Structural and Functional Characterization of New Lipid Transfer Proteins with Chitin-Binding Properties: Insights from Protein Structure Prediction, Molecular Docking, and Antifungal Activity.

Faced with the emergence of multiresistant microorganisms that affect human health, microbial agents have become a serious global threat, affecting human health and plant crops. Antimicrobial peptides have attracted significant attention in research for the development of new microbial control agents. This work's goal was the structural characterization and analysis of antifungal activity of chitin-binding peptides from Capsicum baccatum and Capsicum frutescens seeds on the growth of Candida and Fusarium species. Proteins were initially submitted to extraction in phosphate buffer pH 5.4 and subjected to chitin column chromatography. Posteriorly, two fractions were obtained for each species, Cb-F1 and Cf-F1 and Cb-F2 and Cf-F2, respectively. The Cb-F1 (C. baccatum) and Cf-F1 (C. frutescens) fractions did not bind to the chitin column. The electrophoresis results obtained after chromatography showed two major protein bands between 3.4 and 14.2 kDa for Cb-F2. For Cf-F2, three major bands were identified between 6.5 and 14.2 kDa. One band from each species was subjected to mass spectrometry, and both bands showed similarity to nonspecific lipid transfer protein. Candida albicans and Candida tropicalis had their growth inhibited by Cb-F2. Cf-F2 inhibited the development of C. albicans but did not inhibit the growth of C. tropicalis. Both fractions were unable to inhibit the growth of Fusarium species. The toxicity of the fractions was tested in vivo on Galleria mellonella larvae, and both showed a low toxicity rate at high concentrations. As a result, the fractions have enormous promise for the creation of novel antifungal compounds.

Purification, Structural Characterization, and Anticandidal Activity of a Chitin-Binding Peptide with High Similarity to Hevein and Endochitinase Isolated from Pepper Seeds.

With the emergence of multidrug-resistant microorganisms, microbial agents have become a serious global threat, affecting human health and various plants. Therefore, new therapeutic alternatives, such as chitin-binding proteins, are necessary. Chitin is an essential component of the fungal cell wall, and chitin-binding proteins exhibit antifungal activity. In the present study, chitin-binding peptides isolated from Capsicum chinense seeds were characterized and evaluated for their in vitro antimicrobial effect against the growth of Candida and Fusarium fungi. Proteins were extracted from the seeds and subsequently the chitin-binding proteins were separated by chitin affinity chromatography. After chromatography, two fractions, Cc-F1 (not retained on the column) and Cc-F2 (retained on the column), were obtained. Electrophoresis revealed major protein bands between 6.5 and 26.6 kDa for Cc-F1 and only a ~ 6.5 kDa protein band for Cc-F2, which was subsequently subjected to mass spectrometry. The protein showed similarity with hevein-like and endochitinase and was then named Cc-Hev. Data are available via ProteomeXchange with identifier PXD054607. Next, we predicted the three-dimensional structure of the peptides and performed a peptide docking with (NAG)3. Subsequently, growth inhibition assays were performed to evaluate the ability of the peptides to inhibit microorganism growth. Cc-Hev inhibited the growth of C. albicans (up to 75% inhibition) and C. tropicalis (100% inhibition) and induced a 65% decrease in cell viability for C. albicans and 100% for C. tropicalis. Based on these results, new techniques to combat fungal diseases could be developed through biotechnological applications; therefore, further studies are needed.

Design of improved synthetic antifungal peptides with targeted variations in charge, hydrophobicity and chirality based on a correlation study between biological activity and primary structure of plant defensin γ-cores.

Microbial resistance to available drugs is a growing health threat imposing the need for the development of new drugs. The scaffold of plant defensins, including their γ-cores, are particularly good candidates for drug design. This work aimed to improve the antifungal activity of a previous design peptide, named A36,42,44γ32-46VuDef (for short DD) against yeasts by altering its biochemical parameters. We explore the correlation of the biological activity and structure of plant defensins and compared their primary structures by superimposition with VuDef1 and DD which indicated us the favorable position and the amino acid to be changed. Three new peptides with modifications in charge, hydrophobicity (RR and WR) and chirality (D-RR) were designed and tested against pathogenic yeasts. Inhibition was determined by absorbance. Viability of mammalian cells was determined by MTT. The three designed peptides had better inhibitory activity against the yeasts with better potency and spectrum of yeast species inhibition, with low toxicity to mammalian cells. WR, the most hydrophobic and cationic, exhibited better antifungal activity and lower toxicity. Our study provides experimental evidence that targeted changes in the primary structure of peptides based on plant defensins γ-core primary structures prove to be a good tool for the synthesis of new compounds that may be useful as alternative antifungal drugs. The method described did not have the drawback of synthesis of several peptides, because alterations are guided. When compared to other methods, the design process described is efficient and viable to those with scarce resources.

A synthetic peptide derived of the ß2-ß3 loop of the plant defensin from Vigna unguiculata seeds induces Leishmania amazonensis apoptosis-like cell death.

For medical use of proteins and peptide-based drugs, it is desirable to have small biologically active sequences because they improve stability, reduce side effects, and production costs. Several plant defensins have their biological activities imparted by a sequence named γ-core. Vu-Def, a Vigna unguiculata defensin, has activity against Leishmania amazonensis, which is one etiological agent of leishmaniasis and for which new drugs are needed. Our intention was to understand if the region comprising the Vu-Def γ-core is responsible for the biological activity against L. amazonensis and to unveil its mechanism of action. Different microbiological assays with L. amazonensis in the presence of the synthetic peptide A36,42,44γ32-46Vu-Def were done, as well as ultrastructural and fluorescent analyses. A36,42,44γ32-46Vu-Def showed biological activity similar to Vu-Def. A36,42,44γ32-46Vu-Def (74 µM) caused 97% inhibition of L. amazonensis culture and parasites were unable to regrow in fresh medium. The cells of the treated parasites showed morphological alterations by ultrastructural analysis and fluorescent labelings that corroborate with the data of the organelles alterations. The general significance of our work is based on the description of a small synthetic peptide, A36,42,44γ32-46Vu-Def, which has activity on L. amazonensis and that the interaction between A36,42,44γ32-46Vu-Def-L. amazonensis results in parasite inhibition by the activation of an apoptotic-like cell death pathway.

Insight into the α-Amylase Inhibitory Activity of Plant Lipid Transfer Proteins.

We previously characterized the inhibitory activity of human salivary α-amylase (HSA) and Callosobruchus maculatus intestinal α-amylases by the plant lipid transfer protein from Vigna unguiculata ( Vu-LTP). Herein, we further study this inhibitory activity. First by an analysis of protein α-amylase inhibitors complexed with α-amylase, we find that positively charged amino acids of inhibitors interact with the active site of α-amylases and we know that Vu-LTP is rich in positively charged amino acid residues. For this reason, we model Vu-LTP, and based on its three-dimensional structure, we choose five peptides to be synthesized. Herein, we report that two peptides of Vu-LTP are responsible for HSA inhibition. A comparison of primary and tertiary structures of LTPs with and without inhibitory activity against α-amylase, superimposed with the sequence of Vu-LTP mapped for HSA inhibition, reinforces our suggestion that positively charged amino acids in loops are responsible for the inhibition. To prove our observation, one modified peptide is synthesized in which Arg39 is replaced by Gln. This modified peptide loses the HSA inhibitory property presented by the unmodified peptide. Therefore, we describe a new biological active for Vu-LTP, i.e. the α-amylase inhibitory activity that is not a fortuitous biological activity and probably has evolved to perform a biological function which is still unknown. A good candidate should be defense against insects. The results of this study also expand the possible biotechnological applications of LTPs.

Interaction between the plant ApDef1 defensin and Saccharomyces cerevisiae results in yeast death through a cell cycle- and caspase-dependent process occurring via uncontrolled oxidative stress.

BACKGROUND: Plant defensins were discovered at beginning of the 90s'; however, their precise mechanism of action is still unknown. Herein, we studied ApDef1-Saccharomyces cerevisiae interaction. METHODS: ApDef1-S. cerevisiae interaction was studied by determining the MIC, viability and death kinetic assays. Viability assay was repeated with hydroxyurea synchronized-yeast and pretreated with CCCP. Plasma membrane permeabilization, ROS induction, chromatin condensation, and caspase activation analyses were assessed through Sytox green, DAB, DAPI and FITC-VAD-FMK, respectively. Viability assay was done in presence of ascorbic acid and Z-VAD-FMK. Ultrastructural analysis was done by electron microscopy. RESULTS: ApDef1 caused S. cerevisiae cell death and MIC was 7.8µM. Whole cell population died after 18h of ApDef1 interaction. After 3h, 98.76% of synchronized cell population died. Pretreatment with CCCP protected yeast from ApDef1 induced death. ApDef1-S. cerevisiae interaction resulted in membrane permeabilization, H2O2 increased production, chromatin condensation and caspase activation. Ascorbic acid prevented yeast cell death and membrane permeabilization. Z-VAD-FMK prevented yeast cell death. CONCLUSIONS: ApDef1-S. cerevisiae interaction caused cell death through cell cycle dependentprocess which requires preserved membrane potential. After interaction, yeast went through uncontrolled ROS production and accumulation, which led to plasma membrane permeabilization, chromatin condensation and, ultimately, cell death by activation of caspase-dependent apoptosis via. GENERAL SIGNIFICANCE: We show novel requirements for the interaction between plant defensin and fungi cells, i.e. cell cycle phase and membrane potential, and we indicate that membrane permeabilization is probably caused by ROS and therefore, it would be an indirect event of the ApDef1-S. cerevisiae interaction.

Anti-Neuroblastoma Properties of a Recombinant Sunflower Lectin.

According to their sugar recognition specificity, plant lectins are proposed as bioactive proteins with potential in cancer treatment and diagnosis. Helja is a mannose-specific jacalin-like lectin from sunflower which was shown to inhibit the growth of certain fungi. Here, we report its recombinant expression in a prokaryotic system and its activity in neurobalstoma cells. Helja coding sequence was fused to the pET-32 EK/LIC, the enterokinase/Ligation-independent cloning vector and a 35 kDa protein was obtained in Escherichia coli representing Helja coupled to thioredoxin (Trx). The identity of this protein was verified using anti-Helja antibodies. This chimera, named Trx-rHelja, was enriched in the soluble bacterial extracts and was purified using Ni+2-Sepharose and d-mannose-agarose chromatography. Trx-rHelja and the enterokinase-released recombinant Helja (rHelja) both displayed toxicity on human SH-SY5Y neuroblastomas. rHelja decreased the viability of these tumor cells by 75% according to the tetrazolium reduction assay, and microscopic analyses revealed that the cell morphology was disturbed. Thus, the stellate cells of the monolayer became spheroids and were isolated. Our results indicate that rHelja is a promising tool for the development of diagnostic or therapeutic methods for neuroblastoma cells, the most common solid tumors in childhood.

Bioinspired peptides induce different cell death mechanisms against opportunistic yeasts.

The management of fungal diseases imposes an urgent need for the development of effective antifungal drugs. Among new drug candidates are the antimicrobial peptides, and especially their derivatives. Here, we investigated the molecular mechanism of action of three bioinspired peptides against the opportunistic yeasts Candida tropicalis and Candida albicans. We assessed morphological changes, mitochondrial functionality, chromatin condensation, ROS production, activation of metacaspases, and the occurrence of cell death. Our results indicated that the peptides induced sharply contrasting death kinetics, of 6 h for RR and 3 h for D-RR to C. tropicalis and 1 h for WR to C. albicans. Both peptide-treated yeasts exhibited increased ROS levels, mitochondrial hyperpolarization, cell size reduction, and chromatin condensation. RR and WR induced necrosis in C. tropicalis and C. albicans, but not D-RR in C. tropicalis. The antioxidant ascorbic acid reverted the toxic effect of RR and D-RR, but not WR, suggesting that instead of ROS there is a second signal triggered that leads to yeast death. Our data suggest that RR induced a regulated accidental cell death in C. tropicalis, D-RR induced a programmed cell death metacaspase-independent in C. tropicalis, while WR induced an accidental cell death in C. albicans. Our results were obtained with the LD100 and within the time that the peptides induce the yeast death. Within this temporal frame, our results allow us to gain clarity on the events triggered by the peptide-cell interaction and their temporal order, providing a better understanding of the death process induced by them.

Defensin-like peptides from Capsicum chinense induce increased ROS, loss of mitochondrial functionality, and reduced growth of the fungus Colletotrichum scovillei.

In the present study, we identified and characterized two defensin-like peptides in an antifungal fraction obtained from Capsicum chinense pepper fruits and inhibited the growth of Colletotrichum scovillei, which causes anthracnose. AMPs were extracted from the pericarp of C. chinense peppers and subjected to ion exchange, molecular exclusion, and reversed-phase in a high-performance liquid chromatography system. We investigated the endogenous increase in reactive oxygen species (ROS), the loss of mitochondrial functioning, and the ultrastructure of hyphae. The peptides obtained from the G3 fraction through molecular exclusion chromatography were subsequently fractionated using reverse-phase chromatography, resulting in the isolation of fractions F1, F2, F3, F4, and F5. The F1-Fraction suppressed C. scovillei growth by 90, 70.4, and 44% at 100, 50, and 25 µg mL-1, respectively. At 24 h, the IC50 and minimum inhibitory concentration were 21.5 µg mL-1 and 200 µg mL-1, respectively. We found an increase in ROS, which may have resulted in an oxidative burst, loss of mitochondrial functioning, and cytoplasm retraction, as well as an increase in autophagic vacuoles. MS/MS analysis of the F1-Fraction indicated the presence of two defensin-like proteins, and we were able to identify the expression of three defensin sequences in our C. chinense fruit extract. The F1-Fraction was also found to inhibit the activity of insect α-amylases. In summary, the F1-Fraction of C. chinense exhibits antifungal activity against a major pepper pathogen that causes anthracnose. These defensin-like compounds are promising prospects for further research into antifungal and insecticide biotechnology applications. © 2024 Society of Chemical Industry.

Trypsin/α-Amylase Inhibitors from Capsicum chinense Seeds: Characterization and Antifungal Activity against Fungi of Agronomic Importance.

BACKGROUND: Protease inhibitors (PIs) have attracted attention due to their important roles in plant defense. OBJECTIVE: The objective of this work was to characterize and evaluate the antimicrobial activity of the peptides of a family of serine PIs from Capsicum chinense Jacq. seeds. METHODS: Initially, PIs were extracted from the seeds and subjected to purification by chromatography, resulting in three different peptide enriched fractions (PEFs) termed PEF1, PEF2 and PEF3. Subsequently, the PEF3 was subjected to trypsin inhibition assays, α-amylase activity assays, antimicrobial activity assays on phytopathogenic fungi, and assays to determine the likely mechanisms of action. RESULTS: The PEF3 was composed of three protein bands with molecular masses ranging between 6 and 14 kDa. The amino acid residues of the ~6 kDa band showed high similarity with serine PIs. PEF3 inhibited the activity of the enzymes trypsin, human salivary α-amylase, and Tenebrio molitor larval α-amylase and inhibited the growth of phytopathogenic fungi, showing 83.7% loss of viability in Fusarium oxysporum. PEF3 induced reactive oxygen species in Colletotrichum lindemuthianum and F. oxysporum to dissipate their mitochondrial membrane potential and activated caspases in C. lindemuthianum. CONCLUSION: Our results reinforce the importance of PIs in plant defense mechanisms against phytopathogenic fungi as well as in their biotechnological applications for the control of plant pathogens.

Inhibition of Serine Protease, α-Amylase and Growth of Phytopathogenic Fungi by Antimicrobial Peptides from Capsicum chinense Fruits.

Plant fungal diseases cause major problems for the global economy. Antimicrobial peptides have aroused great interest in the control of phytopathogens, as they are natural molecules and have a broad spectrum of inhibitory activity. Herein, we have tried to identify and characterize antimicrobial peptides present in fruits of Capsicum chinense and to evaluate their enzymatic and antifungal activities. The retained fraction obtained in the anion exchange chromatography with strong antifungal activity was subjected to molecular exclusion chromatography and obtained four fractions named G1, G2, G3, and G4. The 6.0-kDa protein band of G2 showed similarity with protease inhibitors type II, and it was able to inhibit 100% of trypsin and α-amylase activities. The protein band with approximately 6.5 kDa of G3 showed similarity with sequences of protease inhibitors from genus Capsicum and showed growth inhibition of 48% for Colletotrichum lindemuthianum, 49% for Fusarium lateritium, and 51% for F. solani and F. oxysporum. Additionally, G3 causes morphological changes, membrane permeabilization, and ROS increase in F. oxysporum cells. The 9-kDa protein band of G4 fraction was similar to a nsLTP type 1, and a protein band of 6.5 kDa was similar to a nsLTP type 2. The G4 fraction was able to inhibit 100% of the activities of glycosidases tested and showed growth inhibition of 35 and 50% of F. oxysporum and C. lindemuthianum, respectively. C. chinense fruits have peptides with antifungal activity and enzyme inhibition with biotechnological potential.

Antifungal, Antimycobacterial, Protease and α‒Amylase Inhibitory Activities of a Novel Serine Bifunctional Protease Inhibitor from Adenanthera pavonina L. Seeds.

Antifungal resistance poses a significant challenge to disease management, necessitating the development of novel drugs. Antimicrobial peptides offer potential solutions. This study focused on extraction and characterization of peptides from Adenanthera pavonina seeds with activity against Candida species, Mycobacterium tuberculosis, proteases, and α-amylases. Peptides were extracted in phosphate buffer and heated at 90°C for 10 min to create a peptide rich heated fraction (PRHF). After confirming antimicrobial activity and the presence of peptides, the PRHF underwent ion exchange chromatography, yielding retained and non-retained fractions. These fractions were evaluated for antimicrobial activity and cytotoxicity against murine macrophages. The least toxic and most active fraction underwent reversed-phase chromatography, resulting in ten fractions. These fractions were tested for peptides and antimicrobial activity. The most active fraction was rechromatographed on a reversed-phase column, resulting in two fractions that were assessed for antimicrobial activity. The most active fraction revealed a single band of approximately 6 kDa and was tested for inhibitory effects on proteases and α-amylases. Thermal stability experiments were conducted on the 6 kDa peptide at different temperatures followed by reassessment of antifungal activity and circular dichroism. The 6 kDa peptide inhibited yeasts, M. tuberculosis, human salivary and Tenebrio molitor larvae intestine α-amylases, and proteolytic activity from fungal extracts, and thus named ApPI. Remarkably, ApPI retained antifungal activity and conformation after heating and is primarily composed of α-helices. ApPI is a thermally stable serine protease/α-amylase inhibitor from A. pavonina seeds, offering promise as a foundational molecule for innovative therapeutic agents against fungal infections and tuberculosis.

Structural and Biochemical Characterization of Three Antimicrobial Peptides from Capsicum annuum L. var. annuum Leaves for Anti-Candida Use.

The emergence of resistant microorganisms has reduced the effectiveness of currently available antimicrobials, necessitating the development of new strategies. Plant antimicrobial peptides (AMPs) are promising candidates for novel drug development. In this study, we aimed to isolate, characterize, and evaluate the antimicrobial activities of AMPs isolated from Capsicum annuum. The antifungal potential was tested against Candida species. Three AMPs from C. annuum leaves were isolated and characterized: a protease inhibitor, a defensin-like protein, and a lipid transporter protein, respectively named CaCPin-II, CaCDef-like, and CaCLTP2. All three peptides had a molecular mass between 3.5 and 6.5 kDa and caused morphological and physiological changes in four different species of the genus Candida, such as pseudohyphae formation, cell swelling and agglutination, growth inhibition, reduced cell viability, oxidative stress, membrane permeabilization, and metacaspase activation. Except for CaCPin-II, the peptides showed low or no hemolytic activity at the concentrations used in the yeast assays. CaCPin-II inhibited α-amylase activity. Together, these results suggest that these peptides have the potential as antimicrobial agents against species of the genus Candida and can serve as scaffolds for the development of synthetic peptides for this purpose.

A new bioinspired peptide on defensin from C. annuum fruits: Antimicrobial activity, mechanisms of action and therapeutical potential.

BACKGROUND: Antimicrobial peptides, natural or synthetic, appear as promising molecules for antimicrobial therapy because of their both broad antimicrobial activity and mechanism of action. Herein, we determine the anti-Candida and antimycobacterial activities, mechanism of action on yeasts, and cytotoxicity on mammalian cells in the presence of the bioinspired peptide CaDef2.1G27-K44. METHODS: CaDef2.1G27-K44 was designed to attain the following criteria: high positive net charge; low molecular weight (<3000 Da); Boman index ≤2.5; and total hydrophobic ratio ≥ 40%. The mechanism of action was studied by growth inhibition, plasma membrane permeabilization, ROS induction, mitochondrial functionality, and metacaspase activity assays. The cytotoxicity on macrophages, monocytes, and erythrocytes were also determined. RESULTS: CaDef2.1G27-K44 showed inhibitory activity against Candida spp. with MIC100 values ranging from 25 to 50 µM and the standard and clinical isolate of Mycobacterium tuberculosis with MIC50 of 33.2 and 55.4 µM, respectively. We demonstrate that CaDef2.1G27-K44 is active against yeasts at different salt concentrations, induced morphological alterations, caused membrane permeabilization, increased ROS, causes loss of mitochondrial functionality, and activation of metacaspases. CaDef2.1G27-K44 has low cytotoxicity against mammalian cells. CONCLUSIONS: The results obtained showed that CaDef2.1G27-K44 has great antimicrobial activity against Candida spp. and M. tuberculosis with low toxicity to host cells. For Candida spp., the treatment with CaDef2.1G27-K44 induces a process of regulated cell death with apoptosis-like features. GENERAL SIGNIFICANCE: We show a new AMP bioinspired with physicochemical characteristics important for selectivity and antimicrobial activity, which is a promising candidate for drug development, mainly to control Candida infections.

Bifunctional Inhibitors from Capsicum chinense Seeds with Antimicrobial Activity and Specific Mechanism of Action Against Phytopathogenic Fungi.

BACKGROUND: Antimicrobial peptides (AMPs) are found in the defense system in virtually all life forms, being present in many, if not all, plant species. OBJECTIVE: The present work evaluated the antimicrobial, enzymatic activity and mechanism of action of the PEF2 fraction from Capsicum chinense Jack. seeds against phytopathogenic fungi. METHODS: Peptides were extracted from C. chinense seeds and subjected to reverse-phase chromatography on an HPLC system using a C18 column coupled to a C8 guard column, then the obtained PEF2 fraction was rechromatographed using a C2/C18 column. Two fractions, named PEF2A and PEF2B, were obtained. The fractions were tested for antimicrobial activity on Colletotrichum gloeosporioides, Colletotrichum lindemuthianum, Fusarium oxysporum and Fusarium solani. Trypsin inhibition assays, reverse zymographic detection of protease inhibition and α-amylase activity assays were also performed. The mechanism of action by which PEF2 acts on filamentous fungi was studied through analysis of membrane permeability and production of reactive oxygen species (ROS). Additionally, we investigated mitochondrial functionality and caspase activation in fungal cells. RESULTS: It is possible to observe that PEF2 significantly inhibited trypsin activity and T. molitor larval α-amylase activity. The PEF2 fraction was able to inhibit the growth of C. gloeosporioides, C. lindemuthianum and F. oxysporum. PEF2A inhibited the growth of C. lindemuthianum (75%) and F. solani (43%). PEF2B inhibited C. lindemuthianum growth (66%) and F. solani (94%). PEF2 permeabilized F. solani cell membranes and induced ROS in F. oxysporum and F. solani. PEF2 could dissipate mitochondrial membrane potential but did not cause the activation of caspases in all studied fungi. CONCLUSION: The results may contribute to the biotechnological application of these AMPs in the control of pathogenic microorganisms in plants of agronomic importance.

Inhibition mechanism of human salivary α-amylase by lipid transfer protein from Vigna unguiculata.

VuLTP1.1, a LTP1 from Vigna unguiculata, inhibits 78.1 % of the human salivary α-amylase (HSA) activity at 20 µM. We had performed a correlation study between VuLTP1.1 structure and HSA inhibitory activity and showed that two VuLTP1.1 regions are responsible for HSA inhibition. In one of them we had characterized the crucial importance of an Arg39 for inhibition. In this work, we analyzed the VuLTP1.1-HSA interaction by protein-protein docking to understand the most probable interaction model and the mechanism of HSA inhibition by VuLTP1.1. The VuLTP1.1 tertiary structure quality and refinement as well as the docking assay between VuLTP1.1 and HSA were done by bioinformatic programs. HSA inhibition occurs by direct interaction of the VuLTP1.1 with the HSA causing the obstruction of the carbohydrate biding cleft with Gibbs free energy of -18.5 Kcal/mol and the dissociation constant of 2.6E-14 M. The previously identified Arg39 of VuLTP1.1 is burrowed into the active site of the HSA and there it interacts with the Asp300 of HSA catalytic site by a hydrogen bond. We had confirmed the importance of the Arg39 of VuLTP1.1 for the HSA inhibition which interacts with the Asp300 at the HSA active site. I-2, a LTP-like peptide, presents the same HSA inhibition pattern that VuLTP1.1, which indicates that the inhibition mechanism of the LTPs towards α-amylase is very similar. For the best of our knowledge, it is the first time that the HSA inhibition mechanism was understood and described for the LTP1s using VuLTP1.1 and I-2 as prototype inhibitors.

Antimicrobial peptides from Capsicum chinense fruits: agronomic alternatives against phytopathogenic fungi.

In recent years, the antimicrobial activity of peptides isolated from a wide variety of organs from plant species has been reported. However, a few studies have investigated the potential of antimicrobial peptides (AMPs) found in fruits, especially Capsicum chinense (pepper). The present study aimed to purify and characterize peptides from Capsicum chinense fruits and evaluate their inhibitory activities against different phytopathogenic fungi and also analyze the possible mechanisms of action involved in microbial inhibition. After fruit protein extraction and high-performance liquid chromatography (HPLC), different fractions were obtained, named F1 to F10. Peptides in the F4 and F5 fractions were sequenced and revealed similarity with the plant antimicrobial peptides like non-specific lipid transfer proteins and defensin-like peptide. The F4 and F5 fractions presented strong antimicrobial activity against the fungus Fusarium solani and Fusarium oxysporum, causing toxic effects on these fungi, leading to membrane permeabilization, endogenous reactive oxygen species increase, activation of metacaspase and loss of mitochondrial function.

Identification and Characterization of Two Defensins from Capsicum annuum Fruits that Exhibit Antimicrobial Activity.

Scientific advances have not been enough to combat the growing resistance to antimicrobial medicines. Antimicrobial peptides (AMPs) are effector molecules of the innate immune defense system in plants and could provide an important source of new antimicrobial drugs. The aim of this work was to extract, purify, characterize, and evaluate the antifungal activities present in fractions obtained from Capsicum annum fruits through reversed-phase chromatography. The fractions named F2 and F3 presented the highest inhibitory activity against Candida and Mycobacterium tuberculosis species. In addition, we identified two sequences of AMPs in the F2 and F3 fractions through mass spectrometry that showed similarity to an already well-characterized family of plant defensins. A plasma membrane permeabilization assay demonstrated that the peptides present in F2, F3, and F4 fractions induced changes in the membrane of some yeast strains, culminating in permeabilization. The production of reactive oxygen species was induced by the fractions in some yeast strains. Fractions F2, F3, and F4 also did not show toxicity in macrophage or monocyte cultures. In conclusion, the obtained data demonstrate that the AMPs, especially those present in the fractions F2 and F3, are promising antimicrobial agents that may be useful to enhance the development of new therapeutic agents for the treatment of diseases.

Improved smallest peptides based on positive charge increase of the γ-core motif from PνD1 and their mechanism of action against Candida species.

BACKGROUND: Plant defensins have a hallmark γ-core motif (GXCX3-9C) that is related to their antimicrobial properties. The aim of this work was to design synthetic peptides based on the region corresponding to the PvD1 defensin γ-core that are the smallest amino acid sequences that bear the strongest biological activity. METHODS: We made rational substitutions of negatively charged amino acid residues with positively charged ones, and the reduction in length in the selected PvD1 γ-core sequence to verify whether the increased net positive charges and shortened length are related to the increase in antifungal activity. Herein, we opted to evaluate the action mechanism of γ33-41 PvD1 ++ peptide due to its significant inhibitory effect on tested yeasts. In addition, it is the smallest construct comprising only nine amino acid residues, giving it a better possibility to be a prototype for designing a new antifungal drug, with lower costs to the pharmaceutical industry while still maintaining the strongest antimicrobial properties. RESULTS: The γ33-41 PvD1 ++ peptide caused the most toxic effects in the yeast Candida buinensis, leading to membrane permeabilization, viability loss, endogenous reactive oxygen species increase, the activation of metacaspase, and the loss of mitochondrial functionality, suggesting that this peptide triggers cell death via apoptosis. CONCLUSION: We observed that the antifungal activity of PvD1 is not strictly localized in the structural domain, which comprises the γ-core region and that the increase in the net positive charge is directly related to the increase in antifungal activity.

A modified, hypoallergenic variant of the Ricinus communis Ric c1 protein retains biological activity.

Ric c1, an allergenic protein from Ricinus communis , is an insect α-amylase inhibitor that has become an occupational allergen. Ric c1 can cross-react with allergens from wheat, soybean, peanut, shrimp, fish, gluten, house dust, tobacco, and air fungus, thereby amplifying the concern and risks caused by Ricinus allergens. Two continuous IgE-binding epitopes were identified in Ric c1, both containing glutamic acid residues involved in IgE-binding and allergic challenges. We produced recombinant Ric c1 (rRic c1) in Escherichia coli , using primers from foliar R. communis DNA, and a mutant (Glu-Leu) recombinant protein (mrRic c1) in the same system using synthetic genes. rRic c1 preserved both allergenic and α-amylase inhibitory properties, and mrRic c1 drastically reduced allergenic properties. These results can help to establish meaningful relationships between structure, defense and allergenicity, important steps for producing engineered plants and developing new approaches for immunotherapy.