Wyniki wyszukiwania

1

Zastosowanie FGF1 w terapii cukrzycy

100%

Krowarsch D.

Postępy Mikrobiologii. Suplement

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2017

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tom 56

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nr 2

2

Squash inhibitor family of serine proteinases

84%

Otlewski J., Krowarsch D.

Acta Biochimica Polonica

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1996

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tom 43

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nr 3

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.

3

Design, expression and characterization of a highly stable tetratricopeptide-based protein scaffold for phage display application

68%

Petters E., Krowarsch D., Otlewski J.

Acta Biochimica Polonica

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2013

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tom 60

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nr 4

Tetratricopeptide repeat (TPR) is a structural motif mediating variety of protein-protein interactions. It has a high potential to serve as a small, stable and robust, non-immunoglobulin ligand binding scaffold. In this study, we showed the consensus approach to design the novel protein called designed tetratricopeptide repeat (dTPR), composed of three repeated 34 amino-acid tetratricopeptide motifs. The designed sequence was efficiently overexpressed in E. coli and purified to homogeneity. Recombinant dTPR is monomeric in solution and preserves its secondary structure within the pH range from 2.0 to 11.0. Its denaturation temperature at pH 7.5 is extremely high (104.5°C) as determined by differential scanning calorimetry. At extreme pH values the protein is still very stable: denaturation temperature is 90.1°C at pH 2.0 and 60.4°C at pH 11. Chemical unfolding of the dTPR is a cooperative, two-state process both at pH 7.5 and 2.0. The free energy of denaturation in the absence of denaturant equals to 15.0 kcal/mol and 13.5 kcal/mol at pH 7.5 and 2.0, respectively. Efficient expression and extraordinary biophysical properties make dTPR a promising framework for a biotechnological application, such as generation of specific ligand- binding molecules.

4

Protein inhibitors of serine proteinases

68%

Otlewski J., Krowarsch D., Apostoluk W.

Acta Biochimica Polonica

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1999

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tom 46

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nr 3

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 effect of incomplete colostrum milking on its content and on trypsin inhibitor level

68%

Sroka K., Krowarsch D., Szulc T.

Electronic Journal of Polish Agricultural Universities. Series Animal Husbandry

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1998

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tom 01

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nr 1

Analyses on variability of trypsine inhibitor content in cow colostrum and its composition during first 5 milkings after parturition, in dependence with method of milking were performed. Cows were distributed into 3 groups: I - complete colostrum milking in successive milkings after parturition - control (18 cows); II - leaving approx. 30% of colostrum (more complete milking) in first 5 milkings after parturition (18 cows); III - leaving approx. 60% of colostrum (less complete milking) in first 5 milkings after parturition (18 cows).

6

Residues of the C-terminal region (T-box) of the Ultraspiracle nuclear receptor DNA-binding domain crucial to specific interaction with the hsp27 response element

51%

Kowalska A., Rymarczyk G., Grad I., Orlowski M., Krowarsch D., Ozyhar A.

Acta Biochimica Polonica

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2003

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tom 50

|

nr Supplement 1

7

Mutational analysis of the EcRDBD recognition alpha-helix and T-box region uncovers critical determinants of the functional ecdysteroid receptor interaction with the response element

51%

Orlowski M., Szyszka M., Grand I., Kowalska A., Krowarsch D., Ozyhar A.

Acta Biochimica Polonica

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2003

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tom 50

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nr Supplement 1

8

Structure-function relationship of serine protease-protein inhibitor interaction

43%

Otlewski J., Jaskolski M., Buczek O., Cierpicki T., Czapinska H., Krowarsch D., Smalas A. O., Stachowiak D., Szpineta A., Dadlez M.

Acta Biochimica Polonica

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2001

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tom 48

|

nr 2

We re port our prog ress in un der stand ing the struc ture-function re la tion ship of the interaction between protein inhibitors and several serine proteases. Recently, we have de ter mined high res o lu tion so lu tion struc tures of two in hib i tors Apis mellifera chymotrypsin in hib i tor-1 (AMCI-I) and Linum usitatissimumtrypsin in hib i tor (LUTI) in the free state and an ul tra high res o lu tion X-ray struc ture of BPTI. All three in hib i tors, de spite to tally dif fer ent scaf folds, con tain a sol vent ex posed loop of sim i lar con- for ma tion which is highly com ple men tary to the en zyme ac tive site. Iso ther mal calorim e try data show that the in ter ac tion be tween wild type BPTI and chymotrypsin is entropy driven and that the enthalpy com po nent op poses com plex for ma tion. Our research is fo cused on ex ten sive mu ta gen e sis of the four po si tions from the pro te ase bind ing loop of BPTI: P1, P1', P3, and P4. We mu tated these res i dues to dif fer ent amino ac ids and the vari ants were char ac ter ized by de ter mi na tion of the as so ci a tion con stants, sta bil ity pa ram e ters and crys tal struc tures of pro te ase–in hib i tor complexes. Ac com mo da tion of the P1 res i due in the S1 pocket of four pro teas es: chymotrypsin, trypsin, neutrophil elastase and cathepsin G was probed with 18 P1 vari ants. High res o lu tion X-ray struc tures of ten com plexes be tween bo vine trypsin and P1 vari ants of BPTI have been de ter mined and com pared with the cog nate P1 Lysside chain. Mu ta tions of the wild type Ala16 (P1') to larger side chains al ways caused a drop of the as so ci a tion con stant. Ac cord ing to the crys tal struc ture of the Leu16 BPTI–trypsin com plex, in tro duc tion of the larger res i due at the P1' po si tion leads to steric con flicts in the vi cin ity of the mu ta tion. Finally, mu ta tions at the P4 site al lowed an im prove ment of the as so ci a tion with sev eral serine pro teas es in volved in blood clot ting. Con versely, in tro duc tion of Ser, Val, and Phe in place of Gly12 (P4) had invariably a destabilizing ef fect on the com plex with these pro teas es.

Ograniczanie wyników

Zastosowanie FGF1 w terapii cukrzycy

Squash inhibitor family of serine proteinases

Design, expression and characterization of a highly stable tetratricopeptide-based protein scaffold for phage display application

Protein inhibitors of serine proteinases

The effect of incomplete colostrum milking on its content and on trypsin inhibitor level

Residues of the C-terminal region (T-box) of the Ultraspiracle nuclear receptor DNA-binding domain crucial to specific interaction with the hsp27 response element

Mutational analysis of the EcRDBD recognition alpha-helix and T-box region uncovers critical determinants of the functional ecdysteroid receptor interaction with the response element

Structure-function relationship of serine protease-protein inhibitor interaction