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Caspases play essential roles in apoptotic processes, which is necessary for cellular homeostasis. However, over-activation of caspases and subsequent excessive apoptosis is considered a main cause of Parkinson's disease and liver diseases. Here, we found that the insect-derived peptide, CopA3, which has shown antiapoptotic effects in many apoptosis models, directly binds to caspases. The resulting complexes do not dissociate during denaturing polyacrylamide gel electrophoresis, as evidenced by a distinct shift in the migration of caspase reflecting an increase in their molecular weight. Surface plasmon resonance and experiment using cysteine-substituted mutants of CopA3 collectively revealed that binding of CopA3 to caspases is dependent on an internal cysteine residue. Notably, CopA3 binding significantly inhibited proteolytic activation of downstream caspases by upstream caspases. In summary, the demonstration that CopA3 directly binds to caspases and inhibits their activating cleavage suggests a possible therapeutic approach for treating human diseases resulting from uncontrolled apoptosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Periplanetasin-2 from cockroach exhibits broad-spectrum antimicrobial activity. The underlying antibacterial mechanisms rely on the stimulation of reactive oxygen species overproduction to induce apoptotic cell death. A promising strategy to increase the bioavailability of periplanetasin-2 involves reducing the dose through combination therapy with other antibacterials that show synergistic effects. Thus, the synergistic antibacterial activity of periplanetasin-2 with conventional antibacterial agents and its mechanisms was examined against Escherichia coli in this study. Among the agents tested, the combinations of periplanetasin-2 with vancomycin and chloramphenicol exhibited synergistic effects. Periplanetasin-2 in combination with vancomycin and chloramphenicol demonstrated antibacterial activity through the intracellular oxidative stress response. The combination with vancomycin resulted in the enhancement of bacterial apoptosislike death, whereas the combination with chloramphenicol enhanced oxidative stress damage. These synergistic interactions of periplanetasin-2 can help broaden the spectrum of conventional antibiotics. The combination of antimicrobial peptides and conventional antibiotics is proposed as a novel perspective on treatments to combat severe bacterial infection.

Probiotic Gluconacetobacter strains are intestinal microbes with beneficial effects on human health. Recently, researchers have used these strains to biosynthesize metal and non-metal nanoparticles for treating various chronic diseases. Despite their importance in nanotechnology, gold nanoparticles (AuNPs) biosynthesized by Gluconacetobacter species have not been clearly identified for treating inflammation and inflammation-associated diseases. While ginsenoside CK has strong pharmaceutical activity, it also has strong cytotoxicity and hydrophobicity which is hurdle to make formulation. Peptide-nanoparticle hybrids are gaining increasing attention for their potential biomedical applications, including human inflammatory diseases. Herein, we developed peptide CopA3 surface conjugated and ginsenoside compound K (CK) loaded gold nanoparticles (GNP-CK-CopA3), which intracellularly synthesised by the probiotic Gluconacetobacter liquefaciens kh-1, to target lipopolysaccharide (LPS)-activated RAW264.7 macrophages. The synthetic GNP-CK-CopA3 was characterised by various instrumental techniques. The results of our cellular uptake and MTT assays exhibited obvious drug intracellular delivery without significant cytotoxicity. In addition, pre-treatment with GNP-CK-CopA3 significantly ameliorated LPS-induced nitric oxide (NO) and reactive oxygen species (ROS) production and suppressed the mRNA and protein expression of pro-inflammatory cytokines in macrophages. Furthermore, GNP-CK-CopA3 efficiently inhibited the activation of the nuclear factor-κB (NF-κB) and mitogen-activating protein kinase (MAPK) signalling pathways. Taken together, our findings highlight the potential of using peptide-nanoparticle hybrids in the development of anti-inflammatory approaches and providing the experimental foundation for further application.

Nitric oxide (NO) is a potentially powerful weapon against Candida albicans, and the regulation of intracellular NO levels is therefore important for controlling its physiological functions. Lactoferricin B like peptide (LBLP) is a 23-mer antimicrobial peptide (AMP) derived from the Scolopendra subspinipes mutilans. We confirmed that LBLP treatment led to the generation of endogenous NO in C. albicans, which was associated with the NO synthase pathway. Here, we examined the antifungal activity of LBLP with focus on intracellular NO. Total glutathione levels were measured to evaluate cellular defense capacity against NO. LBLP decreased total glutathione levels, leading to nitrosative stress. LBLP also inhibited mitochondrial respiration and altered the NAD +/ NADH ratios. Inhibition of mitochondrial respiration induced mitochondrial membrane depolarization, thus leading to calcium homeostasis disruption and mitochondrial superoxide anion accumulation. Consequently, treatment of C. albicans with LBLP resulted in apoptosis. These physiological changes were attenuated when NO generation was inhibited. Our data strongly indicate that LBLP mediates apoptosis by affecting intracellular NO homeostasis. These results on antifungal activity of LBLP and its mechanism indicate the therapeutic promise of this AMP and support the role of NO in cell death regulation., (© 2020 International Union of Biochemistry and Molecular Biology.)

In a previous work, we performed de novo RNA sequencing of Allomyrina dichotoma using next generation sequencing and identified several antimicrobial peptide candidates based on transcriptome analysis. Among them, a cationic antimicrobial peptide, allomyrinasin, was selected bioinformatically based on its physicochemical properties. Here, we assessed the antimicrobial and anti-inflammatory activities of allomyrinasin against microorganisms and mouse macrophage Raw264.7 cells. Allomyrinasin showed antimicrobial activities against various microbes and decreased the nitric oxide production of the lipopolysaccharide-induced Raw264.7 cells. Furthermore, quantitative RT-PCR and ELISA revealed that allomyrinasin reduced cytokine expression levels in the Raw264.7 cells. We also identified inducible nitric oxide synthase, cyclooxygenase-2 expression, and PGE 2 production through western blot analysis and ELISA. We confirmed that allomyrinasin bound to bacterial cell membranes via a specific interaction with lipopolysaccharides. Taken together, these data indicate that allomyrinasin has antimicrobial and anti-inflammatory activities as exemplified in lipopolysaccharide-induced Raw264.7 cells. We have provided a potentially useful antimicrobial peptide candidate that has both antimicrobial and anti-inflammatory activities.

Communications between various organelle-organelles play an essential role in cell survival. The cross-talk between mitochondria and vacuoles comes up with the vital roles of the intercompartmental process. In this study, we found a couple of cell death features, membrane damage, and apoptosis using antimicrobial peptide from American Cockroach. Periplanetasin-4 (LRHKVYGYCVLGP-NH 2 ) is a 13-mer peptide derived from Periplaneta americana and exhibits phosphatidylserine exposure and caspase activation without DNA fragmentation. Apoptotic features without DNA damage provide evidence that this peptide did not interact with DNA directly and exhibited dysfunction of mitochondria and vacuoles. Superoxide radicals were generated from mitochondria and converted to hydrogen peroxide. Despite the enhancement of catalase and total glutathione contents, oxidative damage disrupted intracellular contents. Periplanetasin-4 induced cell death associated with the production of superoxide radicals, calcium uptake in mitochondria and disorder of vacuoles, such as increased permeability and alkalization. While calcium movement from vacuoles to the mitochondria occurred, the cross-talk with these organelles proceeded and the inherent functionality was impaired. To sum up, periplanetasin-4 stimulates superoxide signal along with undermining the mitochondrial functions and interfering in communication with vacuoles., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Periplanetasin-2 is a 15-mer antimicrobial peptide (AMP), derived from the American cockroach Periplaneta americana. This novel AMP exhibits potent antibacterial effect against several pathogenic bacteria including Escherichia coli. Distinct from the targeting cell membrane, which is the general antibacterial mechanism of AMP, periplanetasin-2 exerts its antibacterial activity via apoptosis-like death, which is physiologically and mechanistically similar to eukaryotic apoptosis. E. coli cells treated with periplanetasin-2 showed features of apoptosis in a concentration-dependent manner, such as membrane depolarization, DNA fragmentation, caspase-like protein activation, and phosphatidylserine externalization. These physiological changes were attenuated by pretreatment with the reactive oxygen species (ROS) scavenger, which demonstrates that periplanetasin-2 induced apoptosis-like death in E. coli by generating ROS. In addition, periplantasin-2-induced apoptotic death was affected by SOS response components. In the absence of RecA, an essential protein for SOS response, apoptosis did not occur and the antibacterial activity of periplanetasin-2 was decreased. In contrast, deletion of the SOS gene dinF caused higher ROS accumulation and apoptotic features were detected. Collectively, these results indicate that the antibacterial mechanism of periplanetasin-2 is ROS-induced apoptosis-like death, which requires RecA for proceeding it, and the role of DinF is assumed to contribute to the ROS defense SOS response.

Numerous studies have reported that enteric neurons involved in controlling neurotransmitter secretion and motility in the gut critically contribute to the progression of gut inflammation. Clostridium difficile toxins, which cause severe colonic inflammation, are also known to affect enteric neurons. Our previous study showed that C. difficile toxin A directly induces neural cell toxicities, such as viability loss and apoptosis. In the current study, we attempted to identify a potent inhibitor of toxin A-induced neural cell toxicity that may aid in managing toxin A-induced gut inflammation. In our recent study, we found that the Korea dung beetle-derived antimicrobial peptide CopA3 completely blocked neural cell apoptosis caused by okadaic acid or 6-OHDA. Here, we examined whether the antimicrobial peptide CopA3 inhibited toxin A-induced neural cell damage. In neuroblastoma SH-SY5Y cells, CopA3 treatment protected against both apoptosis and viability loss caused by toxin A. CopA3 also completely inhibited activation of the pro-apoptotic factor, caspase-3. Additionally, CopA3 rescued toxin A-induced downregulation of neural cell proliferation. However, CopA3 had no effect on signaling through ROS/p38 MAPK/p27 kip1 , suggesting that CopA3 inhibits toxin A-induced neural cell toxicity independent of this well-characterized toxin A pathway. Our data further suggest that ability of CopA3 to rescue toxin A-induced neural cell damage may also ameliorate the gut inflammation caused by toxin A.

In oriental medicine, centipede Scolopendra subspinipes mutilans has long been used as a remedy for rheumatoid arthritis (RA), a well-known chronic autoimmune disorder. However, the molecular identities of its bioactive components have not yet been extensively investigated. We sought to identify bioactive molecules that control RA with a centipede. A novel antimicrobial peptide (AMP) (scolopendrasin IX) was identified from Scolopendra subspinipes mutilans. Scolopendrasin IX markedly activated mouse neutrophils, by enhancing cytosolic calcium increase, chemotactic cellular migration, and generation of superoxide anion in neutrophils. As a target receptor for scolopendrasin IX, formyl peptide receptor (FPR)2 mediates neutrophil activation induced by the AMP. Furthermore, scolopendrasin IX administration strongly blocked the clinical phenotype of RA in an autoantibody-injected model. Mechanistically, the novel AMP inhibited inflammatory cytokine synthesis from the joints and neutrophil recruitment into the joint area. Collectively, we suggest that scolopendrasin IX is a novel potential therapeutic agent for the control of RA via FPR2.

In this study, we identified scolopendrasin X, a novel antimicrobial peptide (AMP), from centipede Scolopendra subspinipes mutilans. Scolopendrasin X strongly stimulated mouse neutrophils, resulting in intracellular calcium increase, chemotactic migration through pertussis toxin-sensitive G-protein and phospholipase C pathway, and increased superoxide anion production in neutrophils. Target receptor for scolopendrasin X, formyl peptide receptor (FPR)2 mediated scolopendrasin X-induced neutrophil activation. Moreover, scolopendrasin X significantly blocked inflammatory cytokine production induced by lipopolysaccharide in mouse neutrophils. Taken together, our results suggest that the novel AMP scolopendrasin X can be used as a material to regulate neutrophil activity through FPR2., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We previously reported that the CopA3 peptide (LLCIALRKK, D -form) originally isolated from the Korean dung beetle has antimicrobial and immunosuppressive effects. However, the high cost of producing the synthetic peptide, especially the D-form, has limited the development of CopA3 for therapeutic purposes. Here, we investigated whether the CopA3 deletion derivative, CopA5, which is composed of only five amino acids (LLCIA) and has the L-form structure, could inhibit the lipopolysaccharide (LPS)-induced activation of macrophages. Peritoneal exudate macrophages (PEM) were isolated from mice and exposed to LPS in the presence or absence of CopA5, and biomarkers of macrophage activation were measured. Our results revealed that LPS-induced nitric oxide (NO) production, tumor necrosis factor (TNF)-α secretion, and phagocytic activity of PEM were significantly inhibited by CopA5 treatment. Similar to CopA3, the structurally modified CopA5 peptide had no cell toxicity (as assessed by measurement of cell viability loss and apoptosis) in PEM. Moreover, the LPS-induced upregulation of the activating phosphorylation of signal transducer and activator of transcription 1 (STAT1) was markedly inhibited by CopA5 treatment. These results suggest that, similar to CopA3, CopA5 inhibits macrophage activation by inhibiting STAT1 phosphorylation and blocking the release of NO and TNF-α. CopA5 may therefore prove therapeutically useful in the realm of immune suppression.

Centipedes, a type of arthropod, reportedly produce antimicrobial peptides as part of an innate immune response. Scolopendin (SPSEKAGLQPVGRIGRMLKK) is a novel antimicrobial peptide derived from Scolopendra subspinipes mutilans Many antifungal agents have more than one type of cell death mechanism. Although scolopendin is involved in membrane perturbation, the corresponding intracellular changes require further investigation. Therefore, we assessed the cell morphology and calcium ion concentration of the cytosol and mitochondria of scolopendin-treated cells. The treated cells were shrunken, and calcium ion homeostasis was disrupted in both the cytosol and mitochondria. These conditions attenuated mitochondrial homeostasis, disrupting mitochondrial membrane potential and cytochrome c levels. Fungal cells treated with scolopendin exhibited various apoptotic phenotypes such as reactive oxygen species accumulation, phosphatidylserine exposure, chromatin condensation, and nuclear fragmentation. Scolopendin-induced cell death also triggered metacaspase activation. In conclusion, treatment of Candida albicans with scolopendin induced the apoptotic response, which in turn led to mitochondrial dysfunction, metacaspase activation, and cell death. The antimicrobial peptide scolopendin from the centipede S.s. mutilans demonstrated a novel apoptotic mechanism as an antifungal agent., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Antimicrobial peptides/proteins (AMPs) are present in all types of organisms, from microbes and plants to vertebrates and invertebrates such as insects. The grasshopper Oxya chinensis sinuosa is an insect species that is widely consumed around the world for its broad medicinal value. However, the lack of available genetic information for this species is an obstacle to understanding the full potential of its AMPs. Analysis of the O. chinensis sinuosa transcriptome and expression profile is essential for extending the available genetic information resources. In this study, we determined the whole-body transcriptome of O. chinensis sinuosa and analyzed the potential AMPs induced by bacterial immunization. A high-throughput RNA-Seq approach generated 94,348 contigs and 66,555 unigenes. Of these unigenes, 36,032 (54.14%) matched known proteins in the NCBI database in a BLAST search. Functional analysis demonstrated that 38,219 unigenes were clustered into 5,499 gene ontology terms. In addition, 26 cDNAs encoding novel AMPs were identified by an in silico approach using public databases. Our transcriptome dataset and AMP profile greatly improve our understanding of O. chinensis sinuosa genetics and provide a huge number of gene sequences for further study, including genes of known importance and genes of unknown function.

We investigated the effects of two antimicrobial peptides (AMPs) isolated from Scolopendra subspinipes mutilans on neutrophil activity. Stimulation of mouse neutrophils with the two AMPs elicited chemotactic migration of the cells in a pertussis toxin-sensitive manner. The two AMPs also stimulated activation of ERK and Akt, which contribute to chemotactic migration of neutrophils. We found that AMP-stimulated neutrophil chemotaxis was blocked by a formyl peptide receptor (FPR) 1 antagonist (cyclosporin H); moreover the two AMPs stimulated the chemotactic migration of FPR1-expressing RBL-2H3 cells but not of vector-expressing RBL-2H3 cells. We also found that the two AMPs stimulate neutrophil migration in vivo, and that this effect is blocked in FPR1-deficient mice. Taken together, our results suggest that the two AMPs stimulate neutrophils, leading to chemotactic migration through FPR1, and the two AMPs will be useful for the study of FPR1 signaling and neutrophil activation. [BMB Reports 2016; 49(9): 520-525].

Centipedes, a kind of arthropod, have been reported to produce antimicrobial peptides as part of an innate immune response. Scolopendin 2 (AGLQFPVGRIGRLLRK) is a novel antimicrobial peptide derived from the body of the centipede Scolopendra subspinipes mutilans by using RNA sequencing. To investigate the intracellular responses induced by scolopendin 2, reactive oxygen species (ROS) and glutathione accumulation and lipid peroxidation were monitored over sublethal and lethal doses. Intracellular ROS and antioxidant molecule levels were elevated and lipids were peroxidized at sublethal concentrations. Moreover, the Ca(2+) released from the endoplasmic reticulum accumulated in the cytosol and mitochondria. These stress responses were considered to be associated with yeast apoptosis. Candida albicans cells exposed to scolopendin 2 were identified using diagnostic markers of apoptotic response. Various responses such as phosphatidylserine externalization, chromatin condensation, and nuclear fragmentation were exhibited. Scolopendin 2 disrupted the mitochondrial membrane potential and activated metacaspase, which was mediated by cytochrome c release. In conclusion, treatment of C. albicans with scolopendin 2 induced the apoptotic response at sublethal doses, which in turn led to mitochondrial dysfunction, metacaspase activation, and cell death. The cationic antimicrobial peptide scolopendin 2 from the centipede is a potential antifungal peptide, triggering the apoptotic response.

Cockroaches are surrogate hosts for microbes that cause many human diseases. In spite of their generally destructive nature, cockroaches have recently been found to harbor potentially beneficial and medically useful substances such as drugs and allergens. However, genomic information for the American cockroach (Periplaneta americana) is currently unavailable; therefore, transcriptome and gene expression profiling is needed as an important resource to better understand the fundamental biological mechanisms of this species, which would be particularly useful for the selection of novel antimicrobial peptides. Thus, we performed de novo transcriptome analysis of P. americana that were or were not immunized with Escherichia coli. Using an Illumina HiSeq sequencer, we generated a total of 9.5 Gb of sequences, which were assembled into 85,984 contigs and functionally annotated using Basic Local Alignment Search Tool (BLAST), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) database terms. Finally, using an in silico antimicrobial peptide prediction method, 86 antimicrobial peptide candidates were predicted from the transcriptome, and 21 of these peptides were experimentally validated for their antimicrobial activity against yeast and gram positive and -negative bacteria by a radial diffusion assay. Notably, 11 peptides showed strong antimicrobial activities against these organisms and displayed little or no cytotoxic effects in the hemolysis and cell viability assay. This work provides prerequisite baseline data for the identification and development of novel antimicrobial peptides, which is expected to provide a better understanding of the phenomenon of innate immunity in similar species.

The CopA3 dimer peptide is a coprisin analog that has an anticancer effect against human cancer cells in vitro. In this study, we investigated the anticancer activity of the enantiomeric CopA3 dimer peptide in human gastric cancer cell lines as well as in an in vivo tumor xenograft model. Enantiomeric CopA3 reduced gastric cancer cell viability and exhibited cytotoxicity against cancer cells. Enantiomeric CopA3-induced cell death was mediated by specific interactions with phosphatidylserine and phosphatidylcholine, membrane components that are enriched in cancer cells, in a calcein leakage assay. Moreover, acridine orange/ethidium bromide staining, flow cytometric analysis, and Western blot analysis showed that enantiomeric CopA3 induced apoptotic and necrotic gastric cancer cell death. The antitumor effect was also observed in a mouse tumor xenograft model in which intratumoral inoculation of the peptide resulted in a significant decrease in the SNU-668 gastric cancer tumor volume. In addition, periodic acid-Schiff and hematoxylin staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay revealed apoptotic and necrotic cell death in tumor masses treated with greater than 150 μg CopA3. Collectively, these results indicate that the enantiomeric CopA3 dimer peptide induces apoptosis and necrosis of gastric cancer cells in vitro and in vivo, indicating that the peptide is a potential candidate for the treatment of gastric cancer, which is a common cause of cancer and cancer deaths worldwide.

The epithelial cells of the gut form a physical barrier against the luminal contents. The collapse of this barrier causes inflammation, and its therapeutic restoration can protect the gut against inflammation. EGF enhances mucosal barrier function and increases colonocyte proliferation, thereby ameliorating inflammatory responses in the gut. Based on our previous finding that the insect peptide CopA3 promotes neuronal growth, we herein tested whether CopA3 could increase the cell proliferation of colonocytes, enhance mucosal barrier function, and ameliorate gut inflammation. Our results revealed that CopA3 significantly increased epithelial cell proliferation in mouse colonic crypts and also enhanced colonic epithelial barrier function. Moreover, CopA3 treatment ameliorated Clostridium difficile toxin As-induced inflammation responses in the mouse small intestine (acute enteritis) and completely blocked inflammatory responses and subsequent lethality in the dextran sulfate sodium-induced mouse model of chronic colitis. The marked CopA3-induced increase of colonocyte proliferation was found to require rapid protein degradation of p21(Cip1/Waf1), and an in vitro ubiquitination assay revealed that CopA3 directly facilitated ubiquitin ligase activity against p21(Cip1/Waf1). Taken together, our findings indicate that the insect peptide CopA3 prevents gut inflammation by increasing epithelial cell proliferation and mucosal barrier function., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

A novel antimicrobial peptide (AMP) was identified from the centipede Scolopendra subspinipes mutilans by RNA sequencing, and the amino acid sequences predicted from the sequenced mRNAs were compared with those of known AMPs. We named this peptide scolopendin, according to its origin, and investigated the molecular mechanisms underlying its antimicrobial activity. Our findings showed that scolopendin had antimicrobial activity against several pathogenic microorganisms, but did not produce hemolysis of human erythrocytes. In addition, disturbances in the cell membrane potential, induction of potassium release from the cytosol, and increased membrane permeability of the microbes Candida albicans and Escherichia coli O157 were detected by the use of 3,3'-dipropylthiacarbocyanine iodide [DiSC3(5)] dye, potassium leakage assay, and propidium iodide influx assay, respectively, following scolopendin treatment. Further evidence to support the membrane-targeted action of scolopendin was obtained using artificial liposomes as models of the cell membrane. Use of calcein and FITC-labeled dextran leakage assays from scolopendin-treated giant unilamellar vesicles and large unilamellar vesicles showed that scolopendin has a pore-forming action on microbial membrane, with an estimated pore radius of 2.3-3.3 nm. In conclusion, scolopendin is a novel and potent AMP with a membrane-targeted mechanism of action., (Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

We recently reported that the antimicrobial peptide Lumbricusin (NH2-RNRRWCIDQQA), isolated from the earthworm, increases cell proliferation in neuroblastoma SH-SY5Y cells. Here, we investigated whether Lumbricusin has neurotropic activity in mouse neural stem cells (MNSCs) and a protective effect in a mouse model of Parkinson's disease (PD). In MNSCs isolated from mouse brains, Lumbricusin treatment significantly increased cell proliferation (up to 12%) and reduced the protein expression of p27(Kip1) through proteasomal protein degradation but not transcriptional regulation. Lumbricusin inhibited the 6-OHDA-induced apoptosis of MNSCs, and also showed neuroprotective effects in a mouse PD model, ameliorating the motor impairments seen in the pole, elevated body swing, and rotation tests. These results suggest that the Lumbricusin-induced promotion of neural cell proliferation via p27(Kip1) degradation has a protective effect in an experimental PD model. Thus, the antimicrobial peptide Lumbricusin could possibly be developed as a potential therapeutic agent for the treatment of PD.

Previously, we performed de novo RNA sequencing of Scolopendra subspinipes mutilans using high-throughput sequencing technology and identified several antimicrobial peptide candidates. Among them, a cationic antimicrobial peptide, scolopendrasin VII, was selected based on its physicochemical properties, such as length, charge, and isoelectric point. Here, we assessed the anticancer activities of scolopendrasin VII against U937 and Jurkat leukemia cell lines. The results showed that scolopendrasin VII decreased the viability of the leukemia cells in MTS assays. Furthermore, flow cytometric analysis and acridine orange/ethidium bromide staining revealed that scolopendrasin VII induced necrosis in the leukemia cells. Scolopendrasin VII-induced necrosis was mediated by specific interaction with phosphatidylserine, which is enriched in the membrane of cancer cells. Taken together, these data indicated that scolopendrasin VII induced necrotic cell death in leukemia cells, probably through interaction with phosphatidylserine. The results provide a useful anticancer peptide candidate and an efficient strategy for new anticancer peptide development.

In this study, we report that one of the antimicrobial peptides scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates actin polymerization and the subsequent chemotactic migration of macrophages through the activation of ERK and protein kinase B (Akt) activity. The scolopendrasin VII-induced chemotactic migration of macrophages is inhibited by the formyl peptide receptor 1 (FPR1) antagonist cyclosporine H. We also found that scolopendrasin VII stimulate the chemotactic migration of FPR1-transfected RBL-2H3 cells, but not that of vector-transfected cells; moreover, scolopendrasin VII directly binds to FPR1. Our findings therefore suggest that the antimicrobial peptide scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates macrophages, resulting in chemotactic migration via FPR1 signaling, and the peptide can be useful in the study of FPR1-related biological responses.

CopA3 is a homodimeric α-helical peptide derived from coprisin which is a defensin-like antimicrobial peptide that was identified from the dung beetle, Copris tripartitus. CopA3 has been reported to have anticancer activity against leukemia cancer cells. In the present study, we investigated the anticancer activity of CopA3 in human gastric cancer cells. CopA3 reduced cell viability and it was cytotoxic to gastric cancer cells in the MTS and LDH release assay, respectively. CopA3 was shown to induce necrotic cell death of the gastric cancer cells by flow cytometric analysis and acridine orange/ethidium bromide staining. CopA3-induced cell death was mediated by specific interactions with phosphatidylserine, a membrane component of cancer cells. Taken together, these data indicated that CopA3 mainly caused necrosis of gastric cancer cells, probably through interactions with phosphatidylserine, which suggests the potential utility of CopA3 as a cancer therapeutic.

Scolopendin 2 is a 16-mer peptide (AGLQFPVGRIGRLLRK) derived from the centipede Scolopendra subspinipes mutilans. We observed that this peptide exhibited antimicrobial activity in a salt-dependent manner against various fungal and bacterial pathogens and showed no hemolytic effect in the range of 1.6 μM to 100 μM. Circular dichroism analysis showed that the peptide has an α-helical properties. Furthermore, we determined the mechanism(s) of action using flow cytometry and by investigating the release of intracellular potassium. The results showed that the peptide permeabilized the membranes of Escherichia coli O157 and Candida albicans, resulting in loss of intracellular potassium ions. Additionally, bis-(1,3-dibutylbarbituric acid) trimethine oxonol and 3,3'-dipropylthiacarbocyanine iodide assays showed that the peptide caused membrane depolarization. Using giant unilamellar vesicles encapsulating calcein and large unilamellar vesicles containing fluorescein isothiocyanate-dextran, which were similar in composition to typical E. coli O157 and C. albicans membranes, we demonstrated that scolopendin 2 disrupts membranes, resulting in a pore size between 4.8 nm and 5.0 nm. Thus, we have demonstrated that a cationic antimicrobial peptide, scolopendin 2, exerts its broad-spectrum antimicrobial effects by forming pores in the cell membrane., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We recently isolated a polypeptide from the earthworm Lumbricus terrestris that is structurally similar to defensin, a well-known antibacterial peptide. An 11-mer antibacterial peptide (NH2-RNRRWCIDQQA), designated Lumbricusin, was synthesized based on the amino acid sequence of the isolated polypeptide. Since we previously reported that CopA3, a dung beetle peptide, enhanced neuronal cell proliferation, we here examined whether Lumbricusin exerted neurotropic and/or neuroprotective effects. Lumbricusin treatment induced a time-dependent increase (∼51%) in the proliferation of human neuroblastoma SH-SY5Y cells. Lumbricusin also significantly inhibited the apoptosis and decreased viability induced by treatment with 6-hydroxy dopamine, a Parkinson's disease-mimicking agent. Immunoblot analyses revealed that Lumbricusin treatment increased ubiquitination of p27(Kip1) protein, a negative regulator of cell-cycle progression, in SH-SY5Y cells, and markedly promoted its degradation. Notably, adenoviral-mediated over-expression of p27(Kip1) significantly blocked the antiapoptotic effect of Lumbricusin in 6-hydroxy dopamine-treated SH-SY5Y cells. These results suggest that promotion of p27(Kip1) degradation may be the main mechanism underlying the neuroprotective and neurotropic effects of Lumbricusin., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The centipede Scolopendra subspinipes mutilans is an environmentally beneficial and medically important arthropod species. Although this species is increasingly applied as a reliable source of new antimicrobial peptides, the transcriptome of this species is a prerequisite for more rational selection of antimicrobial peptides. In this report, we isolated total RNA from the whole body of adult centipedes, S. subspinipes mutilans, that were nonimmunized and immunized against Escherichia coli, and we generated a total of 77,063 pooled contigs and singletons using high-throughput sequencing. To screen putative antimicrobial peptides, in silico analyses of the S. subspinipes mutilans transcriptome were performed based on the physicochemical evidence of length, charge, isoelectric point, and in vitro and in vivo aggregation scores together with the existence of continuous antimicrobial peptide stretches. Moreover, we excluded some transcripts that showed similarity with both previously known antimicrobial peptides and the human proteome, had a proteolytic cleavage site, and had downregulated expression compared with the nonimmunized sample. As a result, we selected 17 transcripts and tested their antimicrobial activity with a radial diffusion assay. Among them, ten synthetic peptides experimentally showed antimicrobial activity against microbes and no toxicity to mouse erythrocytes. Our results provide not only a useful set of antimicrobial peptide candidates and an efficient strategy for novel antimicrobial peptide development but also the transcriptome data of a big centipede as a valuable resource.

The centipede Scolopendra subpinipes mutilans is a medicinally important arthropod species. However, its transcriptome is not currently available and transcriptome analysis would be useful in providing insight into a molecular level approach. Hence, we performed de novo RNA sequencing of S. subpinipes mutilans using next-generation sequencing. We generated a novel peptide (scolopendrasin II) based on a SVM algorithm, and biochemically evaluated the in vitro antimicrobial activity of scolopendrasin II against various microbes. Scolopendrasin II showed antibacterial activities against gram-positive and -negative bacterial strains, including the yeast Candida albicans and antibiotic-resistant gram-negative bacteria, as determined by a radial diffusion assay and colony count assay without hemolytic activity. In addition, we confirmed that scolopendrasin II bound to the surface of bacteria through a specific interaction with lipoteichoic acid and a lipopolysaccharide, which was one of the bacterial cell-wall components. In conclusion, our results suggest that scolopendrasin II may be useful for developing peptide antibiotics.

In our previous study, coprisin, a 43-mer defensin-like peptide, was derived from the dung beetle, Copris tripartitus, and a 9-mer CopA3 (monomer), truncated coprisin analog peptide, was designed. However, the antifungal effects of CopA3 are not known yet. In this study, the antifungal activity and mechanism of CopA3 were investigated and to develop a more effective antimicrobial peptide under physiological conditions, the enantiomeric d-CopA3 was designed. l- and d-CopA3 had a similar antifungal activity without chiral selectivity, and their activity was more potent than that of melittin used as a positive control. Furthermore, l- and d-CopA3 did not even show any hemolysis against human erythrocytes. Membrane studies using propidium iodide and bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)], suggested that the antifungal effect of l- and d-CopA3 was due to the membrane-active mechanism, by contrast with coprisin possessing apoptotic mechanism without membrane permeabilization. Finally, the proteolytic resistance and antifungal activity of l- and d-CopA3 against trypsin was analyzed by HPLC and colony count assay. The results showed that only d-CopA3 maintained a potent antifungal activity despite the proteolytic condition. Therefore, this study suggests that d-CopA3 has potential as a novel antimicrobial agent., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Harmoniasin is a defensin-like antimicrobial peptide identified from the ladybug Harmonia axyridis. Among the synthetic homodimer peptide analogues derived from harmoniasin, HaA4 has been found to have antibacterial activity without hemolytic activity. In this study, we investigated whether HaA4 has anticancer activity against human leukemia cell lines such as U937 and Jurkat cells. HaA4 manifested cytotoxicity and decreased the cell viability of U937 and Jurkat cells in MTS assay and LDH release assay. We found that HaA4 induced apoptotic and necrotic cell death of the leukemia cells using flow cytometric analysis, acridine orange/ethidium bromide staining and nucleosomal fragmentation of genomic DNA. Activation of caspase-7 and -9 and fragmentation of poly (ADP-ribose) polymerase was detected in the HaA4-treated leukemia cells, suggesting induction of a caspase-dependent apoptosis pathway by HaA4. Caspase-dependent apoptosis was further confirmed by reversal of the HaA4-induced viability reduction by treatment of Z-VAD-FMK, a pan-caspase inhibitor. In conclusion, HaA4 caused necrosis and caspase-dependent apoptosis in both U937 and Jurkat leukemia cells, which suggests potential utility of HaA4 as a cancer therapeutic agent.

We recently demonstrated that the antibacterial peptide, CopA3 (a D-type disulfide dimer peptide, LLCIALRKK), inhibits LPS-induced macrophage activation and also has anticancer activity in leukemia cells. Here, we examined whether CopA3 could affect neuronal cell proliferation. We found that CopA3 time-dependently increased cell proliferation by up to 31 ± 2% in human neuroblastoma SH-SY5Y cells, and up to 29 ± 2% in neural stem cells isolated from neonatal mouse brains. In both cell types, CopA3 also significantly inhibited the apoptosis and viability losses caused by 6-hydroxy dopamine (a Parkinson disease-mimicking agent) and okadaic acid (an Alzheimer's disease-mimicking agent). Immunoblotting revealed that the p27Kip1 protein (a negative regulator of cell cycle progression) was markedly degraded in CopA3-treated SH-SY5Y cells. Conversely, an adenovirus expressing p27Kip1 significantly inhibited the antiapoptotic effects of CopA3 against 6-hydroxy dopamine- and okadaic acid-induced apoptosis, and decreased the neurotropic effects of CopA3. These results collectively suggest that CopA3-mediated protein degradation of p27Kip1 may be the main mechanism through which CopA3 exerts neuroprotective and neurotropic effects., (Copyright © 2013. Published by Elsevier Inc.)

Recently, we reported that the synthetic Coprisin analog peptide 9-mer dimer CopA3 (consisted of all-L amino acid sequence) was designed based on a defensin-like peptide, Coprisin isolated from Copris tripartitus. The 9-mer dimer CopA3 (L-CopA3) had antibacterial activity and induced apoptosis in human leukemia cells via a caspase-independent pathway. In this study, all of amino acid sequences of L-CopA3 were modified to all D-form amino acids (D-CopA3) to develop a more effective antimicrobial peptide. We investigated whether D-CopA3 had antimicrobial activities against pathogenic microorganisms and proapoptotic effects in human leukemia cells (U937, Jurkat, and AML-2). The synthetic peptide D-CopA3 had antimicrobial activities against various pathogenic bacteria and yeast fungus with MIC values in the 4~64 microM range. Moreover, D-CopA3 caused cell growth inhibition, and increased the chromosomal DNA fragmentation and the expression of inflammatory cytokines, TNF-alpha and IL1-beta, transcripts in human leukemia cells. The all-D amino acid peptide D-CopA3 proved as effective as the L-CopA3 reported previously. These results provide the basis for developing D-CopA3 as a new antibiotic peptide.

A synthetic coprisin analog peptide, 9-mer dimer CopA3 (CopA3) was designed based on a defensin-like peptide, Coprisin, isolated from the bacteria-immunized dung beetle Copris tripartitus. Here, CopA3 was investigated for its antimicrobial activity and cancer cell growth inhibition. CopA3 showed antimicrobial activities against various pathogenic bacteria and yeast fungus with MIC values in 2~32 μM ranges, and inhibited the cell viabilities of pancreatic and hepatocellular cancer cells, except MIAPaca2, Hep3B, and HepG2 cells, in a dose-dependent manner. The average IC(50) values of CopA3 against pancreatic and hepatocellular cancer cells were 61.7 μM and 67.8 μM, respectively. The results indicate that CopA3 has potential in the treatments of pancreatic and hepatocellular cancers as well as microorganism infection disease.

Papiliocin is a novel 37-residue cecropin-like peptide isolated recently from the swallowtail butterfly, Papilio xuthus. With the aim of identifying a potent antimicrobial peptide, we tested papiliocin in a variety of biological and biophysical assays, demonstrating that the peptide possesses very low cytotoxicity against mammalian cells and high bacterial cell selectivity, particularly against Gram-negative bacteria as well as high anti-inflammatory activity. Using LPS-stimulated macrophage RAW264.7 cells, we found that papiliocin exerted its anti-inflammatory activities by inhibiting nitric oxide (NO) production and secretion of tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-2, producing effects comparable with those of the antimicrobial peptide LL-37. We also showed that the innate defense response mechanisms engaged by papiliocin involve Toll-like receptor pathways that culminate in the nuclear translocation of NF-κB. Fluorescent dye leakage experiments showed that papiliocin targets the bacterial cell membrane. To understand structure-activity relationships, we determined the three-dimensional structure of papiliocin in 300 mm dodecylphosphocholine micelles by NMR spectroscopy, showing that papiliocin has an α-helical structure from Lys(3) to Lys(21) and from Ala(25) to Val(36), linked by a hinge region. Interactions between the papiliocin and LPS studied using tryptophan blue-shift data, and saturation transfer difference-NMR experiments revealed that Trp(2) and Phe(5) at the N-terminal helix play an important role in attracting papiliocin to the cell membrane of Gram-negative bacteria. In conclusion, we have demonstrated that papiliocin is a potent peptide antibiotic with both anti-inflammatory and antibacterial activities, and we have laid the groundwork for future studies of its mechanism of action.

Protaetiamycine is an insect defensin, derived from the larvae of the beetle Protaetia brevitarsis. In our previous work, we designed 9-mer peptide analogs of protaetiamycine, including 9Pbw2 (RLWLAIKRR-NH(2) ), 9Pbw3 (RLWLAIWRR-NH(2) ), and 9Pbw4 (RLWLAWKRR-NH(2) ). 9Pbw2 and 9Pbw4 showed high antimicrobial activity without cytotoxicity, while 9Pbw3 with higher hydrophobicity compared to 9Pbw2 and 9Pbw4 showed high cytotoxicity as well as high antimicrobial activity (Shin et al., J. Pept. Sci. 2009; 15: 559-568). In this study, we investigated the anti-inflammatory activities of 9Pbw2, 9Pbw3, and 9Pbw4 by quantitation of NO production in LPS-stimulated RAW264.7 cells. The results showed that only 9Pbw3 has strong inhibition of NO production, implying that Trp(7) as well as optimum level of hydrophobicity may play key roles in the anti-inflammatory activity of 9Pbw3. In order to design potent anti-inflammatory peptide with lower cytotoxicity as well as high stability from cleavage by protease compared to 9Pbw3, we synthesized 9Pbw3-D, the all-D-amino acid analog of 9Pbw3. 9Pbw3-D showed less cytotoxicity against RAW264.7 cells as well as considerably stronger inhibition of NO production and inflammation-induced cytokine production in LPS-stimulated RAW264.7 cells than 9Pbw3. 9Pbw3-D inhibited the gene expression of inflammatory-induced cytokine significantly more than 9Pbw3 and showed high resistance to proteolytic digestion. Binding of 9Pbw3-D with LPS caused higher enhancement of the FITC fluorescence as a result of its stronger interaction with LPS compared to that of 9Pbw3 and this result is in good agreement with their anti-inflammatory activities. 9Pbw3-D with higher anti-inflammatory activity as well as lower cytotoxicity against mammalian cell compared to 9Pbw3 can be a potent noncytotoxic antibiotic candidates., (Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.)

Papiliocin is a 37-residue peptide isolated from the swallowtail butterfly Papilio xuthus. In this study, we found that Papiliocin induced the accumulation of reactive oxygen species (ROS) and hydroxyl radicals known to be important regulators of apoptosis in Candida albicans. To examine the relationship between the accumulation of ROS and the induction of apoptosis, we investigated the apoptotic effects of Papiliocin using apoptotic markers. Cells treated with Papiliocin showed a series of cellular changes normally seen in cells undergoing apoptosis: plasma membrane translocation of phosphatidylserine from the inner to the outer membrane leaflet, measured by Annexin V staining, dissipation of the mitochondrial membrane potential, observed by DiOC(6)(3) staining; and the presence of active metacaspases, measured using the CaspACE FITC-VAD-FMK, as early apoptotic events. In addition, DNA condensation and fragmentation, which is important marker of late stage apoptosis, was seen by DAPI and TUNEL assay. Therefore, these results suggest that Papiliocin leads to apoptosis in C. albicans via ROS accumulation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Papiliocin is a 37-residue peptide isolated from the swallowtail butterfly Papilio xuthus. In this study, the antifungal effects and the mechanism of actions of papiliocin were investigated. First of all, papiliocin was shown to exert fungicidal activity. To understand the antifungal mechanism(s), propidium iodide influx into Candida albicans cells, induced by papiliocin, was examined. The result indicated that papiliocin perturbed and disrupted the fungal plasma membrane. Furthermore, calcein leakage from large unilamellar vesicles and rhodamine leakage from giant unilamellar vesicles further confirmed and visualized the membrane-disruptive action of papiliocin in the fungal model membrane, respectively. In summary, the present study suggests that papiliocin exerts its antifungal activity by a membrane-active mechanism and that this peptide can be developed into novel potent antifungal agents., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Psacotheasin is a 34-mer knottin-type peptide that is derived from Psacothea hilaris larvae. In this study, the antifungal activity and mechanism(s) by which psacotheasin affects human fungal pathogens were investigated. Psacotheasin shows remarkable antifungal properties without hemolytic activity against human erythrocytes. To understand the antifungal mechanism(s) of psacotheasin in Candida albicans, flow cytometric analysis with DiBAC(4)(3) and PI was conducted. The results showed that psacotheasin depolarized and perturbed the plasma membrane of the C. albicans. Three-dimensional (3D)-flow cytometric contour-plot analysis, accompanied by decreased forward scatter (FS), which indicates cell size, confirmed that psacotheasin exerted antifungal effects via membrane permeabilization. The membrane studies, using a single GUV and FITC-dextran (FD) loaded liposomes, indicate that psacotheasin acts as a pore-forming peptide in the model membrane of C. albicans and the radius of pores were presumed to be anywhere from 2.3 to 3.3nm. Therefore, the current study suggests that the mechanism(s) of psacotheasin's antifungal properties function within the membrane., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Previously, papiliocin was isolated from the swallowtail butterfly Papilio xuthus and its antimicrobial activity was suggested. In this study, the antifungal mechanism of papiliocin was investigated against Candida albicans. Confocal laser scanning microscopy (CLSM) and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence analysis indicated that papiliocin disturbed the fungal plasma membrane. Moreover, the assessment of the release of FITC-dextran (FD) from liposome further demonstrated that the antifungal mechanism of papiliocin could have originated from the pore-forming action and that the radius of the pores was presumed to be anywhere from 2.3 nm and 3.3 nm.

Cecropin is a well-studied antimicrobial peptide that is synthesized in fat body cells and hemocytes of insects in response to hypodermic injury or bacterial infection. A 503 bp cDNA encoding for a cecropin-like peptide was isolated by employing annealing control primer (ACP)-based differential display PCR and 5'-RACE with immunized Papilio xuthus larvae. The open reading frame of the isolated cDNA encoded for a 62-amino acid prepropeptide with a putative 22-residue signal peptide, a 2-residue propeptide, and a 38-residue mature peptide with a theoretical mass of 4060.89 Da. The deduced amino acid sequence of the peptide evidenced a significant degree of identity with other lepidopteran cecropins. This peptide was named papiliocin. RTPCR results revealed that the papiliocin transcript was detected at significant levels after injection with bacterial lipopolysaccharide (LPS). On the basis of the deduced amino acid sequence of papiliocin, a 38-mer mature peptide was chemically synthesized via the Fmoc method, and its antimicrobial activity was analyzed. The synthetic papiliocin peptide evidenced a broad spectrum of activity against fungi, Gram-positive and Gram-negative bacteria, and also evidenced no hemolytic activity against human red blood cells.

We report the isolation and characterization of a novel knottin-type antimicrobial peptide from the yellow-spotted long-horned beetle Psacothea hilaris. A cDNA encoding a 56-mer knottin-type propeptide was identified and its predicted molecular mass and pI was 5.92 kDa and 8.28, respectively. A 34-mer mature peptide was also selected and named herein as psacotheasin. The antimicrobial activity of chemically synthesized psacotheasin against human bacterial pathogens was subsequently investigated. The results showed that psacotheasin exerted potent activities against both Gram-positive and Gram-negative bacterial strains. The present study suggests that psacotheasin can be applied to develop novel therapeutic agents.

Protaetiamycine is an insect defensin, a naturally occurring 43-amino-acid-residue antimicrobial peptide derived from the larvae of the beetle Protaetia brevitarsis. In a previous work that aimed at developing short antibiotic peptides, we designed 9-mer peptide analogs of protaetiamycine. Among them, RLWLAIGRG-NH2 showed good antifungal activity against Candida albicans. In this study, we designed four 9-mer peptide analogs based on the sequence of RLWLAIGRG-NH2, in which Gly or Ile was substituted with Arg, Lys, or Trp to optimize the balance between the hydrophobicity and cationicity of the peptides and to increase bacterial cell selectivity. We measured their toxicity to bacteria and mammalian cells as well as their ability to permeabilize model phospholipid membranes. Substitution of Arg for Gly9 at the C-terminus (9Pbw1) resulted in two- to fourfold improvement in antibacterial activity. Further substitution of Gly7 with Lys (9Pbw2 and 9Pbw4) caused four- to eightfold improvement in the antibacterial activity without increase in cytotoxocity, while substitution of Gly7 with Trp (9Pbw3) increased cytotoxicity as well as antibacterial activity. The peptides 9Pbw2 and 9Pbw4 with the highest bacterial cell selectivity were not effective in depolarizing the membrane of Staphylococcus aureus cytoplasmic membranes and showed almost no leakage of a fluorescent dye entrapped within the vesicles. Gel-retardation experiments indicated that 9Pbw2 and 9Pbw4 inhibited the migration of DNA at concentrations >20 microM. Three positively charged residues at the C-terminus in 9Pbw2 and 9Pbw4 may facilitate effective penetration into the negatively charged phospholipid membrane of bacteria. The results obtained in this study suggest that the bactericidal action of our potent antibacterial peptides, namely 9Pbw2 and 9Pbw4, may be attributed to the inhibition of the functions of intracellular components after penetration of the bacterial cell membrane., (Copyright 2009 European Peptide Society and John Wiley & Sons, Ltd.)