Quest for a stable Cu-ligand complex with a high catalytic activity to produce reactive oxygen species.

Metal ion-catalyzed overproduction of reactive oxygen species (ROS) is believed to contribute significantly to oxidative stress and be involved in several biological processes, from immune defense to development of diseases. Among the essential metal ions, copper is one of the most efficient catalysts in ROS production in the presence of O2 and a physiological reducing agent such as ascorbate. To control this chemistry, Cu ions are tightly coordinated to biomolecules. Free or loosely bound Cu ions are generally avoided to prevent their toxicity. In the present report, we aim to find stable Cu-ligand complexes (Cu-L) that can efficiently catalyze the production of ROS in the presence of ascorbate under aerobic conditions. Thermodynamic stability would be needed to avoid dissociation in the biological environment, and high ROS catalysis is of interest for applications as antimicrobial or anticancer agents. A series of Cu complexes with the well-known tripodal and tetradentate ligands containing a central amine linked to three pyridyl-alkyl arms of different lengths were investigated. Two of them with mixed arm length showed a higher catalytic activity in the oxidation of ascorbate and subsequent ROS production than Cu salts in buffer, which is an unprecedented result. Despite these high catalytic activities, no increased antimicrobial activity toward Escherichia coli or cytotoxicity against eukaryotic AGS cells in culture related to Cu-L-based ROS production could be observed. The potential reasons for discrepancy between in vitro and in cell data are discussed.

Antibacterial Photodynamic Therapy in the Near-Infrared Region with a Targeting Antimicrobial Peptide Connected to a π-Extended Porphyrin.

The increase of antimicrobial resistance to conventional antibiotics is worldwide a major health problem that requires the development of new bactericidal strategies. Antimicrobial photodynamic therapy (a-PDT) that generates reactive oxygen species acting on multiple cellular targets is unlikely to induce bacterial resistance. This localized treatment requires, for safe and efficient treatment of nonsuperficial infections, a targeting photosensitizer excited in the near IR. To this end, a new conjugate consisting of an antimicrobial peptide linked to a π-extended porphyrin photosensitizer was designed for a-PDT. Upon irradiation at 720 nm, the conjugate has shown at micromolar concentration strong bactericidal action on both Gram-positive and Gram-negative bacteria. Moreover, this conjugate allows one to reach a low minimum bactericidal concentration with near IR excitation without inducing toxicity to skin cells.

Antimicrobial peptides: mechanism of action and lipid-mediated synergistic interactions within membranes.

Biophysical and structural studies of peptide-lipid interactions, peptide topology and dynamics have changed our view of how antimicrobial peptides insert and interact with membranes. Clearly, both peptides and lipids are highly dynamic, and change and mutually adapt their conformation, membrane penetration and detailed morphology on a local and a global level. As a consequence, peptides and lipids can form a wide variety of supramolecular assemblies in which the more hydrophobic sequences preferentially, but not exclusively, adopt transmembrane alignments and have the potential to form oligomeric structures similar to those suggested by the transmembrane helical bundle model. In contrast, charged amphipathic sequences tend to stay intercalated at the membrane interface. Although the membranes are soft and can adapt, at increasing peptide density they cause pronounced disruptions of the phospholipid fatty acyl packing. At even higher local or global concentrations the peptides cause transient membrane openings, rupture and ultimately lysis. Interestingly, mixtures of peptides such as magainin 2 and PGLa, which are stored and secreted naturally as a cocktail, exhibit considerably enhanced antimicrobial activities when investigated together in antimicrobial assays and also in pore forming experiments applied to biophysical model systems. Our most recent investigations reveal that these peptides do not form stable complexes but act by specific lipid-mediated interactions and the nanoscale properties of phospholipid bilayers.

Different Biological Activities of Histidine-Rich Peptides Are Favored by Variations in Their Design.

The protein transduction and antimicrobial activities of histidine-rich designer peptides were investigated as a function of their sequence and compared to gene transfection, lentivirus transduction and calcein release activities. In membrane environments, the peptides adopt helical conformations where the positioning of the histidine side chains defines a hydrophilic angle when viewed as helical wheel. The transfection of DNA correlates with calcein release in biophysical experiments, being best for small hydrophilic angles supporting a model where lysis of the endosomal membrane is the limiting factor. In contrast, antimicrobial activities show an inverse correlation suggesting that other interactions and mechanisms dominate within the bacterial system. Furthermore, other derivatives control the lentiviral transduction enhancement or the transport of proteins into the cells. Here, we tested the transport into human cell lines of luciferase (63 kDa) and the ribosome-inactivating toxin saporin (30 kDa). Notably, depending on the protein, different peptide sequences are required for the best results, suggesting that the interactions are manifold and complex. As such, designed LAH4 peptides assure a large panel of biological and biophysical activities whereby the optimal result can be tuned by the physico-chemical properties of the sequences.

The Reversible Non-covalent Aggregation Into Fibers of PGLa and Magainin 2 Preserves Their Antimicrobial Activity and Synergism.

Magainin 2 and PGLa are antimicrobial peptides found together in frog skin secretions. When added as a mixture they show an order of magnitude increase in antibacterial activity and in model membrane permeation assays. Here we demonstrate that both peptides can form fibers with beta-sheet/turn signature in ATR-FTIR- and CD-spectroscopic analyses, but with different morphologies in EM images. Whereas, fiber formation results in acute reduction of the antimicrobial activity of the individual peptides, the synergistic enhancement of activity remains for the equimolar mixture of PGLa and magainin 2 also after fibril formation. The biological significance and potential applications of such supramolecular aggregates are discussed.

Copper-binding motifs Xxx-His or Xxx-Zzz-His (ATCUN) linked to an antimicrobial peptide: Cu-binding, antimicrobial activity and ROS production.

Depending on the coordination, copper ions can have a very high activity in catalyzing the production of reactive oxygen species. Thus interest arose in increasing the activity of antimicrobial peptides (AMPs) by equipping them with a Cu-binding unit. Several examples, native and engineered, have been investigated with the motif Xxx-Zzz-His, called Amino Terminal Cu(II)- and Ni(II)-binding (ATCUN) motif. Here we investigate a short AMP that was equipped either with Xxx-Zzz-His or Xxx-His. Xxx-His is a shorter motif and yields a more redox active copper complex. The control AMP, Xxx-His-AMP and Xxx-Zzz-His-AMP were investigated toward Cu-binding, Reactive Oxygen Species (ROS) production and antimicrobial activity in E. coli. The data indicate that these Cu-binding motifs have very limited impact on antimicrobial activity and low ROS production capability.

Revealing the Mechanisms of Synergistic Action of Two Magainin Antimicrobial Peptides.

The study of peptide-lipid and peptide-peptide interactions as well as their topology and dynamics using biophysical and structural approaches have changed our view how antimicrobial peptides work and function. It has become obvious that both the peptides and the lipids arrange in soft supramolecular arrangements which are highly dynamic and able to change and mutually adapt their conformation, membrane penetration, and detailed morphology. This can occur on a local and a global level. This review focuses on cationic amphipathic peptides of the magainin family which were studied extensively by biophysical approaches. They are found intercalated at the membrane interface where they cause membrane thinning and ultimately lysis. Interestingly, mixtures of two of those peptides namely magainin 2 and PGLa which occur naturally as a cocktail in the frog skin exhibit synergistic enhancement of antimicrobial activities when investigated together in antimicrobial assays but also in biophysical experiments with model membranes. Detailed dose-response curves, presented here for the first time, show a cooperative behavior for the individual peptides which is much increased when PGLa and magainin are added as equimolar mixture. This has important consequences for their bacterial killing activities and resistance development. In membranes that carry unsaturations both peptides align parallel to the membrane surface where they have been shown to arrange into mesophases involving the peptides and the lipids. This supramolecular structuration comes along with much-increased membrane affinities for the peptide mixture. Because this synergism is most pronounced in membranes representing the bacterial lipid composition it can potentially be used to increase the therapeutic window of pharmaceutical formulations.

Investigations of the Structure, Topology, and Interactions of the Transmembrane Domain of the Lipid-Sorting Protein p24 Being Highly Selective for Sphingomyelin-C18.

The p24 proteins play an important role in the secretory pathway where they selectively connect various cargo to other proteins, thereby being involved in the controlled assembly and disassembly of the coat protein complexes and lipid sorting. Recently, a highly selective lipid interaction motif has been identified within the p24 transmembrane domain (TMD) that recognizes the combination of the sphingomyelin headgroup and the exact length of the C18 fatty acyl chain (SM-C18). Here, we present investigations of the structure, dynamics, and sphingomyelin interactions of the p24 transmembrane region using circular dichroism, tryptophan fluorescence, and solid-state nuclear magnetic resonance (NMR) spectroscopies of the polypeptides and the surrounding lipids. Membrane insertion and/or conformation of the TMD is strongly dependent on the membrane lipid composition where the transmembrane helical insertion is strongest in the presence of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) and SM-C18. By analyzing solid-state NMR angular restraints from a large number of labeled sites, we have found a tilt angle of 19° for the transmembrane helical domain at a peptide-to-lipid ratio of 1 mol %. Only minor changes in the solid-state NMR spectra are observed due to the presence of SM-C18; the only visible alterations are associated with the SM-C18 recognition motif close to the carboxy-terminal part of the hydrophobic transmembrane region in the proximity of the SM headgroup. Finally, the deuterium order parameters of POPC- d31 were nearly unaffected by the presence of SM-C18 or the polypeptide alone but decreased noticeably when the sphingomyelin and the polypeptide were added in combination.

Structural Characterization of the Amyloid Precursor Protein Transmembrane Domain and Its γ-Cleavage Site.

Alzheimer's disease is the most common form of dementia that affects about 50 million of sufferers worldwide. A major role for the initiation and progression of Alzheimer's disease has been associated with the amyloid ß-peptide (Aß), which is a protease cleavage product of the amyloid precursor protein. The amyloid precursor protein is an integral membrane protein with a single transmembrane domain. Here, we assessed the structural integrity of the transmembrane domain within oriented phosphatidylcholine lipid bilayers and determined the tilt angle distribution and dynamics of various subdomains using solid-state NMR and attenuated total reflectance Fourier transform infrared spectroscopies. Although the overall secondary structure of the transmembrane domain is α-helical, pronounced conformational and topological heterogeneities were observed for the γ- and, to a lesser extent, the ζ-cleavage site, with pronounced implications for the production of Aß and related peptides, the development of the disease, and pharmaceutical innovation.

Magainin 2-PGLa Interactions in Membranes - Two Peptides that Exhibit Synergistic Enhancement of Antimicrobial Activity.

The structural requirements for the synergistic enhancement of antimicrobial activities of the cationic linear peptides PGLa and magainin 2 were investigated. In a first step the antimicrobial activities were evaluated for a number of modifications of the sequences and equimolar mixtures thereof. In particular fluorophore labelled peptides maintain a high degree of antimicrobial activity and considerable synergism when tested conjointly. Thereafter, circular dichroism spectroscopy indicated that these extended sequences adopt helical conformations in the presence of model membranes similar to the unmodified sequences. Energy transfer between the fluorophores suggested that the peptides reside in close proximity to each other when bound to the membrane surface at high concentrations. The fluorophore interactions quickly diminish at lower peptide-to-lipid ratios indicating that the peptide-peptide interactions are weak. Furthermore, (15)N solid-state NMR measurements of the membrane topology of [(15)N-Ala14]-PGLa and of its fluorophorecarrying analogue reconstituted into supported 1, 2-didecanoyl-sn-glycero-3-phosphocholine bilayers were performed. These experiments revealed no correlation between the topological state of PGLa and the observed synergistic enhancement of antimicrobial activities due to the presence of magainins. These results suggest that lipid mediated interactions rather than the formation of tight peptide-peptide complexes in the membrane are responsible for synergistic activities of the mixtures.

Investigations of the synergistic enhancement of antimicrobial activity in mixtures of magainin 2 and PGLa.

Magainins are antimicrobial peptides isolated from the African clawed frog Xenopus laevis. They interact with bacterial membranes where they cause channel formation and membrane disruption. When added as a cocktail magainin 2 and PGLa are considerably more efficient when compared to the corresponding amounts of individual components. In order to investigate this synergistic interaction of PGLa and magainin a number of magainin variants have been prepared and investigated in biological and biophysical assays. In particular we report on the antimicrobial activities and solid-state NMR investigations of magainins that have been extended by a carboxyterminal GGC tripeptide to form covalently linked dimers. Notably, when the formation of the covalent linkage is prevented by exchanging the cystein by serine or alanine no loss in efficiency was observed indicating that the covalent interaction is not necessary for synergistic interaction. In a next step peptides labelled with (15)N and (2)H were reconstituted into oriented membranes and their topology studied by solid-state NMR spectroscopy. The tendency of some of these peptides to adopt membrane-spanning alignments does not correlate with their synergistic activities in antimicrobial assays. In contrast, the stable alignment of PGLa parallel to the surface of membranes made of Escherichia coli lipid extracts is strongly suggestive that the peptides develop synergistic activities when in an in-planar configuration. Notably, the phospholipid head groups of these samples show a high degree of disturbance suggesting that the synergistic interactions between the magainin peptides could be mediated through the lipid phase.

Endogenous morphine levels are increased in sepsis: a partial implication of neutrophils.

BACKGROUND: Mammalian cells synthesize morphine and the respective biosynthetic pathway has been elucidated. Human neutrophils release this alkaloid into the media after exposure to morphine precursors. However, the exact role of endogenous morphine in inflammatory processes remains unclear. We postulate that morphine is released during infection and can be determined in the serum of patients with severe infection such as sepsis. METHODOLOGY: The presence and subcellular immunolocalization of endogenous morphine was investigated by ELISA, mass spectrometry analysis and laser confocal microscopy. Neutrophils were activated with Interleukin-8 (IL-8) or lipopolysaccharide (LPS). Morphine secretion was determined by a morphine-specific ELISA. mu opioid receptor expression was assessed with flow cytometry. Serum morphine concentrations of septic patients were determined with a morphine-specific ELISA and morphine identity was confirmed in human neutrophils and serum of septic patients by mass spectrometry analysis. The effects of the concentration of morphine found in serum of septic patients on LPS-induced release of IL-8 by human neutrophils were tested. PRINCIPAL FINDINGS: We confirmed the presence of morphine in human neutrophil extracts and showed its colocalisation with lactoferrin within the secondary granules of neutrophils. Morphine secretion was quantified in the supernatant of activated human polymorphonuclear neutrophils in the presence and absence of Ca(2+). LPS and IL-8 were able to induce a significant release of morphine only in presence of Ca(2+). LPS treatment increased mu opioid receptor expression on neutrophils. Low concentration of morphine (8 nM) significantly inhibited the release of IL-8 from neutrophils when coincubated with LPS. This effect was reversed by naloxone. Patients with sepsis, severe sepsis and septic shock had significant higher circulating morphine levels compared to patients with systemic inflammatory response syndrome and healthy controls. Mass spectrometry analysis showed that endogenous morphine from serum of patient with sepsis was identical to poppy-derived morphine. CONCLUSIONS: Our results indicate that morphine concentrations are increased significantly in the serum of patients with systemic infection and that morphine is, at least in part, secreted from neutrophils during sepsis. Morphine concentrations equivalent to those found in the serum of septic patients significantly inhibited LPS-induced IL-8 secretion in neutrophils.

Characterization of human and bovine phosphatidylethanolamine-binding protein (PEBP/RKIP) interactions with morphine and morphine-glucuronides determined by noncovalent mass spectrometry.

BACKGROUND: The phosphatidylethanolamine-binding protein (PEBP/RKIP), initially found to bind phosphatidylethanolamine (PE), has been shown to be associated with morphine derivatives. Our recent study on bovine primary chromaffin cells showed that inside secretory granules, PEBP is noncovalently associated to endogenous morphine-6-glucuronide (M6G), a highly analgesic morphine metabolite. During stress, M6G-PEBP complexes may be released into circulation to target peripheral opioid receptors. We now report the investigation of PEBP binding properties towards morphine and morphine analogs. MATERIAL/METHODS: Noncovalent electrospray ionization mass spectrometry (ESI-MS) was used to investigate bovine and human PEBP binding properties towards morphine and morphine-glucuronides. RESULTS: We describe for the first time that: (i) PEBP directly interacts with morphine glucuronides (M3G and M6G) but not with morphine, (ii) that the presence of a glucuronide group either on the 3rd or the 6th morphine's carbon does not affect these interactions, (iii) that M6G binds PEBP in a similar manner as the reference ligand PE and (iv) that PEBP displays a similar affinity for PE, M6G and M3G. CONCLUSIONS: Our results suggest that PEBP might protect M6G following its secretion into blood, leading to a longer half life. This study highlights the potentialities of ESI-MS to validate / invalidate the formation of protein: ligand noncovalent complexes when low affinity binders (i.e., compounds with affinities lower than 10(3) M(-1)) are concerned.

Endogenous morphine in SH-SY5Y cells and the mouse cerebellum.

BACKGROUND: Morphine, the principal active agent in opium, is not restricted to plants, but is also present in different animal tissues and cell types, including the mammalian brain. In fact, its biosynthetic pathway has been elucidated in a human neural cell line. These data suggest a role for morphine in brain physiology (e.g., neurotransmission), but this hypothesis remains a matter of debate. Recently, using the adrenal neuroendocrine chromaffin cell model, we have shown the presence of morphine-6-glucuronide (M6G) in secretory granules and their secretion products, leading us to propose that these endogenous alkaloids might represent new neuroendocrine factors. Here, we investigate the potential function of endogenous alkaloids in the central nervous system. METHODOLOGY AND PRINCIPAL FINDINGS: Microscopy, molecular biology, electrophysiology, and proteomic tools were applied to human neuroblastoma SH-SY5Y cells (i) to characterize morphine and M6G, and (ii) to demonstrate the presence of the UDP-glucuronyltransferase 2B7 enzyme, which is responsible for the formation of M6G from morphine. We show that morphine is secreted in response to nicotine stimulation via a Ca(2+)-dependent mechanism involving specific storage and release mechanisms. We also show that morphine and M6G at concentrations as low as 10(-10) M are able to evoke specific naloxone-reversible membrane currents, indicating possible autocrine/paracrine regulation in SH-SY5Y cells. Microscopy and proteomic approaches were employed to detect and quantify endogenous morphine in the mouse brain. Morphine is present in the hippocampus, cortex, olfactory bulb, and cerebellum at concentration ranging from 1.45 to 7.5 pmol/g. In the cerebellum, morphine immunoreactivity is localized to GABA basket cells and their termini, which form close contacts on Purkinje cell bodies. CONCLUSIONS/SIGNIFICANCE: The presence of morphine in the brain and its localization in particular areas lead us to conclude that it has a specific function in neuromodulation and/or neurotransmission. Furthermore, its presence in cerebellar basket cell termini suggests that morphine has signaling functions in Purkinje cells that remain to be discovered.

Characterization of natural vasostatin-containing peptides in rat heart.

Chromogranin A (CGA) is a protein that is stored and released together with neurotransmitters and hormones in the nervous, endocrine and diffuse neuroendocrine systems. As human vasostatins I and II [CGA(1-76) and CGA(1-113), respectively] have been reported to affect vessel motility and exert concentration-dependent cardiosuppressive effects on isolated whole heart preparations of eel, frog and rat (i.e. negative inotropism and antiadrenergic activity), we investigated the presence of vasostatin-containing peptides in rat heart. Rat heart extracts were purified by RP-HPLC, and the resulting fractions analyzed for the presence of CGA N-terminal fragments using dot-blot analysis. CGA-immunoreactive fractions were submitted to western blot and MS analysis using the TOF/TOF technique. Four endogenous N-terminal CGA-derived peptides [CGA(4-113), CGA(1-124), CGA(1-135) and CGA(1-199)] containing the vasostatin sequence were characterized. The following post-translational modifications of these fragments were identified: phosphorylation at Ser96, O-glycosylation (trisaccharide, NAcGal-Gal-NeuAc) at Thr126, and oxidation at three methionine residues. This first identification of CGA-derived peptides containing the vasostatin motif in rat heart supports their role in cardiac physiology by an autocrine/paracrine mechanism.

Rethinking the opiate system? Morphine and morphine-6-glucuronide as new endocrine and neuroendocrine mediators.

Since the 80s, intrigued by presence of morphine precursors in some mammalian cells, different laboratories were able to characterize morphine and morphine precursors in animal tissues. Endogenous morphine studies continued during 90s and this alkaloid was successfully characterized from more organs and fluids of vertebrates, including brain, adrenal gland, heart, cerebrospinal fluid and urine. Then, in the last three years a high rate of publications dealing with this topic emerged, leading to a better understanding of the endogenous morphine system. In this regard, this article comment all the new data recently collected on this rising subject and replace the morphine and its derivative, morphine-6-glucuronide, in the mammalian physiology.

Identification of morphine-6-glucuronide in chromaffin cell secretory granules.

We report for the first time that morphine-6-glucuronide, a highly analgesic morphine-derived molecule, is present in adrenal chromaffin granules and secreted from chromaffin cells upon stimulation. We also demonstrate that phosphatidylethanolamine-binding protein (alternatively named Raf-1 kinase inhibitor protein or RKIP) acts as an endogenous morphine-6-glucuronide-binding protein. An UDP-glucuronosyltransferase 2B-like enzyme, described to transform morphine into morphine-6-glucuronide, has been immunodetected in the chromaffin granule matrix, and morphine-6-glucuronide de novo synthesis has been characterized, demonstrating the possible involvement of intragranular UDP-glucuronosyltransferase 2B-like enzyme in morphine-6-glucuronide metabolism. Once secreted into the circulation, morphine-6-glucuronide may mediate several systemic actions (e.g. on immune cells) based on its affinity for mu-opioid receptors. These activities could be facilitated by phosphatidylethanolamine-binding protein (PEBP), acting as a molecular shield and preventing morphine-6-glucuronide from rapid clearance. Taken together, our data represent an important observation on the role of morphine-6-glucuronide as a new endocrine factor.