Amphipathic Α-helical Structure Research Articles

Leptodactylus latrans Amphibian Skin Secretions as a Novel Source for the Isolation of Antibacterial Peptides.

Amphibians´ skin produces a diverse array of antimicrobial peptides that play a crucial role as the first line of defense against microbial invasion. Despite the immense richness of wild amphibians in Argentina, current knowledge about the presence of peptides with antimicrobial properties is limited to a only few species. Here we used LC-MS-MS to identify antimicrobial peptides with masses ranging from 1000 to 4000 Da from samples of skin secretions of Leptodactylus latrans (Anura: Leptodactylidae). Three novel amino acid sequences were selected for chemical synthesis and further studies. The three synthetic peptides, named P1-Ll-1577, P2-Ll-1298, and P3-Ll-2085, inhibited the growth of two ATCC strains, namely Escherichia coli and Staphylococcus aureus. P3-Ll-2085 was the most active peptide. In the presence of trifluoroethanol (TFE) and anionic liposomes, it adopted an amphipathic α-helical structure. P2-Ll-1298 showed slightly lower activity than P3-Ll-2085. Comparison of the MIC values of these two peptides revealed that the addition of seven amino acid residues (GLLDFLK) on the N-terminal of P2-Ll-1298 significantly improved activity against both strains. P1-Ll-1577, which remarkably is an anionic peptide, showed interesting antimicrobial activity against E. coli and S. aureus strain, showing marked membrane selectivity and non-hemolysis. Due to this, P1-L1-1577 emerges as a potential candidate for the development of new antibacterial drugs.

Investigation of cationicity and structure of pseudin-2 analogues for enhanced bacterial selectivity and anti-inflammatory activity

Pseudin-2 (Ps), isolated from the frog Pseudis paradoxa, exhibits potent antibacterial activity and cytotoxicity. To develop antimicrobial peptides with anti-inflammatory activity and low cytotoxicity, we designed Ps analogues with Lys substitutions, resulting in elevated amphipathic α-helical structure and cationicity. We further substituted Gly11 with Pro (Ps-P analogues) to increase bacterial cell selectivity. Ps analogues retained antimicrobial activity and exhibited reduced cytotoxicity, whereas Ps-P analogues exhibited lower cytotoxicity and antimicrobial activity. Tertiary structures revealed that Ps has a linear α-helix from Leu2 to Glu24, whereas Ps-P has a bend at Pro11 between two short α-helixes. Using various biophysical experiments, we found that Ps analogues produced much higher membrane depolarization than Ps-P analogues, whereas Ps-P analogues may penetrate bacterial cell membranes. Ps and its analogue Ps-K18 exhibited potent anti-inflammatory activity in LPS-stimulated RAW264.7 and mouse dendritic cells via a mechanism involving the Toll-like receptor 4 (TLR4) pathway. These activities may arise from their direct inhibition of the formation of TLR4-MD-2_LPS complex, implying that amphipathic α-helical structure with an optimum balance between enhanced cationicity and hydrophobicity may be essential for their anti-inflammatory activity. The bent structure provided by Pro substitution plays an important role in enhancing bacterial cell selectivity and cell penetration.

Sarkosyl-Induced Helical Structure of an Antimicrobial Peptide GW-Q6 Plays an Essential Role in the Binding of Surface Receptor OprI in Pseudomonas aeruginosa.

The emergence of antibiotic-resistant microbial strains has become a public health issue and there is an urgent need to develop new anti-infective molecules. Although natural antimicrobial peptides (AMPs) can exert bactericidal activities, they have not shown clinical efficacy. The limitations of native peptides may be overcome with rational design and synthesis. Here, we provide evidence that the bactericidal activity of a synthetic peptide, GW-Q6, against Pseudomonas aeruginosa is mediated through outer membrane protein OprI. Hyperpolarization/depolarization of membrane potential and increase of membrane permeability were observed after GW-Q6 treatment. Helical structure as well as hydrophobicity was induced by an amphipathic surfactant, sarkosyl, for binding to OprI and possible to membrane. NMR studies demonstrated GW-Q6 is an amphipathic α-helical structure in DPC micelles. The paramagnetic relaxation enhancement (PRE) approach revealed that GW-Q6 orients its α-helix segment (K7-K17) into DPC micelles. Additionally, this α-helix segment is critical for membrane permeabilization and antimicrobial activity. Moreover, residues K3, K7, and K14 could be critical for helical formation and membrane binding while residues Y19 and W20 for directing the C-terminus of the peptide to the surface of micelle. Taken together, our study provides mechanistic insights into the mode of action of the GW-Q6 peptide and suggests its applicability in modifying and developing potent AMPs as therapeutic agents.

Emerging Roles of Apolipoprotein E and Apolipoprotein A-I in the Pathogenesis and Treatment of Lung Disease.

Emerging roles are being recognized increasingly for apolipoproteins in the pathogenesis and treatment of lung diseases on the basis of their ability to suppress inflammation, oxidative stress, and tissue remodeling, and to promote adaptive immunity and host defense. Apolipoproteins, such as apolipoprotein E (apoE) and apolipoprotein A-I (apoA-I), are important components of lipoprotein particles that facilitate the transport of cholesterol, triglycerides, and phospholipids between plasma and cells. ApoE-containing lipoprotein particles are internalized into cells by low-density lipoprotein receptors (LDLRs), whereas apoA-I can interact with the ATP-binding cassette subfamily A member 1 (ABCA1) transporter to efflux cholesterol and phospholipids out of cells. ApoE and apoA-I also mediate receptor-independent effects, such as binding to and neutralizing LPS. Both apoE and apoA-I are expressed by lung cells, which allows apoE/LDLR- and apoA-I/ABCA1-dependent pathways to modulate normal lung health and the pathogenesis of respiratory diseases, including asthma, acute lung injury, cancer, emphysema, pulmonary fibrosis, and pulmonary hypertension. Data from human studies and research using experimental murine model systems have shown that both apoE and apoA-I pathways play primarily protective roles in lung biology and respiratory disease. Furthermore, apolipoprotein mimetic peptides, corresponding to the LDLR-binding domain of apoE or the class A amphipathic α-helical structure of apoA-I, have antiinflammatory and antioxidant effects that attenuate the severity of lung disease in murine models. Thus, the development of inhaled apolipoprotein mimetic peptides as a novel treatment paradigm could represent a significant advance for patients with respiratory disease who do not respond to current therapies.

Structural Changes in Lipid Model Membranes Induced by the Fungal Peptide Toxin Ece1-III

Ece1-III is the first peptide toxin identified in a human pathogenic fungus. This peptide consists of 32 amino acids and it is processed from the protein, Ece1p, which is exclusively expressed by the invasive hyphal form of the opportunistic fungus Candida albicans. The peptide of our interest triggers the host danger response pathway and activates epithelial immunity at low concentrations and shows a pronounced cytolytic effect on epithelial membranes with increasing concentration.We propose that Ece1-III partitions directly into the lipid matrix of biological cell membranes. The association with the membrane leads to its permeabilization and disintegration by one of three different pore-forming mechanisms: (i) the ‘barrel-stave model’, (ii) the ‘toroidal-pore model’, or (iii) the ‘detergent-like carpet model’. We suggest that the membrane perturbation is mainly driven by its characteristic amphipathic α-helical secondary structure and electrostatic interactions play only a minor role as the peptide exhibits a comparably low cationic net charge. Our aim is to reveal the interactions of the peptide with phospholipid model membranes either by assigning it to one of the models described so far or to evolve a new one. The understanding of the interaction mechanisms is of crucial importance with view to a potential protection of the integrity of human epithelial cell membranes and epithelial immunity.Here, we will present results of different biophysical techniques that characterize the lipid-peptide interaction. We will show that the peptide activity towards membranes containing the fungal lipid component ergosterol is reduced as compared to membranes with cholesterol which indicates that ergosterol adapts a self-protective function in C. albicans.

A hemocyanin-derived antimicrobial peptide from the penaeid shrimp adopts an alpha-helical structure that specifically permeabilizes fungal membranes

BackgroundHemocyanins are respiratory proteins with multiple functions. In diverse crustaceans hemocyanins can release histidine-rich antimicrobial peptides in response to microbial challenge. In penaeid shrimp, strictly antifungal peptides are released from the C-terminus of hemocyanins. MethodsThe three-dimensional structure of the antifungal peptide PvHCt from Litopenaeus vannamei was determined by NMR. Its mechanism of action against the shrimp pathogen Fusarium oxysporum was investigated using immunochemistry, fluorescence and transmission electron microscopy. ResultsPvHCt folded into an amphipathic α-helix in membrane-mimicking media and displayed a random conformation in aqueous environment. In contact with F. oxysporum, PvHCt bound massively to the surface of fungal hyphae without being imported into the cytoplasm. At minimal inhibitory concentrations, PvHCt made the fungal membrane permeable to SYTOX-green and fluorescent dextran beads of 4kDa. Higher size beads could not enter the cytoplasm. Therefore, PvHCt likely creates local damages to the fungal membrane. While the fungal cell wall appeared preserved, gradual degeneration of the cytoplasm most often resulting in cell lysis was observed in fungal spores and hyphae. In the remaining fungal cells, PvHCt induced a protective response by the formation of daughter hyphae. ConclusionThe massive accumulation of PvHCt at the surface of fungal hyphae and subsequent insertion into the plasma membrane disrupt its integrity as a permeability barrier, leading to disruption of internal homeostasis and fungal death. General significanceThe histidine-rich antimicrobial peptide PvHCt derived from shrimp hemocyanin is a strictly antifungal peptide, which adopts an amphipathic α-helical structure, and selectively binds to and permeabilizes fungal cells.

Structural Dissection of Crotalicidin, a Rattlesnake Venom Cathelicidin, Retrieves a Fragment with Antimicrobial and Antitumor Activity.

In silico dissection of crotalicidin (Ctn), a cathelicidin from a South American pit viper, yielded fragments Ctn[1-14] and Ctn[15-34], which were tested to ascertain to what extent they reproduced the structure and activity of the parent peptide. NMR data showing Ctn to be α-helical at the N-terminus and unstructured at the C-terminus were matched by similar data from the fragments. The peptides were tested against Gram-positive and -negative bacteria and for toxicity against both tumor and healthy cells. Despite its amphipathic α-helical structure, Ctn[1-14] was totally inert toward bacteria or eukaryotic cells. In contrast, unstructured Ctn[15-34] replicated the activity of parent Ctn against Gram-negative bacteria and tumor cells while being significantly less toxic toward eukaryotic cells. This selectivity for bacteria and tumor cells, plus a stability to serum well above that of Ctn, portrays Ctn[15-34] as an appealing candidate for further development as an anti-infective or antitumor lead.

An Extra Arm May Impair the Open Hand.

In the paper, “Inhibin Biosynthesis and Activity Are Limited by a Prodomain-Derived Peptide,” Walton et al (1) purport that processing of inhibin-α can result in a 43-amino acid pro-α peptide from an alternate cleavage site. This propeptide is projected to have an amphipathic α-helical structure that can wrap around the open hand or butterfly-shaped mature inhibin protein, thereby hindering inhibin association with β-glycan. The function of this proinhibin α-peptide is investigated as an inhibitor of mature inhibin action. The inhibins and activins are produced as prepro-proteins in which nonactive domains that are important to protein folding and dimerization of the molecules are cleaved, releasing the final active products. The β-subunits for inhibin and activin have only one classical cleavage site, but the α-inhibin subunit has another cleavage site between the pro-α-peptide and the N-peptide (Figure 1A). Uncleaved or incompletely cleaved inhibins have been described for many years as high-molecular-weight or big inhibins, with varying demonstrations of bioactivity (2,–4). A free α-subunit in association with the C portion, without the N portion, has also been described and is produced at high levels by a variety of normal and abnormal tissues (5, 6), but this is the first evaluation of a functional small prodomain cleavage peptide for inhibin. Figure 1. A, Inhibin precursor with the cleavage sites. Note the two cleavage sites on the α-subunit and the single cleavage site on the β-subunit. B, Inhibin A/B binding to β-glycan. C, Pro-α-peptide intercalating between inhibin ... The authors also noted some time ago that three hydrophobic amino acids in the prodomain of the inhibin α-subunit were critical to proper folding and secretion of mature inhibins (7). In that work they used mutagenesis evidence to propose a model of interaction of the N-terminal prodomain with a region near the C terminus of the proprotein to hold it in a coiled conformation that would allow the covalent bonds with the similarly held β-proprotein subunits and promote further processing. In that work they also proposed that the entire cleaved prodomain could remain associated with the active inhibin in a noncovalent way, such as the fact that the latency-associated peptide remains associated with TGFβ (8). In this work they have focused on the role of the small 43-amino acid propeptide. Not only do they demonstrate the necessity of cleavage of this promolecule from the N and C regions of inhibin for production of mature peptide to occur, but they also provide strong evidence for a role of this unassuming peptide in modulating the bioactivity of the mature inhibins. Yet it does not seem to interfere with activin activity. Thus, inhibin protein may come along with its own tiny inhibitor after processing, with the little associated prodomain peptide intercalating itself between the interface with inhibin A or B and the β-glycan receptor (Figure 1, B and C). Yet a function of β-glycan is to inhibit the activation of the activin receptor signaling cascade (9, 10). So the inhibitor is packaged with its own inhibitor, like a functional mini-me, providing direct modulation of inhibin function. This has profound implications biologically if this relationship is shown to occur in vivo. Activin action is already known to be tightly regulated on many levels, both directly and indirectly. From the expression of excess inhibin α-subunits (favoring inhibin production over activin), to follistatin or α-macroglobin binding, to production of inhibitory smads that can directly modulate the activin signaling cascade; the action of activin is tightly regulated in tissue-specific ways, and its effects are profoundly concentration and tissue specific (11, 12). Entry into this already complex system regulating activin, the action of a small amphipathic molecule that could be delivered to a specific site to block inhibin and thereby increase activin action and possibly result in increased unbound β-glycan may have great usefulness clinically and as a research tool across several overlapping fields of endocrinology.

Role of phenylalanine and valine10 residues in the antimicrobial activity and cytotoxicity of piscidin-1.

Piscidin-1 (Pis-1) is a linear antibacterial peptide derived from mast cells of aquacultured hybrid striped bass that comprises 22 amino acids with a phenylalanine-rich amino-terminus. Pis-1 exhibits potent antibacterial activity against pathogens but is not selective for distinguishing between bacterial and mammalian cells. To determine the key residues for its antibacterial activity and those for its cytotoxicity, we investigated the role of each Phe residue near the N-terminus as well as the Val10 residue located near the boundary of the hydrophobic and hydrophilic sectors of the helical wheel diagram. Fluorescence dye leakage and tryptophan fluorescence experiments were used to study peptide-lipid interactions, showing comparable depths of insertion of substituted peptides in different membranes. Phe2 was found to be the most deeply inserted phenylalanine in both bacterial- and mammalian-mimic membranes. Each Phe was substituted with Ala or Lys to investigate its functional role. Phe2 plays key roles in the cytotoxicity as well as the antibacterial activities of Pis-1, and Phe6 is essential for the antibacterial activities of Pis-1. We also designed and synthesized a piscidin analog, Pis-V10K, in which Lys was substituted for Val10, resulting in an elevated amphipathic α-helical structure. Pis-V10K showed similar antibacterial activity (average minimum inhibitory concentration (MIC) = 1.6 µM) to Pis-1 (average MIC = 1.5 µM). However, it exhibited much lower cytotoxicity than Pis-1. Lys10-substituted analogs, Pis-F1K/V10K, Pis-F2K/V10K, and Pis-F6K/V10K in which Lys was substituted for Phe retained antibacterial activity toward standard and drug-resistant bacterial strains with novel bacterial cell selectivity. They exert anti-inflammatory activities via inhibition of nitric oxide production, TNF-α secretion, and MIP-1 and MIP-2 production. They may disrupt the binding of LPS to toll-like receptors, eventually suppressing MAPKs-mediated signaling pathways. These peptides may be good candidates for the development of peptide antibiotics with potent antibacterial activity but without cytotoxicity.

Expression analysis and biological activity of moronecidin from rock bream, Oplegnathus fasciatus

The piscidin-family, one of antimicrobial peptides (AMPs) mainly distributed in fish, is crucial effectors of fish innate immune response. Piscidin-family typically has broad-spectrum antimicrobial activity and the ability to modulate the immune response. In this study, we identified moronecidin (Rbmoro) included in piscidin-family from rock bream and investigated its gene expression using quantitative real-time PCR and biological activity (including antimicrobial and cytotoxic activity). The coding region of Rbmoro was 204 bp encoding 67 amino acid residues. Tertiary structure prediction of Rbmoro showed an amphipathic α-helical structure. Rbmoro gene was widely expressed in different tissues of healthy fish. Additionally, Rbmoro gene expression was induced in all tested tissues after infection with Edwardsiella tarda, Streptococcus iniae and red seabream iridovirus. We synthesized mature peptide of Rbmoro based on amino acid sequence of its AMP 12 domain, and the synthetic peptide appeared broad-spectrum antimicrobial activity to various bacteria. However, the synthetic peptide has weak haemolytic activity against fish erythrocytes. These results suggest that Rbmoro might play an important role in innate immune response of rock bream.

Antimicrobial Peptides from Skin Secretions of Hypsiboas pulchellus (Anura: Hylidae)

The skin of many amphibians produces a large repertoire of antimicrobial peptides that are crucial in the first line of defense against microbial invasion. Despite the immense richness of wild amphibians in Argentina, knowledge about peptides with antimicrobial properties is limited to a few species. Here we used LC-MS-MS to analyze samples of Hypsiboas pulchellus skin with the aim to identify antimicrobial peptides in the mass range of 1000 to 2000 Da. Twenty-three novel sequences were identified by MS, three of which were selected for chemical synthesis and further studies. The three synthetic peptides, named P1-Hp-1971, P2-Hp-1935, and P3-Hp-1891, inhibited the growth of two ATCC strains: Escherichia coli (MIC: 16, 33, and 17 μM, respectively) and Staphylococcus aureus (MIC: 8, 66, and 17 μM, respectively). P1-Hp-1971 and P3-Hp-1891 were the most active peptides. P1-Hp-1971, which showed the highest therapeutic indices (40 for E. coli and 80 for S. aureus), is a proline-glycine-rich peptide with a highly unordered structure, while P3-Hp-1891 adopts an amphipathic α-helical structure in the presence of 2,2,2-trifluoroethanol and anionic liposomes. This is the first peptidomic study of Hypsiboas pulchellus skin secretions to allow the identification of antimicrobial peptides.

Structure–activity study of macropin, a novel antimicrobial peptide from the venom of solitary bee Macropis fulvipes (Hymenoptera: Melittidae)

A novel antimicrobial peptide, designated macropin (MAC-1) with sequence Gly-Phe-Gly-Met-Ala-Leu-Lys-Leu-Leu-Lys-Lys-Val-Leu-NH2 , was isolated from the venom of the solitary bee Macropis fulvipes. MAC-1 exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l- or d-lysine in selected positions. Furthermore, all-d analog and analogs with d-amino acid residues introduced at the N-terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α-helical secondary structure in the presence of trifluoroethanol or membrane-mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure-activity relationship for the effect of d-amino acid substitutions in MAC-1 using NMR spectroscopy.

Antimicrobial function of SHβAP, a novel hemoglobin β chain-related antimicrobial peptide, isolated from the liver of skipjack tuna, Katsuwonus pelamis

A 2.3 kDa of antimicrobial peptide was purified from an acidified liver extract of skipjack tuna, Katsuwonus pelamis, by preparative acid-urea–polyacrylamide gel electrophoresis and C18 reversed-phase HPLC. A comparison of the amino acid sequence of the purified peptide with those of other known polypeptides revealed high homology with the C-terminus of hemoglobin β-chain; thus, this peptide was designated as the Skipjack Hemoglobin β chain-related Antimicrobial Peptide (SHβAP). SHβAP showed potent antimicrobial activity against Gram-positive bacteria, such as Bacillus subtilis, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 6.5–57.0 μg/mL), Gram-negative bacteria, such as Escherichia coli D31, Pseudomonas aeruginosa, Salmonella enterica, Shigella sonnei, and two Vibrio parahaemolyticus species (MECs, 2.0–19.0 μg/mL), and against Candida albicans (MEC; 12.0 μg/mL) without significant hemolytic activity. Antimicrobial activity of this peptide was heatstable and pH resistant but is sensitive to proteases and salt. SHβAP did not show membrane permeabilization and killing ability. The secondary structural prediction and the homology modeling expected that this peptide formed an amphipathic α-helical structure. This is the first report the purification of a novel antimicrobial peptide related to the C-terminus of hemoglobin β-chain from marine fish.

Phenol-Soluble Modulins: Novel Virulence-Associated Peptides of Staphylococci

Phenol-soluble modulins (PSMs), a novel class of small peptides with an amphipathic α-helical structure and strong surfactant-like properties, are produced by most staphylococci, especially pathogenic Staphylococcus aureus and Staphylococcus epidermidis. PSMs can: induce the production of proinflammatory cytokines; recruit, activate and lyse neutrophils to help staphylococci evade immune damage; lyse erythrocytes and are associated with the hemolysis of staphylococcal disease; facilitate the structuring and detachment of staphylococcal biofilms and disseminate biofilm-associated infection; and kill competing microbes and act as weapons in interbacterial warfare. Therefore, PSMs are considered to be critical virulence-associated factors and to play important roles in the pathogenesis of staphylococci. This review summarizes the classification, structure, expression regulation and biological functions of PSMs. The possible means to prevent PSM-associated diseases are also outlined in order to emphasize the need to investigate PSMs as potential targets for drug and vaccine design against staphylococcal infections.

Structure-Activity Relations of Myxinidin, an Antibacterial Peptide Derived from the Epidermal Mucus of Hagfish

The structure-activity relations of myxinidin, a peptide derived from epidermal mucus of hagfish, Myxine glutinosa L., were investigated. Analysis of key residues allowed us to design new peptides with increased efficiency. Antimicrobial activity of native and modified peptides demonstrated the key role of uncharged residues in the sequence; the loss of these residues reduces almost entirely myxinidin antimicrobial activity, while insertion of arginine at charged and uncharged position increases antimicrobial activity compared with that of native myxinidin. Particularly, we designed a peptide capable of achieving a high inhibitory effect on bacterial growth. Experiments were conducted using both Gram-negative and Gram-positive bacteria. Nuclear magnetic resonance (NMR) studies showed that myxinidin is able to form an amphipathic α-helical structure at the N terminus and a random coil region at the C terminus.

Design, synthesis and evaluation of antimicrobial activity of N-terminal modified Leucocin A analogues

Class IIa bacteriocins are potent antimicrobial peptides produced by lactic acid bacteria to destroy competing microorganisms. The N-terminal domain of these peptides consists of a conserved YGNGV sequence and a disulphide bond. The YGNGV motif is essential for activity, whereas, the two cysteines involved in the disulphide bond can be replaced with hydrophobic residues. The C-terminal region has variable sequences, and folds into a conserved amphipathic α-helical structure. To elucidate the structure–activity relationship in the N-terminal domain of these peptides, three analogues (1–3) of a class IIa bacteriocin, Leucocin A (LeuA), were designed and synthesized by replacing the N-terminal β-sheet residues of the native peptide with shorter β-turn motifs. Such replacement abolished the antibacterial activity in the analogues, however, analogue 1 was able to competitively inhibit the activity of native LeuA. Native LeuA (37-mer) was synthesized using native chemical ligation method in high yield. Solution conformation study using circular dichroism spectroscopy and molecular dynamics simulations suggested that the C-terminal region of analogue 1 adopts helical folding as found in LeuA, while the N-terminal region did not fold into β-sheet conformation. These structure–activity studies highlight the role of proper folding and complete sequence in the activity of class IIa bacteriocins.

Identification of the amino acid residues and domains in the cysteine‐rich protein of Chinese wheat mosaic virus that are important for RNA silencing suppression and subcellular localization

Cysteine-rich proteins (CRPs) encoded by some plant viruses in diverse genera function as RNA silencing suppressors. Within the N-terminal portion of CRPs encoded by furoviruses, there are six conserved cysteine residues and a Cys-Gly-X-X-His motif (Cys, cysteine; Gly, glycine; His, histidine; X, any amino acid residue) with unknown function. The central domains contain coiled-coil heptad amino acid repeats that usually mediate protein dimerization. Here, we present evidence that the conserved cysteine residues and Cys-Gly-X-X-His motif in the CRP of Chinese wheat mosaic virus (CWMV) are critical for protein stability and silencing suppression activity. Mutation of a leucine residue in the third coiled-coil heptad impaired CWMV CRP activity for suppression of local silencing, but not for the promotion of cell-to-cell movement of Potato virus X (PVX). In planta and in vitro analysis of wild-type and mutant proteins indicated that the ability of the CRP to self-interact was correlated with its suppression activity. Deletion of up to 40 amino acids at the C-terminus did not abolish suppression activity, but disrupted the association of CRP with endoplasmic reticulum (ER), and reduced its activity in the enhancement of PVX symptom severity. Interestingly, a short region in the C-terminal domain, predicted to form an amphipathic α-helical structure, was responsible for the association of CWMV CRP with ER. Overall, our results demonstrate that the N-terminal and central regions are the functional domains for suppression activity, whereas the C-terminal region primarily functions to target CWMV CRP to the ER.

Insight into the antimicrobial activities of coprisin isolated from the dung beetle, Copris tripartitus, revealed by structure–activity relationships

The novel 43-residue, insect defensin-like peptide coprisin, isolated from the dung beetle, Copris tripartitus, is a potent antibiotic with bacterial cell selectivity, exhibiting antimicrobial activities against Gram-positive and Gram-negative bacteria without exerting hemolytic activity against human erythrocytes. Tests against Staphylococcus aureus using fluorescent dye leakage and depolarization measurements showed that coprisin targets the bacterial cell membrane. To understand structure–activity relationships, we determined the three-dimensional structure of coprisin in aqueous solution by nuclear magnetic resonance spectroscopy, which showed that coprisin has an amphipathic α-helical structure from Ala19 to Arg28, and β-sheets from Gly31 to Gln35 and Val38 to Arg42. Coprisin has electropositive regions formed by Arg28, Lys29, Lys30, and Arg42 and ITC results proved that coprisin and LPS have electrostatically driven interactions. Using measurements of nitric oxide release and inflammatory cytokine production, we provide the first verification of the anti-inflammatory activity and associated mechanism of an insect defensin, demonstrating that the anti-inflammatory actions of the defensin-like peptide, coprisin, are initiated by suppressing the binding of LPS to toll-like receptor 4, and subsequently inhibiting the phosphorylation of p38 mitogen-activated protein kinase and nuclear translocation of NF-kB. In conclusion, we have demonstrated that an amphipathic helix and an electropositive surface in coprisin may play important roles in its effective interaction with bacterial cell membranes and, ultimately, in its high antibacterial activity and potent anti-inflammatory activity. In addition to elucidating the antimicrobial action of coprisin, this work may provide insight into the mechanism of action of insect defense systems.

The Nature of Membrane Curvature-Induction by Amphipathic α-Helices Relies upon Protein Length: Simulations of α-Synuclein and H0

Remodeling of membranes, for example the induction of membrane curvature, is a necessary step in vesicle fusion and fission. One mechanism for curvature induction relies upon the interfacial binding of amphipathic proteins, which act as a wedge in one leaflet of the membrane. In this study, we explore differences between the membrane remodeling effects of two biologically important amphipathic proteins, specifically α-Synuclein (αS) and the N-BAR helix H0. αS is an intrinsically disordered protein that adopts an amphipathic α-helical structure upon binding a membrane. Recently it has been shown that αS can induce tubulation and vesiculation of vesicles (Varkey et al. 2010, J Biol Chem, 285(42):32486). In contrast, the amphipathic helix H0 is unable, on its own, to induce tubulation, as N-BAR relies instead upon its large scaffolding domain (Fernandes et al. 2008, Biophys J, 94(8):3065). Using a combination of x-ray scattering and coarse-grained molecular dynamics simulations, we explored the membrane remodeling effects of αS. Our findings suggest that αS 1) causes a significant thinning of the bilayer; and 2) stabilizes an anisotropic curvature-field of both positive mean curvature and negative Gaussian curvature. Simulations of H0 show a similar magnitude of the local curvature as with αS, however owing to its molecular length the curvature-field around H0 is isotropic. We propose that the difference in curvature-field anisotropy explains the difference in the two proteins’ abilities to induce macroscopic curvature (i.e. tubulation) of lipid vesicles.

Direct and synergistic hemolysis caused by Staphylococcus phenol-soluble modulins: implications for diagnosis and pathogenesis

Phenol-soluble modulins are secreted staphylococcal peptides with an amphipathic α-helical structure. Some PSMs are strongly cytolytic toward human neutrophils and represent major virulence determinants during Staphylococcus aureus skin and blood infection. However, capacities of PSMs to lyse human erythrocytes have not been investigated. Here, we demonstrate that many S. aureus and Staphylococcus epidermidis PSMs lyse human erythrocytes. Furthermore, synergism with S. aureus β-toxin considerably increased the hemolytic capacities of several PSMs. This synergism may be of key importance in PSM and β-toxin-producing S. aureus or in mixed-strain or -species infections with PSM and β-toxin producers. Of specific interest, several PSMs, in particular PSMα peptides, contributed to a considerable extent to synergistic hemolysis with β-toxin or when using the β-toxin-producing strain RN4220 in CAMP assays. Thus, CAMP-type assays should not be used to detect or quantify S. aureus δ-toxin production, but may be used for an overall assessment of Agr functionality. Our study suggests an additional role of PSMs in staphylococcal pathogenesis and demonstrates that the repertoire of staphylococcal hemolysins is not limited to S. aureus and is much larger and diverse than previously thought.