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Dusan Turk

Publications and source records attributed to Dusan Turk.

14 recordsLinked to original sources
PubMed2006

Essential role of proline isomerization in stefin B tetramer formation.

Here we present the tetrameric structure of stefin B, which is the result of a process by which two domain-swapped dimers of stefin B are transformed into tetramers. The transformation involves a previously unidentified process of extensive intermolecular contacts, termed hand shaking, which occurs concurrently with trans to cis isomerization of proline 74. This proline residue is widely conserved throughout the cystatin superfamily, a member of which, human cystatin C, is the key protein in cerebral amyloid angiopathy. These results are consistent with the hypothesis that isomerization of proline residues can play a decisive role in amyloidogenesis.

Amino Acid Sequence↗
PubMed2006

Mouse stefins A1 and A2 (Stfa1 and Stfa2) differentiate between papain-like endo- and exopeptidases.

Stefin A (Stfa) acts as a competitive inhibitor of intracellular papain-like cysteine proteases which play important roles in normal cellular functions such as general protein turnover, antigen processing and ovarian follicular growth and maturation. In the mouse there are at least three different variants of Stfa (Stfa1, Stfa2 and Stfa3). Recent genetic studies identified structural polymorphisms in Stfa1 and Stfa2 as candidates for Aod1b, a locus controlling susceptibility to day three thymectomy (D3Tx)-induced autoimmune ovarian disease (AOD). To evaluate the functional significance of these polymorphisms, recombinant allelic proteins were expressed in Escherichia coli, purified and characterized. The polymorphisms do not markedly alter the folding characteristics of the two proteins. Stfa1 and Stfa2 both act as fast and tight binding inhibitors of endopeptidases papain and cathepsins L and S, however their interaction with exopeptidases cathepsins B, C and H was several orders of magnitude weaker compared to human, porcine and bovine Stfa. Notwithstanding, the K(i) values for the interactions of Stfa1-b from AOD resistant C57BL/6J mice was 10-fold higher than that of the Stfa1-a allele from susceptible A/J mice for papain, cathepsins B, C and H but not L and S. In contrast, the inhibitory activities of Stfa2-a and Stfa2-b were found to be roughly equivalent for all targets peptidases.

Amino Acid Sequence↗
PubMed2006

Folding and amyloid-fibril formation for a series of human stefins' chimeras: any correlation?

To study the influence of whole secondary structure elements to the process of folding and amyloid-fibril formation, chimeras of stefins have been prepared. GdnHCl denaturation curves and folding rates (chevron plots) have been analyzed based on a two-state mechanism. The order of stability is: stefin A > aAbbbb > bAbbbb > stefin B = aBaaaa > bBaaaa, where the make up of chimeric proteins is designated by small letters representing the source of individual strands (a for stefin A, b for stefin B) and a capital letter representing the source of the helix (A for stefin A and B for stefin B). Only the fast folding reactions were included in the analysis and it has been found that stefin B folds the fastest (657 s(-1)). Similarly, fast folders are the chimeric proteins aBaaaa and bBaaaa, both of which contain the alpha-helix of stefin B. Unfolding rates correlate very well with protein stability, with the slowest rate for the most stable protein, stefin A. Amyloid-fibril growth was measured for each protein by monitoring thioflavin T fluorescence and was visualized using electron microscopy. The propensity to form amyloid-fibrils is in the order: stefin B > bAbbbb > aAbbbb > bBaaaa > aBaaaa > stefin A. This order does not correlate with stability, or with the folding or unfolding rates. Instead, the propensity to fibrillize is related to selected parts of structure, such as the beta-sheet of stefin B, and can be predicted reasonably well by calculating the beta-strand propensity of the denatured states.

Amyloid↗
PubMed2005

Crystallization and preliminary X-ray crystallographic analysis of the cysteine protease inhibitor clitocypin.

Clitocypin is a cysteine protease inhibitor from the mushroom Clitocybe nebularis. The protein has been purified from natural sources and crystallized in a variety of non-isomorphous forms belonging to monoclinic and triclinic space groups. A diffraction data set to 1.55 A resolution was obtained from a crystal belonging to space group P2, with unit-cell parameters a = 38.326, b = 33.597, c = 55.568 A, beta = 104 degrees. An inability to achieve isomorphism forced the use of MAD and SAD phasing methods. Phasing is in progress.

Agaricales↗
PubMed2005

Recombinant human cathepsin X is a carboxymonopeptidase only: a comparison with cathepsins B and L.

The S1 and S2 subsite specificity of recombinant human cathepsins X was studied using fluorescence resonance energy transfer (FRET) peptides with the general sequences Abz-Phe-Xaa-Lys(Dnp)-OH and Abz-Xaa-Arg-Lys(Dnp)-OH, respectively (Abz=ortho-aminobenzoic acid and Dnp=2,4-dinitrophenyl; Xaa=various amino acids). Cathepsin X cleaved all substrates exclusively as a carboxymonopeptidase and exhibited broad specificity. For comparison, these peptides were also assayed with cathepsins B and L. Cathepsin L hydrolyzed the majority of them with similar or higher catalytic efficiency than cathepsin X, acting as an endopeptidase mimicking a carboxymonopeptidase (pseudo-carboxymonopeptidase). In contrast, cathepsin B exhibited poor catalytic efficiency with these substrates, acting as a carboxydipeptidase or an endopeptidase. The S1' subsite of cathepsin X was mapped with the peptide series Abz-Phe-Arg-Xaa-OH and the enzyme preferentially hydrolyzed substrates with hydrophobic residues in the P1' position.

Carboxypeptidases↗
PubMed2004

Pore formation by equinatoxin, a eukaryotic pore-forming toxin, requires a flexible N-terminal region and a stable beta-sandwich.

Actinoporins are eukaryotic pore-forming proteins that create 2-nm pores in natural and model lipid membranes by the self-association of four monomers. The regions that undergo conformational change and form part of the transmembrane pore are currently being defined. It was shown recently that the N-terminal region (residues 10-28) of equinatoxin, an actinoporin from Actinia equina, participates in building of the final pore wall. Assuming that the pore is formed solely by a polypeptide chain, other parts of the toxin should constitute the conductive channel and here we searched for these regions by disulfide scanning mutagenesis. Only double cysteine mutants where the N-terminal segment 1-30 was attached to the beta-sandwich exhibited reduced hemolytic activity upon disulfide formation, showing that other parts of equinatoxin, particularly the beta-sandwich and importantly the C-terminal alpha-helix, do not undergo large conformational rearrangements during the pore formation. The role of the beta-sandwich stability was independently assessed via destabilization of a part of its hydrophobic core by mutations of the buried Trp117. These mutants were considerably less stable than the wild-type but exhibited similar or slightly lower permeabilizing activity. Collectively these results show that a flexible N-terminal region and stable beta-sandwich are pre-requisite for proper pore formation by the actinoporin family.

Animals↗
PubMed2004

Crystal structure of NS-134 in complex with bovine cathepsin B: a two-headed epoxysuccinyl inhibitor extends along the entire active-site cleft.

The crystal structure of the inhibitor NS-134 in complex with bovine cathepsin B reveals that functional groups attached to both sides of the epoxysuccinyl reactive group bind to the part of active-site cleft as predicted. The -Leu-Pro-OH side binds to the primed binding sites interacting with the His110 and His111 residues with its C-terminal carboxy group, whereas the -Leu-Gly-Meu (-Leu-Gly-Gly-OMe) part (Meu, methoxycarbonylmethyl) binds along the non-primed binding sites. Comparison with the propeptide structures of cathepsins revealed that the binding of the latter part is least similar to the procathepsin B structure; this result, together with the two-residue shift in positioning of the Leu-Gly-Gly part, suggests that the propeptide structures of the cognate enzymes may not be the best starting point for the design of reverse binding inhibitors.

Animals↗
PubMed2004

Different propensity to form amyloid fibrils by two homologous proteins-Human stefins A and B: searching for an explanation.

By using ThT fluorescence, X-ray diffraction, and atomic force microscopy (AFM), it has been shown that human stefins A and B (subfamily A of cystatins) form amyloid fibrils. Both protein fibrils show the 4.7 A and 10 A reflections characteristic for cross beta-structure. Similar height of approximately 3 nm and longitudinal repeat of 25-27 nm were observed by AFM for both protein fibrils. Fibrils with a double height of 5.6 nm were only observed with stefin A. The fibril's width for stefin A fibrils, as observed by transmission electron microscopy (TEM), was in the same range as previously reported for stefin B (Zerovnik et al., Biochem Biophys Acta 2002;1594:1-5). The conditions needed to undergo fibrillation differ, though. The amyloid fibrils start to form at pH 5 for stefin B, whereas in stefin A, preheated sample has to be acidified to pH < 2.5. In both cases, adding TFE, seeding, and alignment in a strong magnetic field accelerate the fibril growth. Visual analysis of the three-dimensional structures of monomers and domain-swapped dimers suggests that major differences in stability of both homologues stem from arrangement of specific salt bridges, which fix alpha-helix (and the alpha-loop) to beta-sheet in stefin A monomeric and dimeric forms.

Amyloid↗
PubMed2004

The role of cathepsin C in Papillon-Lefèvre syndrome, prepubertal periodontitis, and aggressive periodontitis.

We have previously reported that loss-of-function mutations in the cathepsin C gene (CTSC) result in Papillon-Lefèvre syndrome, an autosomal recessive condition characterized by palmoplantar keratosis and early-onset, severe periodontitis. Others have also reported CTSC mutations in patients with severe prepubertal periodontitis, but without any skin manifestations. The possible role of CTSC variants in more common types of non-mendelian, early-onset, severe periodontitis ("aggressive periodontitis") has not been investigated. In this study, we have investigated the role of CTSC in all three conditions. We demonstrate that PLS is genetically homogeneous and the mutation spectrum that includes three novel mutations (c.386T>A/p.V129E, c.935A>G/p.Q312R, and c.1235A>G/p.Y412C) in 21 PLS families (including eight from our previous study) provides an insight into structure-function relationships of CTSC. Our data also suggest that a complete loss-of-function appears to be necessary for the manifestation of the phenotype, making it unlikely that weak CTSC mutations are a cause of aggressive periodontitis. This was confirmed by analyses of the CTSC activity in 30 subjects with aggressive periodontitis and age-sex matched controls, which demonstrated that there was no significant difference between these two groups (1,728.7 +/- SD 576.8 micro moles/mg/min vs. 1,678.7 +/- SD 527.2 micro moles/mg/min, respectively, p = 0.73). CTSC mutations were detected in only one of two families with prepubertal periodontitis; these did not form a separate functional class with respect to those observed in classical PLS. The affected individuals in the other prepubertal periodontitis family not only lacked CTSC mutations, but in addition did not share the haplotypes at the CTSC locus. These data suggest that prepubertal periodontitis is a genetically heterogeneous disease that, in some families, just represents a partially penetrant PLS.

Adult↗
PubMed2003

Crystal structure of Stefin A in complex with cathepsin H: N-terminal residues of inhibitors can adapt to the active sites of endo- and exopeptidases.

Binding of cystatin-type inhibitors to papain-like exopeptidases cannot be explained by the stefin B-papain complex. The crystal structure of human stefin A bound to an aminopeptidase, porcine cathepsin H, has been determined in monoclinic and orthorhombic crystal forms at 2.8A and 2.4A resolutions, respectively. The asymmetric unit of each form contains four complexes. The structures are similar to the stefin B-papain complex, but with a few distinct differences. On binding, the N-terminal residues of stefin A adopt the form of a hook, which pushes away cathepsin H mini-chain residues and distorts the structure of the short four residue insertion (Lys155A-Asp155D) unique to cathepsin H. Comparison with the structure of isolated cathepsin H shows that the rims of the cathepsin H structure are slightly displaced (up to 1A) from their position in the free enzyme. Furthermore, comparison with the stefin B-papain complex showed that molecules of stefin A bind about 0.8A deeper into the active site cleft of cathepsin H than stefin B into papain. The approach of stefin A to cathepsin H induces structural changes along the interaction surface of both molecules, whereas no such changes were observed in the stefin B-papain complex. Carboxymethylation of papain seems to have prevented the formation of the genuine binding geometry between a papain-like enzyme and a cystatin-type inhibitor as we observe it in the structure presented here.

Animals↗
PubMed2003

Lysosomal cysteine proteases (cathepsins): promising drug targets.

Papain-like lysosomal cysteine proteases are processive and digestive enzymes expressed in organisms from bacteria to humans. Their ubiquity alone makes them potential drug targets, with the assumption that appropriate specificities may be achieved. These enzymes have rather short active-site clefts, comprising three well defined substrate-binding subsites (S2, S1 and S1') and additionally have comparatively broad binding areas (S4, S3, S2', S3'). This geometry distinguishes them from other protease classes, such as serine and aspartic proteases, with six and eight substrate-binding sites, respectively. Exopeptidases (cathepsins B, C, H and X), in contrast to endopeptidases (such as cathepsins L, S, V and F), possess structural features that facilitate binding of N- and C-terminal groups of substrates in the active-site cleft. Other than a clear preference for free chain termini in the case of exopeptidases, the substrate-binding sites exhibit no strict specificities. Instead, their subsite preferences arise more from specific exclusions of substrate type. This presents a challenge for the design of inhibitors to target a specific cathepsin: only the cumulative effect of an assembly of inhibitor fragments can produce the desired result. The small number of papain-like lysosomal cysteine proteases (11 human enzymes are known) and the small number of substrate-binding sites calls for a innovative and empirical approach.

Amino Acid Sequence↗
PubMed2002

Two-step membrane binding by Equinatoxin II, a pore-forming toxin from the sea anemone, involves an exposed aromatic cluster and a flexible helix.

Equinatoxin II (EqtII) belongs to a unique family of 20-kDa pore-forming toxins from sea anemones. These toxins preferentially bind to membranes containing sphingomyelin and create cation-selective pores by oligomerization of 3-4 monomers. In this work we have studied the binding of EqtII to lipid membranes by the use of lipid monolayers and surface plasmon resonance (SPR). The binding is a two-step process, separately mediated by two regions of the molecule. An exposed aromatic cluster involving tryptophans 112 and 116 mediates the initial attachment that is prerequisite for the next step. Steric shielding of the aromatic cluster or mutation of Trp-112 and -116 to phenylalanine significantly reduces the toxin-lipid interaction. The second step is promoted by the N-terminal amphiphilic helix, which translocates into the lipid phase. The two steps were distinguished by the use of a double cysteine mutant having the N-terminal helix fixed to the protein core by a disulfide bond. The kinetics of membrane binding derived from the SPR experiments could be fitted to a two-stage binding model. Finally, by using membrane-embedded quenchers, we showed that EqtII does not insert deeply in the membrane. The first step of the EqtII binding is reminiscent of the binding of the evolutionarily distant cholesterol-dependant cytolysins, which share a similar structural motif in the membrane attachment domain.

Animals↗
PubMed2002

Regulating cysteine protease activity: essential role of protease inhibitors as guardians and regulators.

Cysteine proteases are widespread in nature. Their implication in numerous vital processes and pathologies make them highly attractive targets for drug design. The proper functioning and regulation of activity of cysteine proteases is a delicate balance of many factors, one of the most crucial being the protease inhibitors. In this review the basic principles of physiological protease inhibition by protein inhibitors are discussed with the focus on papain-like lysosomal cysteine proteases and the caspases, and their inhibitors.

Animals↗