Natriuretic peptide signalling in the gills of freshwater and seawater fishes

The gills are considered major targets for cardiac natriuretic peptides with studies confirming natriuretic peptide receptor presence on vascular and sometimes epithelial tissues. Natriuretic peptide intracellular signalling is via guanylyl cyclase receptors and the cGMP pathway, and via inhibitory G-proteins linked to cyclic AMP pathways. Natriuretic peptides in the gills alter branchial blood flow and may also alter ion transport in various salinities. We present an overview of natriuretic peptide cGMP and cAMP signalling in fishes and consider the implications of the recent discovery of several CNPs and BNP in bony fishes on natriuretic peptide receptor studies.

Amoebic gill disease and natriuretic peptide receptors in the gills of Atlantic salmon

Amoebic gill disease (AGD) is a problem in the farming of Atlantic salmon, and may compromise osmoregulatory, cardiovascular and respiratory functions. We examined the effects of AGD on atrial and C-type natriuretic peptide (ANP and CNP) stimulated branchial cyclic GMP formation, since natriuretic peptides (NPs) are involved in cardiovascular function and osmoregulation. NPs act via guanylyl cyclase receptors (NPR), which stimulate cGMP formation. NPR activity was measured by ANP and CNP stimulation of branchial cGMP formation, and compared between diseased and healthy salmon over an 11 day AGD infection. We also measured plasma osmolality. Osmolality increased in AGD infected salmon from an initial 355 mmol.kg-1 to 411 mmol.kg-1 at 11 days. There was no evidence that branchial cGMP formation changed in response to AGD. In all groups, CNP stimulation of guanylyl cyclase was 190% of basal rate, whereas ANP was 150% of basal. After 11 days, all groups were given a 4 h freshwater bath, the usual treatment for AGD. Another group was given a seawater to seawater transfer, to control for handling. In this group, plasma osmolality at 11 days was the same as in AGD fish. This elevation may be due to these fish experiencing disturbance for the first time in 11 days. ANP and CNP branchial NPR activity at the conclusion of the 4 h transfers was elevated in all groups compared to that at 11 days. The increased cGMP formation in the handling control suggests a NPR response to the transfer/handling stress. AGD fish demonstrated the greatest elevation in ANP and CNP guanylyl cyclase activity immediately following the bath; these values were greater than in the control groups. The AGD infected salmon, therefore, responded more emphatically to the freshwater treatment, suggesting that the NP system is involved in some aspects of AGD.

Potassium channel modulation by a toxin domain in matrix metalloprotease

Peptide toxins found in a wide array of venoms block K+ channels, causing profound physiological and pathological effects. Here we describe the first functional K+ channel-blocking toxin domain in a mammalian protein. MMP23 (matrix metalloprotease 23) contains a domain (MMP23TxD) that is evolutionarily related to peptide toxins from sea anemones. MMP23TxD shows close structural similarity to the sea anemone toxins BgK and ShK. Moreover, this domain blocks K+ channels in the nanomolar to low micromolar range (Kv1.6 > Kv1.3 > Kv1.1 = Kv3.2 > Kv1.4, in decreasing order of potency) while sparing other K+ channels (Kv1.2, Kv1.5, Kv1.7, and KCa3.1). Full-length MMP23 suppresses K+ channels by co-localizing with and trapping MMP23TxD-sensitive channels in the ER. Our results provide clues to the structure and function of the vast family of proteins that contain domains related to sea anemone toxins. Evolutionary pressure to maintain a channel-modulatory function may contribute to the conservation of this domain throughout the plant and animal kingdoms.

Protonectin (1-6): A novel chemotactic peptide from the venom of the social wasp Agelaia pallipes pallipes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Crystal Structure of Bucain, a Three-Fingered Toxin from the Venom of the Malayan Krait (Bungarus candidus)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Validation of a mutant of the pore-forming toxin sticholysin-I for the construction of proteinase-activated immunotoxins

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Design and synthesis of peptides from bacterial ParE toxin as inhibitors of topoisomerases

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Model peptides mimic the structure and function of the N-terminus of the pore-forming toxin sticholysin II

To investigate the role of the N-terminal region in the lytic mechanism of the pore-forming toxin sticholysin II (St II), we studied the conformational and functional properties of peptides encompassing the first 30 residues of the protein. Peptides containing residues 1-30 (P1-30) and 11-30 (P11-30) were synthesized and their conformational properties were examined in aqueous solution as a function of peptide concentration, pH, ionic strength, and addition of the secondary structure-inducing solvent trifluoroethanol (TFE). CD spectra showed that increasing concentration, pH, and ionic strength led to aggregation of P1-30; as a consequence, the peptide acquired beta-sheet conformation. In contrast, P11-30 exhibited practically no conformational changes under the same conditions, remaining essentially structureless. Moreover, this peptide did not undergo aggregation. These differences clearly point to the modulating effect of the first 10 hydrophobic residues on the peptides aggregation and conformational properties. In TFE both the first ten hydrophobic peptides acquired alpha-helical conformation, albeit to a different extent, P11-30 displayed lower alpha-helical content. P1-30 presented a larger-fraction of residues in alpha-helical conformation in TFE than that found in St II's crystal structure for that portion of the protein. Since TFE mimics the membrane em,, such increase in helical content could also occur upon toxin binding to membranes and represent a step in the mechanism of pore formation. The peptides conformational properties correlated well with their functional behaviour. Thus, P1-30 exhibited much higher hemolytic activity than P11-30. In addition, P11-30 was able to block the toxin's hemolytic activity. The size of pores formed in red blood cells by P 1-30 was estimated by measuring the permeability PEGs of different molecular mass. The pore radius (0.95 +/- 0.01 nm) was very similar to that of the PEGs of different pore formed by the toxin. The results demonstrate that the synthetic peptide P1-30 is a good model of St 11 conformation and function and emphasize the contribution of the toxin's N-terminal region, and, in particular, the hydrophobic residues 1-10 to pore formation. (c) 2005 Wiley Periodicals, Inc.

Fmoc-POAC: [(9-fluorenylmethyloxycarbonyl)-2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-carboxylic acid]: A novel protected spin labeled beta-amino acid for peptide and protein chemistry

The stable free radical 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) is the only spin labeled amino acid that has been used to date to successfully label peptide sequences for structural studies. However, severe difficulty in coupling the subsequent amino acid has been the most serious shortcoming of this paramagnetic marker. This problem stems from the low nucleophilicity of TOAC's amine group towards the acylation reaction during peptide chain elongation. The present report introduces the alternative beta -amino acid 2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-carboxylic acid (POAC), potentially useful in peptide and protein chemistry. Investigations aimed at addressing the stereochemistry of this cyclic molecule through X-ray diffraction measurements of crystalline and bulk samples revealed that it consists only of the trans conformer. The 9-fluorenylmethyloxyearbonyl group (Fmoc) was chosen for temporary protection of the POAC amine function, allowing insertion of the probe at any position in a peptide sequence. The vasoactive octapeptide angiotensin II (AII, DRVYIHPF) was synthesized by replacing Pro(7) with POAC. The reaction of Fmoc-POAC with the peptidyl-resin occurred smoothly, and the coupling of the subsequent amino acid showed a much faster reaction when compared with TOAC. POAC(7)-AII was obtained in good yield, demonstrating that, in addition to TOAC, POAC is a convenient amino acid for the synthesis of spin labeled peptide analogues. The present findings open the possibility of a wide range of chemical and biological applications for this novel beta -amino acid derivative, including structural investigations involving its differentiated bend-inducing characteristics.

Involvement of the atrial natriuretic peptide in cardiovascular pathophysiology and its relationship with exercise

In this minireview we describe the involvement of the atrial natriuretic peptide (ANP) in cardiovascular pathophysiology and exercise. The ANP has a broad homeostatic role and exerts complex effects on the cardio-circulatory hemodynamics, it is produced by the left atrium and has a key role in regulating sodium and water balance in mammals and humans. The dominant stimulus for its release is atrial wall tension, commonly caused by exercise. The ANP is involved in the process of lipolysis through a cGMP signaling pathway and, as a consequence, reducing blood pressure by decreasing the sensitivity of vascular smooth muscle to the action of vasoconstrictors and regulate fluid balance. The increase of this hormone is associated with better survival in patients with chronic heart failure (CHF). This minireview provides new evidence based on recent studies related to the beneficial effects of exercise in patients with cardiovascular disease, focusing on the ANP. © 2012 de Almeida et al; licensee BioMed Central Ltd.

Dynamics and Conformational Studies of TOAC Spin Labeled Analogues of Ctx(Ile21)-Ha Peptide from Hypsiboas albopunctatus

Antimicrobial peptides (AMPs) isolated from several organisms have been receiving much attention due to some specific features that allow them to interact with, bind to, and disrupt cell membranes. The aim of this paper was to study the interactions between a membrane mimetic and the cationic AMP Ctx(Ile21)-Ha as well as analogues containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) incorporated at residue positions n = 0, 2, and 13. Circular dichroism studies showed that the peptides, except for [TOAC13]Ctx(Ile21)-Ha, are unstructured in aqueous solution but acquire different amounts of α-helical secondary structure in the presence of trifluorethanol and lysophosphocholine micelles. Fluorescence experiments indicated that all peptides were able to interact with LPC micelles. In addition, Ctx(Ile21)-Ha and [TOAC13]Ctx(Ile21)-Ha peptides presented similar water accessibility for the Trp residue located near the N-terminal sequence. Electron spin resonance experiments showed two spectral components for [TOAC0]Ctx(Ile21)-Ha, which are most likely due to two membrane-bound peptide conformations. In contrast, TOAC2 and TOAC13 derivatives presented a single spectral component corresponding to a strong immobilization of the probe. Thus, our findings allowed the description of the peptide topology in the membrane mimetic, where the N-terminal region is in dynamic equilibrium between an ordered, membrane-bound conformation and a disordered, mobile conformation; position 2 is most likely situated in the lipid polar head group region, and residue 13 is fully inserted into the hydrophobic core of the membrane. © 2013 Vicente et al.

Angiotensin-(3-4) counteracts the Angiotensin II inhibitory action on renal Ca2+-ATPase through a cAMP/PKA pathway

We recently demonstrated that Angiotensin-(3-4) [Ang-(3-4)], an Ang II-derived dipeptide, overcomes inhibition of plasma membrane Ca2+-ATPase promoted by nanomolar concentrations of Ang II in basolateral membranes of renal proximal tubule cells, with involvement of a so far unknown AT(2)R-dependent and NO-independent mechanism. The present study investigates the signaling pathway triggered by Ang-(3-4) that is responsible for counteracting the inhibitory effect of Ang II, and attempts to elucidate the functional interaction of the dipeptide with Ang II at the level of AT(2)R. Stimulation by cholera toxin of G(s)alpha protein structurally linked to AT(2)R as revealed by their co-immunoprecipitation mimicked the effect of Ang-(3-4) on Ca2+-ATPase activity. Furthermore, addition of dibutyril-cAMP (db-cAMP) mimicked Ang-(3-4), whereas the specific PKA inhibitor, PKAi((5-24)) peptide, suppressed the counter-regulatory effect of Ang-(3-4) and the AT(2)R agonist, CGP42112A. Membrane-associated PKA activity was stimulated by Ang-(3-4) or CGP42112A to comparable levels as db-cAMP, and the Ang-(3-4) effect was abrogated by the AT(2)R antagonist PD123319, whereas the AT(1)R antagonist Losartan had no effect. Ang-(3-4) stimulated PKA-mediated phosphorylation of Ca2+-ATPase and activated PKA to comparable levels. Binding assays demonstrated that Ang-(3-4) could not displace H-3-Ang II from HEK 293T cells expressing AT(2)R, but 10(-10) mol/L Ang-(3-4) resulted in the appearance of a probable higher-affinity site (picomolar range) for Ang II. The results presented herein demonstrate that Ang-(3-4), acting as an allosteric enhancer, suppresses Ang II-mediated inhibition of Ca2+-ATPase through an AT(2)R/cAMP/PKA pathway, after inducing conformational changes in AT(2)R that results in generation of higher-affinity sites for Ang II. (C) 2012 Elsevier B.V. All rights reserved.

Synthesis and properties of cyclic gomesin and analogues

Gomesin (Gm) was the first antimicrobial peptide (AMP) isolated from the hemocytes of a spider, the Brazilian mygalomorph Acanthoscurria gomesiana. We have been studying the properties of this interesting AMP, which also displays anticancer, antimalarial, anticryptococcal and anti-Leishmania activities. In the present study, the total syntheses of backbone-cyclized analogues of Gm (two disulfide bonds), [Cys(Acm)2,15]-Gm (one disulfide bond) and [Thr2,6,11,15,d-Pro9]-Gm (no disulfide bonds) were accomplished, and the impact of cyclization on their properties was examined. The consequence of simultaneous deletion of pGlu1 and Arg16-Glu-Arg18-NH2 on Gm antimicrobial activity and structure was also analyzed. The results obtained showed that the synthetic route that includes peptide backbone cyclization on resin was advantageous and that a combination of 20% DMSO/NMP, EDC/HOBt, 60?degrees C and conventional heating appears to be particularly suitable for backbone cyclization of bioactive peptides. The biological properties of the Gm analogues clearly revealed that the N-terminal amino acid pGlu1 and the amidated C-terminal tripeptide Arg16-Glu-Arg18-NH2 play a major role in the interaction of Gm with the target membranes. Moreover, backbone cyclization practically did not affect the stability of the peptides in human serum; it also did not affect or enhanced hemolytic activity, but induced selectivity and, in some cases, discrete enhancements of antimicrobial activity and salt tolerance. Because of its high therapeutic index, easy synthesis and lower cost, the [Thr2,6,11,15,d-Pro9]-Gm analogue remains the best active Gm-derived AMP developed so far; nevertheless, its elevated instability in human serum may limit its therapeutic potential. Copyright (c) 2012 European Peptide Society and John Wiley & Sons, Ltd.