Profiling the Linear Peptides of Venom from the Brazilian Scorpion Tityus serrulatus: Structural and Functional Characterization.

Scorpion venoms are a rich source of bioactive peptides, most of which are neurotoxic, with 30 to 70 amino acid residues in their sequences. There are a scarcity of reports in the literature concerning the short linear peptides found in scorpion venoms. This type of peptide toxin may be selectively extracted from the venom using 50% (v/v) acetonitrile. The use of LC-MS and MS/MS enabled the detection of 12 bioactive short linear peptides, of which six were identified as cryptides. These peptides were shown to be multifunctional, causing hemolysis, mast cell degranulation and lysis, edema, pain, and anxiety, increasing the complexity of the envenomation mechanism. Apparently, the natural functions of these peptide toxins are to induce inflammation and discomfort in the victims of scorpion stings.

Cytotoxic Cyclotides from Anchietea pyrifolia, a South American Plant Species.

Cyclotides are mini-proteins with potent bioactivities and outstanding potential for agricultural and pharmaceutical applications. More than 450 different plant cyclotides have been isolated from six angiosperm families. In Brazil, studies involving this class of natural products are still scarce, despite its rich floristic diversity. Herein were investigated the cyclotides from Anchietea pyrifolia roots, a South American medicinal plant from the family Violaceae. Fourteen putative cyclotides were annotated by LC-MS. Among these, three new bracelet cyclotides, anpy A-C, and the known cycloviolacins O4 (cyO4) and O17 (cyO17) were sequenced through a combination of chemical and enzymatic reactions followed by MALDI-MS/MS analysis. Their cytotoxic activity was evaluated by a cytotoxicity assay against three human cancer cell lines (colorectal carcinoma cells: HCT 116 and HCT 116 TP53-/- and breast adenocarcinoma, MCF 7). For all assays, the IC50 values of isolated compounds ranged between 0.8 and 7.3 µM. CyO17 was the most potent cyclotide for the colorectal cancer cell lines (IC50, 0.8 and 1.2 µM). Furthermore, the hemolytic activity of anpy A and B, cyO4, and cyO17 was assessed, and the cycloviolacins were the least hemolytic (HD50 > 156 µM). This work sheds light on the cytotoxic effects of the anpy cyclotides against cancer cells. Moreover, this study expands the number of cyclotides obtained to date from Brazilian plant biodiversity and adds one more genus containing these molecules to the list of the Violaceae family.

The Antimicrobial Peptide MK58911-NH2 Acts on Planktonic, Biofilm, and Intramacrophage Cells of Cryptococcus neoformans.

Cryptococcosis is associated with high rates of morbidity and mortality, especially in AIDS patients. Its treatment is carried out by combining amphotericin B and azoles or flucytosine, which causes unavoidable toxicity issues in the host. Thus, the urgency in obtaining new antifungals drives the search for antimicrobial peptides (AMPs). This study aimed to extend the understanding of the mechanism of action of an AMP analog from wasp peptide toxins, MK58911-NH2, on Cryptococcus neoformans. We also evaluated if MK58911-NH2 can act on cryptococcal cells in macrophages, biofilms, and an immersion zebrafish model of infection. Finally, we investigated the structure-antifungal action and the toxicity relationship of MK58911-NH2 fragments and a derivative of this peptide (MH58911-NH2). The results demonstrated that MK58911-NH2 did not alter the fluorescence intensity of the cell wall-binding dye calcofluor white or the capsule-binding dye 18b7 antibody-fluorescein isothiocyanate (FITC) in C. neoformans but rather reduced the number and size of fungal cells. This activity reduced the fungal burden of C. neoformans in both macrophages and zebrafish embryos as well as within biofilms. Three fragments of the MK58911-NH2 peptide showed no activity against Cryptococcus and not toxicity in lung cells. The derivative peptide MH58911-NH2, in which the lysine residues of MK58911-NH2 were replaced by histidines, reduced the activity against extracellular and intracellular C. neoformans. On the other hand, it was active against biofilms and showed reduced toxicity. In summary, these results showed that peptide MK58911-NH2 could be a promising agent against cryptococcosis. This work also opens a perspective for the verification of the antifungal activity of other derivatives.

Salivary glands in workers of Ruptitermes spp. (Blattaria, Isoptera, Termitidae, Apicotermitinae): a morphological and preoteomic approach.

Salivary glands are omnipresent in termites and occur in all developmental stages and castes. They function to produce, store, and secrete compounds, ranging from a feeding function to defensive mechanisms. Here, we provide a complete morphological overview of the salivary glands in the soldierless species Ruptitermes reconditus and R. xanthochiton, and the first proteomic profile of the salivary glands in a Neotropical Apicotermitinae representative, R. reconditus. Salivary glands from both species were composed of several acini, roughly spherical structures composed of two types of central cells (type I and II) and peripheral parietal cells, as well as transporting ducts and two salivary reservoirs. Central cells were richly supplied with electron-lucent secretory vesicles and rough endoplasmic reticulum, a feature of protein-secreting cells. Parietal cells of Ruptitermes spp. had conspicuous characteristics such as electron-lucent secretory vesicles surrounded by mitochondria and well-developed microvilli. Moreover, different individuals showed variation in the secretory cycle of salivary acini, which may be related to polyethism. Ultrastructural analysis evidenced a high synthesis of secretion and also the occurrence of lysosomes and autophagic structures in central cells. Proteomic analysis of the salivary glands revealed 483 proteins divided into functional groups, highlighting toxins/defensins and compounds related to alarm communication and colony asepsis. Soldierless termites are quite successful, especially due to morphological adaptations of the workers, including unknown modifications of exocrine glands. Thus, according to our morphological and proteomic findings, we discuss the potential roles of the salivary gland secretion in different social aspects of the sampled species.

The effect of acidic pH on the adsorption and lytic activity of the peptides Polybia-MP1 and its histidine-containing analog in anionic lipid membrane: a biophysical study by molecular dynamics and spectroscopy.

Antimicrobial peptides (AMPs) are part of the innate immune system of many species. AMPs are short sequences rich in charged and non-polar residues. They act on the lipid phase of the plasma membrane without requiring membrane receptors. Polybia-MP1 (MP1), extracted from a native wasp, is a broad-spectrum bactericide, an inhibitor of cancer cell proliferation being non-hemolytic and non-cytotoxic. MP1 mechanism of action and its adsorption mode is not yet completely known. Its adsorption to lipid bilayer and lytic activity is most likely dependent on the ionization state of its two acidic and three basic residues and consequently on the bulk pH. Here we investigated the effect of bulk acidic (pH 5.5) and neutral pH (7.4) solution on the adsorption, insertion, and lytic activity of MP1 and its analog H-MP1 to anionic (7POPC:3POPG) model membrane. H-MP1 is a synthetic analog of MP1 with lysines replaced by histidines. Bulk pH changes could modulate this peptide efficiency. The combination of different experimental techniques and molecular dynamics (MD) simulations showed that the adsorption, insertion, and lytic activity of H-MP1 are highly sensitive to bulk pH in opposition to MP1. The atomistic details, provided by MD simulations, showed peptides contact their N-termini to the bilayer before the insertion and then lay parallel to the bilayer. Their hydrophobic faces inserted into the acyl chain phase disturb the lipid-packing.

In Situ Metabolomics of the Honeybee Brain: The Metabolism of l-Arginine through the Polyamine Pathway in the Proboscis Extension Response (PER).

The proboscis extension response (PER) reflex may be used to condition the pairing of an odor with sucrose, which is applied to the antennae, in experiments to induce learning, where the odor represents a conditioned stimulus, while sucrose represents an unconditioned stimulus. A series of studies have been conducted on honeybees, relating learning and memory acquisition/retrieval using the PER as a strategy for accessing their ability to exhibit an unconditioned stimulus; however, the major metabolic processes involved in the PER are not well known. Thus, the aim of this investigation is profiling the metabolome of the honeybee brain involved in the PER. In this study, a semiquantitative approach of matrix-assisted laser desorption ionization (MALDI) mass spectral imaging (MSI) was used to profile the most abundant metabolites of the honeybee brain that support the PER. It was reported that execution of the PER requires the metabolic transformations of arginine, ornithine, and lysine as substrates for the production of putrescine, cadaverine, spermine, spermidine, 1,3-diaminopropane, and γ-aminobutyric acid (GABA). Considering the global metabolome of the brain of honeybee workers, the PER requires the consumption of large amounts of cadaverine and 1,3-diaminopropane, in parallel with the biosynthesis of high amounts of spermine, spermidine, and ornithine. To exhibit the PER, the brain of honeybee workers processes the conversion of l-arginine and l-lysine through the polyamine pathway, with different regional metabolomic profiles at the individual neuropil level. The outcomes of this study using this metabolic route as a reference are indicating that the antennal lobes and the calices (medial and lateral) were the most active brain regions for supporting the PER.

Revealing the Venomous Secrets of the Spider's Web.

Orb-weaving spiders use a highly strong, sticky and elastic web to catch their prey. These web properties alone would be enough for the entrapment of prey; however, these spiders may be hiding venomous secrets in the web, which current research is revealing. Here, we provide strong proteotranscriptomic evidence for the presence of toxin/neurotoxin-like proteins, defensins, and proteolytic enzymes on the web silk from Nephila clavipes spider. The results from quantitative-based transcriptomic and proteomic approaches showed that silk-producing glands produce an extensive repertoire of toxin/neurotoxin-like proteins, similar to those already reported in spider venoms. Meanwhile, the insect toxicity results demonstrated that these toxic components can be lethal and/or paralytic chemical weapons used for prey capture on the web, and the presence of fatty acids in the web may be a responsible mechanism opening the way to the web toxins for accessing the interior of prey's body, as shown here. Comparative phylogenomic-level evolutionary analyses revealed orthologous genes among two spider groups, Araneomorphae and Mygalomorphae, and the findings showed protein sequences similar to toxins found in the taxa Scorpiones and Hymenoptera in addition to Araneae. Overall, these data represent a valuable resource to further investigate other spider web toxin systems and also suggest that N. clavipes web is not a passive mechanical trap for prey capture, but it exerts an active role in prey paralysis/killing using a series of neurotoxins.

Comparing activity, toxicity and model membrane interactions of Jelleine-I and Trp/Arg analogs: analysis of peptide aggregation.

Increasing resistance in antibiotic and chemotherapeutic treatments has been pushing studies of design and evaluation of bioactive peptides. Designing relies on different approaches from minimalist sequences and endogenous peptides modifications to computational libraries. Evaluation relies on microbiological tests. Aiming a deeper understanding, we chose the octapeptide Jelleine-I (JI) for its selective and low toxicity profile, designed small modifications combining the substitutions of Phe by Trp and Lys/His by Arg and tested the antimicrobial and anticancer activity on melanoma cells. Biophysical methods identified environment-dependent modulation of aggregation, but critical aggregation concentrations of JI and analogs in buffer show that peptides start membrane interactions as monomers. The presence of model membranes increases or reduces the partial aggregation of peptides. Compared to JI, analog JIF2WR shows the lowest tendency to aggregation on bacterial model membranes. JI and analogs are lytic to model membranes. Their composition-dependent performance indicates preference for the higher charged anionic bilayers in line with their superior performance toward Staphylococcus aureus and Streptococcus pneumoniae. JIF2WR presented the higher partitioning, higher lytic activity and lower aggregated contents. Despite these increased membranolytic activities, JIF2WR exhibited comparable antimicrobial activity in relation to JI at the expenses of some loss in selectivity. We found that the substitution Phe/Trp (JIF2W) tends to decrease antimicrobial but to increase anticancer activity and aggregation on model membranes and the toxicity toward human cells. However, the concomitant substitution Lys/His by Arg (JIF2WR) modulates some of these tendencies, increasing both the antimicrobial and the anticancer activity while decreasing the aggregation tendency.

Worker Defensive Behavior Associated with Toxins in the Neotropical Termite Neocapritermes braziliensis (Blattaria, Isoptera, Termitidae, Termitinae).

Termite societies are abundant in the tropics, and are therefore exposed to multiple enemies and predators, especially during foraging activity. Soldiers constitute a specialized defensive caste, although workers also participate in this process, and even display suicidal behavior, which is the case with the species Neocapritermes braziliensis. Here we describe the morphology, mechanisms of action, and proteomics of the salivary weapon in workers of this species, which due to the autothysis of the salivary glands causes their body rupture, in turn releasing a defensive secretion, observed during aggressiveness bioassays. Salivary glands are paired, composed of two translucent reservoirs, ducts and a set of multicellular acini. Histological and ultrastructural techniques showed that acini are composed of two types of central cells, and small parietal cells located in the acinar periphery. Type I central cells were abundant and filled with a large amount of secretion, while type II central cells were scarce and presented smaller secretion. Parietal cells were often paired and devoid of secretion. The gel-free proteomic approach (shotgun) followed by mass spectrometry revealed 235 proteins in the defensive secretion, which were classified into functional groups: (i) toxins and defensins, (ii) folding/conformation and post-translational modifications, (iii) salivary gland detoxification, (iv) housekeeping proteins and (v) uncharacterized and hypothetical proteins. We highlight the occurrence of neurotoxins previously identified in arachnid venoms, which are novelties for termite biology, and contribute to the knowledge regarding the defense strategies developed by termite species from the Neotropical region.

MALDI Imaging Analysis of Neuropeptides in Africanized Honeybee ( Apis mellifera) Brain: Effect of Aggressiveness.

Aggressiveness in honeybees seems to be regulated by multiple genes, under the influence of different factors, such as polyethism of workers, environmental factors, and response to alarm pheromones, creating a series of behavioral responses. It is suspected that neuropeptides seem to be involved with the regulation of the aggressive behavior. The role of allatostatin and tachykinin-related neuropeptides in honeybee brain during the aggressive behavior is unknown, and thus worker honeybees were stimulated to attack and to sting leather targets hung in front of the colonies. The aggressive individuals were collected and immediately frozen in liquid nitrogen; the heads were removed and sliced at sagittal plan. The brain slices were submitted to MALDI spectral imaging analysis, and the results of the present study reported the processing of the precursors proteins into mature forms of the neuropeptides AmAST A (59-76) (AYTYVSEYKRLPVYNFGL-NH2), AmAST A (69-76) (LPVYNFGL-NH2), AmTRP (88-96) (APMGFQGMR-NH2), and AmTRP (254-262) (ARMGFHGMR-NH2), which apparently acted in different neuropils of the honeybee brain during the aggressive behavior, possibly taking part in the neuromodulation of different aspects of this complex behavior. These results were biologically validated by performing aggressiveness-related behavioral assays using young honeybee workers that received 1 ng of AmAST A (69-76) or AmTRP (88-96) via hemocele. The young workers that were not expected to be aggressive individuals presented a complete series of aggressive behaviors in the presence of the neuropeptides, corroborating the hypothesis that correlates the presence of mature AmASTs A and AmTRPs in the honeybee brain with the aggressiveness of this insect.

Exposure to a sublethal concentration of imidacloprid and the side effects on target and nontarget organs of Apis mellifera (Hymenoptera, Apidae).

The use of insecticides has become increasingly frequent, and studies indicate that these compounds are involved in the intoxication of bees. Imidacloprid is a widely used neonicotinoid; thus, we have highlighted the importance of assessing its oral toxicity to Africanized bees and used transmission electron microscopy to investigate the sublethal effects in the brain, the target organ, and the midgut, responsible for the digestion/absorption of food. In addition, the distribution of proteins involved in important biological processes in the brain were evaluated on the 1st day of exposure by MALDI-imaging analysis. Bioassays were performed to determine the Median Lethal Concentration (LC50) of imidacloprid to bees, and the value obtained was 1.4651 ng imidacloprid/µL diet. Based on this result, the sublethal concentration to be administered at 1, 4 and 8 days was established as a hundredth (1/100) of the LC50. The results obtained from the ultrastructural analysis showed alterations in the midgut cells of bees as nuclear and mitochondrial damage and an increase of vacuoles. The insecticide caused spacing among the Kenyon cells in the mushroom bodies, chromatin condensation and loss of mitochondrial cristae. The MALDI-imaging analysis showed an increase in the expression of such proteins as vascular endothelial growth factor receptor, amyloid protein precursor and protein kinase C, which are related to oxygen supply, neuronal degeneration and memory/learning, and a decrease in the expression of the nicotinic acetylcholine receptor alpha 1, which is fundamental to the synapses. These alterations demonstrated that imidacloprid could compromise the viability of the midgut epithelium, as well as inhibiting important cognitive processes in individuals, and may be reflected in losses of the colony.

Structural characterization of the major ampullate silk spidroin-2 protein produced by the spider Nephila clavipes.

Major ampullate spidroin-2 (MaSp2) is one of the most important spider silk protein, but up to now no information is available regarding the post-translational modifications (PTMs) of this protein. A gel-based mass spectrometry strategy using collision-induced dissociation (CID) and electron-transfer dissociation (ETD) fragmentation methods was used to sequence Nephila clavipes MaSp2 (including the N- and C-terminal non-repetitive domains, and the great part of the central core), and to assign a series of post-translational modifications (PTMs) on to the MaSp2 sequence. Two forms of this protein were identified, with different levels of phosphorylation along their sequences. These findings provide a basis for understanding mechanoelastic properties and can support the future design of recombinant spider silk proteins for biotechnological applications.

Structural studies of the protein endostatin in fusion with BAX BH3 death domain, a hybrid that presents enhanced antitumoral activity.

Endostatin (ES) is an antiangiogenic protein that exhibits antitumor activity in animal models. However, the activity observed in animals was not observed in human clinical trials. ES-BAX is a fusion protein composed of two functional domains: ES, which presents specificity and is internalized by activated endothelial cells and the proapoptotic BH3 domain of the protein BAX, a peptide inductor of cellular death when internalized. We have previously shown (Chura-Chambi et al., Cell Death Dis, 5, e1371, 2014) that ES-BAX presents improved antitumor activity in relation to wild-type ES. Secondary and tertiary structures of ES-BAX are similar to ES, as indicated by homology-modeling studies and molecular dynamics simulations. Tryptophan intrinsic fluorescence and circular dichroism spectroscopy corroborate these data. 15 N HSQC NMR indicates that ES-BAX is structured, but some ES residues have suffered chemical shift perturbations, suggesting that the BH3 peptide interacts with some parts of the ES protein. ES and ES-BAX present similar stability to thermal denaturation. The production of stable hybrid proteins can be a new approach to the development of therapeutic agents presenting specificity for tumoral endothelium and improved antitumor effect.

Silkomics: Insight into the Silk Spinning Process of Spiders.

The proteins from the silk-producing glands were identified using both a bottom-up gel-based proteomic approach as well as from a shotgun proteomic approach. Additionally, the relationship between the functions of identified proteins and the spinning process was studied. A total of 125 proteins were identified in the major ampullate, 101 in the flagelliform, 77 in the aggregate, 75 in the tubuliform, 68 in the minor ampullate, and 23 in aciniform glands. On the basis of the functional classification using Gene Ontology, these proteins were organized into seven different groups according to their general function: (i) web silk proteins-spidroins, (ii) proteins related to the folding/conformation of spidroins, (iii) proteins that protect silk proteins from oxidative stress, (iv) proteins involved in fibrillar preservation of silks in the web, (v) proteins related to ion transport into and out of the glands during silk fiber spinning, (vi) proteins involved in prey capture and pre-digestion, and (vii) housekeeping proteins from all of the glands. Thus, a general mechanism of action for the identified proteins in the silk-producing glands from the Nephila clavipes spider was proposed; the current results also indicate that the webs play an active role in prey capture.

Walker 256 Tumor Growth Suppression by Crotoxin Involves Formyl Peptide Receptors and Lipoxin A4.

We investigated the effects of Crotoxin (CTX), the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, on Walker 256 tumor growth, the pain symptoms associated (hyperalgesia and allodynia), and participation of endogenous lipoxin A4. Treatment with CTX (s.c.), daily, for 5 days reduced tumor growth at the 5th day after injection of Walker 256 carcinoma cells into the plantar surface of adult rat hind paw. This observation was associated with inhibition of new blood vessel formation and decrease in blood vessel diameter. The treatment with CTX raised plasma concentrations of lipoxin A4 and its natural analogue 15-epi-LXA4, an effect mediated by formyl peptide receptors (FPRs). In fact, the treatment with Boc-2, an inhibitor of FPRs, abolished the increase in plasma levels of these mediators triggered by CTX. The blockage of these receptors also abolished the inhibitory action of CTX on tumor growth and blood vessel formation and the decrease in blood vessel diameter. Together, the results herein presented demonstrate that CTX increases plasma concentrations of lipoxin A4 and 15-epi-LXA4, which might inhibit both tumor growth and formation of new vessels via FPRs.

PE and PS Lipids Synergistically Enhance Membrane Poration by a Peptide with Anticancer Properties.

Polybia-MP1 (MP1) is a bioactive host-defense peptide with known anticancer properties. Its activity is attributed to excess serine (phosphatidylserine (PS)) on the outer leaflet of cancer cells. Recently, higher quantities of phosphatidylethanolamine (PE) were also found at these cells' surface. We investigate the interaction of MP1 with model membranes in the presence and absence of POPS (PS) and DOPE (PE) to understand the role of lipid composition in MP1's anticancer characteristics. Indeed we find that PS lipids significantly enhance the bound concentration of peptide on the membrane by a factor of 7-8. However, through a combination of membrane permeability assays and imaging techniques we find that PE significantly increases the susceptibility of the membrane to disruption by these peptides and causes an order-of-magnitude increase in membrane permeability by facilitating the formation of larger transmembrane pores. Significantly, atomic-force microscopy imaging reveals differences in the pore formation mechanism with and without the presence of PE. Therefore, PS and PE lipids synergistically combine to enhance membrane poration by MP1, implying that the combined enrichment of both these lipids in the outer leaflet of cancer cells is highly significant for MP1's anticancer action. These mechanistic insights could aid development of novel chemotherapeutics that target pathological changes in the lipid composition of cancerous cells.

Structural Model for the Spider Silk Protein Spidroin-1.

Most reports about the 3-D structure of spidroin-1 have been proposed for the protein in solid state or for individual domains of these proteins. A gel-based mass spectrometry strategy using collision-induced dissociation (CID) and electron-transfer dissociation (ETD) fragmentation methods was used to completely sequence spidroins-1A and -1B and to assign a series of post-translational modifications (PTMs) on to the spidroin sequences. A total of 15 and 16 phosphorylation sites were detected on spidroin-1A and -1B, respectively. In this work, we present the nearly complete amino acid sequence of spidroin-1A and -1B, including the nonrepetitive N- and C-terminal domains and a highly repetitive central core. We also described a fatty acid layer surrounding the protein fibers and PTMs in the sequences of spidroin-1A and -1B, including phosphorylation. Thus, molecular models for phosphorylated spidroins were proposed in the presence of a mixture fatty acids/water (1:1) and submitted to molecular dynamics simulation. The resulting models presented high content of coils, a higher percentage of α-helix, and an almost neglected content of 310-helix than the previous models. Knowledge of the complete structure of spidroins-1A and -1B would help to explain the mechanical features of silk fibers. The results of the current investigation provide a foundation for biophysical studies of the mechanoelastic properties of web-silk proteins.

Structure-function relationships of the peptide Paulistine: a novel toxin from the venom of the social wasp Polybia paulista.

BACKGROUND: The peptide Paulistine was isolated from the venom of wasp Polybia paulista. This peptide exists under a natural equilibrium between the forms: oxidised - with an intra-molecular disulphide bridge; and reduced - in which the thiol groups of the cysteine residues do not form the disulphide bridge. The biological activities of both forms of the peptide are unknown up to now. METHODS: Both forms of Paulistine were synthesised and the thiol groups of the reduced form were protected with the acetamidemethyl group [Acm-Paulistine] to prevent re-oxidation. The structure/activity relationships of the two forms were investigated, taking into account the importance of the disulphide bridge. RESULTS: Paulistine has a more compact structure, while Acm-Paulistine has a more expanded conformation. Bioassays reported that Paulistine caused hyperalgesia by interacting with the receptors of lipid mediators involved in the cyclooxygenase type II pathway, while Acm-Paullistine also caused hyperalgesia, but mediated by receptors involved in the participation of prostanoids in the cyclooxygenase type II pathway. CONCLUSION: The acetamidemethylation of the thiol groups of cysteine residues caused small structural changes, which in turn may have affected some physicochemical properties of the Paulistine. Thus, the dissociation of the hyperalgesy from the edematogenic effect when the actions of Paulistine and Acm-Paulistine are compared to each other may be resulting from the influence of the introduction of Acm-group in the structure of Paulistine. GENERAL SIGNIFICANCE: The peptides Paulistine and Acm-Paulistine may be used as interesting tools to investigate the mechanisms of pain and inflammation in future studies.

Combining experimental evidence and molecular dynamic simulations to understand the mechanism of action of the antimicrobial octapeptide jelleine-I.

Jelleines are four naturally occurring peptides that comprise approximately eight or nine C-terminal residues in the sequence of the major royal jelly protein 1 precursor (Apis mellifera). The difference between these peptides is limited to one residue in the sequence, but this residue has a significant impact in their efficacy as antimicrobials. In peptide-bilayer experiments, we demonstrated that the lytic, pore-forming activity of Jelleine-I is similar to that of other cationic antimicrobial peptides, which exhibit stronger activity on anionic bilayers. Results from molecular dynamics simulations suggested that the presence of a proline residue at the first position is the underlying reason for the higher efficacy of Jelleine-I compared with the other jelleines. Additionally, simulations suggested that Jelleine-I tends to form aggregates in water and in the presence of mimetic membrane environments. Combined experimental evidence and simulations showed that the protonation of the histidine residue potentiates the interaction with anionic palmitoyl-oleoyl-phosphatidylcholine/palmitoyl-oleoyl-phosphatidylglycerol (POPC/POPG) (70:30) bilayers and reduces the free energy barrier for water passage. The interaction is driven by electrostatic interactions with the headgroup region of the bilayer with some disturbance of the acyl chain region. Our findings point to a mechanism of action by which aggregated Jelleine-I accumulates on the headgroup region of the membrane. Remaining in this form, Jelleine-I could exert pressure to accommodate its polar and nonpolar residues on the amphiphilic environment of the membrane. This pressure could open pores or defects, could disturb the bilayer continuity, and leakage would be observed. The agreement between experimental data and simulations in mimetic membranes suggests that this approach may be a valuable tool to the understanding of the molecular mechanisms of action.