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Biomedical subjects

Roman Jerala

Publications and source records attributed to Roman Jerala.

17 recordsLinked to original sources
PubMed2005

Structural origin of endotoxin neutralization and antimicrobial activity of a lactoferrin-based peptide.

Treatment of Gram-negative bacterial infections with antimicrobial agents can cause release of the endotoxin lipopolysaccharide (LPS), the potent initiator of sepsis, which is the major cause of mortality in intensive care units worldwide. Structural information on peptides bound to LPS can lead to the development of more effective endotoxin neutralizers. Short linear antimicrobial and endotoxin-neutralizing peptide LF11, based on the human lactoferrin, binds to LPS, inducing a peptide fold with a "T-shaped" arrangement of a hydrophobic core and two clusters of basic residues that match the distance between the two phosphate groups of LPS. Side chain arrangement of LF11 bound to LPS extends the previously proposed LPS binding pattern, emphasizing the importance of both electrostatic and hydrophobic interactions in a defined geometric arrangement. In anionic micelles, the LF11 forms amphipathic conformation with a smaller hydrophobic core than in LPS, whereas in zwitterionic micelles, the structure is even less defined. Protection of tryptophan fluorescence quenching in the order SDS>LPS>DPC and hydrogen exchange protection indicates the decreasing extent of insertion of the N terminus and potential role of peptide plasticity in differentiation between bacterial and eukaryotic membranes.

Acrylamide↗
PubMed2005

Enhancement of endotoxin neutralization by coupling of a C12-alkyl chain to a lactoferricin-derived peptide.

Antibacterial peptide acylation, which mimics the structure of the natural lipopeptide polymyxin B, increases antimicrobial and endotoxin-neutralizing activities. The interaction of the lactoferricin-derived peptide LF11 and its N-terminally acylated analogue, lauryl-LF11, with different chemotypes of bacterial lipopolysaccharide (LPS Re, Ra and smooth S form) was investigated by biophysical means and was related to the peptides' biological activities. Both peptides exhibit high antibacterial activity against the three strains of Salmonella enterica differing in the LPS chemotype. Lauryl-LF11 has one order of magnitude higher activity against Re-type, but activity against Ra- and S-type bacteria is comparable with that of LF11. The alkyl derivative peptide lauryl-LF11 shows a much stronger inhibition of the LPS-induced cytokine induction in human mononuclear cells than LF11. Although peptide-LPS interaction is essentially of electrostatic nature, the lauryl-modified peptide displays a strong hydrophobic component. Such a feature might then explain the fact that saturation of the peptide binding takes place at a much lower peptide/LPS ratio for LF11 than for lauryl-LF11, and that an overcompensation of the negative LPS backbone charges is observed for lauryl-LF11. The influence of LF11 on the gel-to-liquid-crystalline phase-transition of LPS is negligible for LPS Re, but clearly fluidizing for LPS Ra. In contrast, lauryl-LF11 causes a cholesterol-like effect in the two chemotypes, fluidizing in the gel and rigidifying of the hydrocarbon chains in the liquid-crystalline phase. Both peptides convert the mixed unilamellar/non-lamellar aggregate structure of lipid A, the 'endotoxic principle' of LPS, into a multilamellar one. These data contribute to the understanding of the mechanisms of the peptide-mediated neutralization of endotoxin and effect of lipid modification of peptides.

Alkylation↗
PubMed2005

Mixed-valence Cu(II)/Cu(I) complex of quinolone ciprofloxacin isolated by a hydrothermal reaction in the presence of L-histidine: comparison of biological activities of various copper-ciprofloxacin compounds.

A new quinolone-metal complex was prepared by a hydrothermal reaction in the presence of L-histidine that served as a reducing agent for a metal. The title compound [Cu(II)(cfH)(2)(Cu(I)Cl(2))(2)] (1) is a mixed-valence Cu(II)-Cu(I) complex, which contains two ciprofloxacin (cfH) molecules bonded to the central copper(II) atom and two almost planar [Cu(I)Cl(2)](-) moieties. Both metal centers are connected through two bridging atoms (chloride and quinolone oxygen). The electrochemical methods (differential-pulse polarography and cyclovoltammetric measurements) confirmed the presence of various copper-ciprofloxacin complex species in aqueous solution at low concentrations used in biological activity tests and also indicated that the equilibria in this system are very complex. The biological properties of the title compound and some previously isolated copper-ciprofloxacin complexes ([Cu(cfH)(2)Cl(2)].6H(2)O (2) and [CuCl(cfH)(phen)]Cl.2H(2)O (3)) (phen=1, 10-phenantroline) were determined and compared. The DNA gyrase inhibition tests and antibacterial activity tests have shown that the effect of copper complexes is comparable to that of free quinolone. Additionally, an interesting DNA cleavage activity of the title compound was also discovered.

Anti-Bacterial Agents↗
PubMed2005

Combination of antimicrobial and endotoxin-neutralizing activities of novel oleoylamines.

A combination of antimicrobial and endotoxin-neutralizing activities is desired in order to prevent progression from infection to sepsis due to the release of lipopolysaccharide from dying gram-negative bacteria. Lipopolyamines have emerged as a new type of endotoxin-neutralizing compound, but their antimicrobial activity has not been investigated. We synthesized a series of 10 oleoylamines differing in the polyamino head group, particularly in the number and separation between nitrogen atoms and the position of the oleoyl moiety. Compounds showed activity against both gram-negative and gram-positive bacteria in the micromolar range. Compounds were able to provide penetration of ethidium bromide into bacteria, indicating effects on the bacterial membrane. Oleoylamines neutralized endotoxin in Limulus amoebocyte lysate assays and by neutralization of tumor necrosis factor alpha release in human blood. Comparison of biological activities of compounds identified structural properties responsible for antimicrobial activity, and quantitative structure-property relationship analysis provided a quantitative model for prediction of activity of oleoylamines.

Anti-Bacterial Agents↗
PubMed2005

Similarities and specificities of fungal keratinolytic proteases: comparison of keratinases of Paecilomyces marquandii and Doratomyces microsporus to some known proteases.

Based on previous screening for keratinolytic nonpathogenic fungi, Paecilomyces marquandii and Doratomyces microsporus were selected for production of potent keratinases. The enzymes were purified and their main biochemical characteristics were determined (molecular masses, optimal temperature and pH for keratinolytic activity, N-terminal amino acid sequences). Studies of substrate specificity revealed that skin constituents, such as the stratum corneum, and appendages such as nail but not hair, feather, and wool were efficiently hydrolyzed by the P. marquandii keratinase and about 40% less by the D. microsporus keratinase. Hydrolysis of keratin could be increased by the presence of reducing agents. The catalytic properties of the keratinases were studied and compared to those of some known commercial proteases. The profile of the oxidized insulin B-chain digestion revealed that both keratinases, like proteinase K but not subtilisin, trypsin, or elastase, possess broad cleavage specificity with a preference for aromatic and nonpolar amino acid residues at the P-1 position. Kinetic studies were performed on a synthetic substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The keratinase of P. marquandii exhibited the lowest Km among microbial keratinases reported in the literature, and its catalytic efficiency was high in comparison to that of D. microsporus keratinase and proteinase K. All three keratinolytic enzymes, the keratinases of P. marquandii and D. microsporus as well as proteinase K, were significantly more active on keratin than subtilisin, trypsin, elastase, chymotrypsin, or collagenase.

Amino Acid Sequence↗
PubMed2005

MD-2 and Der p 2 - a tale of two cousins or distant relatives?

MD-2, an LPS-binding protein is essential for the recognition of LPS by TLR4. MD-2 belongs to the ML superfamily of lipid-binding proteins. The tertiary structure of mite allergen protein Der p 2 was identified as having the protein fold most compatible with the sequence of MD-2. Comparison of MD-2 and Der p 2 reveals that they have many common biochemical characteristics: they are both rich in beta-structure and they are both very stable proteins as they both unfold only above 90 degrees C. In Der p 2, six cysteine residues form three disulfide bridges. We determined one free cysteine residue per recombinant biologically active MD-2 molecule, supporting similar disulfide topology with three disulfides bridges as in Der p 2. MD-2 binds LPS with high affinity; however, only weak binding of LPS was detected with Der p 2. Comparison of electrostatic potentials of the structural model of MD-2 and Der p 2 indicates a region of high positive potential on MD-2 and its absence in Der p 2, which may be the reason for its weak binding of LPS. We suggest that Der p 2 and its homologues probably do not have a role in response to Gram-negative bacteria in insects and that MD-2 family members with their specific role in innate immunity probably evolved from an ML ancestor only in higher vertebrates.

Animals↗
PubMed2004

Structural model of MD-2 and functional role of its basic amino acid clusters involved in cellular lipopolysaccharide recognition.

The receptor complex resulting from association of MD-2 and the ectodomain of Toll-like receptor 4 (TLR4) mediates lipopolysaccharide (LPS) signal transduction across the cell membrane. We prepared a tertiary structure model of MD-2, based on the known structures of homologous lipid-binding proteins. Analysis of circular dichroic spectra of purified bacterially expressed MD-2 indicates high content of beta-type secondary structure, in agreement with the structural model. Bacterially expressed MD-2 was able to confer LPS responsiveness to cells expressing TLR4 despite lacking glycosylation. We identified several clusters of basic residues on the surface of MD-2. Mutation of each of two clusters encompassing the residues Lys(89)-Arg(90)-Lys(91) and Lys(125)-Lys(125) significantly decreased the signal transduction of the respective MD-2 mutants either upon co-expression with TLR4 or upon addition as soluble protein into the supernatant of cells overexpressing TLR4. These basic clusters lie at the edge of the beta-sheet sandwich, which in cholesterol-binding protein connected to Niemann-Pick disease C2 (NPC2), dust mite allergen Der p2, and ganglioside GM2-activator protein form a hydrophobic pocket. In contrast, mutation of another basic cluster composed of Arg(69)-Lys(72), which according to the model lies further apart from the hydrophobic pocket only weakly decreased MD-2 activity. Furthermore, addition of the peptide, comprising the surface loop between Cys(95) and Cys(105), predicted by model, particularly in oxidized form, decreased LPS-induced production of tumor necrosis factor alpha and interleukin-8 upon application to monocytic cells and fibroblasts, respectively, supporting its involvement in LPS signaling. Our structural model of MD-2 is corroborated by biochemical analysis and contributes to the unraveling of molecular interactions in LPS recognition.

Amino Acid Motifs↗
PubMed2004

Comparison of backbone dynamics of monomeric and domain-swapped stefin A.

Three-dimensional domain swapping has been observed in increasing number of proteins and has been implicated in the initial stages of protein aggregation, including that of the cystatins. Stefin A folds as a monomer under native conditions, while under some denaturing conditions domain-swapped dimer is formed. We have determined the backbone dynamics of the monomeric and domain-swapped dimeric forms of stefin A by (15)N relaxation using a model-free approach. The overall correlation times of the molecules were determined to be 4.6 +/- 0.1 ns and 9.2 +/- 0.2 ns for the monomer and the dimer, respectively. In the monomer, decreased order parameters indicate an increased mobility for the N-terminal trunk, the first and the second binding loops. At the opposite side of the molecule, the loop connecting the alpha-helix with strand B, the beginning of strand B and the loop connecting strands C and D show increased localized mobility. In the domain-swapped dimer, a distinctive feature of the structure is the concatenation of strands B and C into a single long beta-strand. The newly formed linker region between strands B and C, which substitutes for the first binding loop in the monomer, has order parameters typical for the remainder of the beta-strands. Thus, the interaction between subunits that occurs on domain-swapping has consequences for the dynamics of the protein at long-range from the site of conformational change, where an increased rigidity in the newly formed linker region is accompanied by an increased mobility of loops remote from that site.

Anisotropy↗
PubMed2004

Biophysical characterization of the interaction of Limulus polyphemus endotoxin neutralizing protein with lipopolysaccharide.

Endotoxin-neutralizing protein (ENP) of the horseshoe crab is one of the most potent neutralizers of endotoxins [bacterial lipopolysaccharide (LPS)]. Here, we report on the interaction of LPS with recombinant ENP using a variety of physical and biological techniques. In biological assays (Limulus amebocyte lysate and tumour necrosis factor-alpha induction in human mononuclear cells), ENP causes a strong reduction of the immunostimulatory ability of LPS in a dose-dependent manner. Concomitantly, the accessible negative surface charges of LPS and lipid A (zeta potential) are neutralized and even converted into positive values. The gel to liquid crystalline phase transitions of LPS and lipid A shift to higher temperatures indicative of a rigidification of the acyl chains, however, the only slight enhancement of the transition enthalpy indicates that the hydrophobic moiety is not strongly disturbed. The aggregate structure of lipid A is converted from a cubic into a multilamellar phase upon ENP binding, whereas the secondary structure of ENP does not change due to the interaction with LPS. ENP contains a hydrophobic binding site to which the dye 1-anilino-8-sulfonic acid binds at a K(d) of 19 micro m, which is displaced by LPS. Because lipopolysaccharide-binding protein (LBP) is not able to bind to LPS when ENP and LPS are preincubated, tight binding of ENP to LPS can be deduced with a K(d) in the low nonomolar range. Importantly, ENP is able to incorporate by itself into target phospholipid liposomes, and is also able to mediate the intercalation of LPS into the liposomes thus acting as a transport protein in a manner similar to LBP. Thus, LPS-ENP complexes might enter target membranes of immunocompetent cells, but are not able to activate due to the ability of ENP to change LPS aggregates from an active into an inactive form.

Animals↗
PubMed2004

Endotoxin neutralizing peptides.

Neutralization and sequestration of bacterial lipopolysaccharide which plays a key role in gram-negative sepsis is required to block the progression of sepsis at early stages in addition to destroying bacteria. Many of the host defense peptides which have antimicrobial activity are also able to bind to and neutralize LPS, however, these two activities do not necessarily correlate. Due to its toxicity application of polymyxin B as the prototype of LPS neutralizing peptide is limited to topical applications and extracorporeal removal of endotoxin. Development of novel endotoxin neutralizing peptides without the toxicity of polymyxin B have been based on the natural host defense peptides, fragments of LPS binding proteins and engineered peptides. Neutralization of LPS can be achieved through several different peptide fold motifs, which are reviewed in this article. Endogenous host defense peptides, fragments of endotoxin-binding proteins and synthetic anti-endotoxin peptides fold into alpha-helical, beta-hairpin, extended and compact conformations without regular secondary structure. In animal models many of the peptides have demonstrated good in vitro and in vivo endotoxin neutralizing activity but up to now none of the peptides has been approved for clinical application with an anti-endotoxin indication. Recent developments include preparation of novel types of endotoxin neutralizing compounds such as peptides modified by lipophilic moieties and non-peptidic molecules, particularly lipopolyamines and on the other hand additional medical applications such as extracorporeal endotoxin removal, targeting to inflammation sites or endotoxoid based vaccines.

Amino Acid Sequence↗
PubMed2003

Monoclonal antibody against a peptide of human prion protein discriminates between Creutzfeldt-Jacob's disease-affected and normal brain tissue.

Current methods for diagnosing transmissible spongiform encephalopathies rely on the degradation of the cellular prion protein (PrP(C)) and the subsequent detection of the protease-resistant remnant of the pathological prion isoform PrP(Sc) by antibodies that react with all forms of PrP. We report on a monoclonal antibody, V5B2, raised against a peptide from the C-terminal part of PrP, which recognizes an epitope specific to PrP(Sc). In cryostat sections from Creutzfeldt-Jacob's disease (CJD) patients' brains, V5B2 selectively labels various deposits of PrP(Sc) without any pretreatment for removal of PrP(C). V5B2 does not bind to non-CJD brain samples or to recombinant PrP, either in its native or denatured form. Specificity for PrP is confirmed by a sandwich enzyme-linked immunosorbent assay utilizing V5B2, which discriminates between CJD and normal samples without proteinase K treatment, and by immunoprecipitation from CJD brain homogenate. The PrP(Sc)-specific epitope is disrupted by denaturation. We conclude that the C-terminal part of PrP in disease-associated PrP(Sc) aggregates forms a structural epitope whose conformation is distinct from that of PrP(C).

Animals↗
PubMed2003

Protein inhibitors form complexes with procathepsin L and augment cleavage of the propeptide.

The proregion fits tightly into the active site in the tertiary structure of procathepsin L and prevents its activity. We show that complexes between enzyme precursor and its endogenous protein inhibitors-the cystatins-can be formed without prior proteolytic removal of the propeptide. Complexes between cystatins and procathepsin L are formed at acidic pH and their formation is facilitated by acidic oligosaccharides. Binding of the inhibitor to the proenzyme is reversible and the slow dissociation of complex around neutral pH may serve as a pool for the sustained release of the enzyme. Formation of the complex between cystatin and procathepsin L increases the susceptibility of the proregion to proteolytic cleavage. This process may constitute an alternative mechanism of formation of the complex between enzyme and inhibitor without prior activation of the proenzyme.

Amino Acid Substitution↗
PubMed2003

Characterization of quercetin binding site on DNA gyrase.

Gyrases are DNA topology modifying enzymes present only in prokaryotes which makes them an attractive target for antibacterial drugs. Quercetin, one of the most abundant natural flavonoids, inhibits supercoiling activity of bacterial gyrase and induces DNA cleavage. It has been generally assumed that the mechanism of flavonoid inhibition is based on interaction with DNA. We show that quercetin binds to the 24 kDa fragment of gyrase B of Escherichia coli with a K(D) value of 15 microM and inhibits ATPase activity of gyrase B. Its binding site overlaps with ATP binding pocket and could be competitively replaced by either ATP or novobiocin. The structural model of quercetin-gyrase complex was prepared, based on the close similarity with ATP and quercetin binding sites of the src family tyrosine kinase Hck. We propose that quercetin inhibits gyrases through two different mechanisms based either on interaction with DNA or with ATP binding site of gyrase.

Adenosine Triphosphatases↗
PubMed2003

Enhancement of antibacterial and lipopolysaccharide binding activities of a human lactoferrin peptide fragment by the addition of acyl chain.

Cationic antibacterial peptides are potentially therapeutic in the treatment of sepsis, because of their amalgamated antibacterial and lipopolysaccharide-binding activities. We prepared acyl analogues of the peptide fragment of human lactoferrin, which originally had weak antibacterial activity. It was found that 12 carbon units constitute the optimal acyl chain length, enhancing the antibacterial activity and binding of lipopolysaccharide by up to two orders of magnitude. Lactoferrin-based lipopeptides approached the activity of polymyxin B, a lipopeptide of natural origin, but were also active against Gram-positive bacteria.

Acylation↗
PubMed2002

Identification of LPS-binding peptide fragment of MD-2, a toll-receptor accessory protein.

Members of the toll-like receptor family are crucial in recognition of microbial pathogens as part of innate immune response. MD-2, an accessory protein to TLR4, present on the extracellular side of the membrane is needed to initiate the signal transduction. We have identified a 15 amino acid region of human MD-2 that contains several features of other lipopolysaccharide (LPS) binding proteins and peptides. In vitro LPS neutralization by this peptide was observed and confirmed by 2D transferred NOESY NMR experiments. NMR experiments have also shown binding of the MD-2 peptide to lipoteichoic acid (LTA) but not to peptidoglycan. Furthermore this peptide inhibited growth of gram-negative and to a lower extent of some gram-positive bacteria. Our results indicate that this region of MD-2 might be responsible for binding of LPS and confirms the role of MD-2 as an accessory protein in LPS signaling bestowing the Toll receptors their specificity.

Antigens, Surface↗
PubMed2002

Semiautomatic sequence-specific assignment of proteins based on the tertiary structure--the program st2nmr.

The sequence-specific assignment of resonances is still the most time-consuming procedure that is necessary as the first step in high-resolution NMR studies of proteins. In many cases a reliable three-dimensional (3D) structure of the protein is available, for example, from X-ray spectroscopy or homology modeling. Here we introduce the st2nmr program that uses the 3D structure and Nuclear Overhauser Effect spectroscopy (NOESY) peak list(s) to evaluate and optimize trial sequence-specific assignments of spin systems derived from correlation spectra to residues of the protein. A distance-dependent target function that scores trial assignments based on the presence of expected NOESY crosspeaks is optimized in a Monte Carlo fashion. The performance of the program st2nmr is tested on real NMR data of an alpha-helical (cytochrome c) and beta-sheet (lipocalin) protein using homology models and/or X-ray structures; it succeeded in completely reproducing the correct sequence-specific assignments in most cases using 2D and/or 15N/13C Nuclear Overhauser Effect (NOE) data. Additionally to amino acid residues the program can also handle ligands that are bound to the protein, such as heme, and can be used as a complementary tool to fully automated assignment procedures.

Algorithms↗