Adhesion and protease activity in cell lines from human salivary gland tumors are regulated by the laminin-derived peptide AG73, syndecan-1 and beta 1 integrin

We studied the induction of protease activity by the laminin alpha 1-derived peptide AG73 in cells from adenoid cystic carcinoma (CAC2) and myoepithelioma (M1), respectively a malignant and a benign salivary gland tumors. Laminin alpha 1 chain and MMP9 were immunolocalized in adenoid cystic carcinoma and myoepithelioma in vivo and in vitro. Cells grown inside AG73-enriched laminin-111 exhibited large spaces in the extracellular matrix, suggestive of remodeling. The broad spectrum MMP inhibitor GM6001 decreased spaces induced by AG73 in CAC2 and M I cells. This result strongly suggests that AG73-mediated matrix remodeling involves matrix metalloproteinases. CAC2 and M1 cells cultured on AG73 showed a dose-dependent increase of MMP9 secretion, as detected by zymography. Furthermore, siRNA silencing of MMP9 decreased remodeling in 3D cultures. We searched for AG73 receptors regulating MMP9 activity in our cell lines. CAC2 and M1 cells grown on AG73 exhibited colocalization of syndecan-1 and beta 1 integrin. siRNA knockdown of syndecan-1 expression in these cells resulted in decreased adhesion to AG73 and reduced protease and remodeling activity. We investigated syndecan-1 co-receptors in both cell lines. Silencing beta 1 integrin inhibited adhesion to AG73, matrix remodeling and protease activity. Double-knockdown experiments were carried out to further explore syndecan-1 and beta 1 integrin cooperation. CAC2 cells transfected with both syndecan-1 and beta 1 integrin siRNA oligos showed significant decrease in adhesion to AG73. Simultaneous silencing of receptors also induced a decrease in protease activity. Our results suggest that syndecan-1 and beta 1 integrin signaling downstream of AG73 regulate adhesion and MMP production by CAC2 and M1 cells. (c) 2008 Elsevier B.V./International Society of Matrix Biology. All rights reserved.

Study of the mechanism of action of anoplin, a helical antimicrobial decapeptide with ion channel-like activity, and the role of the amidated C-terminus

Anoplin, an antimicrobial, helical decapeptide from wasp venom, looses its biological activities by mere deamidation of its C-terminus. Secondary structure determination, by circular dichroism spectroscopy in amphipathic environments, and lytic activity in zwitterionic and anionic vesicles showed quite similar results for the amidated and the carboxylated forms of the peptide. The deamidation of the C-terminus introduced a negative charge at an all-positive charged peptide, causing a loss of amphipathicity, as indicated by molecular dynamics simulations in TFE/water mixtures and this subtle modification in a peptide`s primary structure disturbed the interaction with bilayers and biological membranes. Although being poorly lytic, the amidated form, but not the carboxylated, presented ion channel-like activity on anionic bilayers with a well-defined conductance step; at approximately the same concentration it showed antimicrobial activity. The pores remain open at trans-negative potentials, preferentially conducting cations, and this situation is equivalent to the interaction of the peptide with bacterial membranes that also maintain a high negative potential inside. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.

Immunomodulatory effects of crotoxin isolated from Crotalus durissus terrificus venom in mice immunised with human serum albumin

Crotalus durissus terrificus venom and its main component, crotoxin (CTX), have the ability to down-modulate the immune system. Certain mechanisms mediated by cells and soluble factors of the immune system are responsible for the elimination of pathogenic molecules to ensure the specific protection against subsequent antigen contact. Accordingly, we evaluated the immunomodulatory effects of CTX on the immune response of mice that had been previously primed by immunisation with human serum albumin (HSA). CTX inoculation after HSA immunisation, along with complete Freund`s adjuvant (CFA) or Aluminium hydroxide (Alum) immunisation, was able to suppress anti-HSA IgG1 and IgG2a antibody production. We showed that the inhibitory effects of this toxin are not mediated by necrosis or apoptosis of any lymphoid cell population. Lower proliferation of T lymphocytes from mice immunised with HSA/CFA or HSA/Alum that received the toxin was observed in comparison to the mice that were only immunised. In conclusion, CTX is able to exert potent inhibitory effects on humoural and cellular responses induced by HSA immunisation, even when injected after an innate immune response has been initiated. (C) 2011 Elsevier Ltd. All rights reserved.

Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells

Carraro-Lacroix LR, Malnic G, Girardi AC. Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells. Am J Physiol Renal Physiol 297: F1647-F1655, 2009. First published September 23, 2009; doi:10.1152/ajprenal.00082.2009.-The gut incretin hormone glucagon-like peptide 1 (GLP-1) is released in response to ingested nutrients and enhances insulin secretion. In addition to its insulinotropic properties, GLP-1 has been shown to have natriuretic actions paralleled by a diminished proton secretion. We therefore studied the role of the GLP-1 receptor agonist exendin-4 in modulating the activity of Na(+)/H(+) exchanger NHE3 in LLC-PK(1) cells. We found that NHE3-mediated Na(+)-dependent intracellular pH (pH(i)) recovery decreased similar to 50% after 30-min treatment with 1 nM exendin-4. Pharmacological inhibitors and cAMP analogs that selectively activate protein kinase A (PKA) or the exchange protein directly activated by cAMP (EPAC) demonstrated that regulation of NHE3 activity by exendin-4 requires activation of both cAMP downstream effectors. This conclusion was based on the following observations: 1) the PKA antagonist H-89 completely prevented the effect of the PKA activator but only partially blocked the exendin-4-induced NHE3 inhibition; 2) the MEK1/2 inhibitor U-0126 abolished the effect of the EPAC activator but only diminished the exendin-4-induced NHE3 inhibition; 3) combination of H-89 and U-0126 fully prevented the effect of exendin-4 on NHE3; 4) no additive effect in the inhibition of NHE3 activity was observed when exendin-4, PKA, and EPAC activators were used together. Mechanistically, the inhibitory effect of exendin-4 on pHi recovery was associated with an increase of NHE3 phosphorylation. Conversely, this inhibition took place without changes in the surface expression of the transporter. We conclude that GLP-1 receptor agonists modulate sodium homeostasis in the kidney, most likely by affecting NHE3 activity.

Interactions of mast cell degranulating peptides with model membranes: A comparative biophysical study

In the last decade, there has been renewed interest in biologically active peptides in fields like allergy, autoimmume diseases and antibiotic therapy. Mast cell degranulating peptides mimic G-protein receptors, showing different activity levels even among homologous peptides. Another important feature is their ability to interact directly with membrane phospholipids, in a fast and concentration-dependent way. The mechanism of action of peptide HR1 on model membranes was investigated comparatively to other mast cell degranulating peptides (Mastoparan, Eumenitin and Anoplin) to evidence the features that modulate their selectivity. Using vesicle leakage, single-channel recordings and zeta-potential measurements, we demonstrated that HR1 preferentially binds to anionic bilayers, accumulates, folds, and at very low concentrations, is able to insert and create membrane spanning ion-selective pores. We discuss the ion selectivity character of the pores based on the neutralization or screening of the peptides charges by the bilayer head group charges or dipoles. (C) 2009 Elsevier Inc. All rights reserved.

The interaction of an antiparasitic peptide active against African Sleeping Sickness with cell membrane models

Zwitterionic peptides with trypanocidal activity are promising lead compounds for the treatment of African Sleeping Sickness, and have motivated research into the design of compounds capable of disrupting the protozoan membrane. In this study, we use the Langmuir monolayer technique to investigate the surface properties of an antiparasitic peptide, namely S-(2,4-dinitrophenyl)glutathione di-2-propyl ester, and its interaction with a model membrane comprising a phospholipid monolayer. The drug formed stable Langmuir monolayers. whose main feature was a phase transition accompanied by a negative surface elasticity. This was attributed to aggregation upon compression due to intermolecular bond associations of the molecules, inferred from surface pressure and surface potential isotherms. Brewster angle microscopy (BAM) images, infrared spectroscopy and dynamic elasticity measurements. When co-spread with dipalmitoyl phosphatidyl choline (DPPC). the drug affected both the surface pressure and the monolayer morphology, even at high surface pressures and with low amounts of the drug. The results were interpreted by assuming a repulsive, cooperative interaction between the drug and DPPC molecules. Such repulsive interaction and the large changes in fluidity arising from drug aggregation may be related to the disruption of the membrane, which is key for the parasite killing property. (C) 2009 Elsevier B.V. All rights reserved.

The lipid composition of a cell membrane modulates the interaction of an antiparasitic peptide at the air-water interface

The antiparasitic property of peptides is believed to be associated with their interactions with the protozoan membrane, which calls for research on the identification of membrane sites capable of peptide binding. In this study we investigated the interaction of a lipophilicglutathioine peptide known to be effective against the African Sleeping Sickness (ASS - African Trypanosomiasis) and cell membrane models represented by Langmuir monolayers. It is shown that even small amounts of the peptide affect the monolayers of some phospholipids and other lipids, which points to a significant interaction. The latter did not depend on the electrical charge of the monolayer-forming molecules but the peptide action was particularly distinctive for cholesterol + sphingomyelin monolayers that roughly resemble rafts on a cell membrane. Using in situ polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), we found that the orientation of the peptide is affected by the phospholipids and dioctadecyldimethylammonium bromide (DODAB), but not in monolayers comprising cholesterol + sphingomyelin. In this mixed monolayer resembling rafts, the peptide still interacts and has some induced order, probably because the peptide molecules are fitted together into a compact monolayer. Therefore, the lipid composition of the monolayer modulates the interaction with the lipophilic glutathioine peptide, and this may have important implications in understanding how the peptide acts on specific sites of the protozoan membrane. (C) 2011 Elsevier B.V. All rights reserved.

Jararhagin, a snake venom metalloprotease-disintegrin, activates the Rac1 GTPase and stimulates neurite outgrowth in neuroblastoma cells

It has been shown previously that the snake venom metalloprotease-disintegrin jararhagin stimulates cell migration and cytoskeletal rearrangement, independently of its effects on cellular adhesion but possibly associated with the activation of small GTP-binding proteins from the Rho family [Costa, E.P., Santos, M.F., 2004. Toxicon 44(8), 861-870.] Here we show that jararhagin stimulates spreading, actin dynamics and neurite outgrowth in neuroblastoma cells, and that this effect is accompanied by the translocation of the Rac1 small GTPase to the membrane fraction, suggesting its activation. Stimulation of neurite outgrowth was observed within minutes and was dependent on the proteolytic activity of the toxin. These results suggest that jararhagin may stimulate neuronal differentiation, being potential tool for neuronal regeneration studies. (C) 2008 Elsevier Ltd. All rights reserved.

Interaction of a C-terminal peptide of Bos taurus diacylglycerol acyltransferase 1 with model membranes

Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final and dedicated step in the synthesis of triacylglycerol, which is believed to involve the lipids oleoyl coenzyme A (OCoA) and dioleoyl-sn-glycerol (DOG) as substrates. In this work we investigated the interaction of a specific peptide, referred to as SIT2, on the C-terminal of DGAT1 (HKWCIRHFYKP) with model membranes made with OCoA and DOG in Langmuir monolayers and liposomes. According to the circular dichroism and fluorescence data, conformational changes on SIT2 were seen only on liposomes containing OCoA and DOG. In Langmuir monolayers, SIT2 causes the isotherms of neat OCoA and DOG monolayers to be expanded, but has negligible effect on mixed monolayers of OCoA and DOG. This synergistic interaction between SIT2 and DOG + OCoA may be rationalized in terms of a molecular model in which SIT2 may serve as a linkage between the two lipids. Our results therefore provide molecular-level evidence for the interaction between this domain and the substrates OCoA and DOG for the synthesis of triacylglycerol. (C) 2009 Elsevier B.V. All rights reserved.

The three-dimensional structure of bothropasin, the main hemorrhagic factor from Bothrops jararaca venom: Insights for a new classification of snake venom metalloprotease subgroups

Bothropasin is a 48 kDa hemorrhagic PIII snake venom metalloprotease (SVMP) isolated from Bothrops jararaca, containing disintegrin/cysteine-rich adhesive domains. Here we present the crystal structure of bothropasin complexed with the inhibitor POL647. The catalytic domain consists of a scaffold of two subdomains organized similarly to those described for other SVMPs, including the zinc and calcium-binding sites. The free cysteine residue Cys(189) is located within a hydrophobic core and it is not available for disulfide bonding or other interactions. There is no identifiable secondary structure for the disintegrin domain, but instead it is composed mostly of loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and is structurally related to the RGD region of RGD disintegrins, which are derived from I`ll SVMPs. The ECD motif is stabilized by the Cys(117)_Cys(310) disulfide bond (between the disintegrin and cysteine-rich domains) and by one calcium ion. The side chain of Glu(276) of the ECD motif is exposed to solvent and free to make interactions. In bothropasin, the HVR (hyper-variable region) described for other Pill SVMPs in the cysteine-rich domain, presents a well-conserved sequence with respect to several other Pill members from different species. We propose that this subset be referred to as PIII-HCR (highly conserved region) SVMPs. The differences in the disintegrin-like, cysteine-rich or disintegrin-like cysteine-rich domains may be involved in selecting target binding, which in turn could generate substrate diversity or specificity for the catalytic domain. (C) 2008 Elsevier Ltd. All rights reserved.

Ca(2+)-mediated thiolysis of peptide-resin linkage as an option for preparing protected peptide thioesters

A mild new procedure for preparing protected peptide thioesters, based oil Ca(2+)-assisted thiolysis of peptide-Kaiser oxime resin (KOR) linkage, is described. Ac-Ile-Ser(Bzl)-Asp(OcHx)-SR (Ac: acetyl; Bzl: benzyl; cHx: cyclohexyl), model peptide, was readily released from the resin by incubating the peptide-KOR at 60 degrees C in mixtures of DMF with n-butanethiol [R = (CH(2))(3)CH(3)] or ethyl 3-mercaptopropionate [R = (CH(2))(2)COOCHCH(3)] containing Ca(CH(3)COO)(2). After serine and aspartic acid side-chain deprotection under acid conditions, Ac-Ile-Ser-Asp-S(CH(2))(2)COOCH(2)CH(3) was successfully obtained with good quality and high yield. This type of C-terminal modified peptide may act as an excellent acyl donor in peptide segment condensation by the thioester method, native chemical ligation and enzymatic methods. (c) 2008 Elsevier Ltd. All rights reserved.

A proposed EPR approach to evaluating agonist binding site of a peptide receptor

Angiotensin II (Ang II) and its transmembrane AT(1) receptor were selected in order to test an innovative strategy that might allow the assessment of the agonist binding site in the receptor molecule. With the use of the 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) paramagnetic probe, a biologically active agonist (TOAC(1)-Ang II), as well as an inactive control (TOAC(4)-Ang II) analogs were mixed in solution with various synthesized AT(1) fragments. Comparative intermolecular interactions, as estimated by analyzing the EPR spectra of solutions, suggested the existence of an agonist binding site containing a sequence composed of portions of the N-terminal (13-17) and the third extracellular loop (266-278) fragments of the AT(1) molecule. Therefore, this combined EPR-TOAC approach shows promise as an alternative for use also in other applications related to specific intermolecular association processes.

Effect of cholesterol on the interaction of the amphibian antimicrobial peptide DD K with liposomes

DD K is an antimicrobial peptide previously isolated from the skin of the amphibian Phyllomedusa distincta. The effect of cholesterol on synthetic DD K binding to egg lecithin liposomes was investigated by intrinsic fluorescence of tryptophan residue, measurements of kinetics of 5(6)-carboxyfluorescein (CF) leakage, dynamic light scattering and isothermal titration microcalorimetry. An 8 nm blue shift of tryptophan maximum emission fluorescence was observed when DD K was in the presence of lecithin liposomes compared to the value observed for liposomes containing 43 mol% cholesterol. The rate and the extent of CF release were also significantly reduced by the presence of cholesterol. Dynamic light scattering showed that lecithin liposome size increase from 115 to 140 nm when titrated with DD K but addition of cholesterol reduces the liposome size increments. Isothermal titration microcalorimetry studies showed that DD K binding both to liposomes containing cholesterol as to liposomes devoid of it is more entropically than enthalpically favored. Nevertheless, the peptide concentration necessary to furnish an adjustable titration curve is much higher for liposomes containing cholesterol at 43 mol% (2 mmol L-1) than in its absence (93 mu mol L-1). Apparent binding constant values were 2160 and 10,000 L mol(-1), respectively. The whole data indicate that DD K binding to phosphatidylcholine liposomes is significantly affected by cholesterol, which contributes to explain the low hemolytic activity of the peptide. (C) 2007 Elsevier Inc. All rights reserved.

Design of a Modern Liposome and Bee Venom Formulation for the Traditional VIT-Venom Immunotherapy

Traditional venom immunotherapy uses injections of whole bee venom in buffer or adsorbed in Al (OH)(3) in an expensive, time-consuming way. New strategies to improve the safety and efficacy of this treatment with a reduction of injections would, therefore, be of general interest. It would improve patient compliance and provide socio-economic benefits. Liposomes have a long tradition in drug delivery because they increase the therapeutic index and avoid drug degradation and secondary effects. However, bee venom melittin (Mel) and phospholipase (PLA(2)) destroy the phospholipid membranes. Our central idea was to inhibit the PLA(2) and Mel activities through histidine alkylation and or tryptophan oxidation (with pbb, para-bromo-phenacyl bromide, and/or NBSN-bromosuccinimide, respectively) to make their encapsulations possible within stabilized liposomes. We strongly believe that this formulation will be nontoxic but immunogenic. In this paper, we present the whole bee venom conformation characterization during and after chemical modification and after interaction with liposome by ultraviolet, circular dichroism, and fluorescence spectroscopies. The PLA(2) and Mel activities were, measured indirectly by changes in turbidity at 400(nm), rhodamine leak-out, and hemolysis. The native whole bee venom (BV) presented 78.06% of alpha-helical content. The alkylation (A-BV) and succynilation (S-BV) of BV increased 0.44 and 0.20% of its alpha-helical content. The double-modified venom (S-A-BV) had a 0.74% increase of alpha-helical content. The BV chemical modification induced another change on protein conformations observed by Trp that became buried with respect to the native whole BV. It was demonstrated that the liposomal membranes must contain pbb (SPC:Cho:pbb, 26:7:1) as a component to protect them from aggregation and/or fusion. The membranes containing pbb maintained the same turbidity (100%) after incubation with modified venom, in contrast with pbb-free membranes that showed a 15% size decrease. This size decrease was interpreted as membrane degradation and was corroborated by a 50% rhodamine leak-out. Another fact that confirmed our interpretation was the observed 100% inhibition of the hemolytic activity after venom modification with pbb and NBS (S-A-BV). When S-A-BV interacted with liposomes, other protein conformational changes were observed and characterized by the increase of 1.93% on S-A-BV alpha-helical content and the presence of tryptophan residues in a more hydrophobic environment. In other words, the S-A-BV interacted with liposomal membranes, but this interaction was not effective to cause aggregation, leak-out, or fusion. A stable formulation composed by S-A-BV encapsulated within liposomes composed by SPC:Cho:pbb, at a ratio of 26:7:1, was devised. Large unilamellar vesicles of 202.5 nm with a negative surface charge (-24.29 mV) encapsulated 95% of S-A-BV. This formulation can, now, be assayed on VIT.

Effects of the antimicrobial peptide gomesin on the global gene expression profile, virulence and biofilm formation of Xylella fastidiosa

In the xylem vessels of susceptible hosts, such as citrus trees, Xylella fastidiosa forms biofilm-like colonies that can block water transport, which appears to correlate to disease symptoms. Besides aiding host colonization, bacterial biofilms play an important role in resistance against antimicrobial agents, for instance antimicrobial peptides (AMPs). Here, we show that gomesin, a potent AMP from a tarantula spider, modulates X. fastidiosa gene expression profile upon 60 min of treatment with a sublethal concentration. DNA microarray hybridizations revealed that among the upregulated coding sequences, some are related to biofilm production. In addition, we show that the biofilm formed by gomesin-treated bacteria is thicker than that formed by nontreated cells or cells exposed to streptomycin. We have also observed that the treatment of X. fastidiosa with a sublethal concentration of gomesin before inoculation in tobacco plants correlates with a reduction in foliar symptoms, an effect possibly due to the trapping of bacterial cells to fewer xylem vessels, given the enhancement in biofilm production. These results warrant further investigation of how X. fastidiosa would respond to the AMPs produced by citrus endophytes and by the insect vector, leading to a better understanding of the mechanism of action of these molecules on bacterial virulence.