Synergistic interaction of PMAP-36 and PRW4 with aminoglycoside antibiotics and their antibacterial mechanism

The antimicrobial peptide PMAP-36 is a highly cationic and amphipathic α-helical peptide. PRW4 is a truncated analog that replaces paired lysine residues with tryptophan along the N-terminal and deletes the C-terminal hydrophobic tail of PMAP-36. Studies on the two peptides have already been performed. However, whether there is a synergistic effect with antibiotics has not been investigated, and the study of the antibacterial mechanism of the peptides is inadequate. In this study, antibiotic-peptide combinations were tested against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, and the confocal laser scanning microscopy (LSCM) and DNA gel retardation were measured. The results indicated synergy between the peptides and gentamicin when tested against E. coli [fractional lethal concentration (FLC) < 0.5]; partial synergy was observed between the peptides and gentamicin against S. aureus (0.5 < FLC < 1); and streptomycin showed no reaction with the peptides against E. coli and S. aureus (1 < FLC < 4). LSCM and DNA binding suggest that PMAP-36 was able to translocate across the bacterial membranes and interact with intracellular DNA, but PRW4 presented no DNA-binding ability. These results indicate that the combination of PMAP-36 and PRW4 with aminoglycosides may provide useful information for clinical application, and the antibacterial mechanism of peptides likely does not solely involve cytoplasmic-membrane permeabilization.     

猪源抗菌肽PMAP-36的序列设计及其结构优化的研究进展

抗生素的耐药性和动物源性食品中的药物残留问题严重威胁全球公共卫生系统.因此,开发出不易产生耐药性、抗菌活性高的新型抗菌药物迫在眉睫.抗菌肽因其分子量小、抗菌谱广、不易产生耐药性等优点受到科学家们的广泛关注,但天然抗菌肽具有抗菌活性低、溶血活性和细胞毒性等缺陷.随着抗菌肽序列和结构的不断优化,多种具有显著体内外抗菌活性且...     

Possible role of a PXXP central hinge in the antibacterial activity and membrane interaction of PMAP-23, a member of cathelicidin family

Cathelicidins are essential components of the innate immune system of mammals, providing them a weapon against microbial invasion. PMAP-23 adopting a helix-hinge-helix structure with a central PXXP motif is a member of the cathelicidin family and has potent killing activities against a broad spectrum of microbial organisms. Although the antimicrobial effect of PMAP-23 is believed to be mediated by membrane disruption, many details of this event remain unclear. Here, we try to characterize the interaction between PMAP-23 and membrane phospholipids, focusing on the function of the central PXXP motif. PMAP-PA, in which the Pro residues were substituted by Ala, had significantly more alpha-helical content than PMAP-23, but was less amphipathic and more damaging to human erythrocytes and zwitterionic liposomes. The observed differences in the structures and biological activities of PMAP-23 and PMAP-PA confirmed the functional importance of the central hinge PXXP motif, which enables PMAP-23 to adopt a well-defined amphipathic conformation along its entire length and to have selective antimicrobial activity. CD and Trp fluorescence studies using fragments corresponding to the two helical halves of PMAP-23 revealed that the N-terminal half binds to anionic phospholipids and is more stable than the C-terminal half. In addition, Trp fluorescence quench analyses revealed that the C-terminal helix inserts more deeply into the hydrophobic region of the membrane than the N-terminal helix. Finally, observations made using biosensor technology enabled us to distinguish between the membrane binding and insertion steps, substantiating a proposed kinetic mode of the peptide-membrane interaction in which PMAP-23 first attaches to the membrane via the N-terminal amphipathic helix, after which bending and/or swiveling of the PXXP motif enables insertion of the C-terminal helix into the lipid bilayer.     

Neuropeptide Y and neuropeptide Y18-36. Structural and biological characterization

Neuropeptide Y (NPY), a 36-residue peptide amide, has been shown by numerous studies to be a potent vasoconstrictor. In order to gain an appreciation of the structural requirements for this action, we have previously synthesized a number of fragments of NPY. It had been shown that sequential deletions from the N-terminus resulted in peptides with decreasing hypertensive activity. In the present study we present data supporting the unexpected finding of two fragments, NPY17-36 and NPT18-36 with substantial hypotensive action in vivo. This action was dose dependent (data not shown) and was also observed to a lesser extent with NPY19-36 but not NPY16-36 or NPY20-36. It was, however, slower in onset and of longer duration than the hypertensive action of NPY. These differing kinetics of action may suggest that NPY and NPY18-36 act through different mechanisms. Structural studies using circular dichroism were performed. While NPY was found to assume an ordered helical structure in both aqueous buffer and trifluoroethanol (TFE), 30% TFE in aqueous buffer was required to induce substantial helicity for NPY18-36. This structural investigation suggests that both NPY and NPY18-36 assume an ordered conformation upon reaching the lipid rich receptor environment.     

Structural characteristics and biological properties of Pseudomonas fluorescens lipopolysaccharides

The structure and biological properties of lipopolysaccharides (LPSs) from strains IMB 4125 (=ATCC 13525) and IMB 7769 of the bacterium Pseudomonas fluorescens (biovar I) were studied in vitro. LPSs were similar in the composition of lipid A and the core lipid but differed in the structure of O-specific polysaccharide chains, which was corroborated by the absence of serological relationships between them. The toxicity (LD50) of LPSs of P. fluorescens with respect to D-glucosamine-sensitized mice was 40-50 times lower than the toxicity of the classic endotoxins, LPSs of E. coli. The LPSs studied stimulated the production of tumor necrosis factor (TNF) and nitric oxide (NO) by mouse peritoneal macrophages. The rates of TNF and NO synthesis induced by the LPSs of interest were eight to nine and three to five times lower, respectively, than the corresponding parameters of the control LPSs of E. coli 055:B5 and 026:B6. Additionally, LPS preparations of the P. fluorescens strains induced TNF synthesis by monocytes of human whole-blood preparations. Certain differences in biological properties of these strains have been revealed, which could be due to the characteristic features of LPS structure and composition in different cultures.     

Structural features of distinctin affecting peptide biological and biochemical properties

The antimicrobial peptide distinctin consists of two peptide chains linked by a disulfide bridge; it presents a peculiar fold in water resulting from noncovalent dimerization of two heterodimeric molecules. To investigate the contribution of each peptide chain and the S-S bond to distinctin biochemical properties, different monomeric and homodimeric peptide analogues were synthesized and comparatively evaluated with respect to the native molecule. Our experiments demonstrate that the simultaneous occurrence of both peptide chains and the disulfide bond is essential for the formation of the quaternary structure of distinctin in aqueous media, able to resist protease action. In contrast, distinctin and monomeric and homodimeric analogues exhibited comparable antimicrobial activities, suggesting only a partial contribution of the S-S bond to peptide killing effectiveness. Relative bactericidal properties paralleled liposome permeabilization results, definitively demonstrating that microbial membranes are the main target of distinctin activity. Various biophysical experiments performed in membrane-mimicking media, before and after peptide addition, provided information about peptide secondary structure, lipid bilayer organization, and lipid-peptide orientation with respect to membrane surface. These data were instrumental in the generation of putative models of peptide-lipid supramolecular pore complexes.     

The fibroblast growth factor family: Structural and biological properties

This article sumarizes the structural and biological properties of the family of fibroblast growth factors (FGF). Basic FGF (bFGF) and acidic FGF (aFGF) are the best characterized members of this family. bFGF and aFGF are potent modulators of cell proliferation, motility and differentiation. They are also potent angiogenesis factors in vivo. Some of the important biological characteristics of bFGF and aFGF discussed in the review include the affinity of bFGF and aFGF for heparin, their lack of secretion in culture and their association with extracellular matrix. Recently, several oncogenes, 40–50% homologous in sequence to bFGF and aFGF have been identified. These include int-2, hst, K-fgf and FGF-5. The structural and biological properties of these FGF-related oncogenes are also discussed.     

Structural and biological properties of the Drosophila insulin-like peptide 5 show evolutionary conservation

We report the crystal structure of two variants of Drosophila melanogaster insulin-like peptide 5 (DILP5) at a resolution of 1.85 Å. DILP5 shares the basic fold of the insulin peptide family (T conformation) but with a disordered B-chain C terminus. DILP5 dimerizes in the crystal and in solution. The dimer interface is not similar to that observed in vertebrates, i.e. through an anti-parallel β-sheet involving the B-chain C termini but, in contrast, is formed through an anti-parallel β-sheet involving the B-chain N termini. DILP5 binds to and activates the human insulin receptor and lowers blood glucose in rats. It also lowers trehalose levels in Drosophila. Reciprocally, human insulin binds to the Drosophila insulin receptor and induces negative cooperativity as in the human receptor. DILP5 also binds to insect insulin-binding proteins. These results show high evolutionary conservation of the insulin receptor binding properties despite divergent insulin dimerization mechanisms.     

Amphibian cathelicidin fills the evolutionary gap of cathelicidin in vertebrate

Cathelicidins comprise a family of antimicrobial peptides (AMPs) sharing a highly conserved cathelin domain, and play a central role in the innate defense against infection in most of vertebrates. But so far it has not yet been found in amphibians although a large number of other groups of AMPs have been identified. In the current work, the first amphibian cathelicidin (cathelicidin-AL) has been characterized from the frog skin of Amolops loloensis. Cathelicidin-AL (RRSRRGRGGGRRGGSGGRGGRGGGGRSGAGSSIAGVGSRGGGGGRHYA) is a cationic peptide containing 48 amino acid residues (aa) with 12 basic aa and no acidic aa. The chemical synthesized peptide efficiently killed bacteria and some fungal species including clinically isolated drug-resistance microorganisms. The cDNA encoding cathelicidin-AL precursor was cloned from the skin cDNA library of A. loloensis. As other cathelicidins, the precursor of cathelicidin-AL also contains highly conserved anionic cathelin domain of cysteine proteinase inhibitor followed by the AMP fragment at C-terminus. Phylogenetic analysis revealed that as connecting link, the amphibian cathelicidin predates reptilia but postdates fish cathelicidin. The peptide purification combined with gene cloning results confirms the presence of cathelicidin in amphibians and filled the evolutionary gap of cathelicidin in vertebrate, considering amphibians' special niche as the animals bridging the evolutionary land-water gap.     

Differences between alcohol dehydrogenases. Structural properties and evolutionary aspects.

Comparisons of the primary structures of yeast and horse liver alcohol dehydrogenases reveal that the enzymes are homologous but distantly related. The overall positional identity is 25% between common regions, and several deletions/insertions occur in either enzyme, the longest apparently corresponding to 21 residues, showing that the different subunit sizes are largely explained by internal differences. Variabilities in the structural similarities can be coupled with functional requirements but not directly with whole domains in the previously known tertiary structure of the horse protein. The two most similar regions of the enzymes affect active-site segments and the two most dissimilar regions seem to affect a loop structure without known function, and a segment participating in subunit interactions. The dissimilarities may probably be correlated with changes in zinc-binding properties and quaternary structures. The extra region corresponding to the large internal chain-length difference shows an apparent coincidence in sequence to a following segment of the horse enzyme, and additional elements of internal coincidences, or superficial similarities with other dehydrogenases, are noticed. These characteristics are not fully distinguishable from chance distributions but in view of the extensive species variations in alcohol dehydrogenases some evolutionary considerations may not be excluded, in which case a model relating all regions of these and associated enzymes to a common ancestor is shown to be compatible with all known observations.     

Structural aspects of the distinct biochemical properties of glutaredoxin 1 and glutaredoxin 2 from Saccharomyces cerevisiae

Glutaredoxins (Grxs) are small (9-12 kDa) heat-stable proteins that are ubiquitously distributed. In Saccharomyces cerevisiae, seven Grx enzymes have been identified. Two of them (yGrx1 and yGrx2) are dithiolic, possessing a conserved Cys-Pro-Tyr-Cys motif. Here, we show that yGrx2 has a specific activity 15 times higher than that of yGrx1, although these two oxidoreductases share 64% identity and 85% similarity with respect to their amino acid sequences. Further characterization of the enzymatic activities through two-substrate kinetics analysis revealed that yGrx2 possesses a lower K_M for glutathione and a higher turnover than yGrx1. To better comprehend these biochemical differences, the pK_a of the N-terminal active-site cysteines (Cys27) of these two proteins and of the yGrx2-C30S mutant were determined. Since the pK_a values of the yGrx1 and yGrx2 Cys27 residues are very similar, these parameters cannot account for the difference observed between their specific activities. Therefore, crystal structures of yGrx2 in the oxidized form and with a glutathionyl mixed disulfide were determined at resolutions of 2.05 and 1.91 Å, respectively. Comparisons of yGrx2 structures with the recently determined structures of yGrx1 provided insights into their remarkable functional divergence. We hypothesize that the substitutions of Ser23 and Gln52 in yGrx1 by Ala23 and Glu52 in yGrx2 modify the capability of the active-site C-terminal cysteine to attack the mixed disulfide between the N-terminal active-site cysteine and the glutathione molecule. Mutagenesis studies supported this hypothesis. The observed structural and functional differences between yGrx1 and yGrx2 may reflect variations in substrate specificity.     

Evolutional and biological aspects of placentophagia

Over the last 30 years it could be observed that a very small minority of modern humans consumes the human placenta that belongs to the afterbirth. This behavior, which is known by the technical term of placentophagia, is the focal point of the present essay. Placentophagia is a behavior common among almost all kinds of mammals, even primates can be observed ingesting at least amniotic fluid during the birth. However, humans of traditional human cultures consume the afterbirth only in rare cases. In detail, the present essay discusses the hypothesis that human placentophagia has a phylogenetic basis and the hypothesis that human placentophagia is physiologically reasonable. It is concluded that the behavior of the human placentophagia neither possesses a phylogenetic basis nor can be regarded as physiologically reasonable.     

Structural and functional aspects of stomata

Differential cell wall thickening in developing guard cells of Polypodium vulgare L. has been studied with particular reference to guard cell protoplast deformation and the eventual formation of the stomatal pore. Concomitant studies on the development of guard cell chloroplasts and their starch inclusions during ontogeny of the stomatal complex have provided data which have been incorporated into a model to account for the formation of the pore. Guard cell starch inclusions reach a maximum density per unit volume at the same time as the guard cell walls achieve maximum differential thickening. These events coincide with the development of the pore. It is suggested that, whilst pore formation is initiated enzymatically, the mechanical forces required to bring about the separation of the two guard cells are of an osmotic nature derived from starch hydrolysis. The development of the mesophyll in relation to the epidermis is examined in respect of the formation of substomatal chambers.     

Structural and functional aspects of filamins

Filamins are a family of high molecular mass cytoskeletal proteins that organize filamentous actin in networks and stress fibers. Over the past few years it has become clear that filamins anchor various transmembrane proteins to the actin cytoskeleton and provide a scaffold for a wide range of cytoplasmic signaling proteins. The recent cloning of three human filamins and studies on filamin orthologues from chicken and Drosophila revealed unexpected complexity of the filamin family, the biological implications of which have just started to be addressed. Expression of dysfunctional filamin-A leads to the genetic disorder of ventricular heterotopia and gives reason to expect that abnormalities in the other isogenes may also be connected with human disease. In this review aspects of filamin structure, its splice variants, binding partners and biological function will be discussed.     

Structural studies of porcine myeloid antibacterial peptide PMAP-23 and its analogues in DPC micelles by NMR spectroscopy.

PMAP-23 is a cathelicidin-derived antimicrobial peptide identified from porcine leukocytes. PMAP-23 was reported to show potent antimicrobial activity against Gram-negative and Gram-positive bacteria without hemolytic activity. To study the structure-antibiotic activity relationships of PMAP-23, two analogues by replacing Trp with Ala were synthesized and their tertiary structures bound to DPC micelles have been studied by NMR spectroscopy. PMAP-23 has two alpha-helices, one from Arg1 to Arg10 in the N-terminal region and the other from Phe18 to Arg23 in the C-terminal region. PMAP-1 (Trp(7)-->Ala) shows similar structure to PMAP-23, while PMAP-2 (Trp(21)-->Ala) has a random structure in the C-terminus. PMAP-2 was found to show less antibacterial and vesicle-disrupting activities than PMAP-23 and PMAP-1 [J. H. Kang, S. Y. Shin, S. Y. Jang, K. L. Kim, and K.-S. Hahm (1999) Biochem. Biophys. Res. Commun. 264, 281-286]. Trp(21) in PMAP-23 which induces an alpha-helical structure in the second alpha-helix is essential for the antibacterial activity of PMAP-23. Also, the fluorescence data proved that Trp(21) at the second alpha-helix is buried deep into the phospholipid in the membrane. Therefore, it implies that Trp(21) in the second alpha-helix at the C-terminus of PMAP-23 may play an important role on the interactions with the membrane and the flexible region including two proline residues may allow this alpha-helix to span the lipid bilayer.