ABSTRACT
The emergence of clinically drug-resistant malaria parasites requires the urgent development of new drugs. Mosquitoes are vectors of multiple pathogens and have developed resistance mechanisms against them, which often involve antimicrobial peptides (AMPs). An-cecB is an AMP of the malaria-transmitting mosquito genus Anopheles, and we herein report its antimalarial activity against Plasmodium falciparum 3D7, the artemisinin-resistant strain 803, and the chloroquine-resistant strain Dd2 in vitro. We also demonstrate its anti-parasite activity in vivo, using the rodent malaria parasite Plasmodium berghei (ANKA). We show that An-cecB displays potent antimalarial activity and that its mechanism of action may occur through direct killing of the parasite or through interaction with infected red blood cell membranes. Unfortunately, An-cecB was found to be cytotoxic to mammalian cells and had poor antimalarial activity in vivo. However, its truncated peptide An-cecB-1 retained most of its antimalarial activity and avoided its cytotoxicity in vitro. An-cecB-1 also showed better antimalarial activity in vivo. Mosquito-derived AMPs may provide new ideas for the development of antimalarial drugs against drug-resistant parasites, and An-cecB has potential use as a template for antimalarial peptides.
Subject(s)
Anopheles , Antimalarials , Plasmodium berghei , Plasmodium falciparum , Animals , Antimalarials/pharmacology , Anopheles/drug effects , Anopheles/parasitology , Plasmodium falciparum/drug effects , Plasmodium berghei/drug effects , Mice , Cecropins/pharmacology , Antimicrobial Peptides/pharmacology , Antimicrobial Peptides/chemistry , Malaria/drug therapy , Malaria/parasitology , Erythrocytes/drug effects , Erythrocytes/parasitology , Humans , Mosquito Vectors/drug effects , Mosquito Vectors/parasitology , Female , Insect Proteins/pharmacology , Drug Resistance/drug effects , Chloroquine/pharmacology , Parasitic Sensitivity TestsABSTRACT
BACKGROUND: The world faces a major infectious disease challenge. Interest in the discovery, design, or development of antimicrobial peptides (AMPs) as an alternative approach for the treatment of bacterial infections has increased. Insects are a good source of AMPs which are the main effector molecules of their innate immune system. Black Soldier Fly Larvae (BSFL) are being developed for large-scale rearing for food sustainability, waste reduction and as sustainable animal and fish feed. Bioinformatic studies have suggested that BSFL have the largest number of AMPs identified in insects. However, most AMPs identified in BSF have not yet undergone antimicrobial evaluation but are promising leads to treat critical infections. RESULTS: Jg7197.t1, Jg7902.t1 and Jg7904.t1 were expressed into the haemolymph of larvae following infection with Salmonella enterica serovar Typhimurium and were predicted to be AMPs using the computational tool ampir. The genes encoding these proteins were within 2 distinct clusters in chromosome 1 of the BSF genome. Following removal of signal peptides, predicted structures of the mature proteins were superimposed, highlighting a high degree of structural conservation. The 3 AMPs share primary sequences with proteins that contain a Kunitz-binding domain; characterised for inhibitory action against proteases, and antimicrobial activities. An in vitro antimicrobial screen indicated that heterologously expressed SUMO-Jg7197.t1 and SUMO-Jg7902.t1 did not show activity against 12 bacterial strains. While recombinant SUMO-Jg7904.t1 had antimicrobial activity against a range of Gram-negative and Gram-positive bacteria, including the serious pathogen Pseudomonas aeruginosa. CONCLUSIONS: We have cloned and purified putative AMPs from BSFL and performed initial in vitro experiments to evaluate their antimicrobial activity. In doing so, we have identified a putative novel defensin-like AMP, Jg7904.t1, encoded in a paralogous gene cluster, with antimicrobial activity against P. aeruginosa.
Subject(s)
Anti-Bacterial Agents , Defensins , Diptera , Larva , Animals , Defensins/pharmacology , Defensins/genetics , Defensins/chemistry , Defensins/isolation & purification , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Diptera/genetics , Larva/drug effects , Larva/genetics , Microbial Sensitivity Tests , Amino Acid Sequence , Insect Proteins/genetics , Insect Proteins/pharmacology , Insect Proteins/chemistry , Antimicrobial Peptides/pharmacology , Antimicrobial Peptides/genetics , Antimicrobial Peptides/chemistry , Salmonella typhimurium/drug effects , Salmonella typhimurium/genetics , Gram-Negative Bacteria/drug effectsABSTRACT
The proteins were mainly derived from Protaetia brevitarsis larval extracts obtained using two empty intestine methods (traditional static method: TSM or salt immersion stress method: SISM) and extraction solvents (water: W or 50 % water-ethanol: W:E), and the proteins were used as objects to investigate the effect of emptying intestine methods on hypolipidemic peptides. The results revealed that the F-2 fractions of protein hydrolysate had stronger in vitro hypolipidemic activity, with the peptides obtained by SISM possessing a stronger cholesterol micelle solubility inhibition rate, especially in SISM-W:E-P. Moreover, a total of 106 peptides were tentatively identified, among which SISM identified more peptides with an amino acid number < 8. Meanwhile, five novel peptides (YPPFH, YPGFGK, KYPF, SPLPGPR and VPPP) exhibited good hypolipidemic activity in vitro and in vivo, among which YPPFH, VPPP and KYPF had strong inhibitory activities on pancreatic lipase (PL) and cholesteryl esterase (CE), and KYPF, SPLPGPR and VPPP could significantly reduce the TG content in Caenorhabditis elegans. Thus, P. brevitarsis can be developed as a naturally derived hypolipidemic component for the development and application in functional foods.
Subject(s)
Coleoptera , Protein Hydrolysates , Animals , Larva/chemistry , Protein Hydrolysates/pharmacology , Protein Hydrolysates/metabolism , Coleoptera/chemistry , Peptides/pharmacology , Peptides/metabolism , Water/metabolism , Insect Proteins/pharmacology , Insect Proteins/metabolismABSTRACT
Developing self-assembled biomedical materials based on insect proteins is highly desirable due to their advantages of green, rich, and sustainable characters as well as excellent biocompatibility, which has been rarely explored. Herein, salt-induced controllable self-assembly, antibacterial performance, and infectious wound healing performance of an insect cuticle protein (OfCPH-2) originating from the Ostrinia furnacalis larva head capsule are investigated. Interestingly, the addition of salts could trigger the formation of beaded nanofibrils with uniform diameter, whose length highly depends on the salt concentration. Surprisingly, the OfCPH-2 nanofibrils not only could form functional films with broad-spectrum antibacterial abilities but also could promote infectious wound healing. More importantly, a possible wound healing mechanism was proposed, and it is the strong abilities of OfCPH-2 nanofibrils in promoting vascular formation and antibacterial activity that facilitate the process of infectious wound healing. Our exciting findings put forward instructive thoughts for developing innovative bioinspired materials based on insect proteins for wound healing and related biomedical fields.
Subject(s)
Wound Healing , Wound Infection , Animals , Biocompatible Materials , Anti-Bacterial Agents/pharmacology , Insect Proteins/pharmacology , Insecta , HydrogelsABSTRACT
λ-Cyhalothrin (λ-cyh) is widely used in agricultural production and has been reported to cause damages to numerous nontarget insects. As an important economic and model insect of Lepidoptera, Bombyx mori was extremely sensitive to λ-cyh, and pesticide drift often leads to silkworm poisoning. However, little is known about the persistence of sublethal effects or the potential recovery from short-term exposure to sublethal doses of pesticides. In this study, we estimated the sublethal effects caused by short-term exposure (24 h) of λ-cyh LC1 , LC10 , LC25 , and LC50 , respectively, and investigated the persistent negative effects on the growth, survival, and pupal metamorphosis of silkworm larvae. Silkworm growth was mostly retarded after λ-cyh exposure, with dose-dependent recovery observed at delayed time points. Relative to the control, the treatment groups showed significantly higher larval mortalities and abnormal pupa rates. Additionally, transcriptome sequencing was conducted to investigate the effects of λ-cyh LC10 on the normal physiological functions in the midgut of B. mori. A total of 2697 differentially expressed genes were identified, and 57.1% of DEGs were down-regulated. Gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis further revealed that energy and nutrient metabolisms were negatively affected. Moreover, we demonstrated that sublethal λ-cyh inhibited the oxidative phosphorylation pathway by reducing the expression of mitochondrial electron transport chain complex genes and consequently the synthesis of ATP. This study has provided useful transcriptome-wide expression resources to facilitate the overall knowledge of the molecular basis of sublethal toxicity caused by λ-cyh in the midgut of B. mori.
Subject(s)
Bombyx , Pesticides , Pyrethrins , Animals , Transcriptome , Pyrethrins/toxicity , Nitriles/toxicity , Larva , Insect Proteins/genetics , Insect Proteins/metabolism , Insect Proteins/pharmacologyABSTRACT
Bee venom is a rich source of biologically and pharmacologically active proteins. Waprin is a protein component of venoms; however, waprin has yet to be identified in bee venom. Moreover, the biological functions of waprin in venoms remain poorly characterized. Thus, in this study, we have identified and characterized waprin: a novel protein component from the venom of honeybees (Apis mellifera). The waprin in A. mellifera venom (Amwaprin) was found to consist of an 80-amino acid mature peptide, in which the whey acidic protein domain contains four conserved disulfide bonds. We discovered the presence of the Amwaprin protein in secreted venom by using an antibody against recombinant Amwaprin produced in baculovirus-infected insect cells. Recombinant Amwaprin exhibited inhibitory activity against microbial serine proteases and elastases but not thrombin or plasmin. It recognized carbohydrates in the microbial cell wall molecules and bound to the live microbial surfaces. The binding action of Amwaprin produced its microbicidal activity by inducing structural damage to bacterial and fungal cell walls. In addition, recombinant Amwaprin is heat-stable and contains no hemolytic activity. These findings demonstrate that Amwaprin acts as a microbicidal and anti-elastolytic agent.
Subject(s)
Anti-Infective Agents , Bee Venoms , Insect Proteins , Animals , Bee Venoms/pharmacology , Bees , Insect Proteins/pharmacology , Anti-Infective Agents/pharmacologyABSTRACT
The commercial value of silkworms has been widely explored and the effects of fluoride exposure on silkworms' breeding and silk production cannot be ignored. Bombyx mori is a commonly used model to explore the mechanisms of fluorosis. In the present study, we analyzed the differences in physiological and biochemical indicators after exposing larva to NaF, then evaluated differential genes and proteins. Compared to control, larvae exposed to 600 mg L-1 NaF presented decreased bodyweight, damaged midgut tissue, and were accompanied by oxidative stress. The RNA-seq showed 1493 differentially expressed genes (574 upregulated and 919 downregulated). Meanwhile, the TMT detected 189 differentially expressed proteins (133 upregulated and 56 downregulated). The integrative analysis led to 4 upregulated and 9 downregulated genes and proteins. Finally, we hypothesized that fluoride exposure might affect the intestinal digestion of silkworms, inhibit the gene expression of detoxification enzymes and stimulate cellular immune responses. Our current findings provided new insights into insect fluorosis.
Subject(s)
Bombyx , Environmental Exposure , Environmental Pollutants , Fluorides , Insect Proteins , Sodium Fluoride , Animals , Bombyx/drug effects , Bombyx/genetics , Bombyx/metabolism , Digestive System/drug effects , Digestive System/metabolism , Fluorides/toxicity , Insect Proteins/genetics , Insect Proteins/pharmacology , Larva/drug effects , Larva/genetics , Larva/metabolism , Silk/biosynthesis , Sodium Fluoride/toxicity , Environmental Pollutants/toxicity , Gene Expression Regulation/drug effectsABSTRACT
SignificanceSimilar to mammalian TLR4/MD-2, the Toll9/MD-2-like protein complex in the silkworm, Bombyx mori, acts as an innate pattern-recognition receptor that recognizes lipopolysaccharide (LPS) and induces LPS-stimulated expression of antimicrobial peptides such as cecropins. Here, we report that papiliocin, a cecropin-like insect antimicrobial peptide from the swallowtail butterfly, competitively inhibits the LPS-TLR4/MD-2 interaction by directly binding to human TLR4/MD-2. Structural elements in papiliocin, which are important in inhibiting TLR4 signaling via direct binding, are highly conserved among insect cecropins, indicating that its TLR4-antagonistic activity may be related to insect Toll9-mediated immune response against microbial infection. This study highlights the potential of papiliocin as a potent TLR4 antagonist and safe peptide antibiotic for treating gram-negative sepsis.
Subject(s)
Anti-Infective Agents, Local/pharmacology , Antimicrobial Peptides/pharmacology , Butterflies/immunology , Immunity, Innate/drug effects , Insect Proteins/pharmacology , Toll-Like Receptor 4/antagonists & inhibitors , Animals , Anti-Infective Agents, Local/chemistry , Antimicrobial Peptides/chemistry , Antimicrobial Peptides/metabolism , Escherichia coli Infections/drug therapy , Female , Insect Proteins/chemistry , Insect Proteins/metabolism , Lipopolysaccharides/metabolism , Mice , Mice, Inbred ICR , Molecular Docking Simulation , Protein Binding , Protein Conformation , Toll-Like Receptor 4/metabolismABSTRACT
We analyzed, for the first time, the major components and biological properties of the venom of Vespa bicolor, a wasp from South China. Using HPLC and SDS-PAGE, combined with LC-MS/MS, MALDI-TOF-MS, and NMR data to analyze V. bicolor venom (VBV), we found that VBV contains three proteins (hyaluronidase A, phospholipase A1 (two isoforms), and antigen 5 protein) with allergenic activity, two unreported proteins (proteins 5 and 6), and two active substances with large quantities (mastoparan-like peptide 12a (Vb-MLP 12a), and 5-hydroxytryptamine (5-HT)). In addition, the antimicrobial activity of VBV was determined, and results showed that it had a significant effect against anaerobic bacteria. The minimum inhibitory concentration and minimum bactericidal concentration for Propionibacterium acnes were 12.5 µg/mL. Unsurprisingly, VBV had strong antioxidant activity because of the abundance of 5-HT. Contrary to other Vespa venom, VBV showed significant anti-inflammatory activity, even at low concentrations (1 µg/mL), and we found that Vb-MLP 12a showed pro-inflammatory activity by promoting the proliferation of RAW 264.7 cells. Cytotoxicity studies showed that VBV had similar antiproliferative effects against all tested tumor cell lines (HepG2, Hela, MCF-7, A549, and SASJ-1), with HepG2 being the most susceptible. Overall, this study on VBV has high clinical importance and promotes the development of Vespa bicolor resources.
Subject(s)
Insect Proteins , Wasp Venoms , Wasps/chemistry , A549 Cells , Animals , China , HeLa Cells , Hep G2 Cells , Humans , Insect Proteins/chemistry , Insect Proteins/pharmacology , MCF-7 Cells , Microbial Sensitivity Tests , Wasp Venoms/chemistry , Wasp Venoms/pharmacologyABSTRACT
Cryoprotection is of interest in many fields of research, necessitating a greater understanding of different cryoprotective agents. Antifreeze proteins have been identified that have the ability to confer cryoprotection in certain organisms. Antifreeze proteins are an evolutionary adaptation that contributes to the freeze resistance of certain fish, insects, bacteria and plants. These proteins adsorb to an ice crystal's surface and restrict its growth within a certain temperature range. We investigated the ability of an antifreeze protein from the desert beetle Anatolica polita, ApAFP752, to confer cryoprotection in the frog Xenopus laevis. Xenopus laevis eggs and embryos microinjected with ApAFP752 exhibited reduced damage and increased survival after a freeze-thaw cycle in a concentration-dependent manner. We also demonstrate that ApAFP752 localizes to the plasma membrane in eggs and embryonic blastomeres and is not toxic for early development. These studies show the potential of an insect antifreeze protein to confer cryoprotection in amphibian eggs and embryos.
Subject(s)
Antifreeze Proteins , Coleoptera , Embryo, Nonmammalian , Insect Proteins , Ovum , Animals , Antifreeze Proteins/metabolism , Antifreeze Proteins/pharmacology , Coleoptera/chemistry , Cryoprotective Agents/pharmacology , Embryo, Nonmammalian/drug effects , Insect Proteins/metabolism , Insect Proteins/pharmacology , Ovum/drug effects , Xenopus laevisABSTRACT
Acute ischemic stroke triggers complex systemic pathological responses for which the exploration of drug resources remains a challenge. Wasp venom extracted from Vespa magnifica (Smith, 1852) is most commonly used to treat rheumatoid arthritis as well as neurological disorders. Vespakinin-M (VK), a natural peptide from wasp venom, has remained largely unexplored for stroke. Herein, we first confirmed the structure, stability, toxicity and distribution of VK as well as its penetration into the blood-brain barrier. VK (150 and 300 µg/kg, i.p.) was administered to improve stroke constructed by middle cerebral artery occlusion in mice. Our results indicate that VK promote functional recovery in mice after ischemia stroke, including an improvement of neurological impairment, reduction of infarct volume, maintenance of blood-brain barrier integrity, and an obstruction of the inflammatory response and oxidative stress. In addition, VK treatment led to reduced neuroinflammation and apoptosis associated with the activation of PI3K-AKT and inhibition of IκBα-NF-κB signaling pathways. Simultaneously, we confirmed that VK can combine with bradykinin receptor 2 (B2R) as detected by molecular docking, the B2R antagonist HOE140 could counteract the neuro-protective effects of VK on stroke in mice. Overall, targeting the VK-B2R interaction can be considered as a practical strategy for stroke therapy.
Subject(s)
Bradykinin/analogs & derivatives , Ischemic Stroke/drug therapy , Neuroprotective Agents/pharmacology , Recovery of Function/drug effects , Wasps/chemistry , Animals , Blood-Brain Barrier/physiopathology , Bradykinin/chemistry , Bradykinin/pharmacology , Insect Proteins/chemistry , Insect Proteins/pharmacology , Male , Mice , Neuroprotective Agents/chemistryABSTRACT
Apoptosis of ovarian granular cells is closely related with weakening fertility of women. Hence, resisting apoptosis of human ovarian granular cells is of important significance. According to previous studies, DAPI fluorescence staining experiment and Western Blot test of Caspase-3 demonstrate that small peptides from Periplaneta americana (SPPA) can improve hydrogen peroxide (H2O2) -induced apoptosis of human ovarian granular cells (KGN cells). However, the molecular mechanism of SPPA resistance against apoptosis of granular cells still remains unknown. In this study, key genes and signaling pathways for SPPA to resist H2O2-induced apoptosis of KGN cells were determined through transcriptome sequencing (RNA-seq). Experiments were divided into three groups, namely, the control group, H2O2 group and H2O2 + SPPA group. A total of 1196 differentially expressed genes (DEGs) were screened by comparing the control group and the H2O2 group, and 2805 DEGs were screened by comparing the H2O2 group and H2O2 + SPPA group. It is important to note that 87 overlapping genes were identified upregulating in H2O2 exposure, but downregulating in SPPA repair. Another 151 overlapping genes were identified downregulating in H2O2 exposure, but upregulating in SPPA repair. These 238 overlapping genes have significant enrichment in multiple KEGG pathways. Among them, 13 genes play significant roles in SPPA resistance process of cell apoptosis: EIF3D, RAN, UPF1 and EIF2B4 participate in RNA transport; ACTG1, SIPA1 and CTNND1 participate in Leukocyte transendothelial migration; S100A7, S100A9, RELA and IL17RE participate in IL-17 signaling pathway; BCL2L13, EIF2AK3 and RELA participate in Mitophapy-animal. Ten genes were selected for florescence quantitative PCR (qPCR) verification and the expression level was consistent with sequencing results. Finally, a control network of SPPA resistance against the H2O2-induced KGN cell apoptosis was built based on the target genes screened by the RNA-seq technology. This study provides a direction and some references to further understand the molecular mechanism of SPPA resistance against the H2O2-induced KGN cell apoptosis.
Subject(s)
Granulosa Cells/drug effects , Hydrogen Peroxide/pharmacology , Peptides/pharmacology , Periplaneta/chemistry , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cell Death/drug effects , Cell Line , Drug Interactions , Eukaryotic Initiation Factor-3/genetics , Female , Gene Expression , Granulosa Cells/cytology , Granulosa Cells/metabolism , Humans , Insect Proteins/chemistry , Insect Proteins/pharmacology , Oxidative Stress/drug effects , Peptides/chemistry , RNA-Seq , Signal Transduction , TranscriptomeABSTRACT
With the wide cultivation of transgenic plants throughout the world and the rising risk of resistance to Bacillus thuringiensis crystal (Cry) toxins, it is essential to design an adaptive resistance management strategy for continued use. Neuropeptide F (NPF) of insects has proven to be valuable for the production of novel-type transgenic plants via its important role in the control of feeding behavior. In this study, the gene encoding NPF was cloned from the diamondback moth, Plutella xylostella, an important agricultural pest. Real-time quantitative reverse transcription-polymerase chain reaction and in situ hybridization showed a relatively high expression of P. xylostella-npf (P. x-npf) in endocrine cells of the midgut of fourth instar larvae, and it was found to participate in P. xylostella feeding behavior and Cry1Ac-induced feeding inhibition. Prokaryotic expression and purification provided structure unfolded P. x-npf from inclusion bodies for diet surface overlay bioassays and the results demonstrated a significant synergistic effect of P. x-npf on Cry1Ac toxicity by increasing intake of noxious food which contains Cry toxins, especially quick death at an early stage of feeding. Our findings provided a potential new way to efficiently control pests by increasing intake of lower dose Cry toxins and a novel hint for the complex Cry toxin mechanism.
Subject(s)
Bacillus thuringiensis Toxins , Endotoxins , Hemolysin Proteins , Moths , Neuropeptides , Animals , Bacillus thuringiensis Toxins/pharmacology , Endotoxins/pharmacology , Feeding Behavior/physiology , Gene Expression , Genes, Insect , Hemolysin Proteins/pharmacology , Insect Proteins/genetics , Insect Proteins/metabolism , Insect Proteins/pharmacology , Moths/drug effects , Moths/genetics , Moths/metabolism , Moths/physiology , Neuropeptides/genetics , Neuropeptides/metabolism , Neuropeptides/pharmacology , Pest Control/methodsABSTRACT
Pathogenesis of C. difficile in the intestine is associated with the secretion of toxins which can damage the intestinal epithelial layer and result in diseases such as diarrhoea. Treatment for C. difficile infections consists of antibiotics which, however, have non-specific microbiocidal effects and may cause intestinal dysbiosis which results in subsequent health issues. Therefore, alternative treatments to C. difficile infections are required. We investigated whether different black soldier fly- and mealworm-derived fractions, after applying the INFOGEST digestion protocol, could counteract C. difficile toxin A-mediated barrier damage of small intestinal Caco-2 cells. Treatment and pre-treatment with insect-derived fractions significantly (p < 0.05) mitigated the decrease of the transepithelial electrical resistance (TEER) of Caco-2 cells induced by C. difficile toxin A. In relation to these effects, RNA sequencing data showed an increased transcription of cell junctional and proliferation protein genes in Caco-2 cells. Furthermore, the transcription of genes regulating immune signalling was also increased. To identify whether this resulted in immune activation we used a Caco-2/THP-1 co-culture model where the cells were only separated by a permeable membrane. However, the insect-derived fractions did not change the basolateral secreted IL-8 levels in this model. To conclude, our findings suggest that black soldier fly- and mealworm-derived fractions can attenuate C. difficile induced intestinal barrier disruption and they might be promising tools to reduce the symptoms of C. difficile infections.
Subject(s)
Bacterial Toxins/toxicity , Cell Proliferation/genetics , Enterotoxins/toxicity , Insecta , Intercellular Junctions/genetics , Intestinal Mucosa/physiology , Intestine, Small/cytology , Transcription, Genetic , Animals , Caco-2 Cells , Clostridioides difficile , Coculture Techniques , Coleoptera , Diptera , Epithelial Cells/physiology , Humans , Immunity/genetics , Immunomodulation , Insect Proteins/pharmacology , Intestinal Mucosa/cytology , Intestine, Small/physiology , Macrophages , RNA-Seq , THP-1 CellsABSTRACT
Cockroaches live in places where various pathogens exist and thus are more likely to use antimicrobial compounds to defend against pathogen intrusions. We previously performed an in silico analysis of the Periplaneta americana transcriptome and detected periplanetasin-5 using an in silico antimicrobial peptide prediction method. In this study, we investigated whether periplanetasin-5 has anticancer activity against the human leukemia cell line K562. Cell growth and survival of K562 cells treated with periplanetasin-5 were decreased in a dose-dependent manner. By using flow cytometric analysis, acridine orange/ethidium bromide (AO/EB) staining and DNA fragmentation, we found that periplanetasin-5 induced apoptotic and necrotic cell death in leukemia cells. In addition, these events were associated with increased levels of the pro-apoptotic proteins Fas and cytochrome c and reduced levels of the anti-apoptotic protein Bcl-2. Periplanetasin-5 induces the cleavage of pro-caspase-9, pro-caspase-8, pro-caspase-3, and poly (ADP-ribose) polymerase (PARP). The above data suggest that periplanetasin-5 induces apoptosis via both the intrinsic and extrinsic pathways. Moreover, caspase-related apoptosis was further confirmed by using the caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD-FMK), which reversed the periplanetasin-5-induced reduction in cell viability. In conclusion, periplanetasin-5 caused apoptosis in leukemia cells, suggesting its potential utility as an anticancer therapeutic agent.
Subject(s)
Antimicrobial Peptides/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis , Insect Proteins/pharmacology , Periplaneta/chemistry , Animals , Biological Products/pharmacology , Humans , K562 CellsABSTRACT
Lectins are renowned hemagglutinins and multivalent proteins with a well known quality for sugar-binding specificity that participate significantly in invertebrate defense functions. Studies on biological activity of lectin from coleopteran insect are very scarce. In this study, lectin from the hemolymph in the grub of banana pest, Odoiporus longicollis was subjected to purification, biochemical and functional characterizations. The lectin was purified by PEG precipitation and ion-exchange chromatography using Q-Sepharose as a matrix. The purified lectin showed hemagglutination activity against rat erythrocytes, heat-labile, cation independent and insensitive to EDTA. Further, the carbohydrate affinity of this lectin was found with mannitol, adonitol, L-arabinose, L-rhamnose, D-galactose and sorbitol. The native form of purified lectin was calculated as 360 kDa by FPLC system. Denatured gel electrophoresis of the purified lectin consisted of five distinct polypeptides with molecular weights approximately 160, 60, 52, 40 and 38 kDa, respectively. The amino acid sequences obtained through peptide mass fingerprinting analysis exhibited homologies to the known conserved regions of galactose binding lectins. Further, the purified lectin exhibited bacterial inhibition with LPS from Serratia marcescens. In addition, isolated lectin also exerted bacterial agglutination, antibacterial and anti-proliferative activity against Mycobacterium smegmatis, Bacillus pumilus and Neuro 2a cell line, respectively.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/pharmacology , Coleoptera/metabolism , Galectins/pharmacology , Animals , Anti-Bacterial Agents/isolation & purification , Antineoplastic Agents/isolation & purification , Bacillus pumilus/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Chromatography, Ion Exchange , Dose-Response Relationship, Drug , Galectins/isolation & purification , Humans , Insect Proteins/isolation & purification , Insect Proteins/pharmacology , Mice , Mycobacterium smegmatis/drug effects , RatsABSTRACT
Cell-cycle arrest reflects an accumulation of responses to DNA damage that sequentially affects cell growth and division. Herein, we analyzed the effect of the 9-mer dimer defensin-like peptide, CopA3, against colorectal cancer cell growth and proliferation in a dose-dependent manner upon 96 h of treatment. As observed, CopA3 treatment significantly affected cancer cell growth, reduced colony formation ability, increased the number of SA-ß-Gal positive cells, and remarkably reduced Ki67 protein expression. Notably, in HCT-116 cells, CopA3 (5 µM) treatment effectively increased oxidative stress and, as a result, amplified the endogenous ROS, mitochondrial ROS, and NO content in the cells, which further activated the DNA damage response and caused cell-cycle arrest at the G1 phase. The prolonged cell-cycle arrest elevated the release of inflammatory cytokines in the cell supernatant. Nevertheless, mechanistically, NAC treatment effectively reversed the CopA3 effect and significantly reduced the oxidative stress; subsequently rescuing the cells from G1 phase arrest. Overall, CopA3 treatment can inhibit the growth and proliferation of colorectal cancer cells by inducing cell-cycle arrest through the ROS-mediated pathway.
Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Cell Cycle Checkpoints/drug effects , Cell Proliferation/drug effects , Colorectal Neoplasms , Insect Proteins/pharmacology , Oxidative Stress/drug effects , Antineoplastic Agents/pharmacology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/metabolism , Cytokines/analysis , Dose-Response Relationship, Drug , Growth Inhibitors/pharmacology , HCT116 Cells , Humans , Ki-67 Antigen/analysis , Reactive Oxygen Species/analysis , Treatment Outcome , Tumor Stem Cell Assay/methodsABSTRACT
Cecropins form a family of amphipathic α-helical cationic peptides with broad-spectrum antibacterial properties and potent anticancer activity. The emergence of bacteria and cancer cells showing resistance to cationic antimicrobial peptides (CAMPs) has fostered a search for new, more selective and more effective alternatives to CAMPs. With this goal in mind, we looked for cecropin homologs in the genome and transcriptome of the spruce budworm, Choristoneura fumiferana. Not only did we find paralogs of the conventional cationic cecropins (Cfcec+ ), our screening also led to the identification of previously uncharacterized anionic cecropins (Cfcec- ), featuring a poly-l-aspartic acid C-terminus. Comparative peptide analysis indicated that the C-terminal helix of Cfcec- is amphipathic, unlike that of Cfcec+ , which is hydrophobic. Interestingly, molecular dynamics simulations pointed to the lower conformational flexibility of Cfcec- peptides, relative to that of Cfcec+ . Phylogenetic analysis suggests that the evolution of distinct Cfcec+ and Cfcec- peptides may have resulted from an ancient duplication event within the Lepidoptera. Finally, we found that both anionic and cationic cecropins contain a BH3-like motif (G-[KQR]-[HKQNR]-[IV]-[KQR]) that could interact with Bcl-2, a protein involved in apoptosis; this observation is congruent with previous reports indicating that cecropins induce apoptosis. Altogether, our observations suggest that cecropins may provide templates for the development of new anticancer drugs. We also estimated the antibacterial activity of Cfcec-2 and a ∆Cfce-2 peptide as AMPs by testing directly their ability in inhibiting bacterial growth in a disk diffusion assay and their potential for development of novel therapeutics.
Subject(s)
Anti-Bacterial Agents/chemistry , Antineoplastic Agents/chemistry , Cecropins/chemistry , Insect Proteins/chemistry , Peptides/chemistry , Proto-Oncogene Proteins c-bcl-2/chemistry , Amino Acid Sequence , Animals , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Binding Sites , Cecropins/genetics , Cecropins/metabolism , Cecropins/pharmacology , Escherichia coli/drug effects , Escherichia coli/growth & development , Evolution, Molecular , Humans , Hydrophobic and Hydrophilic Interactions , Insect Proteins/genetics , Insect Proteins/metabolism , Insect Proteins/pharmacology , Molecular Dynamics Simulation , Moths/chemistry , Moths/physiology , Peptides/metabolism , Phylogeny , Protein Binding , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Static ElectricityABSTRACT
Wasps, members of the order Hymenoptera, are distributed in different parts of the world, including Brazil, Thailand, Japan, Korea, and Argentina. The lifestyles of the wasps are solitary and social. Social wasps use venom as a defensive measure to protect their colonies, whereas solitary wasps use their venom to capture prey. Chemically, wasp venom possesses a wide variety of enzymes, proteins, peptides, volatile compounds, and bioactive constituents, which include phospholipase A2, antigen 5, mastoparan, and decoralin. The bioactive constituents have anticancer, antimicrobial, and anti-inflammatory effects. However, the limited quantities of wasp venom and the scarcity of advanced strategies for the synthesis of wasp venom's bioactive compounds remain a challenge facing the effective usage of wasp venom. Solid-phase peptide synthesis is currently used to prepare wasp venom peptides and their analogs such as mastoparan, anoplin, decoralin, polybia-CP, and polydim-I. The goal of the current review is to highlight the medicinal value of the wasp venom compounds, as well as limitations and possibilities. Wasp venom could be a potential and novel natural source to develop innovative pharmaceuticals and new agents for drug discovery.
Subject(s)
Anti-Infective Agents/pharmacology , Anti-Inflammatory Agents/pharmacology , Antineoplastic Agents/pharmacology , Insect Proteins/pharmacology , Nanotechnology , Wasp Venoms/pharmacology , Animals , Anti-Infective Agents/chemical synthesis , Anti-Inflammatory Agents/chemical synthesis , Antineoplastic Agents/chemical synthesis , Humans , Insect Proteins/chemical synthesis , Wasp Venoms/chemical synthesisABSTRACT
Increased amino acid availability acutely stimulates protein synthesis partially via activation of mechanistic target of rapamycin complex 1 (mTORC1). Plant-and insect-based protein sources matched for total protein and/or leucine to animal proteins induce a lower postprandial rise in amino acids, but their effects on mTOR activation in muscle are unknown. C57BL/6J mice were gavaged with different protein solutions: whey, a pea-rice protein mix matched for total protein or leucine content to whey, worm protein matched for total protein, or saline. Blood was drawn 30, 60, 105 and 150 min after gavage and muscle samples were harvested 60 min and 150 min after gavage to measure key components of the mTORC1 pathway. Ingestion of plant-based proteins induced a lower rise in blood leucine compared to whey, which coincided with a dampened mTORC1 activation, both acutely and 150 min after administration. Matching total leucine content to whey did not rescue the reduced rise in plasma amino acids, nor the lower increase in mTORC1 compared to whey. Insect protein elicits a similar activation of downstream mTORC1 kinases as plant-based proteins, despite lower postprandial aminoacidemia. The mTORC1 response following ingestion of high-quality plant-based and insect proteins is dampened compared to whey in mouse skeletal muscle.