Peptide Mastoparan Research Articles

A Rare Case Report of Corneal Wasp Sting

Corneal sting by wasp is a relatively rare phenomenon. Wasp, bees and yellow jackets belong to Hymenoptera species of insects. A triad of mechanical, toxic and immunologic damage has been proposed to explain the spectrum of injuries from wasp stings. Wasp venom is a complex mixture of toxic substances such as Phospholipase A, Phospholipase B, Apamine, Hyalouronidase, Mast cell degranulating peptide and Mastoparan peptide, which is implicated in direct mast cell degranulation leading to histamine release. A wide range of immunologically mediated ocular sequel have been described which include focal or diffuse corneal edema, bullous keratopathy, panuveitis, hyphema, anterior polar cataract, papillodema, lens subluxation and optic neuritis. The author hereby tends to report a case of Striate Keratitis following wasp sting, its expected pathogenesis, treatment and outcome.

The Gαo Activator Mastoparan-7 Promotes Dendritic Spine Formation in Hippocampal Neurons.

Mastoparan-7 (Mas-7), an analogue of the peptide mastoparan, which is derived from wasp venom, is a direct activator of Pertussis toxin- (PTX-) sensitive G proteins. Mas-7 produces several biological effects in different cell types; however, little is known about how Mas-7 influences mature hippocampal neurons. We examined the specific role of Mas-7 in the development of dendritic spines, the sites of excitatory synaptic contact that are crucial for synaptic plasticity. We report here that exposure of hippocampal neurons to a low dose of Mas-7 increases dendritic spine density and spine head width in a time-dependent manner. Additionally, Mas-7 enhances postsynaptic density protein-95 (PSD-95) clustering in neurites and activates Gα o signaling, increasing the intracellular Ca2+ concentration. To define the role of signaling intermediates, we measured the levels of phosphorylated protein kinase C (PKC), c-Jun N-terminal kinase (JNK), and calcium-calmodulin dependent protein kinase IIα (CaMKIIα) after Mas-7 treatment and determined that CaMKII activation is necessary for the Mas-7-dependent increase in dendritic spine density. Our results demonstrate a critical role for Gα o subunit signaling in the regulation of synapse formation.

A fusion protein containing the adenovirus type 2 fiber trimerizing and knob domains linked to HIV-1 gp120 exhibits increased nasal immunogenicity in rabbits (VAC8P.1046)

Abstract We previously reported that the inclusion of the trimerizing and knob domains from adenovirus type 2 fiber (Ad2F) in protein immunogens enhanced their mucosal binding and immunogenicity when delivered nasally. This study was performed to determine if Ad2F would enhance the nasal immunogenicity of HIV-1 gp120 from the Clade C Env C.1086. A fusion protein with Ad2F at the C-terminus of HIV-1 gp120 was used as gp120-Ad2F. NZW rabbits were nasally immunized with 100, 200 or 300 µg of gp120 or equimolar doses of gp120-Ad2F fusion proteins adjuvanted with the cationic mast cell activating peptide mastoparan 7 on days 0 and boosted on days 28 - 34. Intramuscular immunization with 100 µg gp120 adjuvanted with an MF59-like adjuvant served as the positive control. Serum anti-HIV-1 gp120 IgG titers were measured 2 weeks after the final boost. While only the 200 and 300 µg doses of gp120 delivered intranasally induced serum anti-gp120 IgG similar to those induced by IM immunization, all doses of gp120-Ad2F fusion proteins induced serum anti-gp120 IgG titers similar to those induced by IM immunization. While the 100 µg dose of gp120 delivered nasally induced serum anti-gp120 IgG titers significantly lower than that induced by 300 µg of gp120 delivered nasally, all doses of gp120-Ad2F induced similar anti-gp120 IgG titers. Our results demonstrate that the addition of Ad2F to HIV-1 gp120 enhances its nasal immunogenicity, lessening the amount of gp120 required for vaccination.

Quantification of leakage from large unilamellar lipid vesicles by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique that in recent years has found numerous applications for studying biological phenomena. In this article, we scrutinize one of these applications, namely, FCS as a technique for studying leakage of fluorescent molecules from large unilamellar lipid vesicles. Specifically, we derive the mathematical framework required for using FCS to quantify leakage of fluorescent molecules from large unilamellar lipid vesicles, and we describe the appropriate methodology for successful completion of FCS experiments. By use of this methodology, we show that FCS can be used to accurately quantify leakage of fluorescent molecules from large unilamellar lipid vesicles, including leakage of fluorescent molecules of different sizes. To demonstrate the applicability of FCS, we have investigated the antimicrobial peptide mastoparan X. We show that mastoparan X forms transient transmembrane pores in POPC/POPG (3:1) vesicles, resulting in size-dependent leakage of molecules from the vesicles. We conclude the paper by discussing some of the advantages and limitations of FCS as compared to other existing methods to measure leakage from large unilamellar lipid vesicles.

Mastoparan peptide causes mitochondrial permeability transition not by interacting with specific membrane proteins but by interacting with the phospholipid phase.

The mastoparan peptide is known as an inducer of the mitochondrial permeability transition. Although mastoparan was suggested to interact with a proteinaceous target in mitochondria to induce this transition, the action sites of mastoparan have not yet been investigated. To clarify whether specific interactions of mastoparan with receptors or enzymes are associated with the induction of this permeability transition, we examined the effects of d-isomeric peptides, which were synthesized using d-amino acids assembled in endogenous (inverso mastoparan) and reverse (retro-inverso mastoparan) orientations. When we added inverso mastoparan to isolated mitochondria, the peptide caused the permeability transition in a partially cyclosporin A-sensitive manner at lower doses and in a cyclosporin A-insensitive manner at higher ones. The manners of action and the potencies of inverso mastoparan were close to those of parent mastoparan, indicating that the targets of mastoparan for induction of the permeability transition were neither receptors, nor enzymes in the mitochondria. Retro-inverso mastoparan also had the same effect on the mitochondria as mastoparan, although the potencies of the effect were weaker. Not only on mitochondria, but also on phospholipid vesicles, mastoparan and inverso mastoparan showed massive permeabilization effects at the same potencies, although retro-inverso mastoparan showed weaker ones. These results indicate that mastoparan interacted with the phospholipid phase of the mitochondrial membrane (and not with specific proteins) to induce the permeabilization in cyclosporin A-sensitive and -insensitive manners.

The Role of the Cyclic Nucleotide Binding Homology Domain in Voltage and Calcium Dependent Gating of EAG Potassium Channels

Ether-a-go-go (EAG or KCNH1) potassium channels are inhibited by increases of intracellular calcium concentration [Ca2+]i through a calmodulin (CaM) mediated mechanism. Three CaM binding sites have already been identified on intracellular domains, one close to the N-terminally located eag-domain and two close to the cyclic nucleotide binding homology domain (cNBHD) on the C-terminus. Crystal structures of the cNBHD have revealed that instead of binding cyclic nucleotides the binding pocket is occupied by a short β-strand (intrinsic ligand). In this study we set out to elucidate the role of the cNBHD and intrinsic ligand in CaM regulation of these channels. hEAG1 currents were recorded in Xenopus oocytes using two electrode voltage clamp. Elevation of [Ca2+]i by application of the Ca2+-ionophore ionomycin (5μM) and SERCA inhibitor thapsigargin (5μM) profoundly inhibited wild-type (WT) hEAG1 currents in a manner that was blocked by the CaM binding peptide mastoparan. Mutation of the intrinsic ligand slowed the time to 80% activation at +40 mV (t80%,act) from 256.35 ± 31.05 ms (n=6) in WT hEAG1 to 1046 ± 15.62 ms (n=3) in Y672A:L674A hEAG1. Substitution of binding pocket residues with large residues also caused similar slowing of t80%,act (e.g. 682.6 ± 17.9 ms for A609L, n=3). Despite this, none of these mutations caused a reduction in Ca2+-sensitivity. However, deletion of the cNBHD abolished the response to elevated [Ca2+]i and also had a profound effect on channel gating (t80%,act 426.6 ± 44.67 ms, n=3). Overall, our results suggest that an intact cNBHD is required for Ca2+-dependent inhibition, but reducing the affinity of the intrinsic ligand for its binding pocket is important for gating not Ca2+-sensitivity.

Antimicrobial peptides from the venom gland of the social wasp Vespa tropica

Peptide agents are regarded as potential candidates for overcoming the life-threatening resistance of pathogenic microorganisms to classic antibiotics. Accordingly, a peptidomic and genomic investigation of natural antimicrobial peptides (AMPs) can provide structure–functional information for designing peptide antibiotics with therapeutic potential. In the present study, we identified nine AMPs from the venom gland of the wasp Vespa tropica using combined methods of peptidomics and genomics. These AMPs were classified into two different families based on sequence similarity, mastoparan and vespid chemotactic peptides (VCPs), and thus named mastoparan-VT1 to -VT7, VCP-VT1 and -VT2. Among these nine AMPs, mastoparan-VT1 and VCP-VT1 are identical to peptides from other wasps. These AMPs exerted broad-spectrum antimicrobial activity against standard and clinically isolated strains of bacteria. In addition, they showed weak hemolytic activity toward human erythrocytes. Our results reveal that identical AMPs are widely distributed in different wasp venoms and might provide templates for the development of novel peptide antibiotics.

A mastoparan-derived peptide has broad-spectrum antiviral activity against enveloped viruses

Broad-spectrum antiviral drugs are urgently needed to treat individuals infected with new and re-emerging viruses, or with viruses that have developed resistance to antiviral therapies. Mammalian natural host defense peptides (mNHP) are short, usually cationic, peptides that have direct antimicrobial activity, and which in some instances activate cell-mediated antiviral immune responses. Although mNHP have potent activity in vitro, efficacy trials in vivo of exogenously provided mNHP have been largely disappointing, and no mNHP are currently licensed for human use. Mastoparan is an invertebrate host defense peptide that penetrates lipid bilayers, and we reasoned that a mastoparan analog might interact with the lipid component of virus membranes and thereby reduce infectivity of enveloped viruses. Our objective was to determine whether mastoparan-derived peptide MP7-NH2 could inactivate viruses of multiple types, and whether it could stimulate cell-mediated antiviral activity. We found that MP7-NH2 potently inactivated a range of enveloped viruses. Consistent with our proposed mechanism of action, MP7-NH2 was not efficacious against a non-enveloped virus. Pre-treatment of cells with MP7-NH2 did not reduce the amount of virus recovered after infection, which suggested that the primary mechanism of action in vitro was direct inactivation of virus by MP7-NH2. These results demonstrate for the first time that a mastoparan derivative has broad-spectrum antiviral activity in vitro and suggest that further investigation of the antiviral properties of mastoparan peptides in vivo is warranted.

Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes

BackgroundThe peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics). MethodsHuman erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin–luciferase based real-time luminometry. ResultsExposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%.Erythrocytes pre-exposure to 10μM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux.Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers. ConclusionsMST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers. General significanceKinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.

Identification of the Thiol Isomerase-binding Peptide, Mastoparan, as a Novel Inhibitor of Shear-induced Transforming Growth Factor β1 (TGF-β1) Activation

TGF-β1 is a disulfide-bonded homodimeric protein produced by platelets and other cells that plays a role in many physiologic and pathologic processes. TGF-β1 is secreted as an inactive large latent complex (LLC) comprised of TGF-β1, latency-associated peptide, and latent TGF-β binding protein 1. We previously demonstrated that shear force can activate LLC and that thiol-disulfide exchange contributes to the process. We have now investigated the role of thiol isomerases in the activation of LLC in platelet releasates (PR) and recombinant LLC. The wasp venom peptide mastoparan, which inhibits the chaperone activity of PDI, inhibited stirring- and shear-induced activation of latent TGF-β1 by 90 and 75% respectively. To identify the proteins that bind to mastoparan either directly or indirectly, PR were chromatographed on a mastoparan affinity column. Latent TGF-β binding protein 1, latency-associated peptide, TGF-β1, clusterin, von Willebrand factor, multimerin-1, protein disulfide isomerase (PDI), ERp5, ERp57, and ERp72 eluted specifically from the column. Anti-PDI RL90 attenuated the inhibitory effect of mastoparan on LLC activation. Furthermore, reduced PDI inhibited activation of PR LLC, whereas oxidized PDI had no effect. We conclude that thiol isomerases and thiol-disulfide exchange contribute to TGF-β1 activation and identify a number of molecules that may participate in the process.

Proteomic profiling of the molecular targets of interactions of the mastoparan peptide Protopolybia MP‐III at the level of endosomal membranes from rat mast cells

It is well known that the activation of mast cells due to the binding of mastoparan to the G(α) subunit of trimeric G proteins involves exocytosis regulation. However, experimental evidence in the literature indicates that mastoparan can also activate certain regulatory targets of exocytosis at the level of the mast cell endosomal membranes that have not yet been identified. Therefore, the aim of the present investigation was the proteomic identification of these targets. To achieve these objectives, mast cells were activated by the peptide Protopolybia MP-III, and the proteins of the endosomal membranes were converted to proteoliposomes using sonication. Proteins were separated from one another by affinity chromatography using proteoliposomes as analytes and Protopolybia MP III-immobilized Sepharose 4B resin as the ligand. This experimental approach, which used SDS-PAGE, in-gel trypsin digestion and proteomic analysis, permitted the identification of five endosomal proteins: Rho GTPase Cdc 42 and exocyst complex component 7 as components of the Ca(2+) -independent FcεRI-mediated exocytosis pathway, synaptosomal-associated protein 29, and GTP-binding protein Rab3D as components of the Ca(2+) -dependent FcεRI-mediated exocytosis pathway and Ras-related protein M-Ras, a protein that is related to the mediation of cell shaping and proliferation following exocytosis. The identification of the five proteins as targets of mastoparans may contribute in the near future to the use of this family of peptides as novel tools for dissecting the mechanism of exocytosis in mast cells.

Agelaia MP-I: A peptide isolated from the venom of the social wasp, Agelaia pallipes pallipes, enhances insulin secretion in mice pancreatic islets

Peptides isolated from animal venoms have shown the ability to regulate pancreatic beta cell function. Characterization of wasp venoms is important, since some components of these venoms present large molecular variability, and potential interactions with different signal transduction pathways. For example, the well studied mastoparan peptides interact with a diversity of cell types and cellular components and stimulate insulin secretion via the inhibition of ATP dependent K+ (KATP) channels, increasing intracellular Ca2+ concentration. In this study, the insulin secretion of isolated pancreatic islets from adult Swiss mice was evaluated in the presence of synthetic Agelaia MP-I (AMP-I) peptide, and some mechanisms of action of this peptide on endocrine pancreatic function were characterized. AMP-I was manually synthesized using the Fmoc strategy, purified by RP-HPLC and analyzed using ESI-IT-TOF mass spectrometry. Isolated islets were incubated at increasing glucose concentrations (2.8, 11.1 and 22.2 mM) without (Control group: CTL) or with 10 μM AMP-I (AMP-I group). AMP-I increased insulin release at all tested glucose concentrations, when compared with CTL (P < 0.05). Since molecular analysis showed a potential role of the peptide interaction with ionic channels, insulin secretion was also analyzed in the presence of 250 μM diazoxide, a KATP channel opener and 10 μM nifedipine, a Ca2+ channel blocker. These drugs abolished insulin secretion in the CTL group in the presence of 2.8 and 11.1 mM glucose, whereas AMP-I also enhanced insulin secretory capacity, under these glucose conditions, when incubated with diazoxide and nifedipine. In conclusion, AMP-I increased beta cell secretion without interfering in KATP and L-type Ca2+ channel function, suggesting a different mechanism for this peptide, possibly by G protein interaction, due to the structural similarity of this peptide with Mastoparan-X, as obtained by modeling.

Influence of the Bilayer Composition on the Binding and Membrane Disrupting Effect of Polybia-MP1, an Antimicrobial Mastoparan Peptide with Leukemic T-Lymphocyte Cell Selectivity

This study shows that MP-1, a peptide from the venom of the Polybia paulista wasp, is more toxic to human leukemic T-lymphocytes than to human primary lymphocytes. By using model membranes and electrophysiology measurements to investigate the molecular mechanisms underlying this selective action, the porelike activity of MP-1 was identified with several bilayer compositions. The highest average conductance was found in bilayers formed by phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylserine (70:30). The presence of cholesterol or cardiolipin substantially decreases the MP-1 pore activity, suggesting that the membrane fluidity influences the mechanism of selective toxicity. The determination of partition coefficients from the anisotropy of Trp indicated higher coefficients for the anionic bilayers. The partition coefficients were found to be 1 order of magnitude smaller when the bilayers contain cholesterol or a mixture of cholesterol and sphingomyelin. The blue shift fluorescence, anisotropy values, and Stern-Volmer constants are indications of a deeper penetration of MP-1 into anionic bilayers than into zwitterionic bilayers. Our results indicate that MP-1 prefers to target leukemic cell membranes, and its toxicity is probably related to the induction of necrosis and not to DNA fragmentation. This mode of action can be interpreted considering a number of bilayer properties like fluidity, lipid charge, and domain formation. Cholesterol-containing bilayers are less fluid and less charged and have a tendency to form domains. In comparison to healthy cells, leukemic T-lymphocyte membranes are deprived of this lipid, resulting in decreased peptide binding and lower conductance. We showed that the higher content of anionic lipids increases the level of binding of the peptide to bilayers. Additionally, the absence of cholesterol resulted in enhanced pore activity. These findings may drive the selective toxicity of MP-1 to Jurkat cells.

Application of bare gold nanoparticles in open‐tubular CEC separations of polyaromatic hydrocarbons and peptides

In this study, bare gold nanoparticles (GNPs) immobilized in the sol-gel-pretreated fused-silica (FS) capillary as a stationary phase for open-tubular capillary electrochromatography (OT-CEC) are for the first time shown to be able to separate both hydrophobic polyaromatic hydrocarbons (PAHs) as well as hydrophilic cationic antimicrobial peptides. Model mixture of four PAHs, naphthalene, fluorene, phenanthrene, and anthracene, was resolved by OT-CEC in the GNP-modified FS capillaries using the hydro-organic background electrolyte (BGE) composed of 20 mmol/L sodium phosphate buffer, pH 7, modified with ACN at 8:2 v/v ratio. On the other hand, three synthetic analogues of an antimicrobial peptide mastoparan PDD-B, basic tetradecapeptides INWKKLGKKILGAL-NH(2), INSLKLGKKILGAL-NH(2) and NWLRLGRRILGAL-NH(2), were separated in aqueous acidic BGEs, pH 2.1-3.1, composed of weak acids (formic and acetic) or amphoteric amino or imino acids (aspartic or iminodiacetic), utilizing the advantage of a slow reversed (anodic) EOF and slightly positive charge of the GNP-modified FS capillary suppressing the adsorption of cationic peptides on the inner capillary wall and improving their resolution.

Identification of Platelet Releasate Proteins that Bind to Mastoparan, a Peptide that Inhibits Shear-Induced TGF-β1 Activation,

Abstract 3271Transforming growth factor β1 (TGF-β1) is a disulfide-bonded, 25 kD homodimeric protein produced by most cell types, including platelets, that functions as a cytokine in many physiologic and pathologic processes. Platelets contain 40–100 times more TGF-β1 than other cells and release it as an inactive large latent complex (LLC) comprised of TGF-β1 non-covalently associated with its latency-associated peptide (LAP), which is, in turn, disulfide-bonded to latent TGF-β binding protein 1 (LTBP-1). Thrombospondin-1 (TSP1), proteases, and reactive oxygen species have all been shown to activate TGF-β1 in vitro and a role for integrins in vivo has been inferred from studies of transgenic mice. Recently, we discovered that shear force can activate latent TGF-β1 released from platelets in vitro and that thiol-disulfide exchange contributes to shear-dependent TGF-β1 activation. A number of thiol isomerase enzymes that can catalyze thiol-disulfide exchange have been identified in platelets, including protein disulfide isomerase (PDI), ERp5, ERp57, ERp72, ERp44, ERp29, and TMX3. As shear-induced activation of TGF-β1 is partially thiol-dependent, we investigated if thiol isomerases can affect this process. Mastoparan is a non-thiol-containing wasp venom peptide known to inhibit the chaperone activity of PDI, ERp5, and perhaps other thiol isomerases. We recently showed that mastoparan, (INLKALAALAKKIL), inhibits stirring-induced TGF-β1 activation by more than 90% (100 μM; n=3, p=0.03), whereas no inhibition was observed with an inactive mastoparan-like control peptide (INLKAKAALAKKLL) at 100 μM (n=3, p=0.66). To identify the proteins that bind to mastoparan, either directly or indirectly, platelet releasates were chromatographed on a mastoparan affinity column prepared from N-hydroxysuccinimide Sepharose. Two control columns were employed: 1. unconjugated Sepharose, and 2. Sepharose conjugated with the mastoparan-like control peptide. Elution of bound proteins was achieved by increasing the NaCl concentration. Proteins identified by mass spectrometry as specifically binding to the mastoparan peptide column included LTBP-1, TGF-β1 precursor, clusterin, coagulation Factor V, multimerin-1, 14-3-3 protein zeta/delta, and α-actinin 4. These results were confirmed by immunoblotting. Furthermore, the thiol isomerases PDI, ERp5, ERp57, and ERp72 were all found to bind specifically to mastoparan as confirmed by immunoblotting. We conclude that mastoparan affinity chromatography identified a number of proteins in platelet releasates that may contribute to shear-induced TGF-β1 activation. Disclosures:Coller:Centocor/Accumetrics/Rockefeller University:.

New Insight into the Mechanism of Action of Wasp Mastoparan Peptides: Lytic Activity and Clustering Observed with Giant Vesicles

Antimicrobial peptides of the mastoparans family exert their bactericidal activity by binding to lipid membranes, inducing pores or defects and leaking the internal contents of vesicles and cells. However, this does not seem to be the only mechanism at play, and they might be important in the search for improved peptides with lower undesirable side effects. This work deals with three mastoparans peptides, Polybia-MP-1(MP-1), N2-Polybia-MP-1 (N-MP-1), and Mastoparan X (MPX), which exhibit high sequence homology. They all have three lysine residues and amidated C termini, but because of the presence of two, one, and no aspartic acid residues, respectively, they have +2, +3, and +4 net charges at physiological pH. Here we focus on the effects of these mastoparans peptides on anionic model membranes made of palmitoleyoilphosphatidylcholine (POPC) and palmitoleyoilphosphatidylglycerol (POPG) at 1:1 and 3:1 molar ratios in the presence and in the absence of saline buffer. Zeta potential experiments were carried out to measure the extent of the peptides' binding and accumulation at the vesicle surface, and CD spectra were acquired to quantify the helical structuring of the peptides upon binding. Giant unilamellar vesicles were observed under phase contrast and fluorescence microscopy. We found that the three peptides induced the leakage of GUVs at a gradual rate with many characteristics of the graded mode. This process was faster in the absence of saline buffer. Additionally, we observed that the peptides induced the formation of dense regions of phospholipids and peptides on the GUV surface. This phenomenon was easily observable for the more charged peptides (MPX > N-MP-1 > MP-1) and in the absence of added salt. Our data suggest that these mastoparans accumulate on the bilayer surface and induce a transient interruption to its barrier properties, leaking the vesicle contents. Next, the bilayer recovers its continuity, but this happens in an inhomogeneous way, forming a kind of ply with peptides sandwiched between two juxtaposed membranes. Eventually, a peptide-lipid aggregate forming a lump is formed at high peptide-to-lipid ratios.

Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing

Laurdan and di-4-ANEPPDHQ are used as probes for membrane order, with a blue shift in emission for membranes in liquid-ordered (lo) phase relative to membranes in liquid-disordered (ld) phase. Their use as membrane order probes requires that their spectral shifts are unaffected by membrane proteins, which we have examined by using membrane inserting peptides and large unilamellar vesicles (LUVs). The transmembrane polypeptides, mastoparan and bovine prion protein-derived peptide (bPrPp), were added to LUVs of either lo or ld phase, up to 1:10 peptide/total lipid ratio. The excitation and emission spectra of laurdan and di-4-ANEPPDHQ in both lipid phases were unaltered by peptide addition. The integrity and size distribution of the LUVs upon addition of the polypeptides were determined by dynamic light scattering. The insertion efficiency of the polypeptides into LUVs was determined by measuring their secondary structure by circular dichroism. Mastoparan had an α-helical and bPrPp a β-strand conformation compatible with insertion into the lipid bilayer. Our results suggest that the presence of proteins in biological membranes does not influence the spectra of laurdan and di-4-ANEPPDHQ, supporting that the dyes are appropriate probes for assessing lipid order in cells.

The effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and CD spectroscopy

Some mastoparan peptides extracted from social wasps display antimicrobial activity and some are hemolytic and cytotoxic. Although the cell specificity of these peptides is complex and poorly understood, it is believed that their net charges and their hydrophobicity contribute to modulate their biological activities. We report a study, using fluorescence and circular dichroism spectroscopies, evaluating the influence of these two parameters on the lytic activities of five mastoparans in zwitterionic and anionic phospholipid vesicles. Four of these peptides, extracted from the venom of the social wasp Polybia paulista, present both acidic and basic residues with net charges ranging from +1 to +3 which were compared to Mastoparan-X with three basic residues and net charge +4. Previous studies revealed that these peptides have moderate-to-strong antibacterial activity against Gram-positive and Gram-negative microorganisms and some of them are hemolytic. Their affinity and lytic activity in zwitterionic vesicles decrease with the net electrical charges and the dose response curves are more cooperative for the less charged peptides. Higher charged peptides display higher affinity and lytic activity in anionic vesicles. The present study shows that the acidic residues play an important role in modulating the peptides' lytic and biological activities and influence differently when the peptide is hydrophobic or when the acidic residue is in a hydrophilic peptide.

Characterization of two novel polyfunctional mastoparan peptides from the venom of the social wasp Polybia paulista

Hymenoptera venoms are complex mixtures of biochemically and pharmacologically active components such as biogenic amines, peptides and proteins. Polycationic peptides generally constitute the largest group of Hymenoptera venom toxins, and the mastoparans constitute the most abundant and important class of peptides in the venom of social wasps. These toxins are responsible for histamine release from mast cells, serotonin from platelets, and catecholamines and adenylic acids from adrenal chromafin cells. The present work reports the structural and functional characterization of two novel mastoparan peptides identified from the venom of the neotropical social wasp Polybia paulista. The mastoparans Polybia-MP-II and -III were purified, sequenced and synthesized on solid phase using Fmoc chemistry and the synthetic peptides used for structural and functional characterizations. Polybia-MP-II and -III are tetradecapeptides, amidated at their C-termini, and form amphipathic α-helical conformations under membrane-mimetic conditions. Both peptides were polyfunctional, causing pronounced cell lysis of rat mast cells and erythrocytes, in addition to having antimicrobial activity against both Gram-positive and Gram-negative bacteria.

Solution structure of the bb′ domains of human protein disulfide isomerase

Protein disulfide isomerase is the most abundant and best studied of the disulfide isomerases that catalyze disulfide bond formation in the endoplasmic reticulum, yet the specifics of how it binds substrate have been elusive. Protein disulfide isomerase is composed of four thioredoxin-like domains (abb'a'). Cross-linking studies with radiolabeled peptides and unfolded proteins have shown that it binds incompletely folded proteins primarily via its third domain, b'. Here, we determined the solution structure of the second and third domains of human protein disulfide isomerase (b and b', respectively) by triple-resonance NMR spectroscopy and molecular modeling. NMR titrations identified a large hydrophobic surface within the b' domain that binds unfolded ribonuclease A and the peptides mastoparan and somatostatin. Protein disulfide isomerase-catalyzed refolding of reduced ribonuclease A in vitro was inhibited by these peptides at concentrations equal to their affinity to the bb' fragment. Our findings provide a structural basis for previous kinetic and cross-linking studies which have shown that protein disulfide isomerase exhibits a saturable, substrate-binding site.