Helokinestatins: A novel family of bioactive peptides with drug-lead potential fromthe venomof the Gila Monster (Heloderma suspectum)

Venom of the Gila Monster (Heloderma suspectum) has proven to be an unlikely source of lead compounds (exendins) for the development of new injectable peptide therapeutics for the treatment of Type 2 diabetes. However, no systematic searches for new classes of bioactive peptides in lizard venom have appeared until recently. Here we describe the discovery of a new class of peptides – the helokinestatins – from H. suspectum venom, their structural characterisation and that of their biosynthetic precursors from cloned cDNA. In addition, we have subjected members of the family to preliminary pharmacological characterisation. Helokinestatins 1–6 are a family of proline-rich peptides containing 10–15 amino acid residues terminating in a common -Pro-Arg.OH motif. They are encoded in tandem within two virtually identical biosynthetic precursors of 177 and 178 amino acid residues, differing by only a single Pro residue. Each precursor also encodes a single copy of a C-type natriuretic peptide located at the C-terminus. Synthetic replicates of all helokinestatins were shown to be devoid of any direct action on the smooth muscle of rat tail artery but were found to be potent inhibitors of bradykinin-induced relaxation in this preparation in a manner that is suggestive of a non-competitive mechanism. Helokinestatin-3 (VPPPPLQMPLIPR) and helokinestatin-5 (VPPPLQMPLIPR) were found to be most potent in this respect causing almost complete inhibition of bradykinin-induced relaxation. Helokinestatins and BPPs may have a shared evolutionary history but the former do not inhibit ACE. The bradykinin inhibitory potential of helokinestatins may be exploited in the local control of chronic inflammation.

Novel Venom Proteins Produced by Differential Domain-Expression Strategies in Beaded Lizards and Gila Monsters (genus Heloderma)

The origin and evolution of venom proteins in helodermatid lizards were investigated by multidisciplinary techniques. Our analyses elucidated novel toxin types resultant from three unique domain-expression processes: 1) The first full-length sequences of lethal toxin isoforms (helofensins) revealed this toxin type to be constructed by an ancestral monodomain, monoproduct gene (beta-defensin) that underwent three tandem domain duplications to encode a tetradomain, monoproduct with a possible novel protein fold; 2) an ancestral monodomain gene (encoding a natriuretic peptide) was medially extended to become a pentadomain, pentaproduct through the additional encoding of four tandemly repeated proline-rich peptides (helokinestatins), with the five discrete peptides liberated from each other by posttranslational proteolysis; and 3) an ancestral multidomain, multiproduct gene belonging to the vasoactive intestinal peptide (VIP)/glucagon family being mutated to encode for a monodomain, monoproduct (exendins) followed by duplication and diversification into two variant classes (exendins 1 and 2 and exendins 3 and 4). Bioactivity characterization of exendin and helokinestatin elucidated variable cardioactivity between isoforms within each class. These results highlight the importance of utilizing evolutionary-based search strategies for biodiscovery and the virtually unexplored potential of lizard venoms in drug design and discovery.

Arsenic as a food chain contaminant:mechanisms of plant uptake and metabolism and mitigation strategies

Arsenic (As) is an environmental and food chain contaminant. Excessive accumulation of As, particularly inorganic arsenic (As(i)), in rice (Oryza sativa) poses a potential health risk to populations with high rice consumption. Rice is efficient at As accumulation owing to flooded paddy cultivation that leads to arsenite mobilization, and the inadvertent yet efficient uptake of arsenite through the silicon transport pathway. Iron, phosphorus, sulfur, and silicon interact strongly with As during its route from soil to plants. Plants take up arsenate through the phosphate transporters, and arsenite and undissociated methylated As species through the nodulin 26-like intrinsic (NIP) aquaporin channels. Arsenate is readily reduced to arsenite in planta, which is detoxified by complexation with thiol-rich peptides such as phytochelatins and/or vacuolar sequestration. A range of mitigation methods, from agronomic measures and plant breeding to genetic modification, may be employed to reduce As uptake by food crops.

Phytochelatins are involved in differential arsenate tolerance in Holcus lanatus

Arsenate tolerance is conferred by suppression of the high-affinity phosphate/arsenate uptake system, which greatly reduces arsenate influx in a number of higher plant species. Despite this suppressed uptake, arsenate-tolerant plants can still accumulate high levels of As over their lifetime, suggesting that constitutive detoxification mechanisms may be required. Phytochelatins are thiol-rich peptides, whose production is induced by a range of metals and metalloids including arsenate. This study provides evidence for the role of phytochelatins in the detoxification of arsenate in arsenate-tolerant Holcus lanatus. Elevated levels of phytochelatin were measured in plants with a range of tolerance to arsenate at equivalent levels of arsenate stress, measured as inhibition of root growth. The results suggest that arsenate tolerance in H. lanatus requires both adaptive suppression of the high-affinity phosphate uptake system and constitutive phytochelatin production.

LYRIC/AEG-1 is targeted to different subcellular compartments by ubiquitinylation and intrinsic nuclear localization signals

PURPOSE: LYRIC/AEG-1 has been reported to influence breast cancer survival and metastases, and its altered expression has been found in a number of cancers. The cellular function of LYRIC/AEG-1 has previously been related to its subcellular distribution in cell lines. LYRIC/AEG-1 contains three uncharacterized nuclear localization signals (NLS), which may regulate its distribution and, ultimately, function in cells.

EXPERIMENTAL DESIGN: Immunohistochemistry of a human prostate tissue microarray composed of 179 prostate cancer and 24 benign samples was used to assess LYRIC/AEG-1 distribution. Green fluorescent protein-NLS fusion proteins and deletion constructs were used to show the ability of LYRIC/AEG-1 NLS to target green fluorescent protein from the cytoplasm to the nucleus. Immunoprecipitation and Western blotting were used to show posttranslational modification of LYRIC/AEG-1 NLS regions.

RESULTS: Using a prostate tissue microarray, significant changes in the distribution of LYRIC/AEG-1 were observed in prostate cancer as an increased cytoplasmic distribution in tumors compared with benign tissue. These differences were most marked in high grade and aggressive prostate cancers and were associated with decreased survival. The COOH-terminal extended NLS-3 (amino acids 546-582) is the predominant regulator of nuclear localization, whereas extended NLS-1 (amino acids 78-130) regulates its nucleolar localization. Within the extended NLS-2 region (amino acids 415-486), LYRIC/AEG-1 can be modified by ubiquitin almost exclusively within the cytoplasm.

CONCLUSIONS: Changes in LYRIC/AEG-1 subcellular distribution can predict Gleason grade and survival. Two lysine-rich regions (NLS-1 and NLS-3) can target LYRIC/AEG-1 to subcellular compartments whereas NLS-2 is modified by ubiquitin in the cytoplasm.

Liver fluke (Fasciola hepatica)-derived peptides activate rat peritoneal mast cells

Short peptides with sequences derived from those found in the tegumental antigen of Fasciola hepatica have been synthesised. Incubation of some of these peptides with rat peritoneal mast cells resulted in the degranulation of the cells as measured by a histamine release assay. This activity was shown to be associated with the proline-lysine-proline motif, which is responsible for the induction of mast cell degranulation by the mammalian bioactive peptide substance P. Studies on the mode of action of the fluke-derived peptide indicated that it was operating through the same biochemical pathways as substance P. The implications of these findings for the development of immune responses during parasite infections are discussed. (C) 2003 Elsevier Science B.V. All rights reserved.

Antimicrobial peptides in the venom of the European hornet, Vespa crabro, identified as mastoparan C and crabrolin

Amphibian skin secretions are rich sources of cationic amphipathic peptides which often possess potent and broad-spectrum antimicrobial activity. However, the venoms of other animals such as hymenopteran insects, also contain peptides with these characteristics and the literature is unclear as to their antimicrobial potential. Here we subjected the venom of the European hornet, Vespa crabro, to reverse phase HPLC fractionation followed by screening of aliquots of individual fractions in bacterial zonal inhibition assays. Two major peptides possessing activity in these assays were further purified by HPLC and subjected to MALDI-TOF MS analysis and MS/MS fragmentation using an ESI mass spectrometer. The peptides were identified as mastoparan C (LNLKALLAVAKKILamide) and crabrolin (FLPLILRKIVTALamide). Replicates of both peptides were synthesised by solid-phase methodology and mean inhibitory concentrations (MICs) established against Staphylococcus aureus and Escherichia coli. Mastoparan C was found to be a potent antimicrobial with MIC values of 2 µM and 4 µM against S. aureus and E. coli, respectively. Crabrolin was found to be less potent with MIC values of > 160 µM and 40 µM for S. aureus and E. coli. Hornet venom thus contains a potent antimicrobial peptide that has been unambiguously identified as mastoparan C, a peptide that is known to affect profound histamine release from mast cells and to generally activate membrane G protein-linked receptors. It is thus highly probable that its antimicrobial effects, like those previously documented, are a result of a generalized membrane interactive and disruptive function — perhaps reflective of the authentic role of amphibian skin antimicrobials.

Antimicrobial peptides from the skin secretion of the Wuyi Mountain torrent frog, Amolops wuyiensis: An inhabitant of a pristine aquatic environment

The antimicrobial peptides of amphibian skin secretions are proposed to aid survival in microbe-rich environments. While many amphibians inhabit such environments, other such as the Wuyi Mountain torrent frog, Amolops wuyiensis, live in pristine waters flowing from underground mountain springs. This species thus represents an interesting model in which to study antimicrobial peptides. “Shotgun” cloning of a skin-derived cDNA library from this species identified transcripts encoding a brevinin-1 and a ranatuerin-2. Peptides with coincident molecular masses to both predicted mature peptides were identified in HPLC fractions of skin secretion. Synthetic replicates of both peptides were generated by solid-phase peptide synthesis and tested for activity using Staphylococcus aureus, Escherichia coli and Candida albicans. The brevinin was found to be broad-spectrum and potent with minimum inhibitory concentrations (MICs) of 24 µM (Sa), 5 µM (Ec) and 20 µM (Ca). In contrast, the ranatuerin was less effective and of narrower spectrum with an MIC > 200 µM for Sa, 40 µM (Ec) and 120 µM (Ca). Thus this species of amphibian that lives in a pristine environment does indeed possess at least one potent and broad-spectrum antimicrobial peptide in its skin secretion arsenal. This phenomenon could be explained in several ways. Firstly, it may represent an ancestral peptide required when the stem species inhabited microbe-rich environments. However, there is mounting evidence for the second reason, that suggests the function of such peptides is not primarily in antimicrobial defence.

Limnonectins: A new class of antimicrobial peptides from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis)

Amphibian skin secretions are rich sources of biologically-active peptides with antimicrobial peptides predominating in many species. Several studies involving molecular cloning of biosynthetic precursor-encoding cDNAs from skin or skin secretions have revealed that these exhibit highly-conserved domain architectures with an unusually high degree of conserved nucleotide and resultant amino acid sequences within the signal peptides. This high degree of nucleotide sequence conservation has permitted the design of primers complementary to such sites facilitating “shotgun” cloning of skin or skin secretion-derived cDNA libraries from hitherto unstudied species. Here we have used such an approach using a skin secretion-derived cDNA library from an unstudied species of Chinese frog – the Fujian large-headed frog, Limnonectes fujianensis – and have discovered two 16-mer peptides of novel primary structures, named limnonectin-1Fa (SFPFFPPGICKRLKRC) and limnonectin-1Fb (SFHVFPPWMCKSLKKC), that represent the prototypes of a new class of amphibian skin antimicrobial peptide. Unusually these limnonectins display activity only against a Gram-negative bacterium (MICs of 35 and 70 µM) and are devoid of haemolytic activity at concentrations up to 160 µM. Thus the “shotgun” cloning approach described can exploit the unusually high degree of nucleotide conservation in signal peptide-encoding domains of amphibian defensive skin secretion peptide precursor-encoding cDNAs to rapidly expedite the discovery of novel and functional defensive peptides in a manner that circumvents specimen sacrifice without compromising robustness of data

Effects of Charge Location on the Absorptions and Lifetimes of Protonated Tyrosine Peptides in Vacuo

Nearby charges affect the electronic energy levels of chromophores, with the extent of the effect being determined by the magnitude of the charge and degree of charge-chromophore separation. The molecular configuration dictates the charge chromophore distance. Hence, in this study, we aim to assess how the location of the charge influences the absorption of a set of model protonated and diprotonated peptide ions, and whether spectral differences are large enough to be identified. The studied ions were the dipeptide YK, the tripeptide KYK (Y = tyrosine; K = lysine) and their complexes with 18-crown-6-ether (CE). The CE targets the ammonium group by forming internal ionic hydrogen bonds and limits the folding of the peptide. In the tripeptide, the distance between the chromophore and the backbone ammonium is enlarged relative to that in the dipeptide. Experiments were performed in an electrostatic ion storage ring using a tunable laser system, and action spectra based on lifetime measurements were obtained in the range from 210 to 310 nm. The spectra are all quite similar though there seems to be some changes in the absorption band between 210 and 250 nm, while in the lower energy band all ions had a maximum absorption at similar to 275 nm. Lifetimes after photoexcitation were found to shorten upon protonation and lengthen upon CE complexation, in accordance with the increased number of degrees of freedom and an increase in activation energies for dissociation as the mobile proton model is no longer operative.

The outer membrane of Brucella ovis shows increased permeability to hydrophobic probes and is more susceptible to cationic peptides than are the outer membranes of mutant rough Brucella abortus strains

The permeability of the outer membrane (OM) to hydrophobic probes and its susceptibility to bactericidal cationic peptides were investigated for natural rough Brucella ovis and for mutant rough Brucella abortus strains. The OM of B. ovis displayed an abrupt and faster kinetic profile than rough B. abortus during the uptake of the hydrophobic probe N-phenyl-naphthylamine. B. ovis was more sensitive than rough B. abortus to the action of cationic peptides. Bactenecins 5 and 7 induced morphological alterations on the OMs of both rough Brucella strains. B. ovis lipopolysaccharide (LPS) captured considerably more polymyxin B than LPSs from both rough and smooth B. abortus strains. Polymyxin B, poly-L-lysine, and poly-L-ornithine produced a thick coating on the surfaces of both strains, which was more evident in B. ovis than in rough B. abortus. The distinct functional properties of the OMs of these two rough strains correlate with some structural differences of their OMs and with their different biological behaviors in animals and culture cells.

Outer membrane differences between pathogenic and environmental Yersinia enterocolitica biogroups probed with hydrophobic permeants and polycationic peptides

Sensitivities to polycationic peptides and EDTA were compared in Yersinia enterocolitica pathogenic and environmental biogroups. As shown by changes in permeability to the fluorescent hydrophobic probe N-phenylnaphthylamine (NPN), the outer membranes (OMs) of pathogenic and environmental strains grown at 26 degrees C in standard broth were more resistant to poly-L-lysine, poly-L-ornithine, melittin, cecropin P1, polymyxin B, and EDTA than Escherichia coli OMs. At 37 degrees C, OMs of pathogenic biogroups were resistant to EDTA and polycations and OMs of environmental strains were resistant to EDTA whereas E. coli OMs were sensitive to both EDTA and polycations. Similar results were found when testing deoxycholate sensitivity after polycation exposure or when isogenic pairs with or without virulence plasmid pYV were compared. With bacteria grown without Ca++ available, OM permeability to NPN was drastically increased in pathogenic but not in environmental strains or E. coli. Under these conditions, OMs of pYV+ and pYV- cells showed small differences in NPN permeability but differences in polycation sensitivity could not be detected by fluorimetry. O:1,6 (environmental type) lipopolysaccharide (LPS), but not O:3 or O:8 LPS, was markedly rough at 37 degrees C, and this could explain the differences in polycation sensitivity. LPSs from serotypes O:3 and O:8 grown at 37 degrees C were more permeable to NPN than O:1,6 LPS, and O:8 LPS was resistant to polycation-induced permeabilization. These data suggest that LPSs relate to some but not all the OM differences described. It is hypothesized that the different OM properties of environmental and pathogenic biogroups reflect the adaptation of the latter biogroups to pathogenicity.

Ultrashort Cationic Naphthalene-Derived Self-Assembled Peptides as Antimicrobial Nanomaterials

Self-assembling dipeptides conjugated to naphthalene show considerable promise as nanomaterial structures, biomaterials, and drug delivery devices. Biomaterial infections are responsible for high rates of patient mortality and morbidity. The presence of biofilm bacteria, which thrive on implant surfaces, are a huge burden on healthcare budgets, as they are highly resistant to current therapeutic strategies. Ultrashort cationic self-assembled peptides represent a highly innovative and cost-effective strategy to form antibacterial nanomaterials. Lysine conjugated variants display the greatest potency with 2% w/v NapFFKK hydrogels significantly reducing the viable Staphylococcus epidermidis biofilm by 94%. Reducing the size of the R-group methylene chain on cationic moieties resulted in reduction of antibiofilm activity. The primary amine of the protruding R-group tail may not be as readily available to interact with negatively charged bacterial membranes. Cryo-SEM, FTIR, CD spectroscopy, and oscillatory rheology provided evidence of supramolecular hydrogel formation at physiological pH (pH 7.4). Cytotoxicity assays against murine fibroblast (NCTC 929) cell lines confirmed the gels possessed reduced cytotoxicity relative to bacterial cells, with limited hemolysis upon exposure to equine erythrocytes. The results presented in this paper highlight the significant potential of ultrashort cationic naphthalene peptides as future biomaterials.