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Cn-AMP1: A new promiscuous peptide with potential for microbial infections treatment
Abstract
The antimicrobial peptides (AMPs) are evolutionarily ancient molecules that act as components of the innate immune system. Recently, it was demonstrated that a single AMP can perform various functions; this ability is known as "peptide promiscuity." However, little is known about promiscuity in plant AMPs without disulfide bonds. This study was carried out to evaluate the promiscuity of Cn-AMP1: a promising disulfide-free plant peptide with reduced size and cationic and hydrophobic properties. Its activity against human pathogenic bacteria and fungal pathogens, as well as its in vitro immunostimulatory activity and effects on cancerous and healthy mammalian cell proliferation were studied here. Cn-AMP1 exerts antimicrobial effects against Gram-positive bacteria, Gram-negative bacteria, and fungi. Moreover, tumor cell viability activity in Caco-2 cells, as well as immunostimulatory activity by evaluating upregulated inflammatory-cytokine secretion by monocytes was also positively observed. Cn-AMP1 does not exhibit a well-defined conformation in aqueous solution and probably undergoes a 3(10)-helix transition in hydrophobic environments. The experimental results support the promiscuous activity of Cn-AMP1, presenting a wide range of activities, including antibacterial, antifungal, and immunostimulatory activity. In the future, Cn-AMP1 should be used in the development of novel biopharmaceuticals, mainly due to its reduced size and broad spectrum of activity.
... Cn-AMP1 plays a role in a broad range of biological functions including antibacterial, antifungal, and immunostimulatory activity. Furthermore, Cn-AMP1 elicited cytotoxicity against Cancer coli-2 Caucasian colon adenocarcinoma ( Caco-2) [6]. However, Cn-AMP1 cytotoxicity was assessed under 2D cell culture conditions which may not reflect its effect in a more realistic in vivo cellular microenvironment. ...
... Interest in use of cationic antimicrobial peptides in cancer treatment has increased and Cn-AMP1, derived from green coconut milk, previously demonstrated a cytotoxic effect against Caco-2 cell line in in vitro 2D culture models [6]. However, 3D culture models are increasingly used for pre-clinical drug screening. ...
... While Cn-AMP1 had some cytotoxic activity on the A549s in 2D culture, as previously reported for Caco-2 [6], it did not affect the 3D spheroid model. In 2D culture, cationic AMP induced cytotoxicity was involved by several mechanisms. ...
... For Lr-AMP1, peptides were joined at the N-terminus combining four different antimicrobial peptides, i.e. Bombinin, PGLa, Alloferon 1, and Cn-AMP1 in order to increase amphipathicity using the REGEX proposed by [27]. Afterwards, the aminoacid composition was evaluated by three-dimensional helical-wheel structure to predict protein hydrophilicity (EMBOSS pepwindows) [28]. ...
... Rational design was used to predict and modify the physical and chemical properties for this characteristic, including the antimicrobial peptides. Such modifications may be useful for increasing antimicrobial activity [27,39,26,40]. In this study, we modified these peptides in order to increase their amphipathic properties using helical wheel diagrams (Fig. 4). ...
... The modifications performed in Lr-AMP1 followed another strategy. In order to improve the Lr-AMP1 activity, variants were generated linking N-terminus portions of certified AMPs according to the pattern [27]. Lr-AMP1a received bombinin while the Lr-AMP1e was rearranged to show higher activity compared to the original one [41]. ...
Antimicrobial resistance is considered a health issue worldwide. This public health problem underscores the importance of searching for new antimicrobial molecules with different mechanisms of action. Leaf transcriptomes were used to search and develop synthetic antimicrobial peptides derived from mRNA sequences. The in silico search for new AMPs from the L. rotundifolia and L. alba transcriptomes allowed the identification of 120 putative peptide mRNA sequences. Eight of them fitted into optimal parameters and were translated and chemically synthesized antimicrobial peptides. Their biological activity was tested in both Gram-positive and Gram-negative bacteria against which they exhibited antibacterial activity. However, they showed an important hemolytic effect. Afterwards, two active peptides showing bactericidal activity isolated from each plant transcriptome tested were modified and modeled in 11 new variants to increase their antimicrobial activity and stability and to reduce or eliminate their hemolytic effect from their original peptides. The La-AMP1 (MSLLERKLLMHFLRV) the original peptide from L. alba showed a 52% hemolytic effect while the derived peptide La-AMP1a (GLMKLLRELLHMFSRVG) had its hemolytic effect reduced to 0.5% at 128 μg.mL⁻¹. Similarly, we observed that the original peptide from L. rotundifolia, Lr-AMP1 (MRIGLRFVLM), displayed a 71.5% hemolytic effect, while its derived peptide Lr-AMP1f (GSVLRAIMRMFAKLMG) showed 0% hemolysis at 128 μg.mL⁻¹, tested with fresh human erythrocytes. Our results indicate a promising method for the search for novel antimicrobial agents with reduced or zero hemolytic effect, as well as prediction and optimization of their activity from plant mRNA libraries.
... Besides the identification of AMPs, there are other uses for pattern matching related to AMPs. REGEX could be used to explore the variety of functions ( Silva et al., 2012) or even to design novel AMPs ( Loose et al., 2006). ...
... Since some peptides have limited identity with others, but their amino acids present similar physicochemical properties, it is possible to draw a REGEX expanding the residues by their properties and then perform the identification of distantly related peptides in databases ( Silva et al., 2012;Tomita et al., 2008). This approach was used to ana- lyse the multifunctional activity of Cn-AMP1, where initially a similarity was identified to another multifunctional peptide, alloferon-1 ( Chernysh et al., 2002), and then, based on a pairwise alignment and physicochemical properties, a REGEX was designed. ...
... This approach was used to ana- lyse the multifunctional activity of Cn-AMP1, where initially a similarity was identified to another multifunctional peptide, alloferon-1 ( Chernysh et al., 2002), and then, based on a pairwise alignment and physicochemical properties, a REGEX was designed. Through this REGEX, two other multifunctional peptides, in addition to alloferon-1, were retrieved from APD, and in all cases, the REGEX was matched with the N-terminal portion, indicating that the multifunctionality is re- lated to this portion ( Silva et al., 2012). ...
Data mining has been recognized by many researchers as a hot topic in different areas. In the post-genomic era, the growing number of sequences deposited in databases has been the reason why these databases have become a resource for novel biological information. In recent years, the identification of antimicrobial peptides (AMPs) in databases has gained attention. The identification of unannotated AMPs has shed some light on the distribution and evolution of AMPs and, in some cases, indicated suitable candidates for developing novel antimicrobial agents. The data mining process has been performed mainly by local alignments and/or regular expressions. Nevertheless, for the identification of distant homologous sequences, other techniques such as antimicrobial activity prediction and molecular modelling are required. In this context, this review addresses the tools and techniques, and also their limitations, for mining AMPs from databases. These methods could be helpful not only for the development of novel AMPs, but also for other kinds of proteins, at a higher level of structural genomics. Moreover, solving the problem of unannotated proteins could bring immeasurable benefits to society, especially in the case of AMPs, which could be helpful for developing novel antimicrobial agents and combating resistant bacteria.
... All donors signed an informed consent form, and the Human Research Ethics Committee of the Universidade Federal de Pernambuco (CEP/CCS/UFPE No. 145/09) approved the study. Liver hepatocellular carcinoma (HEPG2), human prostate cancer (DU145), acute lymphoblastic leukemia (MOLT-4) and erythromyeloblastoid leukemia (K562) cell lines were obtained from the Banco de Células do Rio de Janeiro of the Universidade Federal do Rio de Janeiro (BCRJ/UFRJ) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U mL −1 penicillin, and 100 g mL −1 streptomycin at 37 • C in an atmosphere with 5% CO 2 [33]. Adherent HEPG2 and DU145 (10 4 cells/well), or nonadherent MOLT-4 and K562 cells (10 5 cells/well) were plated in 96-well microtiter plates (TPP-Techno Plastic Products, Trasadigen, Switzerland). ...
... Human epithelial colorectal adenocarcinoma cells (CACO-2) were maintained in DMEM [33]. To determine the maximum nontoxic concentrations of IVTI, cell viability was further evaluated by MTT (5.0 mg mL −1 in PBS). ...
... Cytotoxicity was determined as a percentage of the maximum value after subtracting the background. The results were expressed as the percentage of each sample compared to the negative control (PBS buffer, pH 7.4) and the cell culture incubated in a lysis buffer (10 mM Tris, pH 7.4, 1 mM ethylenediaminetetraacetic acid (EDTA), and 0.1% Triton X-100) [33,38]. ...
Here, the purification, biochemical and biological properties of a trypsin inhibitor from Inga vera seeds (IVTI) are described. Partial amino acid sequence of IVTI showed that it belongs to the MEROPS I03 Kunitz inhibitor family. Moreover, it is composed of a single 20 kDa polypeptide chain with one disulfide bridge and was capable of inhibiting bovine trypsin at a 1:1 molar ratio with a 1.15 nM inhibition constant. IVTI was stable over a wide range of temperature, pH and concentration of a reducing agent. IVTI also inhibited the trypsin-like enzymes from the midgut of lepidopteran pests, such as Anagasta kuehniella (89%), Spodoptera frugiperda (83%), Corcyra cephalonica (80%), Heliothis virescens (70%) and Helicoverpa zea (64%). Furthermore, bioinsecticidal assays against A. kuehniella demonstrated that IVTI affected larval development by impairing weight gain and survival, as well as altering the duration of the larval cycle. IVTI was also fungicidal to Candida buinensis and bacteriostatic agent to Escherichia coli. Further assays revealed that IVTI is a chemopreventive agent against human epithelial colorectal adenocarcinoma cells (CACO-2), reducing cell viability by 70% at 200 μg.mL−1. In summary, these results demonstrate the multifaceted potential of IVTI as a biotechnological tool for agriculture and healthcare.
... In this context, Cn-AMP1 was also challenged against fungal pathogens and cancerous cells, showing clear activity. Interestingly, an extra in vitro immunostimulatory activity was exerted by Cn-AMP1, showing that coconut peptide was capable of up-regulating inflammatory-cytokine secretion by monocytes [11], suggesting multiple action mechanisms. ...
... These features give the peptide Cn-AMP1 a promiscuous character, even not presenting disulfide bond connection [9,11]. However, little is known about structural properties in plant promiscuous AMPs without disulfide bonds. ...
... Random conformations in the absence of certain hydrophobic conditions like micelles or membranes have been observed before for other linear antimicrobial peptides. Indeed, data reported here corroborate the theoretical data presented for Cn-AMP1 by using molecular modeling [9,11]. Moreover, other AMPs also showed similar structural transitions. ...
Multifunctional and promiscuous antimicrobial peptides (AMPs) can be used as an efficient strategy to control pathogens. However, little is known about the structural properties of plant promiscuous AMPs without disulfide bonds. CD and NMR were used to elucidate the structure of the promiscuous peptide Cn-AMP1, a disulfide-free peptide isolated from green coconut water. Data here reported shows that peptide structure is transitory and could be different according to the micro-environment. In this regard, Cn-AMP1 showed a random coil in a water environment and an α-helical structure in the presence of SDS-d25 micelles. Moreover, deuterium exchange experiments showed that Gly4, Arg5 and Met9 residues are less accessible to solvent, suggesting that flexibility and cationic charges seem to be essential for Cn-AMP1 multiple activities.
... The polypropylene plates (TPP, Switzerland) were incubated at 37°C for 24 h. The MIC was determined as the lowest tested concentration that leads to complete inhibition (100%) in comparison to the negative-control group (18). ...
... The treatment solution was filtered through a 100-m-pore cell strainer, centrifuged, and resuspended in supplemented Dulbecco's modified Eagle's medium (DMEM) (Gibco-Invitrogen) (4 mM L-glutamine, 10% fetal bovine serum [FBS; Gibco-Invitrogen], 2 mM nonessential amino acids [Gibco-Invitrogen], 50 mg ml Ϫ1 gentamicin, and 100 U ml Ϫ1 penicillinstreptomycin [GIBCO-Invitrogen]), seeded into tissue culture flasks (TPP, Switzerland), and incubated at 37°C in 5% CO 2 (19). L929 mouse fibroblasts were also cultured in supplemented DMEM, poured into a tissue culture flask (TPP, Switzerland), and incubated at 37°C in 5% CO 2 (18,20). ...
... Cytokine assays. Proinflammatory cytokines gamma interferon (IFN-␥), interleukin-6 (IL-6), IL-12p70, and tumor necrosis factor alpha (TNF-␣) were measured in the plasma of mice subjected to the lethal E. coli and S. aureus infection model, 24 h after infection, using an enzymelinked immunosorbent assay (ELISA) kit (Peprotech) according to the manufacturer's instructions (18). ...
The rapid increase in the incidence of multi-drug-resistant infections today has led to enormous interest in antimicrobial peptides (AMPs) as suitable compounds for developing unusual antibiotics. In this study clavanin A, an antimicrobial peptide, previously isolated from the marine tunicate Styela clava, was selected as a purposeful molecule that could be used in controlling infection and further synthesized. Clavanin A was in vitro evaluated against Staphylococcus aureus and Escherichia coli as well as toward L929 mice fibroblasts and SPCs cells. Moreover, this peptide was here challenged in an in vivo wound and sepsis model, and immune response was also analysed. Despite displaying clear in vitro antimicrobial activity toward Gram-positive and negative bacteria, clavanin A showed no cytotoxic activities against mammalian cells, and in acute toxicity tests no adverse reaction was observed at any of the concentrations. Moreover, clavanin A significantly reduced the S. aureus colony forming units in an experimental wound model. This same peptide also reduced the mortality of mice with E. coli and S. aureus by 80 % when compared with control animals (PBS-treated), these data suggest that clavanin A prevents the start of sepsis and thereby reduces mortality. These data suggest that clavanin A is an AMP that could improve the development of novel peptide-based strategies for the treatment of wound and sepsis infections.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
... Animals and plants share common elements in their defense processes against pathogens, including direct antimicrobial activities by synthesis of hydrolytic enzymes (chitinases, glucanases, proteinases and oxidases) [109] as well as antimicrobial peptides (AMPs) [13,53]. Furthermore, indirect innate immunity strategies have also been observed; these include some activities in host organisms, like the production of hydrogen peroxide or synthesis of host defense peptides (HDPs) that can cause an immunomodulatory response [145]. AMPs have been isolated from a wide number of sources, including animals, bacteria and plants [73]. ...
... Finally, the AMPs Cn-AMP1 (9 amino acids), Cn-AMP2 (12 amino acids), and Cn-AMP3 (8 amino acids), isolated from Cocos nucifera water, were also described as antimicrobial peptides, revealing bactericidal activities against E. coli, B. subtilis, P. aeruginosa and S. aureus (Table 11). Low homology with the AMPs temporin G and uperin was observed [145]. In addition, immunomodulatory activity has been also reported for Cn-AMP1. ...
... In addition, immunomodulatory activity has been also reported for Cn-AMP1. Its three-dimensional structure has been predicted by homology modeling, exhibiting a poorly defined conformation in aqueous solution, with a possible 3-10 helix in hydrophobic environments [145]. ...
... In addition to its use for AMP identification, REGEX may be utilized to explore different biological functions 166 and to design unconventional AMPs. 167 Once the primary sequence of those peptides has been identified, sequences with similar amino acid composition can be designed, maintaining similar averaged values of physicochemical features, such as mean hydrophobicity or net charge. ...
... Thus, databases can be used in the search for peptides with different sequences but having similar averaged physicochemical and structural proper-ties. 166,168 This approach was used to analyze promiscuous AMPs, 169,170 that is, peptides with multiple biological functions under different environments or conditions. The versatility of such pattern matching was also demonstrated by Loose et al., 167 who developed a different method for rational AMP design, in which the sequences of approximately 700 AMPs were extracted from multiple databases, yielding a varied set of AMPs of similar properties of interest for antimicrobial applications. ...
Antibiotic resistance is one of the greatest challenges of our time. This global health problem originated from a paucity of truly effective antibiotic classes and an increased incidence of multi-drug-resistant bacterial isolates in hospitals worldwide. Indeed, it has been recently estimated that 10 million people will die annually from drug-resistant infections by the year 2050. Therefore, the need to develop out-of-the-box strategies to combat antibiotic resistance is urgent. The biological world has provided natural templates, called antimicrobial peptides (AMPs), which exhibit multiple intrinsic medical properties including the targeting of bacteria. AMPs can be used as scaffolds and, via engineering, can be reconfigured for optimized potency and targetability toward drug-resistant pathogens. Here, we review the recent development of tools for the discovery, design, and production of AMPs and propose that the future of peptide drug discovery will involve the convergence of computational and synthetic biology principles.
... This is important given the regular coconut water intake in some regions of the globe or even considering the perspective of applying the peptide as a biopharmaceutical molecule. In agreement with our findings, Silva and coworkers [12] found no CnAMP1-induced cytotoxicity in murine macrophage-like cells (RAW 264.7). In nondifferentiated Caco-2 cells, however, they observed that CnAMP1 reduced cell viability in a dose-dependent manner [12]. ...
... In agreement with our findings, Silva and coworkers [12] found no CnAMP1-induced cytotoxicity in murine macrophage-like cells (RAW 264.7). In nondifferentiated Caco-2 cells, however, they observed that CnAMP1 reduced cell viability in a dose-dependent manner [12]. When grown for 21 days after confluence, Caco-2 cells differentiate into a small intestinal enterocyte-like phenotype [13]. ...
Coconut antimicrobial peptide-1 (CnAMP1) is a naturally occurring bioactive peptide from green coconut water (Cocos nucifera L.). Although biological activities have been reported, the physiological relevance of these reports remains elusive as it is unknown if CnAMP1 is taken up into intestinal cells. To address this open question, we investigated the cytotoxicity of CnAMP1 in intestinal cells and its cellular uptake into human intestinal cells. Considering the importance of the P-glycoprotein (P-gp) to the intestinal metabolism of xenobiotics, we also investigated the influence of CnAMP1 on P-gp activity and expression. Both cell lines showed intracellular fluorescence after incubation with fluorescein labelled CnAMP1, indicating cellular uptake of the intact or fragmented peptide. CnAMP1 (12.5-400 μmol/L) showed no signs of cytotoxicity in LS180 and differentiated Caco-2 cells and did not affect P-gp expression and activity. Further research is required to investigate the identity of CnAMP1 hydrolysis fragments and their potential biological activities.
... Hydrolysates or bioactive peptides are obtained widely from terrestrial plants. Antioxidant and anticancer properties have been shown by several kind of plants such as Cannabis sativa [75], Medicago sativa [64], Phaseolus vulgaris [76], Amaranthus hypochondriacus [77], Helianthus annuus L. [78], Cicer arietinum L. [50], Santalum álbum [72], Oryza sativa [79], Glycine max [4], Triticum aestivum L. [6], Arachis hypogaea [5], Brassica napus [80], Annona cherimola [81], Gossypium hirsutum [6], Zea mays L. [40], Cocos nucifera [82], Cycas revoluta [83], amongst others. (Table 1) shows a summary of the sequences or active compounds of hydrolysates or peptides against cancer from these terrestrial plants. ...
... Cn-AMP1 peptide is able to reduce the viability of Caco-2, RAW264.7, MCF-7 and HCT-116 cells [82]. Also cherimolacyclopeptide C from Annona cherimola follows this same way presenting in vitro cytotoxicity against KB cells [81]. ...
Cancer is the leading cause of morbidity and mortality worldwide. Therefore, the search for new and less aggressive treatments is currently the focus of the anticancer research. An attractive alternative for this purpose is the use of bioactive peptides from plants. Plants live everywhere on Earth, both on land and in water, and they are a major source of diverse molecules with pharmacological potential as antioxidant peptides. Hence, this review focuses on the importance of the antioxidant activity of terrestrial and aquatic plant peptides against cancer throughout several mechanisms. The influence of the antioxidant activity of peptides by different factors such as molecular weight and amino acid composition as a crucial factor for anticancer activity is also revised. Furthermore, the relation of antioxidant activity with anticancer property as well as safety and legal aspects of protein hydrolysates and bioactive peptides for their use in cancer treatments is discussed.
... They have potential for conservation in the food industry and in agribusiness (Meyer et al., 1996;Agizzio et al., 2003;Pelegrini et al., 2006Pelegrini et al., , 2008Ribeiro et al., 2011;Seo et al., 2014). Their medical use is likely to increase as anti-infective and immunomodulatory therapeutics (Matsuzaki, 1999;Mandal et al., 2009;Maróti et al., 2011;Silva et al., 2012Silva et al., , 2014Jeong et al., 2015;Santana et al., 2015), in intracellular drug delivery, RNA, DNA, and nanoparticles in a non-destructive manner (Nasrollahi et al., 2012) (Figure 2). ...
... Interestingly, an increasing number of works have labeled plant AMPs as promiscuous peptides, due to their different actions associated with the same structure (Franco, 2011). Among these are the peptide Cn-AMP1, considered multifunctional and promiscuous, presenting antibacterial and antifungal activity, also being capable to reduce the viability of tumor cells (Mandal et al., 2009;Silva et al., 2012;Santana et al., 2015). Another example is the peptides isolated from Z. jujuba fruits, with both antibacterial and antifungal properties against S. aureus and Phomopsis azadirachtae, respectively (Daneshmand et al., 2013). ...
Bacterial resistance is a major threat to plant crops, animals and human health, and over the years this situation has increasingly spread worldwide. Due to their many bioactive compounds, plants are promising sources of antimicrobial compounds that can potentially be used in the treatment of infections caused by microorganisms. As well as stem, flowers and leaves, fruits have an efficient defense mechanism against pests and pathogens, besides presenting nutritional and functional properties due to their multifunctional molecules. Among such compounds, the antimicrobial peptides (AMPs) feature different antimicrobials that are capable of disrupting the microbial membrane and of acting in binding to intra-cytoplasmic targets of microorganisms. They are therefore capable of controlling or halting the growth of microorganisms. In summary, this review describes the major classes of AMPs found in fruits, their possible use as biotechnological tools and prospects for the pharmaceutical industry and agribusiness.
... PBS was used as a negative control and lysis buffer (10 mM Tris, pH 7.4, 1 mM EDTA and 0.1% Triton X-100) was used as a positive control. [26][27][28] Statistical analysis ...
... Similar results have been observed with the use of CHL at concentrations 4200 μg ml − 1 , where 50% cytotoxicity was observed. 17,18,25,27 However, when utilizing CHL and CST, in two combinations referent to synergistic effect as 1 μg ml − 1 of CHL+32 μg ml − 1 of CST, or 2 μg ml − 1 of CHL+16 μg ml − 1 of CST, cytotoxic effects on RAW 264.7 and VERO cells were not observed here. Thus, the results obtained here strongly indicate that the combination of these two antibiotics could be a feasible strategy for allowing dose reductions for effective bacterial control with a concomitant reduction in the collateral toxic effects in mammalian cells. ...
Enterobacter cloacae is a Gram-negative bacterium associated with high morbidity and mortality in intensive care patients due to its resistance to multiple antibiotics. Currently, therapy against multi-resistant bacteria consists of using colistin, in spite of its toxic effects at higher concentrations. In this context, colistin-resistant E. cloacae strains were challenged with lower levels of colistin combined with other antibiotics to reduce colistin-associated side effects. Colistin-resistant E. cloacae (ATCC 49141) strains were generated by serial propagation in subinhibitory colistin concentrations. After this, three colistin-resistant and three nonresistant replicates were isolated. The identity of all the strains was confirmed by MALDI-TOF MS, VITEK 2 and MicroScan analysis. Furthermore, cross-resistance to other antibiotics was checked by disk diffusion and automated systems. The synergistic effects of the combined use of colistin and chloramphenicol were observed via the broth microdilution checkerboard method. First, data here reported showed that all strains presented intrinsic resistance to penicillin, cephalosporin (except fourth generation), monobactam, and some associations of penicillin and β-lactamase inhibitors. Moreover, a chloramphenicol and colistin combination was capable of inhibiting the induced colistin-resistant strains as well as two colistin-resistant clinical strains. Furthermore, no cytotoxic effect was observed by using such concentrations. In summary, the data reported here showed for the first time the possible therapeutic use of colistin-chloramphenicol for infections caused by colistin-resistant E. cloacae.The Journal of Antibiotics advance online publication, 6 July 2016; doi:10.1038/ja.2016.77.
... The polypropylene plates (TPP, Switzerland) were incubated at 37 • C for 24 h. MICs were determined as the lowest tested concentration that leads to complete inhibition (100%) compared to the negative control group [33]. ...
... Values of p < 0.05 were considered statistically significant. GraphPad Prism software v5.0 (GraphPad Software, USA) was used for all statistical analyses [33]. ...
The main bacterium associated with skin infection is Staphylococcus aureus, occurring especially in infections acquired via surgical wounds, commonly leading to lethal hospital-acquired infections, emphasizing the importance of identifying new antimicrobial compounds. Among them, cyclotides have gained interest due to their high stability and multifunctional properties. Here, cycloviolacin 2 (CyO2) and kalata B2 (KB2) were evaluated to determinate their anti-staphylococcal activities using a subcutaneous infection model. Anti-staphylococcal activities of 50 mM for KB2 and 25 mM for CyO2 were detected with no cytotoxic activities against RAW 264.7 monocytes. In the in vivo assays, both cyclotides reduced bacterial load and CyO2 demonstrated an increase in the phagocytosis index, suggesting that the CyO2 in vivo anti-staphylococcal activity may be associated with phagocytic activity, additionally to direct anti-pathogenic activity.
... Cells were plated at a density of 2500 cells/well in a 96-well plate and peptides (1 g mL −1 ) were added to medium with a final volume of 250 L. Both adherent and floating cells were harvested by trypsinization with varying time intervals after the addition of peptide fractions. Cells were counted in a hemocytometer, cell proliferation was calculated by computing the increase in the cell number, and the cell proliferation index (p) was calculated using the formula p = N t /N 0 , where N t is the number of cells at time t and N 0 is the number of cells at the time of treatment [48]. ...
... Recent trends in peptidomics are to identify biologically active fragments from larger proteins [30,37,39] and also multifunctional bioactive peptides having two or more different biological activities [32,48]. A few milk-derived peptides have been identified and revealed their multifunctional properties from the fermented milk products [35]. ...
... Cells were plated at a density of 2500 cells/well in a 96-well plate and peptides (1 g mL −1 ) were added to medium with a final volume of 250 L. Both adherent and floating cells were harvested by trypsinization with varying time intervals after the addition of peptide fractions. Cells were counted in a hemocytometer, cell proliferation was calculated by computing the increase in the cell number, and the cell proliferation index (p) was calculated using the formula p = N t /N 0 , where N t is the number of cells at time t and N 0 is the number of cells at the time of treatment [48]. ...
... Recent trends in peptidomics are to identify biologically active fragments from larger proteins [30,37,39] and also multifunctional bioactive peptides having two or more different biological activities [32,48]. A few milk-derived peptides have been identified and revealed their multifunctional properties from the fermented milk products [35]. ...
Pharmaceutical industries have renewed interest in screening multifunctional bioactive peptides as a marketable product in health care applications. In this context, several animal and plant peptides with potential bioactivity have been reported. Milk proteins and peptides have received much attention as a source of health-enhancing components to be incorporated into nutraceuticals and functional foods. By using this source, 24 peptides have been fractionated and purified from human milk using RP-HPLC. Multifunctional roles including antimicrobial, antioxidant and growth stimulating activity have been evaluated in all 24 fractions. Nevertheless, only 4 fractions show multiple combined activities among them. Using a proteomic approach, two of these four peptides have been identified as lactoferrin derived peptide and kappa casein short chain peptide. Lactoferrin derived peptide (f8) is arginine-rich and kappa casein derived (f12) peptide is proline-rich. Both peptides (f8 and f12) showed antimicrobial activity against both Gram-positive and Gram-negative bacteria. Fraction 8 (f8) exhibits growth stimulating activity in 3T3 cell line and f12 shows higher free radical scavenging activity in comparison to other fractions. Finally, both peptides were in silico evaluated and some insights into their mechanism of action were provided. Thus, results indicate that these identified peptides have multiple biological activities which are valuable for the quick development of the neonate and may be considered as potential biotechnological products for for nutraceutical industry.
... Although they are diverse in length and sequence, two physicalchemical features are often the hallmarks of these molecules: they are cationic, often ranging from +2 to +7 at pH 7, and amphipathic, therefore their stereogeometry confers relatively polarized hydrophilic and hydrophobic facets (Nguyen et al., 2011). Despite their structural conservation, they have a broad spectrum of activity such as antibacterial (Okubo et al., 2012; Tavares et al., 2012), antioxidative (Power et al., 2013 ), antihypertensive (Escudero et al., 2012), antifungal (Mandal et al., 2013), antiviral (Findlay et al., 2013), antitumor as well as modulation of the immune response (Silva et al., 2012). The modulation of the immune response role is specific to a group of peptides named host defense peptides (HDPs). ...
... Along with cyclic peptides, many active non-cyclic antitumor peptides have also been isolated from different plants. For instance, the promiscuous peptide Cn-AMP1, isolated and purified from coconut water (Cocos nucifera), was tested against CACO-2-human epithelial colorectal adenocarcinoma cells and showed a 13% reduction of cell viability (Silva et al., 2012). Additionally, the peptide lunasin, isolated from soybeans and other seeds, was capable of suppressing in vitro and in vivo chemical carcinogen-induced tumorigenesis (Hsieh et al., 2010). ...
Cationic antimicrobial peptides (AMPs) and host defense peptides (HDPs) show vast potential as peptide-based drugs. Great effort has been made in order to exploit their mechanisms of action, aiming to identify their targets as well as to enhance their activity and bioavailability. In this review, we will focus on both naturally occurring and designed antiviral and antitumor cationic peptides, including those here called promiscuous, in which multiple targets are associated with a single peptide structure. Emphasis will be given to their biochemical features, selectivity against extra targets, and molecular mechanisms. Peptides which possess antitumor activity against different cancer cell lines will be discussed, as well as peptides which inhibit virus replication, focusing on their applications for human health, animal health and agriculture, and their potential as new therapeutic drugs. Moreover, the current scenario for production and the use of nanotechnology as delivery tool for both classes of cationic peptides, as well as the perspectives on improving them is considered.
... Although they are diverse in length and sequence, two physicalchemical features are often the hallmarks of these molecules: they are cationic, often ranging from +2 to +7 at pH 7, and amphipathic, therefore their stereogeometry confers relatively polarized hydrophilic and hydrophobic facets (Nguyen et al., 2011). Despite their structural conservation, they have a broad spectrum of activity such as antibacterial (Okubo et al., 2012; Tavares et al., 2012), antioxidative (Power et al., 2013 ), antihypertensive (Escudero et al., 2012), antifungal (Mandal et al., 2013), antiviral (Findlay et al., 2013), antitumor as well as modulation of the immune response (Silva et al., 2012). The modulation of the immune response role is specific to a group of peptides named host defense peptides (HDPs). ...
... Along with cyclic peptides, many active non-cyclic antitumor peptides have also been isolated from different plants. For instance, the promiscuous peptide Cn-AMP1, isolated and purified from coconut water (Cocos nucifera), was tested against CACO-2-human epithelial colorectal adenocarcinoma cells and showed a 13% reduction of cell viability (Silva et al., 2012). Additionally, the peptide lunasin, isolated from soybeans and other seeds, was capable of suppressing in vitro and in vivo chemical carcinogen-induced tumorigenesis (Hsieh et al., 2010). ...
Cationic antimicrobial peptides (AMPs) and host defense peptides (HDPs) show vast potential as
peptide-based drugs. Great effort has been made in order to exploit their mechanisms of action,
aiming to identify their targets as well as to enhance their activity and bioavailability. In this
review, we will focus on both naturally occurring and designed antiviral and antitumor cationic
peptides, including those here called promiscuous, in which multiple targets are associated with a
single peptide structure. Emphasis will be given to their biochemical features, selectivity against
extra targets and molecular mechanisms. Peptides which possess antitumor activity against
different cancer cell lines will be discussed, as well as peptides which inhibit virus replication,
focusing on their applications for human health, animal health and agriculture, and their potential
as new therapeutic drugs. Moreover, the current scenario for production and the use of
nanotechnology as delivery tool for both classes of cationic peptides, as well as the perspectives
on improving them is considered.
... Linear peptide Cn-AMP1 isolated from green coconut water (Cocos nucifera) exerted activity against E. coli, B. subtilis, S. aureus, and P. aeruginosa (Mandal et al., 2009). Cn-AMP1 was synthesised by SPPS using the Fmoc strategy, and showed a wide range of activities, including antimicrobial and immunostimulatory (Silva et al., 2012). Anaya et al. (2020) revealed that the synthetic Cn-AMP1 showed little cytotoxicity toward LS180 and Caco-2 cell lines, and had no effect on the expression and activity of P-glycoprotein. ...
Antimicrobial peptides (AMPs) are active short peptides that exist in microorganisms, insects, amphibians, plants, and mammals. Some naturally occurring AMPs have low antimicrobial activity, high haemolysis, potential toxicity toward mammalian cells, and high susceptibility to proteolytic degradation, which limit their practical application. In recent years, many efforts have been made to design and modify AMPs to improve their properties. The present review focuses on site-directed mutation, truncation, hybridisation, capping, and cyclisation of AMPs. The review further introduces the application of solid-phase peptide synthesis technology for AMPs, and summarises the methods for evaluating the antimicrobial activity of AMPs. The in-depth research on AMPs is expected to play an essential role in the fields of agriculture, animal husbandry, food industry, and medicine.
... These peptides showed antimicrobial activity against multiple Gram-positive and Gram-negative bacteria and fungi [73]. Interestingly, further studies investigated the promiscuity of these peptides and demonstrated that in addition to their antimicrobial activities, they also present activity against cancerous cells and immunostimulatory effects [91][92][93]. The immunostimulatory effect is a very important property, particularly in diabetes due to the low-grade inflammation, and it is difficult to have a controlled inflammatory phase of wound healing. ...
Diabetic foot ulcers (DFU) are one of the most serious and devastating complications of diabetes and account for a significant decrease in quality of life and costly healthcare expenses worldwide. This condition affects around 15% of diabetic patients and is one of the leading causes of lower limb amputations. DFUs generally present poor clinical outcomes, mainly due to the impaired healing process and the elevated risk of microbial infections which leads to tissue damage. Nowadays, antimicrobial resistance poses a rising threat to global health, thus hampering DFU treatment and care. Faced with this reality, it is pivotal to find greener and less environmentally impactful alternatives for fighting these resistant microbes. Antimicrobial peptides are small molecules that play a crucial role in the innate immune system of the host and can be found in nature. Some of these molecules have shown broad-spectrum antimicrobial properties and wound-healing activity, making them good potential therapeutic compounds to treat DFUs. This review aims to describe antimicrobial peptides derived from green, eco-friendly processes that can be used as potential therapeutic compounds to treat DFUs, thereby granting a better quality of life to patients and their families while protecting our fundamental bio-resources.
... The Ca-MAP1 peptide was evaluated in the presence of water, 50% trifluoroethanol (TFE), and sodium dodecyl sulphate (SDS), which are hydrophilic, hydrophobic, and anionic environments, respectively for circular dichroism analyses. The band spectrum format ( Figure 6) was characterized by the occurrence of two negative band values approximately between 208 and 222 nm and one positive band at 190 nm, which are characteristic of peptides with a flexible α-helix secondary structure in the presence of SDS and TFE [22,23]. ...
The importance of neuroinflammation in neurology is becoming increasingly apparent. In addition to neuroinflammatory diseases such as multiple sclerosis, the role of neuroinflammation has been identified in many non-inflammatory neurological disorders such as stroke, epilepsy, and cancer. The immune response within the brain involves the presence of CNS resident cells; mainly glial cells, such as microglia, the CNS resident macrophages. We evaluated the peptide Ca-MAP1 bioinspired on the C. albicans immature cytolytic toxin candidalysin to develop a less hemolytic peptide with anti-neuroinflammatory, antibacterial, and cytotoxic activity against tumor cells. In silico and in vitro studies were performed at various concentrations. Ca-MAP1 exhibits low hemolytic activity at lower concentrations and was not cytotoxic to MRC-5 and BV-2 cells. Ca-MAP1 showed activity against Acinetobacter baumannii, Escherichia coli ATCC, E. coli KPC, Klebsiella pneumoniae ATCC, Pseudomonas aeruginosa, and Staphylococcus aureus ATCC. Furthermore, Ca-MAP1 exhibits anti-neuroinflammatory activity in the BV-2 microglia model, with 93.78% inhibition of nitrate production at 18.1 μM. Ca-MAP1 presents cytotoxic activity against tumor cell line NCI-H292 at 36.3 μM, with an IC50 of 38.4 μM. Ca-MAP1 demonstrates results that qualify it to be evaluated in the next steps to promote the control of infections and provide an alternative antitumor therapy.
... The first and widely studied anuran peptides group is the antimicrobial peptides (AMPs) or also called host defense peptides (HDPs), being particularly abundant in skin secretions (Simmaco et al., 1998). These peptides have several functions and targets in terms of biological activities (Wang and Epand, 2016), from antimicrobial, antiviral, and antifungal agents (Reddy et al., 2004;Mello et al., 2011;Torcato et al., 2013aTorcato et al., , 2013b to response modulation immune (Silva et al., 2012). Regarding the latter, the rapid and non-specific interaction of AMPs with the membrane lipids of microbial targets results in the death of the pathogen (Arouri et al., 2009). ...
At the end of 2019, in China, clinical signs and symptoms of unknown etiology have been reported in several patients whose sample sequencing revealed pneumonia caused by the SARS-CoV-2 virus. COVID-19 is a disease triggered by this virus, and in 2020, the World Health Organization declared it a pandemic. Since then, efforts have been made to find effective therapeutic agents against this disease. Identifying novel natural antiviral drugs can be an alternative to treatment. For this reason, antimicrobial peptides secreted by anurans' skin have gained attention for showing a promissory antiviral effect. Hence, this review aimed to elucidate how and which peptides secreted by anurans' skin can be considered therapeutic agents to treat or prevent human viral infectious diseases. Through a literature review, we attempted to identify potential antiviral frogs’ peptides to combat COVID-19. As a result, the Magainin-1 and -2 peptides, from the Magainin family, the Dermaseptin-S9, from the Dermaseptin family, and Caerin 1.6 and 1.10, from the Caerin family, are molecules that already showed antiviral effects against SARS-CoV-2 in silico. In addition to these peptides, this review suggests that future studies should use other families that already have antiviral action against other viruses, such as Brevinins, Maculatins, Esculentins, Temporins, and Urumins. To apply these peptides as therapeutic agents, experimental studies with peptides already tested in silico and new studies with other families not tested yet should be considered.
... Thus, AMPs have also been found in many plant components, such as fruits, flowers, leaves and stems [57,58], most of which contain cysteine residues and form disulphide bonds [57][58][59]. Recently, short disulphide-free AMPs were also found in green coconut water and proven to be multifunctional peptides without any sign of cytotoxicity to human cells [60][61][62][63]. ...
In the last century, conventional antibiotics have played a significant role in global healthcare. Antibiotics support the body in controlling bacterial infection and simultaneously increase the tendency of drug resistance. Consequently, there is a severe concern regarding the regression of the antibiotic era. Despite the use of antibiotics, host defence systems are vital in fighting infectious diseases. In fact, the expression of ribosomal antimicrobial peptides (AMPs) has been crucial in the evolution of innate host defences and has been irreplaceable to date. Therefore, this valuable source is considered to have great potential in tackling the antimicrobial resistance (AMR) crisis. Furthermore, the possibility of bacterial resistance to AMPs has been intensively investigated. Here, we summarize all aspects related to the multiple applications of ribosomal AMPs and their derivatives in combating AMR.
... Besides BAMPs, other small peptides demonstrate anticancer activity in plants (Table 3). For example, Cn-AMP1 derived from Cocos nucifera can reduce cancer cell viability without causing hemolysis [152] and Cr-ACP isolated from Cycas revoluta arrests the Hep2 cell cycle in the G0-G1 phase [153]. Further, the cyclic heptapeptide cherimolacyclopeptide C isolated from Annona cherimola seeds displays in vitro cytotoxicity against KB cells [154], while a cell cycle-inhibiting octapeptide cyclosaplin purified from Santalum album L. inhibits breast cancer cell proliferation in a dose-and time-dependent manner [155]. ...
Botany-derived antimicrobial peptides (BAMPs), a class of small, cysteine-rich peptides produced in plants, are an important component of the plant immune system. Both in vivo and in vitro experiments have demonstrated their powerful antimicrobial activity. Besides in plants, BAMPs have cross-kingdom applications in human health, with toxic and/or inhibitory effects against a variety of tumor cells and viruses. With their diverse molecular structures, broad-spectrum antimicrobial activity, multiple mechanisms of action, and low cytotoxicity, BAMPs provide ideal backbones for drug design, and are potential candidates for plant protection and disease treatment. Lots of original research has elucidated the properties and antimicrobial mechanisms of BAMPs, and characterized their surface receptors and in vivo targets in pathogens. In this paper, we review and introduce five kinds of representative BAMPs belonging to the pathogenesis-related protein family, dissect their antifungal, antiviral, and anticancer mechanisms, and forecast their prospects in agriculture and global human health. Through the deeper understanding of BAMPs, we provide novel insights for their applications in broad-spectrum and durable plant disease prevention and control, and an outlook on the use of BAMPs in anticancer and antiviral drug design.
... One commonly used strategy to identify new drugs involves exploring the chemistry of folk medicinal plant extracts [13]. Among these natural products, peptides and proteins have been described that interact with animal systems, including the uterotonic cyclotides [14], immunomodulatory peptides [15][16][17][18][19], and anti-inflammatory thaumatin proteins [20,21]. ...
Early plants began colonizing earth about 450 million years ago. During the process of coevolution, their metabolic cellular pathways produced a myriad of natural chemicals, many of which remain uncharacterized biologically. Popular preparations containing some of these molecules have been used medicinally for thousands of years. In Brazilian folk medicine, plant extracts from the bamboo plant Guadua paniculata Munro have been used for the treatment of infections and pain. However, the chemical basis of these therapeutic effects has not yet been identified. Here, we performed protein biochemistry and downstream pharmacological assays to determine the mechanisms underlying the anti-inflammatory and antinociceptive effects of an aqueous extract of the G. paniculata rhizome, which we termed AqGP. The anti-inflammatory and antinociceptive effects of AqGP were assessed in mice. We identified and purified a protein (AgGP), with an amino acid sequence similar to that of thaumatins (~20 kDa), capable of repressing inflammation through downregulation of neutrophil recruitment and of decreasing hyperalgesia in mice. In conclusion, we have identified the molecule and the molecular mechanism responsible for the anti-inflammatory and antinociceptive properties of a plant commonly used in Brazilian folk medicine.
... Classification of plant AMP families is largely based on their Cys motifs which exhibit a characteristic Cys pattern with a defined number of non-Cys residues between the two neighboring Cys. The cysteine-rich peptides are the prominently identified AMPs in plants, while the description of cysteine-free AMPs from plants is very limited (Egorov et al. 2005;Silva et al. 2012;Zipfel 2009;Umadevi et al. 2018 Abstract Abiotic stress is one of the major factors limiting the crop production globally. Plants experience diverse abiotic stresses including higher concentration of salt (salinity), temperature extremities, and water shortage (drought or dehydration). ...
Soil is a dynamic and complex biological system comprising of various populations of microbes. These microorganisms enhance soil richness or fertility and water retention ability and have a major impact on the growth of the vegetation present on the ground. The range of diversity of microbes plays an essential role in improvement of soil quality that varies with depth, pH and horizons as well as soil health. Abiotic stresses comprising drought, salinity, high and low temperature and heavy metal (HM) toxicity are the principal restraining factors for microbial inhabitants and agricultural productivity. Several reports have indicated that inoculation of microbes in nutrient-deficient or stressed soil has significantly improved soil as well as plant health and established supportable way of agriculture. Microorganisms interact with plants and regulate systemic and local mechanisms to provide defence under adverse environmental conditions. Besides providing non-nutritional effects such as limiting soil erosion and detoxifying insecticides as well as pesticides, microbes also help in atmospheric nitrogen (N2) fixation; activation of antioxidants, phytohormones and osmolytes; solubilization of mineral phosphates; decomposition of organic wastes; regulation of gene expression; improvement of nutrient cycling and plant-water relation; etc. and assist the plant to survive under unfavourable conditions. In addition, use of harmonious multiple microbial associations (e.g. fungal and bacterial) provides several benefits and is a promising approach towards this direction. This review deals with the potential of soil and its inhabitant microbes to nurture plants, plant-microbe interactions and their roles in abiotic stress management.
... Classification of plant AMP families is largely based on their Cys motifs which exhibit a characteristic Cys pattern with a defined number of non-Cys residues between the two neighboring Cys. The cysteine-rich peptides are the prominently identified AMPs in plants, while the description of cysteine-free AMPs from plants is very limited (Egorov et al. 2005;Silva et al. 2012;Zipfel 2009;Umadevi et al. 2018 ...
Abiotic stress is one of the major factors limiting the crop production globally. Plants experience diverse abiotic stresses including higher concentration of salt (salinity), temperature extremities, and water shortage (drought or dehydration). Such stressors impair the normal metabolic functioning of the plant leading to poor growth and development. A wide range of adaptations and mitigation strategies are required to efficiently manage the deleterious impacts of such stresses. Development of tolerant varieties, shifting the crop calendars, and resource management practices are some examples of such strategies. However, most of these technologies are cost-intensive and are beyond the reach of the small and marginal farmers. Microorganisms are naturally endowed with the ability to sustain extreme environmental conditions and also help other living beings in vicinity/association to cope with such stress to certain extent. Microorganisms through induction of systemic tolerance, modulation of plant defense mechanisms, and improvement of nutrition and growth can effectively alleviate or reduce the effect of stress. Hence, application of stress-alleviating microorganisms for crop production holds considerable potential to become a sustainable option to combat abiotic stresses.
... Due to these limitations, AMPs and their manufactured synthetic analogs have identified alternate therapies to cure cancer. Peptides from Clitoria ternatea (like CT2, CT4, CT7, CT10, and CT12 (cyclotides)) exhibited cytotoxicity and reduced 2 to 4-fold IC 50 value when compared with mitotic inhibitor used in cancer chemotherapy against human lung cancer cells (A549) [148]. Another peptide isolated from Cocos nucifera (coconut: Cn-AMP1) was examined on human epithelial colorectal adenocarcinoma cells (Caco-2), which led to 13% decline in cell viability. ...
Antimicrobial peptides (AMPs) are small peptides (12–50 amino acids), which are active against microbes including bacteria, yeast, molds, fungi, and other parasitic infections. These are ribosomally synthesized peptides secreted by most organisms and play an important role in host defense. They are specific and cause no damage to the host cell/producer cell. AMPs are mostly active on the target membrane by direct binding with the membrane phospholipids and create the pores. These pores disturb proton motive force, releases intracellular molecules causing target cell death. AMPs are structurally classified into α-helical, β-sheet, and extended peptides based on their amino acid sequence. Barrel-stave, toroidal, carpet, and detergent-like models have been proposed for their action mechanism which differs from species to species and within species. AMPs produced by mammals function as immune-modulators and have a crucial role in innate immunity. AMPs also have other bioactivities such as antioxidant, antitumor, antimicrobial, and many more which makes them popular for wide applications.
... Majority of known plant AMPs are stabilized by multiple disulfide bonds, but there are some exceptions which include Cn-AMPs, Cr-ACP1, GRPs Pg-AMP1 and shepherins, which do not contain Cys or contain only one which imparts high structural flexibility. Cn-AMP1 is a 9 amino acid Cys-free AMP identified from green coconut (Cocos nucifera) water with "promiscuous" antibacterial, antifungal and immune stimulatory activity [78] . Cr-ACP1 is a 9 aa AMP from the seeds of Cycas revoluta with pro-apoptotic and antimicrobial activities [79] . ...
Antimicrobial peptides (AMPs) are ubiquitous low-molecular weight host defense peptides, which exhibit broad spectrum resistance against bacteria, fungi and viruses. They are vital components of the plant innate immune system and act in the first line of defense against various pathogens in imparting disease resistance to plants. They exhibit a wide variation in structure, function, mode of action and antimicrobial spectrum which enable them to perform multiple functions. These particular proteins play an important role in display of broad antimicrobial resistance to pathogens and are controlled by various physiological regulators like plant hormones (jasmonates, salicylic acid, gibberellins and ethylene acid). Because of their antimicrobial nature, they can be employed as powerful tools to develop disease resistance against economically important pathogens to improve global food production and security. In addition, they can also act as promising candidates for plant-based bio-manufacturing to develop new antimicrobial agents in pharmaceutical industry. The present study focuses on summarizing the structure, function and biological properties of plant antimicrobial proteins, which opens up novel aspects for their future use as powerful tools in agricultural and medical biotechnology.
... A 10-kDa peptide with antifungal and HIV-1 reverse transcriptase inhibitory activities (Wang and Ng 2005), and three antimicrobial peptides, named CnAMP1, CnAMP2, and CnAMP3, with activity against human pathogenic bacteria such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis (Mandal et al. 2009). CnAMP1 is a small (nine amino acids) disulphide-free peptide, with cationic and hydrophobic portions, which possesses antifungal and immunostimulatory activity (Silva et al. 2012). ...
Plant foods are rich sources of biologically active peptides that may have a role in the prevention of diseases. Coconut water is a valuable beverage due to its nutrient composition and the presence of bioactive compounds, such as the peptide CnAMP1. It is unknown if CnAMP1 can be absorbed into intestinal cells. We, therefore, aimed to develop and validate a simple reversed-phase liquid chromatographic method to quantify the peptide in Caco-2 and LS180 cell lysates. CnAMP1 standards (1–200 µmol/L) and spiked cell lysates were injected onto a Reprosil-Pur 120 C18-AQ column (4.6 × 250 mm) using acetonitrile:water:trifluoroacetic acid (14.0:85.9:0.1, by volume) as mobile phase in isocratic mode at flow rate of 1 mL/min. The method achieved rapid separation (total run time of 6 min), with linear response, good sensitivity (limit of detection, 8.2 ng; lower limit of quantification, 30.6 ng) and no interfering peaks. Best recoveries (84–96%), accuracies (7.6–14.8%) and precision (1.5–8%) were found for LS180 cell lysates spiked with medium (50 µmol/L) and high (100 µmol/L) amounts of the peptide. Uptake assays detected no peptides in the cell lysates; however, after the first 15-min incubation CnAMP1 underwent partial hydrolysis upon incubation with LS180 cells (29%) and extensive hydrolysis with Caco-2 cells (93%).
... Other in vitro biological activity assays have shown that Cn-AMP1 exerts several alternative functions simultaneously to microbial control. The peptide was able to reduce the viability of different types of tumor cells, besides promoting immunomodulatory activity through the stimulation of cytokine secretion by laboratory-grown monocytes, without causing cytotoxicity to human erythrocytes and murine macrophages (Silva et al. 2012). ...
The constant demand for new antibiotic drugs has driven efforts by the scientific community to prospect for peptides with a broad spectrum of action. In this context, antimicrobial peptides (AMPs) have acquired great scientific importance in recent years due to their ability to possess antimicrobial and immunomodulatory activity. In the last two decades, plants have attracted the interest of the scientific community and industry as regards their potential as biofactories of heterologous proteins. One of the most promising approaches is the use of viral vectors to maximize the transient expression of drugs in the leaves of the plant Nicotiana benthamiana. Recently, the MagnifectionTM expression system was launched. This sophisticated commercial platform allows the assembly of the viral particle in leaf cells and the systemic spread of heterologous protein biosynthesis in green tissues caused by Agrobacterium tumefaciens “gene delivery method”. The system also presents increased gene expression levels mediated by potent viral expression machinery. These characteristics allow the mass recovery of heterologous proteins in the leaves of N. benthamiana in 8 to 10 days. This system was highly efficient for the synthesis of different classes of pharmacological proteins and contains enormous potential for the rapid and abundant biosynthesis of AMPs.
... Here, sequences with 19, 20 and 23 amino acids were designed, while Loose et al. [28] reported sequences with 20 amino acids. However, the design of longer sequences is not a matter of concern, since the biotechnological potential of peptides is inversely proportional to the size of their sequences, cases of the 13 mer peptide designed based on the APD database to combat S. aureus [42] or the multifunctional 9 mer peptide Cn-AMP1 from green coconut water [53]. In fact, the Joker algorithm provides new perspectives for the design of antimicrobial peptides, including multifunctional molecules; and also promises new advances in the fight against resistant bacteria. ...
The rise of bacterial resistance makes it necessary to find innovative alternatives to control bacterial infections. Antimicrobial peptides (AMPs) have been considered as the new generation of antimicrobial agents. Based on the fact that AMPs are sequence-dependent, a linguistic model for designing AMPs was previously developed. That model considers AMPs as a formal language with a grammar (patterns or motifs) and a vocabulary (amino acids). Albeit promising, that model has been poorly exploited mainly because it generates thousands of sequences, and the outcome has high similarity to already known AMPs. Here we present Joker, an innovative algorithm that improves the application of the linguistic model for rational design of antimicrobial peptides. Joker designs AMPs by means of insertion of an antimicrobial pattern into a parental sequence, generating a few variants. Our algorithm is capable of improving existing AMPs or even creating new AMPs from inactive peptides. A standalone version of Joker is available for download at <https://github.com/williamfp7/Joker> and requires a Linux 32-bit machine.
... The scanning range was 300-190 nm. The baseline was corrected by subtracting the chloroform as blank (Silva et al. 2012). ...
Amniotic membrane (AM) is frequently used in ophthalmologic surgery for rapid ocular surface reconstruction. Sometimes it may create a major problem with associated infections after biofilm formation over the membrane. To overcome this problem, AM was coated with the antimicrobial peptide clavanin A. The antifungal activity of clavanin A in the native and self-assembled form was determined against the common ocular surface pathogens Candida albicans, Aspergillus fumigatus, Alternaria sp. and Fusarium sp. Biofilm formation over the coated surface was significantly reduced in comparison with the uncoated membrane. The coated membrane revealed effectiveness in terms of biocompatibility, cell attachment colonization when tested in non-cancerous 3T3 and human embryonic kidney (HEK)-293 cell lines. Clavanin A-coated AM also exhibited excellent physical, morphological and antifungal characteristics, indicating potential applicability for ocular surface infection control.
... The active motifs of ACPs are short, so large-scale synthesis is cost-effective. Certain ACPs not only show intrinsic anticancer activity but also enhance the potency of conventional drugs (Gaspar et al. 2013;Hancock et al. 2006;Silva et al. 2012). There are currently 196 entries in the Antimicrobial Peptide Database (APD) (http://aps.unmc. ...
Antimicrobial peptides/proteins (AMPs) are biologically active molecules with diverse structural properties that are produced by mammals, plants, insects, ticks, and microorganisms. They have a range of antibacterial, antifungal, antiviral, and even anticancer activities, and their biological properties could therefore be exploited for therapeutic and prophylactic applications. Cancer and cancer drug resistance are significant current health challenges, so the development of innovative cancer drugs with minimal toxicity toward normal cells and novel modes of action that can evade resistance may provide a new direction for anticancer therapy. The skin is the first line of defense against heat, sunlight, injury, and infection, and skin cancer is thus the most common type of cancer. The skin that has been exposed to sunlight is particularly susceptible, but lesions can occur anywhere on the body. Skin cancer awareness and self-efficacy are necessary to improve sun protection behavior, but more effective preventative approaches are also required. AMPs may offer a new prophylactic approach against skin cancer. In this mini review, we draw attention to the potential use of insect AMPs for the prevention and treatment of skin cancer.
... Furthermore, peptide's production is of lower complexity when compared to other protein-based therapies and thus, cost-effective (Fosgerau and Hoffmann 2015). Antimicrobial peptides (AMPs) are a class of natural occurring peptides with several important targets and activities, from antimicrobial, antiviral, and antifungal (Reddy et al. 2004;Torcato et al. 2013a, b;Mello et al. 2011) to the modulation of the immune response (Silva et al. 2012). As part of immune defense (Iwasaki et al. 2009), AMPs are found in eukaryotic organisms of many different species (Reddy et al. 2004) and their rapid and non-specific interactions with the membrane lipids of the microbial targets results in the pathogen death with very low chance of resistance development (Arouri et al. 2009;Fernebro 2011). ...
Current cancer treatments require improvements in selectivity and efficacy. Surgery, radiation, and chemotherapy approaches result in patient’s suffering over time due to the development of severe side-effects that simultaneously condition adherence to therapy. Biologically active peptides, in particular antimicrobial peptides (AMPs), are versatile molecules in terms of biological activities. The cytotoxic activities of several AMPs turn this group of molecules into an amazing pool of new templates for anticancer drug development. However, several unmet challenges limit application of peptides in cancer therapy. The mechanism(s) of action of the peptides need better description and understanding, and innovative targets have to be discovered and explored, facilitating drug design and development. In this chapter, we explore the natural occurring AMPs as potential new anticancer peptides (ACPs) for cancer prevention and treatment. Their modes of action, selectivity to tumor compared to normal cells, preferential targets, and applications, but also their weaknesses, are described and discussed.
... The experiment was carried out with mice divided into 5 groups of 5 animals [41]. Groups were infected via intraperitoneal (IP) injection with E. coli (ATCC 8739) suspension equalized and diluted in cold PBS to a sub-lethal concentration of 1 × 10 5 UFC (50 μL in each animal) [42]. The protocol to evaluate Pa-MAP2 in a single, 2 or 3 doses was realized [43]. ...
... All of these plant AMPs either do not contain Cys or contain only one and thus, possess high structural flexibility. Cn-AMP1 is a 9 aa Cys-free AMP identified from green coconut (Cocos nucifera) water with "promiscuous" activity, as it is antibacterial, antifungal, and immune stimulatory [291]. Cr-ACP1 is a 9 aa AMP from the seeds of Cycas revoluta with pro-apoptotic and antimicrobial activities [292]. ...
Plant antimicrobial peptides (AMPs) have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs) of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic), lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms.
... For example, the linear peptide Cn-AMP1, isolated and purified from coconut water (Cocos nucifera), was tested against Caco-2, RAW264.7, MCF-7, HCT-116 cells, and human erythrocytes and showed a reduction of cell viability in cancer cells without causing hemolysis [91]. Other examples are the peptides Cr-ACP, isolated from Cycas revoluta, and the acetylated-modified Cr-AcACP1, both repressors of cell proliferation of human epidermoid cancer (Hep2) and colon carcinoma. ...
Antimicrobial peptides (AMPs) are part of the innate immune defense mechanism of many organisms and are promising candidates to treat infections caused by pathogenic bacteria to animals and humans. AMPs also display anticancer activities because of their ability to inactivate a wide range of cancer cells. Cancer remains a cause of high morbidity and mortality worldwide. Therefore, the development of methods for its control is desirable. Attractive alternatives include plant AMP thionins, defensins, and cyclotides, which have anticancer activities. Here, we provide an overview of plant AMPs anticancer activities, with an emphasis on their mode of action, their selectivity, and their efficacy.
... The majority of plant AMPs are cysteine-rich [3,19,24], with few examples of plant disulfide-free AMPs [14,15,20,23,26]. Among the disulfide-free AMPs, Pg-AMP1 is the only plant GRP reported to be active against bacteria so far, although GRPs from animals may also be active against bacteria [9,12]. ...
Glycine-rich proteins (GRPs) derived from plants compose a family of proteins and peptides that share a glycine repeat domain and can perform diverse functions. Two structural conformations have been proposed for GRPs: glycine loops arranged as a Velcro and an anti-parallel β-sheet with several β-strands. The antimicrobial peptide Pg-AMP1 is the only plant GRP with antibacterial activity reported so far and its structure remains unclear. Recently, its recombinant expression was reported, where the recombinant peptide had an additional methionine residue at the N-terminal and a histidine tag at the C-terminal (His6-tag). These changes seem to change the peptide's activity, generating a broader spectrum of antibacterial activity. In this report, through ab initio molecular modelling and molecular dynamics, it was observed that both peptide structures were composed of an N-terminal α-helix and a dynamic loop that represents two-thirds of the protein. In contrast to previous reports, it was observed that there is a tendency to adopt a globular fold instead of an extended one, which could be in both, glycine loops or anti-parallel β-sheet conformation. The recombinant peptide showed a slightly higher solvated potential energy compared to the native form, which could be related to the His6-tag exposition. In fact, the His6-tag could be mainly responsible for the broader spectrum of activity, but it does not seem to cause great structural changes. However, novel studies are needed for a better characterization of its pharmacological properties so that in the future novel drugs may be produced based on this peptide.
... These discoveries have powerfully demonstrated the potential for peptides as alternatives to existing therapeutic drugs. In the past few decades, numerous studies have established several functionalities associated with numerous peptides, such as antioxidative [4], antihypertensive, antioxidant [5], antibacterial [6,7] insecticide [8], antifungal [9], antiviral [10], inhibition of tumor proliferation [11], and modulation of the immune response [12]. ...
Numerous peptides are available on the market as therapeutic drugs for regulating tumor growth, microorganism proliferation, immune response and/or metabolic disorders. Peptides are produced either by chemical synthesis or heterologous expression. Independent of the method chosen, there are challenges to transferring its production from the bench (~mg/L) to the industrial (~g/L) scale. Thus, the main scale-up pitfalls for the two methods of peptide production are reviewed here, including the advantages of each. Moreover, there will be a special focus on the main challenges for large-scale, heterologous production systems. Peptides that are currently available on the market are also described with an emphasis on how their process optimization has been designed in order to develop a cost-effective product.
Introduction:
Antimicrobial peptides (AMP) have received particular attention due to their capacity to kill bacteria. Although much is known about them, peptides are currently being further researched. A large number of AMPs have been discovered, but only a few have been approved for topical use, due to their promiscuity and other challenges, which need to be overcome.
Areas covered:
AMPs are diverse in structure. Consequently, they have varied action mechanisms when targeting microorganisms or eukaryotic cells. Herein, the authors focus on linear peptides, particularly those that are alpha-helical structured, and examine how their charge distribution and hydrophobic amino acids could modulate their biological activity.
Expert opinion:
The world currently needs urgent solutions to the infective problems caused by resistant pathogens. In order to start the race for antimicrobial development from the charge distribution viewpoint, bioinformatic tools will be necessary. Currently, there is no software available that allows discriminate charge distribution in AMPs and predicts the biological effects of this event. Furthermore, there isn't software available that predicts the side chain length of residues and its role in biological functions. More specialized software is necessary.
Coconut water is an ancient, natural beverage and its popularity in the international market has been continuously increasing in recent years. Coconut water in its natural form is a refreshing and nutritious beverage, which is extensively consumed across the globe due to its beneficial properties to health. Various chemical components which contribute to its bioactivity are essential to the plant industry, biotechnology and biomedical fields. Large number of studies have been conducted on the bio chemical composition, properties and reactions of coconut water. This chapter provides extensive information generated on important constituents such as amino acids, antimicrobial peptides, aromatic compounds, electrolytes, enzymes, phytohormones, polyphenols, sugars and vitamins apart from properties and reactions (flavour, rancidity, turbidity, appearance/colour, pH and acidity). Although many reports on the chemical content of coconut water is already available, there may still be unknown constituents which contribute to its special biological effects. Information on the technological advancements made in the instrumental methods of analysis is furnished in a section. Thus, with the development of more advanced detection techniques, screening can be intensified to detect and identify novel compounds of medicinal values present in coconut water.
Cancer is the second leading cause of death worldwide, and the search for specialized therapy options has been a challenge for decades. The emergence of active targeted therapies provides the opportunity to treat cancerous tissues without harming healthy ones due to peculiar physiological changes. Herein, peptides and peptide analogs have been gaining a lot of attention over the last decade, especially for the on-site delivery of therapeutics to target tissues in order to achieve efficient and reliable cancer treatment. Combining peptides with highly efficient drug delivery platforms could potentially eliminate off-target adverse effects encountered during active targeting of conventional chemotherapeutics. Small size, ease of production and characterization, low immunogenicity and satisfactory binding affinity of peptides offer some advantages over other complex targeting moiety, no wonder the market of peptide-based drugs continues to expand expeditiously. It is estimated that the global peptide drug market will be worth around USD 48.04 billion by 2025, with a compound annual growth rate of 9.4%.
In this review, the current state of art of peptide-based therapeutics with special interest on tumor targeting peptides has been discussed. Moreover, various active targeting strategies such as the use functionalized peptides or peptide analogs are also elaborated.
Antimicrobial peptides (AMPs) are known to play important roles in plant development and stress tolerance. The AMPs are involved in the defense reaction of innate plant immunity and are known to increase the transcription level in response to abiotic or biotic stress factors. There are numerous reports available on the structure and the in vitro efficiency of antimicrobial peptides against phytopathogens. This chapter provides the available pipelines to identify the plant AMPs and its role in mediating the defense in plants in particular.
Plant antimicrobial peptides are the interesting source of studies in defense response as they are essential components of innate immunity which exert rapid defense response. In spite of abundant reports on the isolation of antimicrobial peptides (AMPs) from many sources, the profile of AMPs expressed/identified from single crop species under certain stress/physiological condition is still unknown. This work describes the AMP signature profile of black pepper and their expression upon Phytophthora infection using label-free quantitative proteomics strategy. The differential expression of 24 AMPs suggests that a combinatorial strategy is working in the defense network. The 24 AMP signatures belonged to the cationic, anionic, cysteine-rich and cysteine-free group. As the first report on the possible involvement of AMP signature in Phytophthora infection, our results offer a platform for further study on regulation, evolutionary importance and exploitation of theses AMPs as next generation molecules against pathogens.
The extraction and purification of parigidin-br3, a cyclotide analogue belonging to the "bracelet" subfamily, from Palicourea rigida leaves is discussed. Unlike conventional cyclotides, parigidin-br3 has free N- and C-termini, as identified by MALDI-TOF/TOF analysis and confirmed by gene structure elucidation, and is one of a small number of acyclotides discovered during recent years. Parigidin-br3 showed cytotoxic activity against MCF-7 (breast cancer) and CACO2 (colorectal adenocarcinoma) cells, with IC50 values of ∼2.5 μM and less than 10% hemolytic activity. Overall, parigidin-br3 is a promising new molecule with cytotoxic properties against tumor cell lines and, unlike many synthetic acyclic analogues, demonstrates that cytotoxic activity is not limited to conventional (i.e., cyclic) cyclotides.
Antibiotic-resistant infections are predicted to kill 10 million people per year by 2050, costing the global economy $100 trillion. Therefore, there is an urgent need to develop alternative technologies. We have engineered a synthetic peptide called clavanin-MO, derived from a marine tunicate antimicrobial peptide, which exhibits potent antimicrobial and immunomodulatory properties both in vitro and in vivo. Peptide effectively killed a panel of representative bacterial strains, including multidrug-resistant hospital isolates. Antimicrobial activity of the peptide was demonstrated in animal models, reducing bacterial counts by six orders of magnitude, and contributing to infection clearance. In addition, clavanin-MO was capable of modulating innate immunity by stimulating leukocyte recruitment to the site of infection, and production of immune mediators GM-CSF, IFN-γ and MCP-1, while suppressing an excessive and potentially harmful inflammatory response by increasing synthesis of anti-inflammatory cytokines such as IL-10 and repressing the levels of pro53
inflammatory cytokines IL-12 and TNF-α. Finally, treatment with the peptide protected mice against otherwise lethal infections caused by both Gram-negative and -positive drug-resistant strains. Peptide presented here directly kills bacteria and further helps resolve infections through its immune modulatory properties. Peptide anti-infective therapeutics with combined antimicrobial and immunomodulatory properties represent a new approach to treat antibiotic resistant infections.
Viruses are the major cause of disease and mortality worldwide. Nowadays there are treatments based on antivirals or prophylaxis with vaccines. However, the rising number of reports of viral resistance to current antivirals and the emergence of new types of virus has concerned the scientific community. In this scenario, the search for alternative treatments has led scientists to the discovery of antimicrobial peptides (AMPs) derived from many different sources. Since some of them have shown antiviral activities, here we challenged 10 synthetic peptides from different animal and plant sources against, herpes simplex virus 1 (HSV-1) and Aichi virus. Among them, the highlight was Pa-MAP from the polar fish Pleuronectes americanus, which caused around 90% of inhibition of the herpes simplex virus with a selectivity index of 5 and a virucidal mechanism of action. Moreover, LL-37 from human neutrophils showed 96% of inhibition against the Aichi virus, showing a selectivity index of 3.4. The other evaluated peptides did not show significant antiviral activity. In conclusion, the present study demonstrated that Pa-MAP seems to be a reliable candidate for a possible alternative drug to treat HSV-1 infections. This article is protected by copyright. All rights reserved.
We would like to express our appreciation to all the authors for their informative contributions and the reviewers for their support and constructive critiques in making this special issue possible.
Phytopathogens cause economic losses in agribusiness. Plant-derived compounds have been proposed to overcome this problem, including the antimicrobial peptides (AMPs). This paper reports the identification of Ps-AFP1, a novel AMP isolated from the Pisum sativum radicle. Ps-AFP1 was purified and evaluated against phytopathogenic fungi, showing clear effectiveness. In silico analyses were performed, suggesting an unusual fold and disulfide bond pattern. A novel fold and a novel AMP class were here proposed, the αβ-trumpet fold and αβ-trumpet peptides, respectively. The name αβ-trumpet was created due to the peptide's fold, which resembles the musical instrument. The Ps-AFP1 mechanism of action was also proposed. Microscopic analyses revealed that Ps-AFP1 could affect the fungus during the hyphal elongation from spore germination. Furthermore, confocal microscopy performed with Ps-AFP1 labeled with FITC shows that the peptide was localized at high concentration along the fungal cell surface. Due to low cellular disruption rates, it seems that the main target is the fungal cell wall. The binding thermogram and isothermal titration, molecular dynamics and docking analyses were also performed, showing that Ps-AFP1 could bind to chitin producing a stable complex. Data here reported provided novel structural-functional insights into the αβ-trumpet peptide fold.
In recent years, a number of peptides containing a cyclic structural fold have been described. Among them, the cyclotides family was widely reported in different plant tissues, being composed of small cyclic peptides containing 6 conserved cysteine residues connected by disulfide bonds and forming a cysteine-binding cyclic structure known as a cyclic cysteine knot. This structural scaffold is responsible for an enhanced structural stability against chemical, thermal, and proteolytic degradation. Because of the observed stability and multifunctionality, including insecticidal, antimicrobial, and anti-HIV (human immunodeficiency virus) action, much effort has gone into trying to elucidate the structural-function relations of cyclotide compounds. This review focuses on the novelties involving gene structure, precursor formation and processing, and protein folding of the cyclotide family, shedding some light on molecular mechanisms of cyclotide production. Because cyclotides are clear targets for drug development and also biotechnology applications, their chemical synthesis, heterologous systems production, and protein grafting are also addressed.
The search for novel antimicrobial peptides in free databases is a key element to design new antibiotics. Their amino acid
physicochemical features impact into the antimicrobial peptides activities. The relationship between the amino acid physicochemical
properties and the antimicrobial target might have a fuzzy behavior. This study proposes a sequence similarity and physicochemical
search method followed by a fuzzy inference system to find the most appropriated antimicrobial peptides for each domain. The
proposed system was tested with NCBI’s NR protein data file and the obtained peptide sub dataset will be tested in vitro.
The PROCHECK suite of programs provides a detailed check on the stereochemistry of a protein structure. Its outputs comprise a number of plots in PostScript format and a comprehensive residue-by-residue listing. These give an assessment of the overall quality of the structure as compared with well refined structures of the same resolution and also highlight regions that may need further investigation. The PROCHECK programs are useful for assessing the quality not only of protein structures in the process of being solved but also of existing structures and of those being modelled on known structures.
Campylobacter is well recognized as the leading cause of bacterial foodborne diarrheal disease worldwide. Symptoms can range from mild to serious infections of the children and the elderly and permanent neurological symptoms. The organism is a cytochrome oxidase positive, microaerophilic, curved Gram-negative rod exhibiting corkscrew motility and is carried in the intestine of many wild and domestic animals, particularly avian species including poultry. Intestinal colonization results in healthy animals as carriers. In contrast with the most recent published reviews that cover specific aspects of Campylobacter/campylobacteriosis, this broad review aims at elucidating and discussing the (i) genus Campylobacter, growth and survival characteristics; (ii) detection, isolation and confirmation of Campylobacter; (iii) campylobacteriosis and presence of virulence factors; and (iv) colonization of poultry and control strategies.
Author Summary
Wounding of the skin and other epithelial barriers represents an ever-present challenge and poses a potential threat for invasive infection and sepsis. Therefore, it is not surprising that evolutionary pressure has maintained and developed multiple host defense systems, involving initial hemostasis and fibrin formation, and the subsequent action of multiple proteins and peptides of our innate immune system. In humans, the coagulation pathways and those mediating innate immune responses to infections have so far been seen as separate entities. This view is challenged by the present study, which discloses novel host defense functions of C-terminal peptides of thrombin, a key enzyme in the coagulation cascade. The thrombin-derived peptides, which are detected in human wounds and fibrin, effectively kill microbes by membrane lysis, but also exert potent immunomodulatory and anti-endotoxic functions. Importantly, these peptides protect against P. aeruginosa sepsis, as well as lipopolysaccharide-induced shock in animal models. Thus, from the perspective of wounding and infection, thrombin, after fulfilling its primary function by generating a first line of defense, the fibrin clot, serves an additional role by the generation of antimicrobial and anti-endotoxic host-defense peptides.
Due to increasing resistance development among bacteria, antimicrobial peptides (AMPs), are receiving increased attention. Ideally, AMP should display high bactericidal potency, but low toxicity against (human) eukaryotic cells. Additionally, short and proteolytically stable AMPs are desired to maximize bioavailability and therapeutic versatility.
A facile approach is demonstrated for reaching high potency of ultra-short antimicrobal peptides through end-tagging with W and F stretches. Focusing on a peptide derived from kininogen, KNKGKKNGKH (KNK10) and truncations thereof, end-tagging resulted in enhanced bactericidal effect against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Through end-tagging, potency and salt resistance could be maintained down to 4-7 amino acids in the hydrophilic template peptide. Although tagging resulted in increased eukaryotic cell permeabilization at low ionic strength, the latter was insignificant at physiological ionic strength and in the presence of serum. Quantitatively, the most potent peptides investigated displayed bactericidal effects comparable to, or in excess of, that of the benchmark antimicrobial peptide LL-37. The higher bactericidal potency of the tagged peptides correlated to a higher degree of binding to bacteria, and resulting bacterial wall rupture. Analogously, tagging enhanced peptide-induced rupture of liposomes, particularly anionic ones. Additionally, end-tagging facilitated binding to bacterial lipopolysaccharide, both effects probably contributing to the selectivity displayed by these peptides between bacteria and eukaryotic cells. Importantly, W-tagging resulted in peptides with maintained stability against proteolytic degradation by human leukocyte elastase, as well as staphylococcal aureolysin and V8 proteinase. The biological relevance of these findings was demonstrated ex vivo for pig skin infected by S. aureus and E. coli.
End-tagging by hydrophobic amino acid stretches may be employed to enhance bactericidal potency also of ultra-short AMPs at maintained limited toxicity. The approach is of general applicability, and facilitates straightforward synthesis of hydrophobically modified AMPs without the need for post-peptide synthesis modifications.
Infections caused by pathogenic bacteria could cause an expressive negative impact on human health. A significant enhance in resistance to commercial antibiotics has been observed in all kinds of pathogenic bacteria. In order to find novel approaches to control such common infections, a wide number of defense peptides with bactericidal properties have been characterized. In this report, three peptides lower than 3kDa were purified and identified from green coconut (Cocos nucifera L.) water by using reversed phase-high performance liquid chromatography (HPLC), showing molecular masses of 858Da, 1249Da and 950Da. First one, named Cn-AMP1, was extremely efficient against both Gram-positive and Gram-negative bacteria, being MICs calculated for three peptides. All complete sequences were determined by MALDI-ToF analysis showing no identity in databanks. Moreover, peptide net charge and hydrophobicity of each peptide was in silico evaluated. Finally molecular modeling and dynamics were also applied generating peptides three-dimensional structures, indicating a better explanation to probable mechanisms of action. Cn-AMPs here reported show remarkable potential to contribute in the development of novel antibiotics from natural sources.
The antimicrobial peptide database (APD, http://aps.unmc.edu/AP/main.php) has been updated and expanded. It now hosts 1228 entries with 65 anticancer, 76 antiviral (53 anti-HIV), 327 antifungal
and 944 antibacterial peptides. The second version of our database (APD2) allows users to search peptide families (e.g. bacteriocins,
cyclotides, or defensins), peptide sources (e.g. fish, frogs or chicken), post-translationally modified peptides (e.g. amidation,
oxidation, lipidation, glycosylation or d-amino acids), and peptide binding targets (e.g. membranes, proteins, DNA/RNA, LPS or sugars). Statistical analyses reveal
that the frequently used amino acid residues (>10%) are Ala and Gly in bacterial peptides, Cys and Gly in plant peptides,
Ala, Gly and Lys in insect peptides, and Leu, Ala, Gly and Lys in amphibian peptides. Using frequently occurring residues,
we demonstrate database-aided peptide design in different ways. Among the three peptides designed, GLK-19 showed a higher
activity against Escherichia coli than human LL-37.
The particulate enzyme responsible for the synthesis of endothelium-derived relaxing factor has been purified from cultured and native (noncultured) bovine aortic endothelial cells. Purification of the solubilized particulate enzyme preparation by affinity chromatography on adenosine 2',5'-bisphosphate coupled to Sepharose followed by Superose 6 gel filtration chromatography resulted in a single protein band after denaturing polyacrylamide gel electrophoresis that corresponded to approximately 135 kDa. The enzyme activity in the various fractions was assayed by its stimulatory effect on soluble guanylyl cyclase of rat fetal lung fibroblasts (RFL-6 cells), by the formation of L-citrulline from L-arginine, by measuring nitrite/nitrate formation, and by bioassay on endothelium-denuded vascular strips. Endothelium-derived relaxing factor synthase was purified 3419-fold from the crude particulate fraction of cultured bovine aortic endothelial cells with a 12% recovery (RFL-6 assay). Purified endothelium-derived relaxing factor synthase required L-arginine, NADPH, Ca2+, calmodulin, and 5,6,7,8-tetrahydrobiopterin for full activity.
Insects can rapidly clear microbial infections by producing a variety of immune-induced molecules including antibacterial and/or antifungal peptides/polypeptides. In this report, we present the isolation, structural characterization, and biological properties of two variants of a group of bioactive, slightly cationic peptides, referred to as alloferons. Two peptides were isolated from the blood of an experimentally infected insect, the blow fly Calliphora vicina (Diptera), with the following amino acid sequences: HGVSGHGQHGVHG (alloferon 1) and GVSGHGQHGVHG (alloferon 2). Although these peptides have no clear homologies with known immune response modifiers, protein database searches established some structural similarities with proteins containing amino acid stretches similar to alloferon. In vitro experiments reveal that the synthetic version of alloferon has stimulatory activities on natural killer lymphocytes, whereas in vivo trials indicate induction of IFN production in mice after treatments with synthetic alloferon. Additional in vivo experiments in mice indicate that alloferon has antiviral and antitumoral capabilities. Taken together, these results suggest that this peptide, which has immunomodulatory properties, may have therapeutic capacities. The fact that insects may produce cytokine-like materials modulating basic mechanisms for human immunity suggests a source of anti-infection and antitumoral biopharmaceuticals.
LyP-1 is a peptide selected from a phage-displayed peptide library that specifically binds to tumor and endothelial cells of tumor lymphatics in certain tumors. Fluorescein-conjugated LyP-1 and a related peptide, LyP-1b, strongly accumulated in primary MDA-MB-435 breast cancer xenografts and their metastases from i.v. peptide injections, allowing visualization of orthotopic tumors in intact mice. The LyP peptide accumulation coincided with hypoxic areas in tumors. LyP-1 induced cell death in cultured human breast carcinoma cells that bind and internalize the peptide. Melanoma cells that do not bind LyP-1 were unaffected. Systemic LyP-1 peptide treatment of mice with xenografted tumors induced with the breast cancer cells inhibited tumor growth. The treated tumors contained foci of apoptotic cells and were essentially devoid of lymphatics. These results reveal an unexpected antitumor effect by the LyP-1 peptide that seems to be dependent on a proapoptotic/cytotoxic activity of the peptide. As LyP-1 affects the poorly vascularized tumor compartment, it may complement treatments directed at tumor blood vessels.
• phage display
• tumor targeting
• live imaging
• therapy
Motivation: Protein homology detection and sequence alignment are at the basis of protein structure prediction, function prediction and evolution.
Results: We have generalized the alignment of protein sequences with a profile hidden Markov model (HMM) to the case of pairwise alignment of profile HMMs. We present a method for detecting distant homologous relationships between proteins based on this approach. The method (HHsearch) is benchmarked together with BLAST, PSI-BLAST, HMMER and the profile–profile comparison tools PROF_SIM and COMPASS, in an all-against-all comparison of a database of 3691 protein domains from SCOP 1.63 with pairwise sequence identities below 20%.
Sensitivity: When the predicted secondary structure is included in the HMMs, HHsearch is able to detect between 2.7 and 4.2 times more homologs than PSI-BLAST or HMMER and between 1.44 and 1.9 times more than COMPASS or PROF_SIM for a rate of false positives of 10%. Approximately half of the improvement over the profile–profile comparison methods is attributable to the use of profile HMMs in place of simple profiles.
Alignment quality: Higher sensitivity is mirrored by an increased alignment quality. HHsearch produced 1.2, 1.7 and 3.3 times more good alignments (‘balanced’ score >0.3) than the next best method (COMPASS), and 1.6, 2.9 and 9.4 times more than PSI-BLAST, at the family, superfamily and fold level, respectively.
Speed: HHsearch scans a query of 200 residues against 3691 domains in 33 s on an AMD64 2GHz PC. This is 10 times faster than PROF_SIM and 17 times faster than COMPASS.
Availability: HHsearch can be downloaded from http://www.protevo.eb.tuebingen.mpg.de/download/ together with up-to-date versions of SCOP and PFAM. A web server is available at http://www.protevo.eb.tuebingen.mpg.de/toolkit/index.php?view=hhpred
Contact:johannes.soeding@tuebingen.mpg.de
To understand the functional differences between a nontoxic membrane anchor corresponding to the N-terminal sequence of the Escherichia coli enzyme IIA(Glc) and a toxic antimicrobial peptide aurein 1.2 of similar sequence, a series of peptides was designed to bridge the gap between them. An alteration of a single residue of the membrane anchor converted it into an antibacterial peptide. Circular dichroism spectra indicate that all peptides are disordered in water but helical in micelles. Structures of the peptides were determined in membrane-mimetic micelles by solution NMR spectroscopy. The quality of the distance-based structures was improved by including backbone angle restraints derived from a set of chemical shifts ((1)H(alpha), (15)N, (13)C(alpha), and (13)C(beta)) from natural abundance two-dimensional heteronuclear correlated spectroscopy. Different from the membrane anchor, antibacterial peptides possess a broader and longer hydrophobic surface, allowing a deeper penetration into the membrane, as supported by intermolecular nuclear Overhauser effect cross-peaks between the peptide and short chain dioctanoyl phosphatidylglycerol. An attempt was made to correlate the NMR structures of these peptides with their antibacterial activity. The activity of this group of peptides does not correlate exactly with helicity, amphipathicity, charge, the number of charges, the size of the hydrophobic surface, or hydrophobic transfer free energy. However, a correlation is established between the peptide activity and membrane perturbation potential, which is defined by interfacial hydrophobic patches and basic residues in the case of cationic peptides. Indeed, (31)P solid state NMR spectroscopy of lipid bilayers showed that the extent of lipid vesicle disruption by these peptides is proportional to their membrane perturbation potential.
The three-dimensional structure in dodecyl phosphocholine micelles of the 26-mer membrane-permeabilizing bacteriocin-like pheromone plantaricin A (PlnA) has been determined by use of nuclear magnetic resonance spectroscopy. The peptide was unstructured in water but became partly structured upon exposure to micelles. An amphiphilic alpha-helix stretching from residue 12 to 21 (possibly also including residues 22 and 23) was then formed in the C-terminal part of the peptide, whereas the N-terminal part remained largely unstructured. PlnA exerted its membrane-permeabilizing antimicrobial activity through a nonchiral interaction with the target cell membrane because the d-enantiomeric form had the same activity as the natural l-form. This nonchiral interaction involved the amphiphilic alpha-helical region in the C-terminal half of PlnA because a 17-mer fragment that contains the amphiphilic alpha-helical part of the peptide had antimicrobial potency that was similar to that of the l- and d-enantiomeric forms of PlnA. Also the pheromone activity of PlnA depended on this nonchiral interaction because both the l- and d-enantiomeric forms of the 17-mer fragment inhibited the pheromone activity. The pheromone activity also involved, however, a chiral interaction between the N-terminal part of PlnA and its receptor because high concentrations of the l-form (but not the d-form) of a 5-mer fragment derived from the N-terminal part of PlnA had pheromone activity. The results thus reveal a novel mechanism whereby peptide pheromones such as PlnA may function. An initial nonchiral interaction with membrane lipids induces alpha-helical structuring in a segment of the peptide pheromone. The peptide becomes thereby sufficiently structured and properly positioned in the membrane interface, thus enabling it to engage in a chiral interaction with its receptor in or near the membrane water interface. This membrane-interacting mode of action explains why some peptide pheromones/hormones such as PlnA sometimes display antimicrobial activity in addition to their pheromone activity.
Antimicrobial peptides (AmPs) are small proteins that are used by the innate immune system to combat bacterial infection in multicellular eukaryotes. There is mounting evidence that these peptides are less susceptible to bacterial resistance than traditional antibiotics and could form the basis for a new class of therapeutic agents. Here we report the rational design of new AmPs that show limited homology to naturally occurring proteins but have strong bacteriostatic activity against several species of bacteria, including Staphylococcus aureus and Bacillus anthracis. These peptides were designed using a linguistic model of natural AmPs: we treated the amino-acid sequences of natural AmPs as a formal language and built a set of regular grammars to describe this language. We used this set of grammars to create new, unnatural AmP sequences. Our peptides conform to the formal syntax of natural antimicrobial peptides but populate a previously unexplored region of protein sequence space.
A major problem in structural biology is the recognition of errors in experimental and theoretical models of protein structures.
The ProSA program (Protein Structure Analysis) is an established tool which has a large user base and is frequently employed
in the refinement and validation of experimental protein structures and in structure prediction and modeling. The analysis
of protein structures is generally a difficult and cumbersome exercise. The new service presented here is a straightforward
and easy to use extension of the classic ProSA program which exploits the advantages of interactive web-based applications
for the display of scores and energy plots that highlight potential problems spotted in protein structures. In particular,
the quality scores of a protein are displayed in the context of all known protein structures and problematic parts of a structure
are shown and highlighted in a 3D molecule viewer. The service specifically addresses the needs encountered in the validation
of protein structures obtained from X-ray analysis, NMR spectroscopy and theoretical calculations. ProSA-web is accessible
at https://prosa.services.came.sbg.ac.at
The aim of broth and agar dilution methods is to determine the lowest concentration of the assayed antimicrobial agent (minimal inhibitory concentration, MIC) that, under defined test conditions, inhibits the visible growth of the bacterium being investigated. MIC values are used to determine susceptibilities of bacteria to drugs and also to evaluate the activity of new antimicrobial agents. Agar dilution involves the incorporation of different concentrations of the antimicrobial substance into a nutrient agar medium followed by the application of a standardized number of cells to the surface of the agar plate. For broth dilution, often determined in 96-well microtiter plate format, bacteria are inoculated into a liquid growth medium in the presence of different concentrations of an antimicrobial agent. Growth is assessed after incubation for a defined period of time (16-20 h) and the MIC value is read. This protocol applies only to aerobic bacteria and can be completed in 3 d.
This study evaluated the cytotoxic and antiproliferative efficacy of two well-characterized members of the Cecropin-family of antimicrobial peptides against bladder tumor cells and benign fibroblasts.
The antiproliferative and cytotoxic potential of the Cecropins A and B was quantified by colorimetric WST-1-, BrdU- and LDH-assays in four bladder cancer cell lines as well as in murine and human fibroblast cell lines. IC50 values were assessed by logarithmic extrapolation, representing the concentration at which cell viability was reduced by 50%. Scanning electron microscopy (SEM) was performed to visualize the morphological changes induced by Cecropin A and B in bladder tumor cells and fibroblasts.
Cecropin A and B inhibit bladder cancer cell proliferation and viability in a dose-dependent fashion. The average IC50 values of Cecropin A and B against all bladder cancer cell lines ranged between 73.29 mug/ml and 220.05 mug/ml. In contrast, benign fibroblasts were significantly less or not at all susceptible to Cecropin A and B. Both Cecropins induced an increase in LDH release from bladder tumor cells whereas benign fibroblasts were not affected. SEM demonstrated lethal membrane disruption in bladder cancer cells as opposed to fibroblasts.
Cecropin A and B exert selective cytotoxic and antiproliferative efficacy in bladder cancer cells while sparing targets of benign murine or human fibroblast origin. Both peptides may offer novel therapeutic strategies for the treatment of bladder cancer with limited cytotoxic effects on benign cells.
Human alpha-defensin-1 (HNP1), a small antimicrobial peptide, shows cytotoxicity to tumor cells in vitro and inhibitory activity for pathologic neovascularization in vivo. Here, we did a gene therapy with a plasmid that expresses a secretable form of HNP1 for assaying its antitumor activity. The expression and secretion of HNP1 were determined by reverse transcription-PCR and ELISA in vitro. We found that expression of HNP1 in A549 tumor cells caused significant growth inhibition. This effect is most likely cell autonomous, as a significant amount of recombinant HNP1 protein was found to be accumulated in the cytoplasm by immunohistochemical staining using an anti-HNP1 antibody and the supernatant containing secreted HNP1 failed to produce any noticeable antitumor activity. Flow cytometry and Hoechst 33258 staining showed that the number of apoptotic cells among the A549 cells expressing recombinant HNP1 proteins was significantly greater than that of the nontransfected control cultures, suggesting that this growth-inhibitory activity was due to an apoptotic mechanism triggered by the intracellular HNP1. The antitumor activity of intracellularly expressed HNP1 was also shown in vivo. Decreased microvessel density and increased lymphocyte infiltration were observed in tumor tissue from HNP1-treated mice through histologic analysis. These results indicate that intracellularly expressed HNP1 induces tumor cell apoptosis, which inhibits tumor growth. The antiangiogenesis effect of HNP1 may contribute to its inhibitory activity in vivo, and HNP1 might involve the host immune response to tumor. These findings provide a rationale for developing HNP1-based gene therapy for cancer.
The constant alteration of cultural identities accompanies our imaginings of the city. Questions like "What is a city?", "Who am I in it?", and "Who is the Other?" touch on the issue of social boundaries and emotional mapping. Comparisons of the city to the body as expressed in architecture, design, and urban planning vary, but the city as a site of emotions is often left unexplored. City is about the organic urban body. Although high-rise and masculine in architecture, city expresses the female in its desires, memory bites, emotions, and sensuality. In applying the concept of the human body to the city, the awareness of the fact that emotions are visually stored in the city's public sphere is heightened. The city itself becomes the canvas for intimate dialogues.
It is suggested that the experience of a skin-to-skin contact with the physicality of the streets, once we allow ourselves to consciously be in touch with the city, leads to inevitable changes in the construction of our outlook on our urban environment. On the one hand, the occurring transformation can be a positive and liberating experience as depicted in spatial experiences based on the sensuality and ecstasy of the city. On the other hand, there is the intimacy with the street that results in prostitution and homelessness, linked with displacement and disembodiment in society. If the city is regarded as a space that consists of energy forms rather than as a fixed constant, its fluidity and performative actions are expressed in a free-flow of excessive energies that create connections. Such a hybridity is a fluid concept structured around the presence, absence, and ambivalence of roots, and air relates to such a spatial discourse based on accounts of the flesh, fantasies, and dreams that encompass histories and identity formations.
We are emotionally involved when we are in touch with something. Emotions move us, metaphorically and in literary terms, from one place to another, and every journey consists of encounters and actions like departing, meeting, staying. There is a way of mapping space that does not need to fulfill the need to conquer, but rather helps us to gain insight into an inner landscape reflected in outer urban space. Water is about the journey, a scan of emotional spaces, shifting between privacy and publicity, outer and inner space. It is about a fluid voyage that follows an itinerary of emotions framed by existential questions. The city spills over with inscriptions and instabilities. The fluidity of a cultural discourse opens up a discussion of instabilities and insecurities. Because there is no real orientation, only ambivalence, which creates a spatial gap, it resembles a swim in darkness. We do not exactly know where we are going, but we have an inherent idea.
Bauman points out that the question of whether to enter and commit to a relationship is influenced by bouts of fear and desire. The hedonistic pursuit of happiness consists of the act of being on the run rather than of arriving, and therein the pleasure or addiction of compulsion lies, on quests for fulfillment and ecstasy. Anxieties about growing old, about death, about being abandoned and left to one's own devices, reveal the frailty of human bonds. Compulsions and obsessions with the object of one's desires, expressed in activities like speed-dating or mobile phone messaging, stand for the need to belong to a network that creates some sense of safety. The need for security spills over to human relationships with public space and the identity question of who we are in this particular city at this given time. We try to avoid these questions and to escape definitions for our modes-of-being because there are no straightforward answers. An alternative understanding of our existence in relationship with others will open up if we are willing to embrace the uncertainty, or what Bauman terms "liquidity," that is the essence of human existence.
The frailty of an old man named Homer in Wim Wenders's Wings of Desire is used as a symbol for "the tired, perhaps dying city," as opposed to the modern, dynamic, technologically advanced city, an urban fantasy that feeds into the culturally desirable...
Ein aus 24 Aminosäuren bestehendes Polypeptid wurde aus dem Abwehrsekret europäischer Unken isoliert. Abbauversuche machen folgende Primärstruktur wahrscheinlich:
Neutrophil granules contain several antimicrobial peptides (AMPs) that are important effector molecules of innate immunity. In mammals, the main families of these peptides are the cathelicidins and defensins. Several defensins have been characterized in humans, while there is only one human cathelicidin, designated LL-37. This peptide is stored in specific granules of neutrophils in an inactive proform, which is processed extracellularly to the mature active peptide LL-37 and the propart cathelin after neutrophil degranulation. Apart from exhibiting a broad antimicrobial spectra, it is now evident that LL-37 possesses several additional functions that are related to host defense. Examples of such functions are chemotactic, endotoxin neutralizing, angiogenic and wound healing activities. These effects of LL-37 reveal a role as a mediator between innate and adaptive immunity. This review is giving an overview of the different immunological effects exerted by LL-37 and the physiological significance of these functions in immunity.
The circular dichroism (CD) of a protein at any wavelength can be expressed as X = fHXHn̄ + fβXβ + fRfR (1). The f's are the fractions of helix (H), β form, and unordered form (R). n̄ refers to the average number of peptide units per helical segment in a protein molecule. The X parameters are determined from the CD spectra of five or eight proteins of known f's, using a least-squares method. They, in turn, can be used to estimate the fH and fβ of a protein by fitting its CD spectrum (below 245 nm) with eq 1. Results of tests on eight proteins are good, provided that the n̄ of the proteins is close to the overall n̄ of the reference proteins (around 10). This restriction of a single n̄ for all proteins can be removed by introducing a chain-length-dependent factor, k, for the helix, i.e., XHn = XH∞(1 - k/n). The XH∞ is further expressed by three Gaussian bands. Three methods of analysis are proposed by (1) combining the CD spectrum of a protein with its optical rotation at 233 nm, (2) introducing a restriction of constant rotational strength for each CD band of an infinite helix, and (3) combining methods 1 and 2. They determine not only the fractions of helix and β form but also provide an estimate of the number and average size of helical segments in a protein molecule. Results of tests on eight proteins indicate that the proposed methods are superior to the use of eq 1 alone.
Evaluation of the electrostatic properties of biomolecules has become a standard practice in molecular biophysics. Foremost
among the models used to elucidate the electrostatic potential is the Poisson-Boltzmann equation; however, existing methods
for solving this equation have limited the scope of accurate electrostatic calculations to relatively small biomolecular systems.
Here we present the application of numerical methods to enable the trivially parallel solution of the Poisson-Boltzmann equation
for supramolecular structures that are orders of magnitude larger in size. As a demonstration of this methodology, electrostatic
potentials have been calculated for large microtubule and ribosome structures. The results point to the likely role of electrostatics
in a variety of activities of these structures.
Antimicrobial peptides have been found in mollusks and other sea animals. In this report, a crude extract of the marine snail Cenchritis muricatus was evaluated against human pathogens responsible for multiple deleterious effects and diseases. A peptide of 1485.26 Da was purified by reversed-phase HPLC and functionally characterized. This trypsinized peptide was sequenced by MS/MS technology, and a sequence (SRSELIVHQR), named Cm-p1 was recovered, chemically synthesized and functionally characterized. This peptide demonstrated the capacity to prevent the development of yeasts and filamentous fungi. Otherwise, Cm-p1 displayed no toxic effects against mammalian cells. Molecular modeling analyses showed that this peptide possible forms a single hydrophilic α-helix and the probable cationic residue involved in antifungal activity action is proposed. The data reported here demonstrate the importance of sea animals peptide discovery for biotechnological tools development that could be useful in solving human health and agribusiness problems.
Nowadays, novel pharmacies have been screened from plants. Among them are the peptides, which show multiple biotechnological activities. In this report, a small peptide (Ala-Trp-Lys-Leu-Phe-Asp-Asp-Gly-Val) with a molecular mass of 1,050 Da was purified from Cycas revoluta seeds by using reversed-phase liquid chromatography. This peptide shows clear deleterious effects against human epidermoid cancer (Hep2) and colon carcinoma cells (HCT15). It caused inhibition of cancer cell proliferation and further disruption of nucleosome structures, inducing apoptosis by direct DNA binding. A remarkable antibacterial activity was also observed in this same peptide. Nevertheless, no significant lysis of normal RBC cells was observed in the presence of peptide. Additionally, an acetylation at the N-termini portion is able to reduce both activities. Bioinformatics tools were also utilized for construction of a three-dimensional model showing a single amphipathic helix. Since in vitro binding studies show that the target of this peptide seems to be DNA, theoretical docking studies were also performed to better understand the interaction between peptide and nucleic acids and also to shed some light on the acetyl group role. Firstly, binding studies showed that affinity contacts basically occur due to electrostatic attraction. The complex peptide-ssDNA was clearly oriented by residues Ala(1), Lys(3), and Asp(6), which form several hydrogen bonds that are able to stabilize the complex. When acetyl was added, hydrogen bonds are broken, reducing the peptide affinity. In summary, it seems that information here provided could be used to design a novel derivative of this peptide which a clear therapeutic potential.
The solution structure of bovine lactoferricin (LfcinB) has been determined using 2D 1H NMR spectroscopy. LfcinB is a 25-residue antimicrobial peptide released by pepsin cleavage of lactoferrin, an 80 kDa iron-binding glycoprotein with many immunologically important functions. The NMR structure of LfcinB reveals a somewhat distorted antiparallel beta-sheet. This contrasts with the X-ray structure of bovine lactoferrin, in which residues 1-13 (of LfcinB) form an alpha-helix. Hence, this region of lactoferricin B appears able to adopt a helical or sheetlike conformation, similar to what has been proposed for the amyloidogenic prion proteins and Alzheimer's beta-peptides. LfcinB has an extended hydrophobic surface comprised of residues Phe1, Cys3, Trp6, Trp8, Pro16, Ile18, and Cys20. The side chains of these residues are well-defined in the NMR structure. Many hydrophilic and positively charged residues surround the hydrophobic surface, giving LfcinB an amphipathic character. LfcinB bears numerous similarities to a vast number of cationic peptides which exert their antimicrobial activities through membrane disruption. The structures of many of these peptides have been well characterized, and models of their membrane-permeabilizing mechanisms have been proposed. The NMR solution structure of LfcinB may be more relevant to membrane interaction than that suggested by the X-ray structure of intact lactoferrin. Based on the solution structure, it is now possible to propose potential mechanisms for the antimicrobial action of LfcinB.
Bacterial infections directly affect the world's population, and this situation has been aggravated by indiscriminate use of antimicrobial agents, which can generate resistant microorganisms. In this report, an initial screening of proteins with antibacterial activity from corms of 15 species of the Xanthosoma genus was conducted. Since Xanthosoma blandum corms showed enhanced activity toward bacteria, a novel protein with bactericidal activity was isolated from this particular species. Edman degradation was used for protein N-termini determination; the primary structure showed similarities with Kunitz inhibitors, and this protein was named Xb-KTI. This protein was further challenged against serine proteinases from different sources, showing clear inhibitory activities. Otherwise, no hemolytic activity was observed for Xb-KTI. The results demonstrate the biotechnological potential of Xb-KTI, the first proteinase inhibitor with antimicrobial activity described in the Xanthosoma genus.
The phenomenon of protein promiscuity, in which multiple functions are associated with a single peptide structure, has gained attention in several research fields, including the plant defense field. With this in mind, this report intends to link various plant defense peptides with common scaffolds (defensins, cyclotides and 2S albumins), and multiple activities with the processes of promiscuity generation and protein evolvability. This link seems to create an efficient system of plant defense against insect pests and pathogens, and is thus essential to plant survival and evolution. This review also identifies future possibilities for the use of peptide promiscuity in designing novel drugs and synthetic biotechnological products.
Human cathelicidin LL-37, a host defense peptide derived from leukocytes and epithelial cells, plays a crucial role in innate and adaptive immunity. Not only does LL-37 eliminate pathogenic microbes directly but also modulates host immune responses. Emerging evidence from tumor biology studies indicates that LL-37 plays a prominent and complex role in carcinogenesis. Although overexpression of LL-37 has been implicated in the development or progression of many human malignancies, including breast, ovarian and lung cancers, LL-37 suppresses tumorigenesis in gastric cancer. These data are beginning to unveil the intricate and contradictory functions of LL-37. The reasons for the tissue-specific function of LL-37 in carcinogenesis remain to be elucidated. Here, we review the relationship between LL-37, its fragments and cancer progression as well as discuss the potential therapeutic implications of targeting this peptide.
Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.
Several natural antimicrobial peptides including cecropins, magainins and melittins have been found to kill cancer cells. However, their efficacy may not be adequate for their development as anticancer agents. In this study, we used a natural antimicrobial peptide, cecropin B (CB), as a template to generate a novel anticancer peptide. Cecropin B is an amphipathic and polycationic peptide derived from the hemolymph of Hyalophora cecropia with well-known antimicrobial and cytolytic properties. The signature pattern of cecropins is W-x-(0,2)-[KDN]-x-{L}-K-[KRE]-[LI]-E-[RKN] (PROSITE: PS00268), and this signature sequence is located at N-terminus of CB. CB1a was constructed by repeating the N-terminal ten amino acids of CB three times and including a hinge near C-terminus. The circular dichroism spectra showed that CB1a is unstructured in aqueous solution, but adopt a helical conformation in membrane-like environment. The solution structure of CB1a in a polar solvent was also studied by NMR. CB1a formed a helix-hinge-helix in 20% HFIP solution, and it was found the bent angle between two helical segments was induced ranging from 60 degrees to 110 degrees . A heparin-binding motif is located in the central part of helix 1. Isothermal titration calorimetry reveals the association constant of CB1a bound to low molecular weight heparin is 1.66 x 10(5)M(-1) at physiological ionic strength at 25 degrees C. Binding of CB1a to heparin produces a large conformational change toward a more structural state. CB1a demonstrated promising activity against several cancer cells but low toxicity against non-cancer cells. The IC(50) of CB1a on leukemia and stomach carcinoma cells were in the range of 2-8-fold lower than those of CB. Besides, CB1a exhibited low hemolytic activity against human red blood cells. Due to these properties, CB1a has the potential to become a promising anticancer agent.
A family of peptides with broad-spectrum antimicrobial activity has been isolated from the skin of the African clawed frog Xenopus laevis. It consists of two closely related peptides that are each 23 amino acids and differ by two substitutions. These peptides are water soluble, nonhemolytic at their effective antimicrobial concentrations, and potentially amphiphilic. At low concentrations they inhibit growth of numerous species of bacteria and fungi and induce osmotic lysis of protozoa. The sequence of a partial cDNA of the precursor reveals that both peptides derive from a common larger protein. These peptides appear to represent a previously unrecognized class of vertebrate antimicrobial activities.
From the nucleotide sequence of clones isolated from a cDNA library constructed from skin of Xenopus laevis, the existence of PYLa, a peptide comprised of 24 amino acids, was predicted. This peptide was synthesized by solid-phase methods and purified to homogeneity with an overall yield of 61%. The synthetic peptide was used as reference substance to search for its natural counterpart in skin secretion of Xenopus. Two peptides were found which were very similar to PYLa except for the absence of the first three amino acids. These 21-amino-acid peptides, termed PGLa, can be generated from PYLa by cleavage after the single arginine residue present in the latter. The two forms of PGLa differ in their retention time on HPLC but have identical amino acid compositions and terminal sequences. Tryptic hydrolysis of synthetic PYLa after the single arginine yields exclusively PGLa with the shorter retention time on HPLC. The chemical difference between the two forms of PGLa is currently not known. The possible biological role of these newly discovered constituents of frog skin secretion is discussed.
Xenopus laevis skin secretion contains a mixture of magainins, which are small positively charged oligopeptides with antimicrobial activity. In this study, we show that two of these peptides, i.e. magainin-2 and PGLa, are much more active in biological functions when added together than when added alone. This synergy applies for the antimicrobial activity of these peptides, and for the toxic effects on tumor cells. We show that this peptide combination is also synergistic when permeabilizing protein-free liposomes for glucose, when dissipating the membrane potential in cytochrome oxidase liposomes and Escherichia coli, and, reversibly, when stimulating respiration in the liposomes. The occurrence of synergy in these diverse systems (complex and simple) suggests that the biological synergy results from synergy in the primary activity of the magainin peptides, namely the permeabilization of free-energy transducing membranes, possibly by forming a multimeric transmembrane pore of mixed peptide composition. The antimicrobial activity of X. laevis skin secretions may be greatly enhanced by the application of this binary weapon.
A series of designed peptide 33-mers (betapep peptides) areknown to be bactericidal [Mayo, Haseman, Ilyina and Gray (1998)Biochim. Biophys. Acta 1425, 81-92]. Here dodecapeptides (SC-1-SC-8), which 'walk through' the sequence ofbetapep-25, were investigated for their ability to kill Gram-negativeand -positive bacteria and to neutralize endotoxin. SC-4 (KLFKRHLKWKI I-NH(2); the -NH(2) at the right of each sequenceindicates amidation of the C-terminal carboxylate group) is the mosteffective, more so than betapep-25, at killing Gram-negative bacteriawith nanomolar LD(50) values. Against Gram-positive bacteria,SC-4 also shows good activity with submicromolar LD(50)values. Leakage studies indicate rapid bacterial membrane permeability,with t(1/2) valuesof 10-15 min. SC-4 in the micromolar range also effectivelyneutralizes endotoxin and is not haemolytic below 10(-4)M. For all SC peptides, CD and NMR data indicate the presence of both 3(10)- and alpha-helix. For SC-4, nuclear-Overhauser-effect-based computational modelling yields an amphipathic helix with K1, K4,R5, and K8 arrayed on the same face (K is lysine, R is arginine). Activity differences among SC peptides and single-site variants of SC-4allow some structure-function relationships to be deduced. Relative to other known bactericidal peptides in the linear peptide,helix-forming category, SC-4 is the most potent broad-spectrumantibacterial identified to date. The present study contributes to thedevelopment of agents involved in combating the ever-recurring problemof drug-resistant micro-organisms.
Defensins, a family of small, cationic, antimicrobial peptides, are found in mammals, insects and plants. alpha-defensins are stored in granules of neutrophils and released upon activation by exocytosis. It was shown here that human neutrophil peptide (HNP), at concentrations of 10(-8) -10(-9) M, up-regulated the expression of TNF-alpha and IL-1 beta in monocytes activated with Staphylococcus aureus or PMA, while expression of IL-10 mRNA was down-regulated and production of IL-8 was not affected. HNP alone was unable to induce TNF-alpha or IL-1 beta expression in resting monocytes. At concentrations of 10(-4) -10(-5)M, HNP was cytotoxic for monocytes in serum-free medium. The cytotoxicity was abrogated in the presence of serum, while a cytokine-modulating effect of HNP was observed in the presence of serum and in whole blood, suggesting that this mechanism may function in vivo. Similarly, serum did not abrogate bactericidal activity of HNP. It was also demonstrated herein that HNP at 10 (-8) -10(-9) M, attenuated the inhibitory action of dexamethasone on TNF-alpha production. In parallel to monocyte studies, we have showed that HNP at concentrations ranging from 10(-9)M to 10(-6)M caused about 5-fold suppression of VCAM-1 expression in TNF-alpha-activated human umbilical vein endothelial cells, while the ICAM-1 expression was not affected. Our findings suggest that neutrophil defensins have the potential to modulate the inflammatory responses through regulation of cytokine production and adhesion molecule expression.
Investigation of magainin II amide analogs with cationic charges ranging between +3 and +7 showed that enhancement of the peptide charge up to a threshold value of +5 and conservation of appropriate hydrophobic properties optimized the antimicrobial activity and selectivity. High selectivity was the result of both enhanced antimicrobial and reduced hemolytic activity. Charge increase beyond +5 with retention of other structural motifs led to a dramatic increase of hemolytic activity and loss of antimicrobial selectivity. Selectivity could be restored by reduction of the hydrophobicity of the hydrophobic helix surface (H(hd)), a structural parameter not previously considered to modulate activity. Dye release experiments with lipid vesicles revealed that the potential of peptide charge to modulate membrane activity is limited: on highly negatively charged 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol bilayers, reinforcement of electrostatic interactions had an activity-reducing effect. On neutral 1-palmitoyl-2-oleoylphosphatidylcholine bilayers, the high activity was determined by H(hd). H(hd) values above a certain threshold led to effective permeabilization of all lipid systems and even compensated for the activity-reducing effect of charge increase on highly negatively charged membranes.
A novel bombesin-related peptide was isolated from skin secretions of Chinese red belly toad Bombina maxima. Its primary structure was established as pGlu-Lys-Lys-Pro-Pro-Arg-Pro-Pro-Gln-Trp-Ala-Val-Gly-His-Phe-Met-NH(2.) The amino-terminal (N-terminal) 8-residue segment comprising four prolines and three basic residues is extensively different from bombesins from other Bombina species. The peptide was thus named proline rich bombesin (PR-bombesin). PR-bombesin was found to elicit concentration-dependent contractile effects in the rat stomach strip, with both increased potency and intrinsic activity as compared with those of [Leu(13)]bombesin. Analysis of different bombesin cDNA structures revealed that an 8 to 14- nucleotide fragment replacement in the peptide coding region (TGGGGAAT in the cDNAs of multiple bombesin forms from Bombina orientalis and CACCCCGGCCACCC in the cDNA of PR-bombesin) resulted in an unusual Pro-Pro-Arg-Pro-Pro motif in the N-terminal part of PR-bombesin.
A 5.6 kDa trypsin-chymotrypsin protease inhibitor was isolated from the tubers of the potato (Solanum tuberosum L cv. Gogu) by extraction of the water-soluble fraction, dialysis, ultrafiltration, and C18 reversed-phase high performance liquid chromatography. This inhibitor, which we named potamin-1 (PT-1), was thermostable and possessed antimicrobial activity but lacked hemolytic activity. PT-1 strongly inhibited pathogenic microbial strains, including Candida albicans, Rhizoctonia solani, and Clavibacter michiganense subsp. michiganinse. Automated Edman degradation showed that the N-terminal sequence of PT-1 was NH2-DICTCCAGTKGCNTTSANGAFICEGQSDPKKPKACPLNCDPHIAYA-. The sequence had 62% homology with a serine protease inhibitor belonging to the Kunitz family, and the peptide inhibited chymotrypsin, trypsin, and papain. This protease inhibitor, PT-1, was composed of polypeptide chains joined by disulfide bridge(s). Reduced PT-1 almost completely lost its activity against fungi and proteases indicating that disulfide bridge is essential for its protease inhibitory and antifungal activity. These results suggest that PT-1 is an excellent candidate as a lead compound for the development of novel oral or other anti-infective agents.
In the last decade intensive research has been conducted to determine the role of innate immunity host defense peptides (also termed antimicrobial peptides) in the killing of prokaryotic and eukaryotic cells. Many antimicrobial peptides damage the cellular membrane as part of their killing mechanism. However, it is not clear what makes cancer cells more susceptible to some of these peptides, and what the molecular mechanisms underlying these activities are. Two general mechanisms were suggested: (i) plasma membrane disruption via micellization or pore formation, and (ii) induction of apoptosis via mitochondrial membrane disruption. To be clinically used, these peptides need to combine high and specific anticancer activity with stability in serum. Although so far very limited, new studies have paved the way for promising anticancer host defense peptides with a new mode of action and with a broad spectrum of anticancer activity.
Magainin II belongs to a family of antimicrobial peptides and has been shown to exhibit antibiotic activity in a wide range of organisms. Recent studies have also reported a significant antitumor effect of magainin II against various cancer cell lines and tumor mice models. In this study, we evaluated the cytotoxic and antiproliferative potency of magainin II in bladder tumor cells and normal fibroblasts.
The antiproliferative and cytotoxic effect of magainin II was quantified by colorimetric WST-1-, bromodeoxyuridine (BrdU)-, and lactic dehydrogenase (LDH) assays in three bladder cancer cell lines (RT4, 647V, and 486P) and in the murine fibroblast cell line 3T3 as well as in a primary culture from human fibroblasts. The median inhibitory concentration (IC50) values were determined for each assay, representing the concentration at which cell viability was reduced by 50%. Scanning electron microscopy (SEM) was used to visualize the morphologic effects of magainin II on bladder tumor cells and fibroblasts.
Magainin II inhibited cell proliferation of bladder cancer cells in a dose-dependent manner. The average IC50 of magainin II against all bladder cancer cell lines was 198.1 microM (range, 52.4-484.03 microM) for the WST-1 assay and 75.2 microM (range, 31.0-135.3 microM) for the BrdU assay. The normal murine and human fibroblast cell lines were not affected by magainin II and their IC50 could not be determined at the concentrations of magainin II tested. LDH release was increased in all bladder tumor cell lines in the presence of magainin II, whereas normal fibroblasts showed no cell lysis. SEM demonstrated lethal membrane perforation by peptide pore formation in bladder cancer cells, but not in fibroblasts.
Magainin II peptide exerts cytotoxic and antiproliferative efficacy by pore formation in bladder cancer cells but has no effect on normal murine or human fibroblasts. Magainin II may offer a novel therapeutic strategy in the treatment of bladder cancer with potentially low cytotoxic effects on normal cells.
Nine neuroendocrine tumors of the skin were examined by light and electron microscopy. The patients (7 males and 2 females) had an average age of 57 years. Seven tumors were located in the head and neck region. Light microscopic examination showed sheets of cells. In 4 cases, a tendency to form small groups of cells was observed. Contact with the epidermis was seen in only 2 cases. The dominant ultrastructural feature was the presence of cytoplasmic processes that contained membrane-bound granules 100-200 nm. Three tumors recurred locally, and in 7 patients, regional nodal metastases occurred. Three patients died of disseminated disease.
Functional characterization of a protein sequence is a common goal in biology, and is usually facilitated by having an accurate three-dimensional (3-D) structure of the studied protein. In the absence of an experimentally determined structure, comparative or homology modeling can sometimes provide a useful 3-D model for a protein that is related to at least one known protein structure. Comparative modeling predicts the 3-D structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target-template alignment, model building, and model evaluation. This unit describes how to calculate comparative models using the program MODELLER and discusses all four steps of comparative modeling, frequently observed errors, and some applications. Modeling lactate dehydrogenase from Trichomonas vaginalis (TvLDH) is described as an example. The download and installation of the MODELLER software is also described.
Bacterial pathogens cause an expressive negative impact worldwide on human health, with ever increasing treatment costs. A significant rise in resistance to commercial antibiotics has been observed in pathogenic bacteria responsible for urinary and gastro-intestinal infections. Towards the development of novel approaches to control such common infections, a number of defense peptides with antibacterial activities have been characterized. In this report, the peptide Pg-AMP1 was isolated from guava seeds (Psidium guajava) and purified using a Red-Sepharose Cl-6B affinity column followed by a reversed-phase HPLC (Vydac C18-TP). Pg-AMP1 showed no inhibitory activity against fungi, but resulted in a clear growth reduction in Klebsiella sp. and Proteus sp., which are the principal pathogens involved in urinary and gastro-intestinal hospital infections. SDS-PAGE and mass spectrometry (MALDI-ToF) characterized Pg-AMP1 a monomer with a molecular mass of 6029.34Da and small quantities of a homodimer. Amino acid sequencing revealed clear identity to the plant glycine-rich protein family, with Pg-AMP1 the first such protein with activity towards Gram-negative bacteria. Furthermore, Pg-AMP1 showed a 3D structural homology to an enterotoxin from Escherichia coli, and other antibacterial proteins, revealing that it might act by formation of a dimer. Pg-AMP1 shows potential, in a near future, to contribute to development of novel antibiotics from natural sources.
Clinical and Laboratory Standards Institute
- Pa Wayne
Wayne, PA: Clinical and Laboratory Standards Institute. 2008;
28: 1–24.
- J Y Kim
- S C Park
- M H Kim
- H T Lim
- Y Park
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K. S. Biochem. Biophys. Res. Commun 2005, 330, 921-927.
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- M E Akerman
- H Biliran
- M Yang
- F Ferrer
- T Karpanen
- R M Hoffman
- E Ruoslahti
Laakkonen, P.; Akerman, M. E.; Biliran, H.; Yang, M.; Ferrer, F.;
Karpanen, T.; Hoffman, R. M.; Ruoslahti, E. Proc Natl Acad Sci
USA 2004, 22, 9381-9386.