Non-targeted proteomics reveals altered immune response in geographically distinct populations of green sea turtles (Chelonia mydas).

All seven species of sea turtle are facing increasing pressures from human activities that are impacting their health. Changes in circulating blood proteins of an individual, or all members of a population, can provide an early indicator of adverse health outcomes. Non-targeted measurement of all detectable proteins in a blood sample can indicate physiological changes. In the context of wildlife toxicology, this technique can provide a powerful tool for discovering biomarkers of chemical exposure and effect. This study presents a non-targeted examination of the protein abundance in sea turtle plasma obtained from three geographically distinct foraging populations of green turtles (Chelonia mydas) on the Queensland coast. Relative changes in protein expression between sites were compared, and potential markers of contaminant exposure were investigated. Blood plasma protein profiles were distinct between populations, with 85 out of the 116 identified proteins differentially expressed (p < 0.001). The most strongly dysregulated proteins were predominantly acute phase proteins, suggestive of differing immune status between the populations. The highest upregulation of known markers of immunotoxicity, such as pentraxin fusion and complement factor h, was observed in the Moreton Bay turtles. Forty-five different organohalogens were also measured in green turtle plasma samples as exposure to some organohalogens (e.g., polychlorinated biphenyls) has previously been identified as a cause for immune dysregulation in marine animals. The few detected organohalogens were at very low (pg/mL) concentrations in turtles from all sites, and are unlikely to be the cause of the proteome differences observed. However, the changes in protein expression may be indicative of exposure to other chemicals or environmental stressors. The results of this study provide important information about differences in protein expression between different populations of turtles, and guide future toxicological and health studies on east-Australian green sea turtles.

Polycyclic Aromatic Hydrocarbons Detected in Processed Meats Cause Genetic Changes in Colorectal Cancers.

Polycyclic aromatic hydrocarbons (PAHs) are commonly ingested via meat and are produced from high-temperature cooking of meat. Some of these PAHs have potential roles in carcinogenesis of colorectal cancer (CRC). We aimed to investigate PAH concentrations in eight types of commonly consumed ready-to-eat meat samples and their potential effects on gene expressions related to CRC. Extraction and clean-up of meat samples were performed using QuEChERS method, and PAHs were detected using GC-MS. Nine different PAHs were found in meat samples. Interestingly, roast turkey contained the highest total PAH content, followed by salami meat. Hams of varying levels of smokedness showed a proportional increase of phenanthrene (PHEN), anthracene (ANTH), and fluorene (FLU). Triple-smoked ham samples showed significantly higher levels of these PAHs compared to single-smoked ham. These three PAHs plus benzo[a]pyrene (B[a]P), being detected in three meat samples, were chosen as treatments to investigate in vitro gene expression changes in human colon cells. After PAH treatment, total RNA was extracted and rtPCR was performed, investigating gene expression related to CRC. B[a]P decreased mRNA expression of TP53. In addition, at high concentrations, B[a]P significantly increased KRAS expression. Treatments with 1 µM PHEN, 25 µM, and 10 µM FLU significantly increased KRAS mRNA expression in vitro, implying the potential basis for PAH-induced colorectal carcinogenesis. Opposingly, the ANTH treatment led to increased TP53 and APC expression and decreased KRAS expression, suggesting an anti-carcinogenic effect. To conclude, PAHs are common in ready-to-eat meat samples and are capable of significantly modifying the expression of key genes related to CRC.

Protein interactions of FAM134B with EB1 and APC/beta-catenin in vitro in colon carcinoma.

FAM134B is an autophagy regulator of endoplasmic reticulum and acts as a cancer suppressor in colon cancer. However, the molecular signaling pathways by which FAM134B interacts within colon carcinogenesis is still unknown. Herein, this study aims to determine the interacting partners of FAM134B for the first time in colon cancer and to explore the precise location of FAM134B in cancer signalling pathways. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) followed by anti-FAM134B co-immune precipitation of FAM134B interacting complex was used to identify the potential interactors of FAM134B in colon cancer cells. Western blot and confocal microscopic analysis were used to validate the physical interactions of FAM134B with the interactors. Lentiviral shRNA mediated silencing of FAM134B was used to examine the modulation of FAM134B interactors in cells. We have identified 29 novel binding partners, including CAP1, RPS28, FTH1, KDELR2, MAP4, EB1, PSMD6, PPIB/CYPB etc. Subsequent immunoassays confirmed the direct physical interactions of FAM134B with CAP1, EB1, CYPB, and KDELR2 in colon cancer cells. Exogenous suppression of FAM134B has led to significant upregulation of EB1 as well as reduction of KDELR2 expression. It was noted that overexpression of EB1 promotes WNT/ß-catenin signaling pathways via inactivating tumor suppressor APC followed by activating ß-catenin in colorectal carcinogenesis. This study has first time reported the gene signaling networks with which FAM134B interacts and noted that FAM134B is involved in the regulation of WNT/ß-catenin pathway by EB1-mediated modulating of APC in colon cancer cells.

Interaction of Tarantula Venom Peptide ProTx-II with Lipid Membranes Is a Prerequisite for Its Inhibition of Human Voltage-gated Sodium Channel NaV1.7.

ProTx-II is a disulfide-rich peptide toxin from tarantula venom able to inhibit the human voltage-gated sodium channel 1.7 (hNaV1.7), a channel reported to be involved in nociception, and thus it might have potential as a pain therapeutic. ProTx-II acts by binding to the membrane-embedded voltage sensor domain of hNaV1.7, but the precise peptide channel-binding site and the importance of membrane binding on the inhibitory activity of ProTx-II remain unknown. In this study, we examined the structure and membrane-binding properties of ProTx-II and several analogues using NMR spectroscopy, surface plasmon resonance, fluorescence spectroscopy, and molecular dynamics simulations. Our results show a direct correlation between ProTx-II membrane binding affinity and its potency as an hNaV1.7 channel inhibitor. The data support a model whereby a hydrophobic patch on the ProTx-II surface anchors the molecule at the cell surface in a position that optimizes interaction of the peptide with the binding site on the voltage sensor domain. This is the first study to demonstrate that binding of ProTx-II to the lipid membrane is directly linked to its potency as an hNaV1.7 channel inhibitor.

Rational design and synthesis of an orally bioavailable peptide guided by NMR amide temperature coefficients.

Enhancing the oral bioavailability of peptide drug leads is a major challenge in drug design. As such, methods to address this challenge are highly sought after by the pharmaceutical industry. Here, we propose a strategy to identify appropriate amides for N-methylation using temperature coefficients measured by NMR to identify exposed amides in cyclic peptides. N-methylation effectively caps these amides, modifying the overall solvation properties of the peptides and making them more membrane permeable. The approach for identifying sites for N-methylation is a rapid alternative to the elucidation of 3D structures of peptide drug leads, which has been a commonly used structure-guided approach in the past. Five leucine-rich peptide scaffolds are reported with selectively designed N-methylated derivatives. In vitro membrane permeability was assessed by parallel artificial membrane permeability assay and Caco-2 assay. The most promising N-methylated peptide was then tested in vivo. Here we report a novel peptide (15), which displayed an oral bioavailability of 33% in a rat model, thus validating the design approach. We show that this approach can also be used to explain the notable increase in oral bioavailability of a somatostatin analog.

Mirror Images of Antimicrobial Peptides Provide Reflections on Their Functions and Amyloidogenic Properties.

Enantiomeric forms of BTD-2, PG-1, and PM-1 were synthesized to delineate the structure and function of these ß-sheet antimicrobial peptides. Activity and lipid-binding assays confirm that these peptides act via a receptor-independent mechanism involving membrane interaction. The racemic crystal structure of BTD-2 solved at 1.45 Å revealed a novel oligomeric form of ß-sheet antimicrobial peptides within the unit cell: an antiparallel trimer, which we suggest might be related to its membrane-active form. The BTD-2 oligomer extends into a larger supramolecular state that spans the crystal lattice, featuring a steric-zipper motif that is common in structures of amyloid-forming peptides. The supramolecular structure of BTD-2 thus represents a new mode of fibril-like assembly not previously observed for antimicrobial peptides, providing structural evidence linking antimicrobial and amyloid peptides.

Approaches to the stabilization of bioactive epitopes by grafting and peptide cyclization.

Peptides are attracting increasing interest from the pharmaceutical industry because of their specificity and ability to address novel targets, including protein-protein interactions. However, typically they require stabilization for therapeutic applications owing to their susceptibility to degradation by proteases. Advances in the ability to chemically synthesize peptides and the development of new side-chain and backbone ligation strategies provide new tools to stabilize bioactive peptide epitopes. Two such epitopes are LyP1, a nine residue peptide that localizes to tumor cells and has potential as an anticancer therapeutic, and RGDS, a tetrapeptide shown to bind to survivin and induce apoptosis. Here we applied a variety of strategies for the stabilization of LyP1 and RGDS, including side-chain cyclization using "click" chemistry and "grafting" the epitopes into two naturally occurring cyclic peptide scaffolds, i.e., θ-defensins and cyclotides. NMR data showed that the three-disulfide θ-defensin and cyclotide scaffolds accommodated the LyP1 and RGDS epitopes but that scaffolds with fewer disulfide bonds were structurally compromised by inclusion of the LyP1 epitope. LyP1, LyP1-, and RGDS-grafted peptides that were largely unstructured also had reduced resistance to degradation in human serum, showing that grafting into a stable cyclic scaffold is an effective strategy for increasing the stability of a bioactive peptide epitope. Overall, the study demonstrates several methods for stabilizing peptide epitopes using side-chain or backbone cyclization and illustrates their potential in peptide drug design.

Anticancer and toxic properties of cyclotides are dependent on phosphatidylethanolamine phospholipid targeting.

Cyclotides, ultrastable disulfide-rich cyclic peptides, can be engineered to bind and inhibit specific cancer targets. In addition, some cyclotides are toxic to cancer cells, though not much is known about their mechanisms of action. Here we delineated the potential mode of action of cyclotides towards cancer cells. A novel set of analogues of kalata B1 (the prototypic cyclotide) and kalata B2 and cycloviolacin O2 were examined for their membrane-binding affinity and selectivity towards cancer cells. By using solution-state NMR, surface plasmon resonance, flow cytometry and bioassays we show that cyclotides are toxic against cancer and non-cancerous cells and their toxicity correlates with their ability to target and disrupt lipid bilayers that contain phosphatidylethanolamine phospholipids. Our results suggest that the potential of cyclotides as anticancer therapeutics might best be realised by combining their amenability to epitope engineering with their ability to bind cancer cell membranes.

Influence of chemical dose and exposure duration on protein synthesis in green sea turtle primary cells.

Understanding the impacts of chemical exposure in marine wildlife is challenging, due to practical and ethical constraints that preclude traditional toxicology research on these animals. This study addressed some of these limitations by presenting an ethical and high throughput cell-based approach to elucidate molecular-level effects of contaminants on sea turtles. The experimental design addressed basic questions of cell-based toxicology, including chemical dose and exposure time. Primary green turtle skin cells were exposed to polychlorinated biphenyl (PCB) 153 and perfluorononanoic acid (PFNA) for 24 and 48 h, at three sub-lethal, environmentally relevant concentrations (1, 10 and 100 µg/L). Sequential window acquisition of all theoretical mass spectra (SWATH-MS) identified over 1000 differentially abundant proteins within the 1% false discovery rate (FDR) threshold. The 24 h exposure resulted in a greater number of differentially abundant proteins, compared to 48 h exposure, for both contaminants. However, there were no statistically significant dose-response relationships for the number of differentially synthesised proteins, nor differences in the proportion of increased vs decreased proteins between or within exposure times. Known in vivo markers of contaminant exposure, superoxide dismutase and glutathione S-transferase, were differentially abundant following exposure to PCB153 and PFNA. SIGNIFICANCE: Cell-based (in vitro) proteomics provides an ethical and high throughput approach to understanding the impacts of chemical contamination on sea turtles. Through investigating effects of chemical dose and exposure duration on unique protein abundance in vitro, this study provides an optimised framework for conducting cell-based studies in wildlife proteomics, and highlights that proteins detected in vitro could act as biomarkers of chemical exposure and effect in vivo.

Field-scale monitoring of green sea turtles (Chelonia mydas): Influence of site characteristics and capture technique on the blood metabolome.

Given their threatened status, there is considerable interest in establishing monitoring techniques that can be used to evaluate the health of sea turtles in the wild. The present study represents a methodological contribution towards field-scale metabolomic assessment of sea turtles, by exploring differences in blood biochemistry associated with site characteristics and capture technique. We compared the metabolome of blood from animals at three locations (two coastal and one reefal), collected from turtles that were either resting or active, and sampled across multiple seasons at one location. Our results show clear differences in the metabolome of turtles from the three locations, some of which are likely attributable to differences in diet or forage quality and others which may reflect differences in other factors (e.g., occurrence of land-based contaminants or other biotic and/or abiotic stressors) between coastal and reefal sites. Our analysis also revealed the influence of capture technique on metabolite profiles, with numerous markers of physical exertion in animals captured while active that were absent in turtles sampled while resting. We observed a modest potential for temporal differences in the metabolome, but controlling for sampling time did not change the overall conclusions of our study. This suggests that temporal differences in the metabolome warrant consideration when designing studies to evaluate the status of sea turtles in the wild, but that site characteristics and capture technique are bigger drivers. However, sample size for this comparison was relatively small and further investigation of seasonal differences in the metabolome are warranted. Research exploring each of these factors more closely will further contribute towards achieving robust metabolomics analysis of sea turtles across large spatial and temporal scales.

Changes in global protein expression in sea turtle cells exposed to common contaminants indicates new biomarkers of chemical exposure.

Non-targeted protein expression at the cellular level can provide insights into mechanistic effects of contaminants in wildlife, and hence new and potentially more accurate biomarkers of exposure and effect. However, this technique has been relatively unexplored in the realm of in vitro biomarker discovery in threatened wildlife, despite the vulnerability of this group of animals to adverse sublethal effects of contaminant exposure. Here we examined the usefulness of non-targeted protein expression for biomarker discovery in green sea turtles (Chelonia mydas) by investigating differences in the response of primary cells from five different tissue types that were exposed to three contaminants known to accumulate in this species. Cells derived from C. mydas skin, liver, kidney, ovary and small intestine were exposed to 100 µg/L of either polychlorinated biphenyl 153 (PCB153), perfluorononanoic acid (PFNA) or phenanthrene for 24 h. The global protein expression was then quantitatively evaluated using sequential window acquisition of all theoretical mass spectra (SWATH-MS). Comparison of the global protein profiles revealed that, while a majority of proteins were mutually expressed in controls of all tissue types (~90%), the response to exposure in terms of protein expression strength was significantly different between tissue types. Furthermore, a comparison to known markers of chemical exposure in sea turtles from the literature indicated that in vitro response can reflect known in vivo responses. In particular, markers such as heat shock protein (HSP) 60, glutathione S-transferases (GSTs) and superoxide dismutases (SODs), cytochrome P450 and catalase were dysregulated in response to exposure. Furthermore, potential new markers of exposure were discovered such as annexin, an important protein in cell signalling processes. While this methodology proved promising further studies are required to confirm the accuracy of in vitro protein expression as a tool for biomarker discovery in wildlife.

Cyclic gomesin, a stable redesigned spider peptide able to enter cancer cells.

Anticancer chemo- and targeted therapies are limited in some cases due to strong side effects and/or drug resistance. Peptides have received renascent interest as anticancer therapeutics and are currently being considered as alternatives and/or as complementary to biologics and small-molecule drugs. Gomesin, a disulfide-rich host defense peptide expressed in the Brazilian spider Acanthoscurria gomesiana selectively targets and disrupts cancer cell membranes. In the current study, we employed a range of biophysical methodologies with model membranes and bioassays to investigate the use of a cyclic analogue of gomesin as a drug scaffold to internalize cancer cells. We found that cyclic gomesin can internalize cancer cells via endocytosis and direct membrane permeation. In addition, we designed an improved non-disruptive and non-toxic cyclic gomesin analogue by incorporating D-amino acids within the scaffold. This improved analogue retained the ability to enter cancer cells and can be used as a scaffold to deliver drugs. Efforts to investigate the internalization mechanism used by host defense peptides, and to improve their stability, potency, selectivity and ability to permeate cancer cell membranes will increase the opportunities to repurpose peptides as templates for designing alternative anticancer therapeutic leads.

Charting a path towards non-destructive biomarkers in threatened wildlife: A systematic quantitative literature review.

Threatened species are susceptible to irreversible population decline caused by adverse sub-lethal effects of chemical contaminant exposure. It is therefore vital to develop the necessary tools to predict and detect these effects as early as possible. Biomarkers of contaminant exposure and effect are widely applied to this end, and a significant amount of research has focused on development and validation of sensitive and diagnostic biomarkers. However, progress in the use biomarkers that can be measured using non-destructive techniques has been relatively slow and there are still many difficulties to overcome in the development of sound methods. This paper systematically quantifies and reviews studies that have aimed to develop or validate non-destructive biomarkers in wildlife, and provides an analysis of the successes of these methods based on the invasiveness of the methods, the potential for universal application, cost, and the potential for new biomarker discovery. These data are then used to infer what methods and approaches appear the most effective for successful development of non-destructive biomarkers of contaminant exposure in wildlife. This review highlights that research on non-destructive biomarkers in wildlife is severely lacking, and suggests further exploration of in vitro methods in future studies.

Redesigned Spider Peptide with Improved Antimicrobial and Anticancer Properties.

Gomesin, a disulfide-rich antimicrobial peptide produced by the Brazilian spider Acanthoscurria gomesiana, has been shown to be potent against Gram-negative bacteria and to possess selective anticancer properties against melanoma cells. In a recent study, a backbone cyclized analogue of gomesin was shown to be as active but more stable than its native form. In the current study, we were interested in improving the antimicrobial properties of the cyclic gomesin, understanding its selectivity toward melanoma cells and elucidating its antimicrobial and anticancer mode of action. Rationally designed analogues of cyclic gomesin were examined for their antimicrobial potency, selectivity toward cancer cells, membrane-binding affinity, and ability to disrupt cell and model membranes. We improved the activity of cyclic gomesin by ∼10-fold against tested Gram-negative and Gram-positive bacteria without increasing toxicity to human red blood cells. In addition, we showed that gomesin and its analogues are more toxic toward melanoma and leukemia cells than toward red blood cells and act by selectively targeting and disrupting cancer cell membranes. Preference toward some cancer types is likely dependent on their different cell membrane properties. Our findings highlight the potential of peptides as antimicrobial and anticancer leads and the importance of selectively targeting cancer cell membranes for drug development.

Optimization of the cyclotide framework to improve cell penetration properties.

Cell penetrating peptides have been regarded as promising vectors to deliver hydrophilic molecules inside cells. Although they are great tools for research and have high potential as drug delivery systems, their application as drugs is impaired by their low stability in serum. Cyclotides, cyclic disulfide-rich peptides from plants, are ultra-stable molecules that have inspired applications in drug design as they can be used as scaffolds to stabilize linear bioactive sequences. Recently, they have also been shown to possess cell-penetrating properties. The combination of their remarkable stability and cell-penetrating properties opens new avenues for the application of peptides to bind to and inhibit intracellular proteins. Nevertheless, for a broader application of these molecules as vectors is of utmost importance to improve their cellular internalization efficiency. In this study we successfully modified MCoTI-II, one of the most widely studied cyclotide scaffolds in drug design, and improved its internalization properties. The internalization of the newly designed MCoTI-II is as efficient as the gold standard cell-penetrating peptide (CPP) TAT and maintains all the required features as a template to graft desired bioactivities.

Exploring experimental and computational markers of cyclic peptides: Charting islands of permeability.

An increasing number of macrocyclic peptides that cross biological membranes are being reported, suggesting that it might be possible to develop peptides into orally bioavailable therapeutics; however, current understanding of what makes macrocyclic peptides cell permeable is still limited. Here, we synthesized 62 cyclic hexapeptides and characterized their permeability using in vitro assays commonly used to predict in vivo absorption rates, i.e. the Caco-2 and PAMPA assays. We correlated permeability with experimentally measured parameters of peptide conformation obtained using rapid methods based on chromatography and nuclear magnetic resonance spectroscopy. Based on these correlations, we propose a model describing the interplay between peptide permeability, lipophilicity and hydrogen bonding potential. Specifically, peptides with very high permeability have high lipophilicity and few solvent hydrogen bond interactions, whereas peptides with very low permeability have low lipophilicity or many solvent interactions. Our model is supported by molecular dynamics simulations of the cyclic peptides calculated in explicit solvent, providing a structural basis for the observed correlations. This prospective exploration into biomarkers of peptide permeability has the potential to unlock wider opportunities for development of peptides into drugs.

The Prototypic Cyclotide Kalata B1 Has a Unique Mechanism of Entering Cells.

Cyclotides combine the stability of disulfide-rich peptides with the intracellular accessibility of cell-penetrating peptides, giving them outstanding potential as drug scaffolds with an ability to inhibit intracellular protein-protein interactions. To realize and optimize the application of cyclotides as a drug framework and delivery system, we studied the ability of the prototypic cyclotide, kalata B1, to enter mammalian cells. We show that kalata B1 can enter cells via both endocytosis and direct membrane translocation. Both pathways are initiated by targeting phosphatidylethanolamine phospholipids at the cell surface and inducing membrane curvature. This unusual approach to initiate internalization might be harnessed to deliver drugs into cells and, in particular, cancer cells, which present a higher proportion of surface-exposed phosphatidylethanolamine phospholipids. Our findings highlight the potential of these peptides as drug leads for the modulation of traditionally "undruggable" targets, such as intracellular protein-protein interactions.

Rapid short term and gradual permanent cardiotoxic effects of vertebrate toxins from Chironex fleckeri (Australian box jellyfish) venom.

The vertebrate cardiotoxic components of the venom produced by the Australian box jellyfish, Chironex fleckeri, have not previously been isolated. We have uncovered for the first time, three distinct cytotoxic crude fractions from within the vertebrate cardiotoxic peak of C. fleckeri venom by monitoring viability of human muscle cells with an impedance based assay (ACEA xCELLigence system) measuring cell detachment as cytotoxicity which was correlated with a reduction in cell metabolism using a cell proliferation (MTS) assay. When the effects of the venom components on human cardiomyocytes and human skeletal muscle cells were compared, two fractions were found to specifically affect cardiomyocytes with distinct temporal profiles (labelled Crude Toxic Fractions (CTF), α and ß). A third fraction (CTF-γ) was toxic to both muscle cell types and therefore not cardio specific. The vertebrate, cardio specific CTF-α and CTF-ß, presented distinct activities; CTF-α caused rapid but short term cell detachment and reduction in cell metabolism with enhanced activity at lower concentrations than CTF-ß. This activity was not permanent, with cell reattachment and subsequent increased metabolism of heart muscle cells observed when exposed to all but the highest concentrations of CTF-α tested. The cytotoxic effect of CTF-ß took twice as long to act on the cells compared to CTF-α, however, the activity was permanent. Furthermore, we showed that the two fractions combined have a synergistic effect causing a much stronger and faster cell detachment (death) when combined than the sum of the individual effects of each toxin. These data presented here improves the current understanding of the toxic mechanisms of the Australian box jellyfish, C. fleckeri, and provides a basis for in vivo research of these newly isolated toxic fractions.

Cyclic Penta- and Hexaleucine Peptides without N-Methylation Are Orally Absorbed.

Development of peptide-based drugs has been severely limited by lack of oral bioavailability with less than a handful of peptides being truly orally bioavailable, mainly cyclic peptides with N-methyl amino acids and few hydrogen bond donors. Here we report that cyclic penta- and hexa-leucine peptides, with no N-methylation and five or six amide NH protons, exhibit some degree of oral bioavailability (4-17%) approaching that of the heavily N-methylated drug cyclosporine (22%) under the same conditions. These simple cyclic peptides demonstrate that oral bioavailability is achievable for peptides that fall outside of rule-of-five guidelines without the need for N-methylation or modified amino acids.