RESUMO
The extensive application of nuclear technology has increased the potential of uncontrolled radiation exposure to the public. Since skin is the largest organ, radiation-induced skin injury remains a serious medical concern. Organisms evolutionally develop distinct strategies to protect against environment insults and the related research may bring novel insights into therapeutics development. Here, 26 increased peptides are identified in skin tissues of frogs (Pelophylax nigromaculatus) exposed to electron beams, among which four promoted the wound healing of irradiated skin in rats. Specifically, radiation-induced frog skin peptide-2 (RIFSP-2), from histone proteolysis exerted membrane permeability property, maintained cellular homeostasis, and reduced pyroptosis of irradiated cells with decreased TBK1 phosphorylation. Subsequently, stearyl-CoA desaturase 1 (SCD1) is identified, a critical enzyme in biogenesis of monounsaturated fatty acids (MUFAs) as a direct target of RIFSP-2 based on streptavidin-biotin system. The lipidomic analysis further assured the restrain of MUFAs biogenesis by RIFSP-2 following radiation. Moreover, the decreased MUFA limited radiation-induced and STING-mediated inflammation response. In addition, genetic depletion or pharmacological inhibition of STING counteracted the decreased pyroptosis by RIFSP-2 and retarded tissue repair process. Altogether, RIFSP-2 restrains radiation-induced activation of SCD1-MUFA-STING axis. Thus, the stress-induced amphibian peptides can be a bountiful source of novel radiation mitigators.
Assuntos
Inflamação , Pele , Animais , Pele/metabolismo , Pele/efeitos da radiação , Pele/efeitos dos fármacos , Ratos , Inflamação/metabolismo , Protetores contra Radiação/farmacologia , Estearoil-CoA Dessaturase/metabolismo , Estearoil-CoA Dessaturase/genética , Peptídeos/farmacologia , Peptídeos/metabolismo , Ranidae/metabolismo , Modelos Animais de Doenças , Cicatrização/efeitos dos fármacos , Anuros/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genéticaRESUMO
Five host-defense peptides (figainin 2PL, hylin PL, raniseptin PL, plasticin PL, and peptide YL) were isolated from norepinephrine-stimulated skin secretions of the banana tree dwelling frog Boana platanera (Hylidae; Hylinae) collected in Trinidad. Raniseptin PL (GVFDTVKKIGKAVGKFALGVAKNYLNS.NH2) and figainin 2PL (FLGTVLKLGKAIAKTVVPMLTNAMQPKQ. NH2) showed potent and rapid bactericidal activity against a range of clinically relevant Gram-positive and Gram-negative ESKAPE + pathogens and Clostridioides difficile. The peptides also showed potent cytotoxic activity (LC50 values < 30 µM) against A549, MDA-MB-231 and HT29 human tumor-derived cell lines but appreciably lower hemolytic activity against mouse erythrocytes (LC50 = 262 ± 14 µM for raniseptin PL and 157 ± 16 µM for figainin 2PL). Hylin PL (FLGLIPALAGAIGNLIK.NH2) showed relatively weak activity against microorganisms but was more hemolytic. The glycine-leucine-rich peptide with structural similarity to the plasticins (GLLSTVGGLVGGLLNNLGL.NH2) and the non-cytotoxic peptide YL (YVPGVIESLL.NH2) lacked antimicrobial and cytotoxic activities. Hylin PL, raniseptinPL and peptide YL stimulated the rate of release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥100 nM. Peptide YL was the most effective (2.3-fold increase compared with basal rate at 1 µM concentration) and may represent a template for the design of a new class of incretin-based anti-diabetic drugs.
Assuntos
Anuros , Pele , Animais , Pele/metabolismo , Humanos , Anuros/metabolismo , Camundongos , Linhagem Celular Tumoral , Proteínas de Anfíbios/farmacologia , Proteínas de Anfíbios/química , Proteínas de Anfíbios/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/química , Hemólise/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Sequência de Aminoácidos , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação , Células HT29 , Musa/químicaRESUMO
In response to seasonal droughts, the green striped burrowing frog Cyclorana alboguttata enters a reversible hypometabolic state called aestivation where heart rate and oxygen consumption can be reduced despite warm (>25C°) ambient temperatures. With a view to understanding molecular mechanisms we profiled aestivating versus control gastrocnemius muscle using mRNA sequencing. This indicated an extensive metabolic reprogramming, with nearly a quarter of the entire transcriptome (3996 of 16,960 mRNA) exhibiting a nominal >2-fold change. Consistent with a physiological adaptation to spare carbohydrate reserves, carbohydrate catabolism was systemically downregulated. A 630-fold downregulation of ENO3 encoding the enolase enzyme was most striking. The 590 frog orthologs of mRNA encoding the mitoproteome were, viewed as a population, significantly downregulated during aestivation, although not to the same extent as mRNA encoding carbohydrate catabolism. Prominent examples include members of the TCA cycle (IDH2), electron transport chain (NDUFA6), the ATP synthase complex (ATP5F1B) and ADP/ATP intracellular transport (SLC25A4). Moreover, mRNA derived from the mt genome itself (e.g. mt-ND1) were also downregulated. Most prominent among the upregulated mRNA are those encoding aspects of regulated proteolysis including the proteosome (e.g. PSME4L), peptidases (USP25), atrogins (FBXO32) and ubiquitination (VCP). Finally, we note the â¼5-fold upregulation of the mRNA EIFG3 that encodes part of the EIF4F complex. This possesses global control of protein synthesis. Given protein synthesis is repressed in aestivating frogs this indicates the skeletal musculature is poised for accelerated translation of mRNA upon emergence, supporting a strategy to rapidly restore function when the summer rains come.
Assuntos
Anuros , Músculo Esquelético , Animais , Músculo Esquelético/metabolismo , Anuros/metabolismo , Carboidratos , Trifosfato de Adenosina/metabolismo , Estivação/fisiologiaRESUMO
Climate change and other factors have contributed to an increased frequency and intensity of global wildfires in recent years. Ammonium-based fire retardants are widely used to suppress or delay the spread of fire and have generally been regarded as presenting a low risk of acute toxicity to fauna. However, studies have raised concerns about their potential to cause indirect or sub-lethal effects, and toxicity information regarding the potential for such impacts in aquatic species is limited. To address these knowledge gaps, we used an untargeted metabolomics approach to evaluate the sub-lethal physiological and metabolic responses of striped marsh frog (Limnodynastes peronii) tadpoles exposed to a concentration gradient of the ammonium polyphosphate (APP)-based fire retardant Phos-Chek LC95W (PC). Acute exposure (96 h) to PC significantly altered the relative abundance of 14 metabolites in whole tadpoles. The overall metabolic response pattern was consistent with effects reported for ammonia toxicity and also suggestive of energy dysregulation and osmotic stress associated with alterations to physicochemical water quality parameters in the PC treatments. Results suggest that run-off or accidental application of this formulation into waterways can have significant sub-lethal consequences on the biochemical profiles (i.e., the metabolome) of aquatic organisms and may be a concern for frog species that breed and develop in small, often ephemeral, waterbodies. Our study highlights the benefits of integrating untargeted metabolomics with other ecological and toxicological endpoints to provide a more holistic characterisation of the sub-lethal impacts associated with bushfire-fighting chemicals and with environmental contaminants more broadly.
Assuntos
Compostos de Amônio , Retardadores de Chama , Poluentes Químicos da Água , Animais , Áreas Alagadas , Retardadores de Chama/toxicidade , Larva , Anuros/metabolismo , Compostos de Amônio/farmacologia , Poluentes Químicos da Água/metabolismoRESUMO
Subtilases play a significant role in microbial pathogen infections by degrading the host proteins. Subtilisin inhibitors are crucial in fighting against these harmful microorganisms. LL-TIL, from skin secretions of Lepidobatrachus laevis, is a cysteine-rich peptide belonging to the I8 family of inhibitors. Protease inhibitory assays demonstrated that LL-TIL acts as a slow-tight binding inhibitor of subtilisin Carlsberg and proteinase K with inhibition constants of 91 pM and 2.4 nM, respectively. The solution structures of LL-TIL and a mutant peptide reveal that they adopt a typical TIL-type fold with a canonical conformation of a reactive site loop (RSL). The structure of the LL-TIL-subtilisin complex and molecular dynamics (MD) simulations provided an in-depth view of the structural basis of inhibition. NMR relaxation data and molecular dynamics simulations indicated a rigid conformation of RSL, which does not alter significantly upon subtilisin binding. The energy calculation for subtilisin inhibition predicted Ile31 as the highest contributor to the binding energy, which was confirmed experimentally by site-directed mutagenesis. A chimeric mutant of LL-TIL broadened the inhibitory profile and attenuated subtilisin inhibition by 2 orders of magnitude. These results provide a template to engineer more specific and potent TIL-type subtilisin inhibitors.
Assuntos
Subtilisina , Subtilisinas , Animais , Subtilisina/genética , Subtilisina/metabolismo , Sequência de Aminoácidos , Subtilisinas/genética , Subtilisinas/metabolismo , Anuros/metabolismo , Peptídeos , Simulação de Dinâmica Molecular , Domínio CatalíticoRESUMO
The amphibian family Leptodactylidae is divided into three sub-families: Leiuperinae, Leptodactylinae, and Paratelmatobiinae. Host-defense peptides (HDPs) present in the skins of frogs belonging to the Leptodactylinae have been studied extensively, but information is limited regarding peptides from Leiuperinae species. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Tungara frog Engystomops pustulosus (Leiuperinae) collected in Trinidad led to the isolation and structural characterization of previously undescribed pustulosin-1 (FWKADVKEIG KKLAAKLAEELAKKLGEQ), [Q28E] pustulosin-1 (pustulosin-2), and pustulosin-3 (DWKETAKELLKKIGAKVAQVISDKLNPAPQ). The primary structures of these peptides do not resemble those of previously described frog skin HDPs. In addition, the secretions contained tigerinin-1EP (GCKTYLIEPPVCT) with structural similarity to the tigerinins previously identified in skin secretions from frogs from the family Dicroglossidae. Pustulosin-1 and -3 adopted extended α-helical conformations in 25% trifluoroethanol-water and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). Pustulosin-1 and -3 displayed cytotoxic activity against a range of human tumor-derived cell lines (A549, MDA-MB-231, and HT29), but their therapeutic potential for development into anti-cancer agents is limited by their comparable cytotoxic activity against non-neoplastic human umbilical vein endothelial cells. The peptides also displayed weak antimicrobial activity against Escherichia coli (MIC = 125 µM) but were inactive against Staphylococcus aureus. Tigerinin-1EP was inactive against both the tumor-derived cells and bacteria.
Assuntos
Antineoplásicos , Neoplasias , Animais , Humanos , Peptídeos Catiônicos Antimicrobianos/química , Células Endoteliais/metabolismo , Proteínas de Anfíbios/química , Anuros/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/metabolismo , Neoplasias/metabolismo , Pele/metabolismo , Testes de Sensibilidade MicrobianaRESUMO
Amphibians' skin is a rich source of natural antimicrobial peptides (AMPs). These AMPs exhibit marked inter- and intraspecific sequence divergence linked to the arms race between host and pathogens. Here, we combine peptidomics, molecular modeling, and phylogenetic analyses to understand the evolution of AMPs in Cophomantini, a diverse clade of neotropical tree frogs, and to investigate their interaction with bacterial membranes. Consistent with results in other amphibians, all species of Cophomantini secrete a mixture of peptides. We selected the hylin peptide family to survey sequence variability and the presence of common amino acid motifs. We found that most species secrete a unique set of hylins that, though variable, share the conserved motif Gly-X-X-X-Pro-Ala-X-X-Gly, with Gly and Pro colocalizing with charged or polar residues. Our modeling revealed that Pro curves the peptide through a hinge, facilitating its insertion into the bacterial membrane and, once inserted, contributes to stabilizing the pore structure. The phylogenetic inference using hylid prepro-peptides showed the need to classify new AMPs using the full-length sequence of the prepro-peptide region and highlighted the complex relationships between peptide families. Our findings revealed that conserved motifs occurred independently in distinct AMP families, suggesting a convergent evolution and a significant role in peptide-membrane interactions.
Assuntos
Peptídeos Antimicrobianos , Peptídeos , Humanos , Animais , Sequência de Aminoácidos , Filogenia , Peptídeos/química , Anuros/metabolismoRESUMO
Cell membranes form barriers for molecule exchange between the cytosol and the extracellular environments. ßγ-CAT, a complex of pore-forming protein BmALP1 (two ßγ-crystallin domains with an aerolysin pore-forming domain) and the trefoil factor BmTFF3, has been identified in toad Bombina maxima. It plays pivotal roles, via inducing channel formation in various intracellular or extracellular vesicles, as well as in nutrient acquisition, maintaining water balance, and antigen presentation. Thus, such a protein machine should be tightly regulated. Indeed, BmALP3 (a paralog of BmALP1) oxidizes BmALP1 to form a water-soluble polymer, leading to dissociation of the ßγ-CAT complex and loss of biological activity. Here, we found that the B. maxima IgG Fc-binding protein (FCGBP), a well-conserved vertebrate mucin-like protein with unknown functions, acted as a positive regulator for ßγ-CAT complex assembly. The interactions among FCGBP, BmALP1, and BmTFF3 were revealed by co-immunoprecipitation assays. Interestingly, FCGBP reversed the inhibitory effect of BmALP3 on the ßγ-CAT complex. Furthermore, FCGBP reduced BmALP1 polymers and facilitated the assembly of ßγ-CAT with the biological pore-forming activity in the presence of BmTFF3. Our findings define the role of FCGBP in mediating the assembly of a pore-forming protein machine evolved to drive cell vesicular delivery and transport.
Assuntos
Cristalinas , Peptídeos , Animais , Peptídeos/metabolismo , Pele/metabolismo , Anuros/metabolismo , Cristalinas/metabolismo , Porinas/metabolismo , Imunoglobulina G/metabolismoRESUMO
Toxin-like proteins and peptides of skin secretions from amphibians play important physiological and pathological roles in amphibians. ßγ-CAT is a Chinese red-belly toad-derived pore-forming toxin-like protein complex that consists of aerolysin domain, crystalline domain, and trefoil factor domain and induces various toxic effects via its membrane perforation process, including membrane binding, oligomerization, and endocytosis. Here, we observed the death of mouse hippocampal neuronal cells induced by ßγ-CAT at a concentration of 5 nM. Subsequent studies showed that the death of hippocampal neuronal cells was accompanied by the activation of Gasdermin E and caspase-1, suggesting that ßγ-CAT induces the pyroptosis of hippocampal neuronal cells. Further molecular mechanism studies revealed that the pyroptosis induced by ßγ-CAT is dependent on the oligomerization and endocytosis of ßγ-CAT. It is well known that the damage of hippocampal neuronal cells leads to the cognitive attenuation of animals. The impaired cognitive ability of mice was observed after intraperitoneal injection with 10 µg/kg ßγ-CAT in a water maze assay. Taken together, these findings reveal a previously unknown toxicological function of a vertebrate-derived pore-forming toxin-like protein in the nerve system, which triggers the pyroptosis of hippocampal neuronal cells, ultimately leading to hippocampal cognitive attenuation.
Assuntos
Proteínas de Anfíbios , Anuros , Neurônios , Piroptose , Animais , Camundongos , Anuros/metabolismo , Cognição , Peptídeos/química , Proteínas de Anfíbios/toxicidade , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacosRESUMO
The host-defense peptide ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH2 ), first isolated from the Caribbean frog Leptodactylus nesiotus, inhibited growth of clinically relevant Gram-positive and Gram-negative bacteria as well as a strain of the major emerging yeast pathogen Candida parapsilosis. Increasing cationicity while maintaining amphipathicity by the substitution Asp4 âLys increased potency against the microorganisms by between 4- and 16-fold (MIC ≤3 µM) compared with the naturally occurring peptide. The substitution Ala18 âLys and the double substitution Asp4 âLys and Ala18 âLys had less effects on potency. The [D4K] analog also showed 2.5- to 4-fold greater cytotoxic potency against non-small-cell lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, and colorectal adenocarcinoma HT-29 cells (LC50 values in the range of 12-20 µM) compared with ocellatin-3N but was less hemolytic to mouse erythrocytes. However, the peptide showed no selectivity for tumor-derived cells [LC50 = 20 µM for human umbilical vein endothelial cells (HUVECs)]. Ocellatin-3N and [D4K]ocellatin-3N stimulated the release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥1 nM, and [A18K]ocellatin-3N, at concentrations ≥0.1 nM. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 µM, indicating that plasma membrane integrity had been preserved. The three peptides produced an increase in intracellular [Ca2+ ] in BRIN-BD11 cells when incubated at a concentration of 1 µM. In view of its high insulinotropic potency and relatively low hemolytic activity, the [A18K] ocellatin analog may represent a template for the design of agents with therapeutic potential for the treatment of patients with type 2 diabetes.
Assuntos
Anti-Infecciosos , Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Diabetes Mellitus Tipo 2 , Neoplasias Pulmonares , Camundongos , Animais , Humanos , Peptídeos Catiônicos Antimicrobianos/química , Lisina , Antibacterianos/química , Diabetes Mellitus Tipo 2/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Células Endoteliais/metabolismo , Proteínas de Anfíbios/farmacologia , Bactérias Gram-Positivas , Bactérias Gram-Negativas , Neoplasias Pulmonares/metabolismo , Insulina/metabolismo , Antineoplásicos/farmacologia , Anuros/metabolismo , Pele/metabolismoRESUMO
Amphibian skin contains wound-healing peptides, antimicrobial peptides, and insulin-releasing peptides, which give their skin a strong regeneration ability to adapt to a complex and harsh living environment. In the current research, a novel wound-healing promoting peptide, PM-7, was identified from the skin secretions of Polypedates megacephalus, which has an amino acid sequence of FLNWRRILFLKVVR and shares no structural similarity with any peptides described before. It displays the activity of promoting wound healing in mice. Moreover, PM-7 exhibits the function of enhancing proliferation and migration in HUVEC and HSF cells by affecting the MAPK signaling pathway. Considering its favorable traits as a novel peptide that significantly promotes wound healing, PM-7 can be a potential candidate in the development of novel wound-repairing drugs.
Assuntos
Peptídeos , Cicatrização , Camundongos , Animais , Peptídeos/química , Anuros/metabolismo , Sequência de Aminoácidos , Transdução de SinaisRESUMO
Maintaining water balance is a real challenge for amphibians in terrestrial environments. Our previous studies with toad Bombina maxima discovered a pore-forming protein and trefoil factor complex ßγ-CAT, which is assembled under tight regulation depending on environmental cues. Here we report an unexpected role for ßγ-CAT in toad water maintaining. Deletion of toad skin secretions, in which ßγ-CAT is a major component, increased animal mortality under hypertonic stress. ßγ-CAT was constitutively expressed in toad osmoregulatory organs, which was inducible under the variation of osmotic conditions. The protein induced and participated in macropinocytosis in vivo and in vitro. During extracellular hyperosmosis, ßγ-CAT stimulated macropinocytosis to facilitate water import and enhanced exosomes release, which simultaneously regulated aquaporins distribution. Collectively, these findings uncovered that besides membrane integrated aquaporin, a secretory pore-forming protein can facilitate toad water maintaining via macropinocytosis induction and exocytosis modulation, especially in responses to osmotic stress.
Assuntos
Peptídeos , Água , Animais , Anuros/metabolismo , Peptídeos/metabolismo , Pele/metabolismo , Água/metabolismoRESUMO
Tryptophyllins constitute a heterogeneous group of peptides that are one of the first classes of peptides identified from amphibian's skin secretions. Here, we report the structural characterization and antioxidant properties of a novel tryptophyllin-like peptide, named PpT-2, isolated from the Iberian green frog Pelophylax perezi. The skin secretion of P. perezi was obtained by electrical stimulation and fractionated using RP-HPLC. De novo peptide sequencing was conducted using MALDI MS/MS. The primary structure of PpT-2 (FPWLLS-NH2 ) was confirmed by Edman degradation and subsequently investigated using in silico tools. PpT-2 shared physicochemical properties with other well-known antioxidants. To test PpT-2 for antioxidant activity in vitro, the peptide was synthesized by solid phase and assessed in the chemical-based ABTS and DPPH scavenging assays. Then, a flow cytometry experiment was conducted to assess PpT-2 antioxidant activity in oxidatively challenged murine microglial cells. As predicted by the in silico analyses, PpT-2 scavenged free radicals in vitro and suppressed the generation of reactive species in PMA-stimulated BV-2 microglia cells. We further explored possible bioactivities of PpT-2 against prostate cancer cells and bacteria, against which the peptide exerted a moderate antiproliferative effect and negligible antimicrobial activity. The biocompatibility of PpT-2 was evaluated in cytotoxicity assays and in vivo toxicity with Galleria mellonella. No toxicity was detected in cells treated with up to 512 µg/ml and in G. mellonella treated with up to 40 mg/kg PpT-2. This novel peptide, PpT-2, stands as a promising peptide with potential therapeutic and biotechnological applications, mainly for the treatment/prevention of neurodegenerative disorders.
Assuntos
Antioxidantes , Fármacos Neuroprotetores , Animais , Antioxidantes/metabolismo , Anuros/metabolismo , Masculino , Camundongos , Microglia/metabolismo , Peptídeos/química , Ranidae/metabolismo , Relação Estrutura-Atividade , Espectrometria de Massas em TandemRESUMO
In COVID-19 infection, the SARS-CoV-2 spike protein S1 interacts to the ACE2 receptor of human host, instigating the viral infection. To examine the competitive inhibitor efficacy of broad spectrum alpha helical AMPs extracted from frog skin, a comparative study of intermolecular interactions between viral S1 and AMPs was performed relative to S1-ACE2p interactions. The ACE2 binding region with S1 was extracted as ACE2p from the complex for ease of computation. Surprisingly, the Spike-Dermaseptin-S9 complex had more intermolecular interactions than the other peptide complexes and importantly, the S1-ACE2p complex. We observed how atomic displacements in docked complexes impacted structural integrity of a receptor-binding domain in S1 through conformational sampling analysis. Notably, this geometry-based sampling approach confers the robust interactions that endure in S1-Dermaseptin-S9 complex, demonstrating its conformational transition. Additionally, QM calculations revealed that the global hardness to resist chemical perturbations was found more in Dermaseptin-S9 compared to ACE2p. Moreover, the conventional MD through PCA and the torsional angle analyses indicated that Dermaseptin-S9 altered the conformations of S1 considerably. Our analysis further revealed the high structural stability of S1-Dermaseptin-S9 complex and particularly, the trajectory analysis of the secondary structural elements established the alpha helical conformations to be retained in S1-Dermaseptin-S9 complex, as substantiated by SMD results. In conclusion, the functional dynamics proved to be significant for viral Spike S1 and Dermaseptin-S9 peptide when compared to ACE2p complex. Hence, Dermaseptin-S9 peptide inhibitor could be a strong candidate for therapeutic scaffold to prevent infection of SARS-CoV-2.
Assuntos
Enzima de Conversão de Angiotensina 2 , Peptídeos Catiônicos Antimicrobianos , Tratamento Farmacológico da COVID-19 , COVID-19 , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Anuros/metabolismo , COVID-19/prevenção & controle , Humanos , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
BACKGROUND: Bacterial resistance to all currently available conventional antibiotics has caused a global public health crisis and led to an imperative search for new agents. Antimicrobial peptides (AMPs) are essential components of host innate immune defense against microbial invasions. OBJECTIVES: The objective of this study was to report a novel AMP, brevinin-2KP, from the skin of the black Kaloula pulchra frog and describe its structural and biological characterization. MATERIALS AND METHODS: The physical and chemical parameters of brevinin-2KP were predicted with the ExPASy Bioinformatics Resource Portal. The assembled sequences were aligned with ClustalW, and the phylogenetic tree was constructed using Mega. Circular dichroism (CD) experiments were carried out to identify the secondary structure and the stability of peptide in different solvent environments. The cytotoxicity of brevinin-2KP was evaluated by the MTT test. To determine antibacterial activity of brevinin- 2KP, a standard two-fold broth dilution method was used. SEM was carried out to observe the morphological change in the bacterial treated by brevinin-2KP. The live/dead bacterial viability was measured with a LIVE/DEAD® BacLight kit. Histamine release and mast cell degranulation assays were performed. RESULTS: The precursor of brevinin-2KP contains 72 amino acid residues, including a conserved signal peptide, acidic propeptide with KR residues, and mature peptide with a sequence of GVITDALKGAAKTVAAELLKKAHCKLTNSC. Phylogenetic analysis based on the amino acid sequences of 34 brevinin-2 peptides from 30 anuran species demonstrates that K. pulchra is genetically closely related to the genus Hylarana. The CD spectra analysis indicates that brevinin-2KP adopts random coil in the water and an organized α-helical conformation in SDS solution. Further, this secondary structure is stable under high salt and high-temperature conditions. Brevinin-2KP is weakly active towards the tested Gram-positive and Gram-negative bacteria as well as fungi due to its membranolytic action. Moreover, brevinin-2KP inhibits the proliferation of several mammal cells with IC50 values ranging from 3.27 to 59.75 µM. In addition, brevinin-2KP promotes degranulation and histamine release of mast cells, indicating that it is involved in the inflammatory response. CONCLUSION: This is the first report on AMP identified from the skin of K. pulchra. Brevinin-2KP adopts a typical amphipathic α-helix conformation in membrane mimic environment and shows antimicrobial and antitumor activities by potential membranolytic mechanism. In addition, brevinin-2KP can promote degranulation and histamine release of mast cells. Brevinin-2KP is expected to become a good drug temple molecule.
Assuntos
Anti-Infecciosos , Peptídeos Catiônicos Antimicrobianos , Animais , Aminoácidos , Antibacterianos/metabolismo , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Antimicrobianos , Anuros/metabolismo , Clonagem Molecular , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Mamíferos/metabolismo , Testes de Sensibilidade Microbiana , Filogenia , Sinais Direcionadores de Proteínas , Pele/metabolismo , Solventes , ÁguaRESUMO
Bombesin mediates several biological activities in the gastrointestinal (GI) tract and central nervous system in mammals, including smooth muscle contraction, secretion of GI hormones and regulation of homeostatic mechanisms. Here, we report a novel bombesin-like peptide isolated from Boana raniceps. Its amino acid sequence, GGNQWAIGHFM-NH2, was identified and structurally confirmed by HPLC, MS/MS and 454-pyrosequencing; the peptide was named BR-bombesin. The effect of BR-bombesin on smooth muscle contraction was assessed in ileum and esophagus, and its anti-secretory activity was investigated in the stomach. BR-bombesin exerted significant contractile activity with a concentration-response curve similar to that of commercially available bombesin in ileum strips of Wistar rats. In esophageal strips, BR-bombesin acted as an agonist, as many other bombesin-related peptides act, although with different behavior compared to the muscarinic agonist carbachol. Moreover, BR-bombesin inhibited stomach secretion by approximately 50% compared to the untreated control group. This novel peptide has 80% and 70% similarity with the 10-residue C-terminal domain of human neuromedin B (NMB) and human gastrin releasing peptide (GRP10), respectively. Molecular docking analysis revealed that the GRP receptor had a binding energy equal to - 7.3 kcal.mol-1 and - 8.5 kcal.mol-1 when interacting with bombesin and BR-bombesin, respectively. Taken together, our data open an avenue to investigate BR-bombesin in disorders that involve gastrointestinal tract motility and acid gastric secretion.
Assuntos
Bombesina , Receptores da Bombesina , Animais , Anuros/metabolismo , Bombesina/metabolismo , Bombesina/farmacologia , Mamíferos/metabolismo , Simulação de Acoplamento Molecular , Peptídeos/farmacologia , Ratos , Ratos Wistar , Receptores da Bombesina/genética , Receptores da Bombesina/metabolismo , Estômago , Espectrometria de Massas em TandemRESUMO
The ß-defensins are important components of the vertebrate innate immune system. While mammalian ß-defensins have wide-ranging antibacterial and immunomodulatory activities, those of amphibians remain largely uncharacterised. In this study, ß-defensin cDNA was identified from the skin transcriptome of the Chinese spiny frog Quasipaa spinosa. This ß-defensin (QS-BD) consists of a signal and a mature peptide. Sequence alignments with other amphibian ß-defensins showed conservation of the functional mature peptide and that its closest relative is ß-defensin from Zhangixalus puerensis. Synthetic QS-BD showed antibacterial activity against Vibrio vulnificus, Vibrio harveyi, Streptococcus iniae, and Aeromonas hydrophila. QS-BD showed bactericidal activity by destroying the cell membrane integrity, but did not hydrolyse genomic DNA. QS-BD treatment promoted respiratory bursts and upregulated the expression of interleukin-1ß and tumour necrosis factor-α in the murine leukemic monocyte/macrophage cell line RAW264.7. This is the first demonstration of immunomodulatory activity by an amphibian ß-defensin.
Assuntos
beta-Defensinas , Animais , Antibacterianos/metabolismo , Anuros/metabolismo , China , Mamíferos , Camundongos , Ranidae/genética , beta-Defensinas/genética , beta-Defensinas/metabolismoRESUMO
The use of acetylcholinesterase (AChE) inhibitors, antioxidants or multitarget compounds are among the main strategies against Alzheimer's disease (AD). Between AChE inhibitors, those targeting the peripheral anionic site (PAS) are of special interest. Here, we describe the rational design and synthesis of peptide analogs of a natural PAS-targeting sequence that we recently discovered, aiming at increasing its activity against AChE. We also tested their radical scavenging and metal chelating properties. Our design strategy was based on the position-specific, computer-aided insertion of aromatic residues. The analog named as W3 showed a 30-fold higher inhibitory activity than the original sequence and an improved antioxidant activity. W3 is the most potent modified natural peptide against Electrophorus electricus AChE ever reported with an IC50 of 10.42 µM (± 1.02). In addition, it showed a radical scavenging activity of 47.00% ± 3.11 at 50 µM and 93.47% ± 1.53 at 400 µM. Since peptides are receiving increasing interest as drugs, we propose the W3 analog as an attractive sequence for the development of new peptide-based multitarget drugs for AD. Besides, this work sheds light on the importance of the aromatic residues in the modulation of AChE activity and their effect on the radical scavenging activity of a peptide.
Assuntos
Doença de Alzheimer , Inibidores da Colinesterase , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Anuros/metabolismo , Inibidores da Colinesterase/química , Simulação de Acoplamento Molecular , Peptídeos/farmacologia , Relação Estrutura-AtividadeRESUMO
Several years have passed since the Zika virus (ZIKV) pandemic reoccurred in 2015-2016. However, there is still a lack of proved protective vaccines or effective drugs against ZIKV. The peptide brevinin-2GHk (BR2GK), pertaining to the brevinin-2 family of antimicrobial peptides, has been reported to exhibit only weak antibacterial activity, and its antiviral effects have not been investigated. Thus, we analyzed the effect of BR2GK on ZIKV infection. BR2GK showed significant inhibitory activity in the early and middle stages of ZIKV infection, with negligible cytotoxicity. Furthermore, BR2GK was suggested to bind with ZIKV E protein and disrupt the integrity of the envelope, thus directly inactivating ZIKV. In addition, BR2GK can also penetrate the cell membrane, which may contribute to inhibition of the middle stage of ZIKV infection. BR2GK blocked ZIKV E protein expression with an IC50 of 3.408 ± 0.738 µΜ. In summary, BR2GK was found to be a multi-functional candidate and a potential lead compound for further development of anti-ZIKV drugs.
Assuntos
Peptídeos Antimicrobianos/farmacologia , Antivirais/farmacologia , Pele/química , Infecção por Zika virus/virologia , Zika virus/efeitos dos fármacos , Animais , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/metabolismo , Antivirais/química , Antivirais/metabolismo , Anuros/metabolismo , Humanos , Simulação de Acoplamento Molecular , Pele/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Zika virus/genética , Zika virus/fisiologiaRESUMO
Polyphenism-in which multiple distinct phenotypes are produced from a single genotype owing to differing environmental conditions-is commonplace, but its molecular bases are poorly understood. Here, we examine the transcriptomic bases of a polyphenism in Mexican spadefoot toads (Spea multiplicata). Depending on their environment, their tadpoles develop into either a default "omnivore" morph or a novel "carnivore" morph. We compared patterns of gene expression among sibships that exhibited high versus low production of carnivores when reared in conditions that induce the carnivore morph versus those that do not. We found that production of the novel carnivore morph actually involved changes in fewer genes than did the maintenance of the default omnivore morph in the inducing environment. However, only body samples showed this pattern; head samples showed the opposite pattern. We also found that changes to lipid metabolism (especially cholesterol biosynthesis) and peroxisome contents and function might be crucial for establishing and maintaining differences between the morphs. Thus, our findings suggest that carnivore phenotype might have originally evolved following the breakdown of robustness mechanisms that maintain the default omnivore phenotype, and that the carnivore morph is developmentally regulated by lipid metabolism and peroxisomal form, function, and/or signaling. This study also serves as a springboard for further exploration into the nature and causes of plasticity in an emerging model system.