinases and their inhibitors by way of affinity chromatography in the presence of high NaCl concentration. It has been shown that some highly specific trypsin inhibitors exhibit also antichymotrypsin activity when high concentration of Na+ but not K+ or Li+ ions are present in the reaction mixture. Taking advantage of this phenomenon the virgin forms of trypsin inhibitors from squash seeds, Kazal-type inhibitor from porcine pancreas and arproteinase inhibitor from human and sheep plasma, as an example, were separated using immobilized chymotrypsin or its inactive derivative methylchymotrypsin in the presence of 5 M NaCl.

9

Engineered resistance against proteinases

51%

Milner M. , Chroboczek J. , Zagorski-Ostoja W.

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tom 54

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nr 3

EN

Exogenous proteinase inhibitors are valuable and economically interesting protective biotechnological tools. We examined whether small proteinase inhibitors when fused to a selected target protein can protect the target from proteolytic degradation without simultaneously affecting the function and activity of the target domain. Two proteinase inhibitors were studied: a Kazal-type silk proteinase inhibitor (SPI2) from Galleria mellonella, and the Cucurbita maxima trypsin inhibitor I (CMTI I). Both inhibitors target serine proteinases, are small proteins with a compact structure stabilized by a network of disulfide bridges, and are expressed as free polypeptides in their natural surroundings. Four constructs were prepared: the gene for either of the inhibitors was ligated to the 5' end of the DNA encoding one or the other of two selected target proteins, the coat protein (CP) of Potato potyvirus Y or the Escherichia coli β-glucuronidase (GUS). CMTI I fused to the target proteins strongly hampered their functions. Moreover, the inhibitory activity of CMTI I was retained only when it was fused to the CP. In contrast, when fused to SPI2, specific features and functions of both target proteins were retained and the inhibitory activity of SPI2 was fully preserved. Measuring proteolysis in the presence or absence of either inhibitor, we demonstrated that proteinase inhibitors can protect target proteins used either free or as a fusion domain. Interestingly, their inhibitory efficiency was superior to that of a commercial inhibitor of serine proteinases, AEBSF.

10

Structural and energetic aspects of protein-protein recognition

51%

Otlewski J. , Apostoluk W.

Acta Biochimica Polonica

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1997

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tom 44

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nr 3

EN

Specific recognition between proteins plays a crucial role in a great number of vital processes. In this review different types of protein-protein complexes are analyzed on the basis of their three-dimensional structures which became available in recent years. The complexes which are analyzed include: those resulting from different types of recognition between proteinase and protein inhibitor (canonical inhibitors of serine proteinases, hirudin, inhibitors of cysteine proteinases, carboxypeptidase inhibitor), barnase-barstar, human growth hormone-receptor and antibody-antigen. It seems obvious that specific and strong protein-protein recognition is achieved in many different ways. To further explore this question, the structural information was analyzed together with kinetic and thermodynamic data available for the respective complexes. It appears that the energy and rates of specific recognition of proteins are influenced by many different factors, including: area of interacting surfaces; complementarity of shapes, charges and hydrogen bonds; water structure at the interface; conformational changes; additivity and cooperativity of individual interactions, steric effects and various (conformational, hydration) entropy changes.