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The outbreak caused by SARS-CoV-2 has taken many lives worldwide. Although vaccination has started, the development of drugs to either alleviate or abolish symptoms of COVID-19 is still necessary. Here, four synthetic peptides were assayed regarding their ability to protect Vero E6 cells from SARS-CoV-2 infection and their toxicity to human cells and zebrafish embryos. All peptides had some ability to protect cells from infection by SARS-CoV-2 with the D614G mutation. Molecular docking predicted the ability of all peptides to interact with and induce conformational alterations in the spike protein containing the D614G mutation. PepKAA was the most effective peptide, by having the highest docking score regarding the spike protein and reducing the SARS-CoV-2 plaque number by 50% (EC

This study aimed to partially characterize the three main serine carboxypeptidases (SCP3, SCP20, and SCP47) from Nepenthes mirabilis. Furthermore, one peptidase (SCP3) was chosen for further heterologous expression in Escherichia coli Shuffle®T7. SCP3 also was characterized in terms of its allergenic potential using bioinformatics tools. SCP3, SCP20, and SCP47 showed very similar 3D structures and mechanistic features to other plant serine peptidases belonging to clan SC and family S10. Although SCP3 was obtained in its soluble form, using 1% ethanol during induction with 0.5 mM IPTG at 16 °C for 18 h, it did not show proteolytic activity by zymography or in vitro analysis. SCP3 presented a few allergenic peptides and several cleavage sites for digestive enzymes. This work describes additional features of these enzymes, opening new perspectives for further studies for characterization and analysis of heterologous expression, as well as their potential biotechnological applications.

Background: The herbivores Danaus plexippus (Lepidoptera), Oncopeltus fasciatus, and Aphis nerii (Hemiptera) are special insects that feed on Calotropis procera (Apocynaceae) (Sodom Apple). At least 35 chemically distinct cardenolides have been reported in C. procera. Objective: We aimed to evaluate the interaction between cardenolides and Na+/K+ ATPases from herbivores. Methods: The Na+/K+ ATPases from these insects were modeled, and docking studies were performed involving cardenolides from C. procera. Results: The replacement of serine in sensitive Na+/K+ ATPase by histidine, phenylalanine, and tyrosine in the structures examined suggested spatial impairment caused by interaction, probably making the herbivorous insects resistant against the cardenolides of C. procera. In addition, the ability of the insects to avoid cardenolide toxicity was not correlated with cardenolide polarity. Therefore, the plant fights predation through molecular diversity, and the insects, regardless of their taxonomy, face this molecular diversity through amino acid replacements at key positions of the enzyme targeted by the cardenolides. Conclusions: The results show the arsenal of chemically distinct cardenolides synthesized by the C. procera.

Vigna unguiculata is an important source of proteins and energy for humans and animals. However, postharvest losses caused by Callosobruchus maculatus can reach from 20 to 100% of stored seeds. In this study, the insecticide potential of compounds extracted from Himatanthus drasticus latex was assessed. The latex was extracted with ethanol (70%) and then partitioned through sequential use of hexane and chloroform. These fractions were investigated by chromatography to determine their chemical composition. Plumieride, identified in a hydroalcoholic subfraction, was tested for insecticidal activity against C. maculatus. The ethanolic fraction (LC50 = 0.109; LC90 = 0.106%) and the plumieride (LC50 = 0.166; LC90 = 0.167%) were lethal to larvae. Plumieride (0.25%) delayed larval development, and mortality reached 100%. Its inhibitory action on intestinal α-amylase from larvae was higher (89.12%) than that of acarbose (63.82%). Plumieride (0.1%) inhibited the enzyme α-amylase in vivo in the larval intestine. This result was confirmed by a zymogram test performed by SDS-PAGE when the enzyme electrophoresed on gel copolymerized with starch. When spread on seeds, the hydroalcoholic fraction (1.0%) reduced infestation. The loss of seed mass was 5.26% compared to the control (44.97%). The results confirm the effect of latex compounds in protecting stored seeds against weevil infestation.

Latex is an endogenous fluid exuded by some plants after injury. It is rich in different molecules and has shown many biological activities against different fungi and insects. This work aimed to isolate a peptide fraction from Calotropis procera (PepCp) latex and to evaluate its activity against two fungi and an insect. The chromatographic profile showed that PepCp is constituted mainly by 7 kDa peptides. PepCp showed a low effect against the spore germination of both fungi Colletotrichum gloeosporioides and Fusarium solani. However, PepCp (1.25 mg mL−1) inhibited the mycelial growth of C. gloeosporioides by 80%. On the other hand, PepCp, 0.05% (w/w), reduced the larval weight of Callosobruchus maculatus by 90%, and at 0.2%, no larva was found. At 0.05%, there was a delay of 2 days in the emergence of adult insects, while at 0.1%, no emergence of adult insects was noticed. Our results corroborate the hypothesis of the defense role played by latex and that PepCp can be an interesting biological source of new fungicidal or insecticidal peptides.

Calotropis procera cysteine peptidases (CpCPs) have presented several potential biotechnological applications. Here, these enzymes were immobilized on glyoxyl-agarose (glyoxyl-CpCPs) with yields of 90-95 % and the recovered activities ranged from 10 % to 15 %, according to enzyme loadings (5, 10, 20, 40, and 50 mgBSAeq/g). Spectrophotometric assays and SDS-PAGE showed that the casein hydrolysis by glyoxyl-CpCPs was similar to soluble CpCPs. In addition, glyoxyl-CpCPs exhibited similar ratio of milk-clotting activity to proteolytic activity in comparison with soluble CpCPs and chymosin. Even after being stored for six months at 8 °C, the residual proteolytic activity of glyoxyl-CpCPs remained close to 100 %. Atomic force microscopy and dynamic light scattering techniques showed that the process of casein micelle aggregation after treatment with glyoxyl-CpCPs was very similar to its soluble form and chymosin. Glyoxyl-CpCPs performed well after five reaction cycles, producing cheeses with yield, moisture, protein, and fat similar to those produced with chymosin.

Vaccines could be the solution to the current SARS-CoV-2 outbreak. However, some studies have shown that the immunological memory only lasts three months. Thus, it is imperative to develop pharmacological treatments to cope with COVID-19. Here, the in silico approach by molecular docking, dynamic simulations and quantum biochemistry revealed that ACE2-derived peptides strongly interact with the SARS-CoV-2 RBD domain of spike glycoprotein (S-RBD). ACE2-Dev-PepI, ACE2-Dev-PepII, ACE2-Dev-PepIII and ACE2-Dev-PepIV complexed with S-RBD provoked alterations in the 3D structure of S-RBD, leading to disruption of the correct interaction with the ACE2 receptor, a pivotal step for SARS-CoV-2 infection. This wrong interaction between S-RBD and ACE2 could inhibit the entry of SARS-CoV-2 in cells, and thus virus replication and the establishment of COVID-19 disease. Therefore, we suggest that ACE2-derived peptides can interfere with recognition of ACE2 in human cells by SARS-CoV-2 in vivo. Bioinformatic prediction showed that these peptides have no toxicity or allergenic potential. By using ACE2-derived peptides against SARS-CoV-2, this study points to opportunities for further in vivo research on these peptides, seeking to discover new drugs and entirely new perspectives to treat COVID-19. Communicated by Ramaswamy H. Sarma

A chitosanase (CvCsn46) from Chromobacterium violaceum ATCC 12472 was produced in Escherichia coli, purified, and partially characterized. When subjected to denaturing polyacrylamide gel electrophoresis, the enzyme migrated as two protein bands (38 and 36 kDa apparent molecular masses), which were both identified as CvCsn46 by mass spectrometry. The enzyme hydrolyzed colloidal chitosan, with optimum catalytic activity at 50 °C, and two optimum pH values (at pH 6.0 and pH 11.0). The chitosanolytic activity of CvCsn46 was enhanced by some ions (Ca2+, Co2+, Cu2+, Sr2+, Mn2+) and DTT, whereas Fe2+, SDS and β-mercaptoethanol completely inhibited its activity. CvCsn46 showed a non-Michaelis-Menten kinetics, characterized by a sigmoidal velocity curve (R2 = 0.9927) and a Hill coefficient of 3.95. ESI-MS analysis revealed that the hydrolytic action of CvCsn46 on colloidal chitosan generated a mixture of low molecular mass chitooligosaccharides, containing from 2 to 7 hexose residues, as well as D-glucosamine. The chitosan oligomers generated by CvCsn46 inhibited in vitro the mycelial growth of Lasiodiplodia theobromae, significantly reducing mycelium extension and inducing hyphal morphological alterations, as observed by scanning electron microscopy. CvCsn46 was characterized as a versatile biocatalyst that produces well-defined chitooligosaccharides, which have potential to control fungi that cause important crop diseases.

Calotropis procera produces a milky sap containing proteolytic enzymes. At low concentrations, they induce milk-clotting (60 µg/ml) and to dehair hides (0.05 and 0.1%). A protocol for obtaining the...

Protozoa is a group of microorganisms that cause neglected tropical diseases, such as malaria, Chagas disease, and Leishmaniasis. Due to the growing demand for new therapeutic agents, antimicrobial peptides (AMPs) have gained attention for antiprotozoal action. A systematic literature review described the current scenario of plant and animal AMPs with action antiprotozoal. The terms "antimicrobial peptides", "plant", and "animal" combined with the names of the etiological agents were used in the search. Boolean and Operator were used to connect the terms. The search found 4,825 articles. However, 79 articles were excluded because they were duplicates, and 4,627 were excluded based on title and abstract. Therefore, 119 were evaluated and included here. Of these, the use of antimicrobial peptides of animal origin was predominant. Still, the works with plant peptides focused on the genus Leishmania. Only antimicrobial peptides of animal origin were described for the other genera of protozoa (Toxoplasma spp, Trypanosoma spp, Plasmodium spp). Antimicrobial peptides are an excellent option as a pharmacological tool to fight these infections due to their aggregation and extravasation of cellular content through the formation of pores in the cell membrane of these microorganisms.

Yeasts belonging to the Candida genus are important human pathogens. Candida biofilm is the most common resistance mechanism, which could increase in 1000 times the resistance to antifungal drugs. This study aimed to evaluate the antibiofilm activity of synthetic peptides, as well as action mechanisms and synergistic effect with Nystatin (NYS) and Itraconazole (ITR) by Scanning Electron Microscopy (SEM) and Fluorescence Microscopy (FM). ITR (1000 µg. mL− 1) inhibited 10% of biofilm formation of C. krusei and NYS (1000 µg. mL− 1) 40% of C. albicans. Regarding synergistic effect, peptides enhance 7-fold the action of ITR to inhibit the biofilm formation of C. krusei and C. albicans, as well as the degradation of formed biofilm of C. krusei. The action mechanism of peptides or in combination with antifungal drugs involved cell wall damage, membrane pore formation, loss of cytoplasmic content, and overproduction of reactive oxygen species (ROS). Docking analysis revealed ionic and hydrophobic interactions between peptides and both drugs, which may explain the synergistic effect. Altogether, our results suggest the high potential of synthetic peptides be employed as adjuvants enhancing the activity of antifungal drugs to overcome the resistance provided by fungal biofilm and decrease the toxicity of drugs.

Caseins (αs1-, αs2, β-, and κ-caseins) form the major protein fraction of milk, irrespective of their origins. They are able to form well-ordered colloidal structures in association with colloidal calcium phosphate, named casein micelles. Chymosin-mediated milk coagulation takes place through loss of casein micelles’ stability by hydrolyzing κ-caseins. This process is critical for the quality of cheese and other milk derivatives. Therefore, many microscopy techniques have been used to understand the structural aspects underlying the integrity of casein micelles during chymosin action. However, these technologies can be costly and laborious. In this study, atomic force microscopy (AFM) and dynamic light scattering (DLS) were used to study milk coagulation by chymosin. Following 15 min of chymosin action, the AFM images showed the start of the formation of casein micelle aggregates. After 30–45 min, the micelles continued aggregating, forming structures such as bunches of grapes. Finally, after 45–60 min, these structures formed large clusters of casein micelles. After 60 min, the zeta potential did not drop to zero when the milk clotted, but still had a negative value, suggesting that the κ-caseins on the micelle surface were not totally hydrolyzed. The results corroborate those previously described and suggest this tool as an alternative to study the milk-clotting process at the ultrastructural level induced by proteases.

Latex, a sap produced by cells called laticifers, occurs in plants of wide taxonomic diversity. Plants exude latex sap in response to physical damage. Questions about the function of latex or the underlying mechanisms persist, but a role in defense is likely. The presence of constitutive peptidases in latex sap in addition to inducible and de novo synthesized pathogenesis-related proteins (PR-proteins), raises the question about the role that each sap component plays to protect plants and how synergism occurs among sap proteins in the course of herbivory or infection. Here we discuss a variety of functions for laticifer and latex in plant defense. We propose that latex peptidases build the front line of defense against herbivores or pathogens.

L: operculata is a plant commonly found in the North and Northeast of Brazil. Although the regional population knows its medicinal potential, there are few scientific studies about its antimicrobial potential. Thus, this study aimed to characterize the proteins from L. operculata seeds extracted using different solutions and evaluate their antimicrobial potentials. The protein extracts obtained with NaCl and sodium acetate buffer presented the best inhibitory activities against Candida albicans and C. krusei. The study of the mechanism of action revealed proteins from L. operculata seeds induced pore formation on the membrane and ROS overaccumulation. Scanning Electron Microscopy images also showed severe morphological changes in Candida albicans and C. krusei. Proteins from L.operculata seeds did not show antibacterial activity. The enzymatic assays revealed the presence of proteolytic enzymes, serine and cysteine protease inhibitors, and chitinases in both protein extracts. Proteomic analysis by LC-ESI-MS/MS identified 57 proteins related to many biological processes, such as defense to (a)biotic stress, energetic metabolism, protein folding, and nucleotide metabolism. In conclusion, the L. operculata seed proteins have biotechnological potential against the human pathogenic yeasts Candida albicans and C. krusei.

Latex proteins extracted from Plumeria pudica (LPPp) have anti-inflammatory activity in different experimental models. Considering this relevant result, we evaluated the toxicological aspects of the treatment of mice with LPPp. Acute and subchronic toxicities were determined by daily intraperitoneal administration of 40 mg/kg of LPPp for 10 or 20 days, respectively, and were followed by behavioral, hematological, biochemical and histopathological evaluation. Results showed no significant changes in body weight and organs of animals treated with LPPp. Total and differential blood leukocyte counts of LPPp groups did not differ from controls. There was no significant difference in aspartate aminotransferase, alanine aminotransferase, creatinine and urea measurements for the group treated for 20 days. The level of glutathione in the kidney was significantly higher in animals treated with LPPp in the acute toxicity test, but no differences were observed for the subchronic evaluation. The concentration of malondialdehyde and myeloperoxidase activity in the organs did not differ from controls. Histopathological examination of kidney, spleen and liver tissue of animals treated with LPPp revealed normal structures or reversible alterations. Intraperitoneal LD50 of LPPp was higher than 2,000 mg/kg. The data obtained reveal that LPPp is nontoxic at 40 mg/kg. Las proteínas extraídas del látex de Plumeria pudica (LPPp) tienen actividad antiinflamatoria en diferentes modelos experimentales. Ante este relevante resultado, evaluamos los aspectos toxicológicos del tratamiento de ratones con LPPp. La toxicidad aguda y subcrónica se determinó mediante la administración intraperitoneal diaria de 40 mg/kg de LPPp durante 10 o 20 días, respectivamente, y fueron seguidas por evaluación conductual, hematológica, bioquímica e histopatológica. Los resultados no mostraron cambios significativos en el peso corporal y los órganos de los animales tratados con LPPp. El recuento de leucocitos total y diferencial en la sangre de los grupos LPPp no difirió de los controles. No hubo diferencias significativas en las mediciones de aspartato aminotransferasa, alanina aminotransferasa, creatinina y urea para el grupo tratado durante 20 días. El nivel de glutatión en los riñones fue significativamente mayor en los animales tratados con LPPp en la prueba de toxicidad aguda, pero no se observaron diferencias para la evaluación subcrónica. La concentración de malondialdehído y la actividad de la mieloperoxidasa en los órganos no difirieron de los controles. El examen histopatológico del tejido renal, el bazo y el hígado de los animales tratados con LPPp reveló estructuras normales o cambios reversibles. La LD50 de LPPp administrada por vía intraperitoneal fue superior a 2000 mg / kg. Los datos obtenidos revelan que LPPp no es tóxico a dosis de 40 mg/kg. Proteínas extraídas do látex de Plumeria pudica (LPPp) apresentam atividade anti-inflamatória em diferentes modelos experimentais. Diante desse relevante resultado, avaliamos os aspectos toxicológicos do tratamento de camundongos com LPPp. A toxicidade aguda e subcrônica foram determinadas pela administração intraperitoneal diária de 40 mg/kg de LPPp por 10 ou 20 dias, respectivamente, e foram seguidas por avaliação comportamental, hematológica, bioquímica e histopatológica. Os resultados não mostraram alterações significativas no peso corporal e órgãos dos animais tratados com LPPp. A contagem total e diferencial de leucócitos no sangue dos grupos LPPp não diferiu dos controles. Não houve diferença significativa nas medidas de aspartato aminotransferase, alanina aminotransferase, creatinina e uréia para o grupo tratado por 20 dias. O nível de glutationa nos rins foram significativamente maior nos animais tratados com LPPp no teste de toxicidade aguda, mas nenhuma diferença foi observada para a avaliação subcrônica. A concentração de malondialdeído e a atividade da mieloperoxidase nos órgãos não diferiram dos controles. O exame histopatológico do tecido renal, do baço e do fígado de animais tratados com LPPp revelou estruturas normais ou alterações reversíveis. A DL50 de LPPp administrada por via intraperitoneal foi superior a 2.000 mg/kg. Os dados obtidos revelam que LPPp não é tóxico na dose de 40 mg/kg.

Background Previous studies have shown that latex proteins from Plumeria pudica (LPPp) have anti-inflammatory and antioxidant activity. Therefore, the aim of this study was to evaluate the effects in rats of LPPp on ligature-induced periodontitis, an inflammatory disease. Methods The animals were divided into groups: saline (animals without induction of periodontitis); periodontitis (induced periodontitis and untreated) and LPPp (induced periodontitis and treated with 40 mg/kg). The following parameters were evaluated after 20 consecutive days of treatment: gingival bleeding index (GBI), probing pocket depth (PPD), alveolar bone height (ABH) and gingival myeloperoxidase (MPO) activity. In the hepatic tissue, malondialdehyde (MDA), glutathione (GSH) and histopathological alterations were evaluated. Blood levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. Results Significant reduction of GBI, PPD and gingival MPO activity and ABH was seen in animals treated with LPPp compared to periodontitis. Values of GSH, MDA, ALT and histopathological evaluation were preserved in animals treated with LPPp. Conclusions Treatment with LPPp improved clinical aspects of periodontitis, reduced the blood and hepatic alterations and prevented alveolar bone loss. Data suggest that LPPp have potential for treatment of periodontitis.

The recent outbreak caused by SARS-CoV-2 continues to threat and take many lives all over the world. The lack of an efficient pharmacological treatments are serious problems to be faced by scientists and medical staffs worldwide. In this work, an in silico approach based on the combination of molecular docking, dynamics simulations, and quantum biochemistry revealed that the synthetic peptides RcAlb-PepI, PepGAT, and PepKAA, strongly interact with the main protease (Mpro) a pivotal protein for SARS-CoV-2 replication. Although not binding to the proteolytic site of SARS-CoV-2 Mpro, RcAlb-PepI, PepGAT, and PepKAA interact with other protein domain and allosterically altered the protease topology. Indeed, such peptide-SARS-CoV-2 Mpro complexes provoked dramatic alterations in the three-dimensional structure of Mpro leading to area and volume shrinkage of the proteolytic site, which could affect the protease activity and thus the virus replication. Based on these findings, it is suggested that RcAlb-PepI, PepGAT, and PepKAA could interfere with SARS-CoV-2 Mpro role in vivo. Also, unlike other antiviral drugs, these peptides have no toxicity to human cells. This pioneering in silico investigation opens up opportunity for further in vivo research on these peptides, towards discovering new drugs and entirely new perspectives to treat COVID-19. Communicated by Ramaswamy H. Sarma

Recently, the dramatic emergence of antimicrobial resistance has received attention from World Health Organization. Synthetic antimicrobial peptides (SAMPs) are considered new weapons to fight against infections caused by multi-drug resistant pathogens. Here, the authors provide an overview of the current research on SAMPs. The focus is SAMPs, how to design them, which features must be considered during design, and comparison with natural peptides. This review also includes a discussion about the natural AMPs, mechanisms of action and applications as new drugs or even as adjuvants molecules to enhance commercial drugs activity. The advances in chemical synthesis have reduced the cost to produce synthetic peptides open ways to achieve new antimicrobial agents. Therefore, synthetic peptides are new promising molecules to safeguard human and animal health.

The global outbreak of COVID-19 (Coronavirus Disease 2019) caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome caused by Coronavirus 2) began in December 2019. Its closest relative, SARS-CoV-1, has a slightly mutated Spike (S) protein, which interacts with ACE2 receptor in human cells to start the infection. So far, there are no vaccines or drugs to treat COVID-19. So, research groups worldwide are seeking new molecules targeting the S protein to prevent infection by SARS-CoV-2 and COVID-19 establishment. We performed molecular docking analysis of eight synthetic peptides against SARS-CoV-2 S protein. All interacted with the protein, but Mo-CBP3-PepII and PepKAA had the highest affinity with it. By binding to the S protein, both peptides led to conformational alterations in the protein, resulting in incorrect interaction with ACE2. Therefore, given the importance of the S protein-ACE2 interaction for SARS-CoV-2 infection, synthetic peptides could block SARS-CoV-2 infection. Moreover, unlike other antiviral drugs, peptides have no toxicity to human cells. Thus, these peptides are potential molecules to be tested against SARS-CoV-2 and to develop new drugs to treat COVID-19., Highlights • Synthetic peptides bind to SARS-CoV-2 Spike protein. • Synthetic peptides induced conformational changes on SARS-CoV-2 spike protein structure. • Synthetic peptides bind to ACE2 protein but did not affect its structure. • Synthetic peptides induced the wrong interaction of SARS-CoV-2 with ACE2 receptor.

BACKGROUND Calotropis procera latex protein fraction (LP) was previously shown to protect animals from septic shock. Further investigations showed that LP modulate nitric oxide and cytokines levels. OBJECTIVES To evaluate whether the protective effects of LP, against lethal bacterial infection, is observed in its subfractions (LPPII and LPPIII). METHODS Subfractions (5 and 10 mg/kg) were tested by i.p. administration, 24 h before challenging with lethal injection (i.p.) of Salmonella Typhimurium. LPPIII (5 mg/kg) which showed higher survival rate was assayed to evaluate bacterial clearance, histopathology, leukocyte recruitment, plasma coagulation time, cytokines and NO levels. FINDINGS LPPIII protected 70% of animals of death. The animals given LPPIII exhibited reduced bacterial load in blood and peritoneal fluid after 24 h compared to the control. LPPIII promoted macrophage infiltration in spleen and liver. LPPIII restored the coagulation time of infected animals, increased IL-10 and reduced NO in blood. MAIN CONCLUSIONS LPPIII recruited macrophages to the target organs of bacterial infection. This addressed inflammatory stimulus seems to reduce bacterial colonisation in spleen and liver, down regulate bacterial spread and contribute to avoid septic shock.

Phytomodulatory proteins from the latex of the medicinal plant Calotropis procera has been shown to be implicated in many pharmacological properties. However there is no current information about their activity on glucose metabolism, although the latex is used in folk medicine for treating diabetes. In this study the phytomodulatory proteins (LP) from C. procera latex were assessed on glycemic homeostasis. Control animals received a single intravenous dose (5 mg/kg) of LP or saline solution (CTL). Four hours after treatment, the animals were euthanized and their livers were excised for analysis by western blot and RT-PCR AMP-activated protein kinase (AMPK), phosphoenolpyruvate carboxykinase (PEPCK) and tumor necrosis factor alpha (TNF-α). In vivo tests of intraperitoneal tolerance to insulin, glucose and pyruvate were also performed, and the effect of LP administration on fed glycemia was studied followed by blood analysis to determine serum insulin levels. Treatment with LP reduced glycemia two hours after glucose administration (LP: 87.2 ± 3.70 mg/dL versus CTL: 115.6 ± 8.73 mg/dL). However, there was no change in insulin secretion (CTL: 14.16 ± 0.68 mUI/mL and LP: 14.96 ± 0.55 mUI/mL). LP improved the insulin sensitivity, represented by a superior glucose decay constant during an insulin tolerance test (kITT) (4.17 ± 0.94%/min) compared to the CTL group (0.82 ± 0.72%/min), and also improved glucose tolerance at 30 min (105.2 ± 12.4 mg/dL versus 154.2 ± 18.51 mg/dL), while it decreased hepatic glucose production at 15 and 30 min (LP: 75.5 ± 9.31 and 52.5 ± 12.05 mg/dL compared to the CTL: 79.0 ± 3.02 and 84.5 ± 7.49 mg/dL). Furthermore, there was a significant inhibition of gene expression of PEPCK (LP: 0.66 ± 0.06 UA and CTL: 1.14 ± 0.22 UA) and an increase of phosphorylated AMPK (LP: 1.342 ± 0.21 UA versus CTL: 0.402 ± 0.09 UA). These findings confirm the effect of LP on glycemic control and suggest LP may be useful in diabetes treatment. However, the pharmacological mechanism of LP in PEPCK modulation still needs more clarification.

Antimicrobial peptides (AMPs) have been reported to be promising alternatives to chemical preservatives. Thus, this study aimed to characterise AMPs generated from the hydrolysis of wheat gluten proteins using latex peptidases of Calotropis procera, Cryptostegia grandiflora, and Carica papaya. The three hydrolysates (obtained after 16 h at 37 °C, using a 1: 25 enzyme: substrate ratio) inhibited the growth of Aspergillus niger, A. chevalieri, Trichoderma reesei, Pythium oligandrum, Penicillium sp., and Lasiodiplodia sp. by 60-90%, and delayed fungal growth on bread by 3 days when used at 0.3 g/kg. Moreover, the specific volume and expansion factor of bread were not affected by the hydrolysates. Of 28 peptides identified, four were synthesised and exhibited activity against Penicillium sp. Fluorescence and scanning electron microscopy suggested that the peptides damaged the fungal plasma membrane. Bioinformatics analysis showed that no peptide was toxic and that the antigenic ones had cleavage sites for trypsin or pepsin.

Transitory allergies to cow milk proteins in infants or adults have become a public health problem. Although extensively or partially hydrolyzed cow milk protein formulas are available, these products are costly. Therefore, studies into innovative enzymes to digest cow milk proteins are needed. Danaus plexippus gut peptidases were purified and examined with regard to cow milk protein hydrolysis. The peptidases hydrolyzed caseins and whey proteins. However, after heat treatment, there was a significant improvement in β-lactoglobulin hydrolysis. The hydrolyzed cow milk proteins were not recognized by anti-casein antibodies and only reacted slightly with antibodies against whey proteins. This performance was better than that of partially hydrolyzed formulas and similar to that of an extensively hydrolyzed formula. These results suggest that D. plexippus gut peptidases are suitable and innovative enzymes to produce hypoallergenic cow milk protein formulas.

Fungal contamination is among the main reasons for food spoilage, affecting food safety and the economy. Among fungi, Penicillium digitatum is a major agent of this problem. Here, the in vitro activity of eight synthetic antimicrobial peptides was assessed against P. digitatum, and their action mechanisms were evaluated. All peptides were able to inhibit fungal growth. Furthermore, atomic force and fluorescence microscopies revealed that all peptides targeted the fungal membrane leading to pore formation, loss of internal content, and death. The induction of high levels of reactive oxygen species (ROS) was also a mechanism employed by some peptides. Interestingly, only three peptides (PepGAT, PepKAA, and Mo-CBP3-PepI) effectively control P. digitatum colonization in orange fruits, at a concentration (50 µg mL−1) 20-fold lower than the commercial food preservative (sodium propionate). Altogether, PepGAT, PepKAA, and Mo-CBP3-PepI showed high biotechnological potential as new food preservatives to control food infection by P. digitatum.

Germin-like proteins (GLPs) have been identified in several plant tissues. However, only one work describes GLP in latex fluids. Therefore, the goal of this study was to investigate GLPs in latex and get new insights concerning the structural and functional aspects of these proteins. Two complete sequences with high identity (>50%) with other GLPs, termed CpGLP1 and CpGLP2, were obtained and consecutively presented 216 and 206 amino acid residues, corresponding to molecular masses of 22.7 and 21.7kDa, pI 6.8 and 6.5. The three-dimensional models revealed overall folding similar to those reported for other plant GLPs. Both deduced sequences were grouped into the GER 2 subfamily. Molecular docking studies indicated a putative binding site consisting of three highly conserved histidines and a glutamate residue, which interacted with oxalate. This interaction was later supported by enzymatic assays. Superoxide dismutase (common activity in GLPs) was not detected for CpGLP1 and CpGLP2 by zymogram. The two proteins were detected in the latex, but not in non-germinated or germinated seeds and calli. These results give additional support that germin-like proteins are broadly distributed in plants and they are tissue-specific. This particularity deserves further studies to better understand their functions in latex.

The latex of Calotropis procera has previously been reported to contain osmotin. This protein (CpOsm) inhibited phytopathogens and this was mechanistically characterized. Here, the time-course profile of CpOsm transcripts was examined in the salt-stressed cultivated callus of C. procera in order to better understand its role in the physiology of the plant. Stressed callus (80 mM NaCl) showed an unbalanced content of organic compounds (proline and total soluble sugar) and inorganic ions (Na+, Cl−, and K+). Under salt treatment, the transcripts of CpOsm were detected after 12 h and slightly increased to a maximum at day seven, followed by reduction. Interestingly, CpOsm was not detected in the soluble protein fraction recovered from the salt-stressed callus as probed by electrophoresis, dot/Western blotting and mass spectrometry. The results suggested that (1) CpOsm is not constitutive in cultivated cells (laticifer-free tissues); (2) CpOsm transcripts appear under salt-stressed conditions; (3) the absence of CpOsm in the protein fractions of stressed cultivated cells indicated that salt-induced transcripts were not used for protein synthesis and this accounts to the belief that CpOsm may be a true laticifer protein in C. procera. More effort will be needed to unveil this process. In this study we show evidences that CpOsm gene is responsive to salt stress. However the corresponding protein is not produced in cultivated cells. Therefore, presently the hypothesis that CpOsm is involved in abiotic stress is not fully supported.

As proteins isolated from the Rhinella schneideri parotoid gland secretion (RsPP) exhibit anti-inflammatory activity, the goal of this work was to investigate their anti-nociceptive effects using acetic acid-induced writhing, formalin, and hot-plate tests. The intraperitoneal administration of RsPP (2.5 or 5 mg/kg) one hour prior to stimuli significantly reduced the abdominal constrictions induced by acetic acid (73.06 and 72.69% inhibition, respectively) and the inflammatory phase of paw licking time induced by formalin (69.3% inhibition, at 2.5 mg/kg). However, RsPP (1, 2.5 or 5 mg/kg) did not change the latency in response at the hot-plate test. The involvement of inflammatory mediators on the anti-nociceptive effect of RsPP was further demonstrated. RsPP (2.5 mg/kg) significantly inhibited the inflammatory peak of paw edema induced by histamine (44.0%), bradykinin (51.3%), or prostaglandin E2 (53.7%). Our data indicate that RsPP may act on the pain process by inhibiting the effect of inflammatory mediators.

Dehairing of crude leather is a critical stage performed at the beginning of its processing to obtain industrially useful pieces. Tanneries traditionally apply a chemical process based on sodium sulfide. Since this chemical reactive is environmentally toxic and inefficiently recycled, innovative protocols for reducing or eliminating its use in leather depilation are welcomed. Therefore, latex peptidases from Calotropis procera (CpLP) and Cryptostegia grandiflora (CgLP) were assayed for this purpose. Enzyme activity on substrates representative of skin such as hide powder azure (UHPA), elastin (UE), azocollagen (UAZOCOL), keratin (UK), and epidermis (UEP) was determined, while depilation activity was assayed on cow hide. Only CpLP was active against keratin (13.4 UK) and only CgLP was active against elastin (0.12 UE). CpLP (93.0 UHPA, 403.6 UAZOCOL, 36.3 UEP) showed higher activity against the other substrates than CgLP (47.6 UHPA, 261.5 UAZOCOL, 8.5 UEP). In pilot assays, CpLP (0.05% w/v with sodium sulfite 0.6% w/v as activator) released hairs from cow hide pieces. Macroscopic and microscopic analyses of the hide revealed that the dehairing process was complete and the leather structure was preserved. The proteolytic system of C. procera is a suitable bioresources to be exploited by tanneries. Fil: Lopez, Laura Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Industrial. Centro de Investigación y Desarrollo del Cuero. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Centíficas. Centro de Investigación y Desarrollo del Cuero; Argentina Fil: Viana, Carolina A.. Universidade Estadual Do Ceara; Brasil Fil: Errasti, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Industrial. Centro de Investigación y Desarrollo del Cuero. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Centíficas. Centro de Investigación y Desarrollo del Cuero; Argentina Fil: Garro, María Laura. Instituto Nacional de Tecnología Industrial. Centro de Investigación y Desarrollo del Cuero. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Centíficas. Centro de Investigación y Desarrollo del Cuero; Argentina Fil: Martegani, José Eduardo. Instituto Nacional de Tecnología Industrial. Centro de Investigación y Desarrollo del Cuero. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Centíficas. Centro de Investigación y Desarrollo del Cuero; Argentina Fil: Mazzilli, German Ariel. Instituto Nacional de Tecnología Industrial. Centro de Investigación y Desarrollo del Cuero. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Centíficas. Centro de Investigación y Desarrollo del Cuero; Argentina Fil: Freitas, Cléverson D. T.. Universidade Estadual Do Ceara; Brasil Fil: Araújo, Ídila M. S.. EMBRAPA Agrotropical; Brasil Fil: da Silva, Rafaela O.. Universidade Estadual Do Ceara; Brasil Fil: Ramos, Márcio V.. Universidade Estadual Do Ceara; Brasil

The emergence of antibiotic-resistant microbes has stimulated research worldwide seeking new biologically active molecules. In this respect, synthetic antimicrobial peptides (SAMPs) have been suggested to overcome this problem. Although there are some online servers that provide putative SAMPs from protein sequences, the choice of the best peptide sequences for further analysis is still difficult. Therefore, the goal of this paper is not to launch a new tool but to provide a friendly workflow to characterize and predict potential SAMPs by employing existing tools. Using this proposed workflow, two peptides (PepGAT and PepKAA) were obtained and extensively characterized. These peptides damaged microbial membranes and cell walls, and induced overproduction of reactive oxygen species (ROS). Both peptides were found to assume random coil secondary structure in aqueous solution, organic solvent, and upon binding to negatively charged lipid systems. Peptides were also able to degrade formed biofilms but not to prevent biofilm formation. PepGAT was not resistant to proteolysis, whereas PepKAA was resistant to pepsin but not to pancreatin. Furthermore, both presented no hemolytic activity against red blood cells, even at a 10-fold higher concentration than the antimicrobial concentration. The pipeline proposed here is an easy way to design new SAMPs for application as alternatives to develop new drugs against human pathogenic microorganisms.

Candida albicans has emerged as a major public health problem in recent decades. The most important contributing factor is the rapid increase in resistance to conventional drugs worldwide. Synthetic antimicrobial peptides (SAMPs) have attracted substantial attention as alternatives and/or adjuvants in therapeutic treatments due to their strong activity at low concentrations without apparent toxicity. Here, two SAMPs, named Mo-CBP3 -PepI (CPAIQRCC) and Mo-CBP3 -PepII (NIQPPCRCC), are described, bioinspired by Mo-CBP3 , which is an antifungal chitin-binding protein from Moringa oleifera seeds. Furthermore, the mechanism of anticandidal activity was evaluated as well as their synergistic effects with nystatin. Both peptides induced the production of reactive oxygen species (ROS), cell wall degradation, and large pores in the C. albicans cell membrane. In addition, the peptides exhibited high potential as adjuvants because of their synergistic effects, by increasing almost 50-fold the anticandidal activity of the conventional antifungal drug nystatin. These peptides have excellent potential as new drugs and/or adjuvants to conventional drugs for treatment of clinical infections caused by C. albicans.

Laticifers are present in plants belonging to a broad range of taxonomic groups, and originated independently during the evolution of species. Although laticifers are cells with specialized metabolism, their ultrastructural aspects are closely similar among different species. They appear early after seed germination and elongate throughout the plant body. Recent evidence suggests that independent clusters of laticifers may arise in different organs and tissues of the plant and then exhibit distinct chemical and biochemical contents. Many proteins and secondary metabolites are synthesized and stored in laticifers. Despite similarities in metabolism, proteomes and the secondary metabolites existing in laticifers are distinct among species even when they are closely related taxonomically. Studies of molecular composition, structural aspects and activities of latex molecules suggest that laticifers and latex are strongly involved in plant defense.

Ethnopharmacological relevance Himatanthus drasticus is an important medicinal plant whose latex is traditionally used in Northeast Brazil to treat various diseases, including diabetes. The use of α-amylase and α-glucosidase inhibitors can be an effective strategy to modulate levels of postprandial hyperglycemia via control of starch metabolism. Aims of the study This study aimed to verify if H. drasticus latex has inhibitory activity against enzymes linked to type 2 diabetes, besides chemically characterizing the metabolites responsible for such activities. In addition, in silico analysis was performed to support the traditional claim of possible antidiabetic activity of this latex. Materials and methods Latex from H. drasticus stems was sequentially partitioned with n-hexane (FHDH), CHCl3 (FHDC) and EtOH (FHDHA). Wash extraction of the FHDHA fraction was performed to obtain the other extract fractions. The FHDHA was submitted to chromatography in a SPE C18 cartridge using gradient elution with MeOH/H2O to produce five fractions: FHDHA1, FHDHA2, FHDHA3, FHDHA4 and FHDHA5. The FHDHA1 was subjected to semi-preparative reverse phase HPLC. Lineweaver-Burk plots were used to investigate the kinetic parameters of α-amylase and α-glucosidase inhibitory activity. The interactions between plumieride and porcine pancreatic α-amylase and α-glucosidase were analyzed through an in silico molecular docking study. Results Phytochemical identification of compounds present in the FHDHA fraction of H. drasticus latex was possible by 1H, 13C NMR analysis and mass spectrometry, and the results were compared with the literature. The identified compounds were α-ethyl glucoside, protocatechuic acid, 3-O-caffeoylquinic acid, 15-demethylplumieride acid, 5-O-caffeoylquinic acid, caffeic acid, vanillic acid, plumieride, and catechin. The inhibition results of the fractions tested against α-amylase and α-glucosidase showed inhibitory activities dependent on the increase of fractions and compound concentrations. The IC50 results obtained from FHDHA, FHDHA1 and plumieride fractions against α-amylase were 36.46, 72.61, 33.87 μg/mL respectively. The IC50 of plumieride was the closest to that of acarbose (22.52 μg/mL), a result similar to that obtained for α-glucosidase. The type of inhibition was competitive for both enzymes. Conclusions There was strong inhibition of α-amylase and α-glucosidase by FHDHA, FHDHA1 and plumieride, suggesting that these enzymes slow glucose absorption.

Plant seeds can exudate active molecules with inhibitory effects against several soil pathogens, including nematodes. This study aimed to characterize and evaluate the nematicidal properties against Meloidogyne incognita of exuded proteins from Moringa oleifera seeds. M. oleifera seeds were soaked in distilled water, and exudates were harvested and analyzed for the presence of defense proteins and anthelmintic activity. Enzymatic assays revealed the existence of PR-proteins such as β-1,3-glucanases (0.18 ± 0.003 nkatal mg−1 of protein), chitinases (0.22 ± 0.004 nkatal mg−1 of protein), proteases (261.30 ± 6.405 AU mg−1 of protein min−1), serine (190.30 ± 5.574 IA mg−1 of protein) and cysteine (231.70 ± 0.923 IA mg−1 of protein), protease inhibitors. The exuded proteins presented ovicidal activity and caused 100% mortality of second-stage juveniles (J2s). Scanning electron microscopy (SEM) revealed deleterious effects on M. incognita eggs, such as invaginations, cracks, scratched surface, and loss of internal content. These findings confirm the presence of anthelmintic proteins in M. oleifera seed exudate, possibly involved in plant defense during seed germination. Besides this, the exuded proteins exhibited strong biotechnological potential for use in the biocontrol of M. incognita infections, which are responsible for millions of dollars in staple crop losses every year.

BACKGROUND Severe bacterial infections initiate inadequate inflammation that leads to disseminated intravascular coagulation and death. OBJECTIVES To evaluate the influence of bacterial infection on blood viscosity and red blood cells (RBCs) morphology, and the ability of Calotropis procera proteins (CpLP) to prevent the patho-hemorheology in infected animals. METHODS Rheology of blood, atomic force microscopy measurements on specific blood elements and blood count were performed to examine changes in blood viscosity, RBCs morphology, platelets activation, and RBCs indices. FINDINGS Infected mice hold their blood rheological behaviour as compared to that of the control group. However, they presented hyperactivated platelets, RBCs at different stages of eryptosis, and variation on RBCs indices. CpLP administration in healthy animals altered blood behaviour from pseudoplastic to Bingham-like fluid. Such effect disappeared over time and by inhibiting its proteases. No alterations were observed in RBCs morphology or platelets. Treatment of infected animals with CpLP prevented the changes in RBCs indices and morphology. MAIN CONCLUSIONS The inflammatory process triggered by bacterial infection induced pathological changes in RBCs and platelets activation. Treatment of infected animals with CpLP prevented the emergence of RBCs abnormal morphology and this may have implications in the protective effect of CpLP, avoiding animal death.

Background: Osmotin-Like Proteins (OLPs) have been purified and characterized from different plant tissues, including latex fluids. Besides its defensive role, tobacco osmotin seems to induce adiponectin-like physiological effects, acting as an agonist. However, molecular information about this agonistic effect on adiponectin receptors has been poorly exploited and other osmotins have not been investigated yet. Objective and Methods: The present study involved the characterization of three OLPs from Plumeria rubra latex and molecular docking studies to evaluate the interaction between them and adiponectin receptors (AdipoR1 and AdipoR2). Results: P. rubra Osmotin-Like Proteins (PrOLPs) exhibited molecular masses from 21 to 25 kDa and isoelectric points ranging from 4.4 to 7.7. The proteins have 16 cysteine residues, which are involved in eight disulfide bonds, conserved in the same positions as other plant OLPs. The threedimensional (3D) models exhibited the three typical domains of OLPs, and molecular docking analysis showed that two PrOLP peptides interacted with two adiponectin receptors similarly to tobacco osmotin peptide. Conclusion: As observed for tobacco osmotin, the latex osmotins of P. rubra exhibited compatible interactions with adiponectin receptors. Therefore, these plant defense proteins (without known counterparts in humans) are potential tools to study modulation of glucose metabolism in type II diabetes, where adiponectin plays a pivotal role in homeostasis.

Chitinases (EC 3.2.1.14) found in the latex of Calotropis procera (Ait) R. Br. were studied. The proteins were homogeneously obtained after two ion exchange chromatography steps. Most proteins were identified individually in 15 spots on 2-D gel electrophoresis with isoelectric points ranging from 4.6 to 6.0 and molecular masses extending from 27 to 30 kDa. Additionally, 66 kDa proteins were identified as chitinases in SDS-PAGE. Their identities were further confirmed by mass spectrometry (MS) analysis of the tryptic digests of each spot and MS analysis of the non-digested proteins. Positive reaction for Schiff's reagent suggested the proteins are glycosylated. The chitinases exhibited high catalytic activity toward to colloidal chitin at pH 5.0, and this activity underwent decay in the presence of increasing amounts of reducing agent dithiothreitol. Spore germination and hyphae growth of two phytopathogenic fungi were inhibited only marginally by the chitinases but were affected differently. This suggested a complex relationship might exist between the specificity of the proteins toward the fungal species. The chitinases showed potent insecticidal activity against the Bruchidae Callosobruchus maculatus, drastically reducing survival, larval weight and adult emergence. It is concluded that closely related chitinases are present in the latex of C. procera, and the first experimental evidence suggests these proteins are involved more efficiently in defence strategies against insects rather than fungi.

Parotoid glands of amphibians are known for the production of several biologically active compounds having pharmacological and toxic effects in mammals. In the present work, a protein fraction obtained from Rhinella schneideri parotoid gland (RsPP) was characterized to study its biological and toxic effects. Rhinella schneideri parotoid secretion is composed of up to 30% (w/w) of soluble proteins. Tandem mass spectrometric analysis of the RsPP identified 104 proteins, including actin, beta-actin, ribosomal proteins, catalase, galectin, and uncharacterized proteins; however, no peptidases were found, and this result was reinforced by the absence of proteolytic activity. In addition, RsPP did not exhibit pro-coagulant or antibacterial effects. However, pretreatment of mice with different doses of RsPP intraperitoneally inhibited carrageenan-induced paw edema and increased tissue myeloperoxidase activity. RsPP also reduced interleukin 1β levels in the peritoneal cavities and cell migration in the peritoneal cavities of an animal model of carrageenan-induced peritonitis. Subchronic treatment of animals with RsPP for 7 consecutive days did not alter the serum biochemical, renal, or liver parameters. However, a significant reduction in blood leukocyte count was observed. Our results showed that R. schneideri parotoid secretion contains proteins with anti-inflammatory and slight toxic effects.

Some amphibian species have developed a breeding strategy in which they deposit their eggs in stable foam nests to protect their eggs and larvae. The frog foam nests are rich in proteins (ranaspumin), especially surfactant proteins, involved in the production of the foam nest. Despite the ecological importance of the foam nests for evolution and species conservation, the biochemical composition, the long-term stability and even the origin of the components are still not completely understood. Recently we showed that Lv-RSN-1, a 23.5-kDa surfactant protein isolated from the nest of the frog Leptodacylus vastus, presents a structural conformation distinct from any protein structures yet reported. So, in the current study we aimed to reveal the protein composition of the foam nest of L. vastus and further characterize the Lv-RSN-1. Proteomic analysis showed the foam nest contains more than 100 of proteins, and that Lv-RSN-1 comprises 45% of the total proteins, suggesting a key role in the nest construction and stability. We demonstrated by Western blotting that Lv-RSN-1 is mainly produced only by the female in the pars convoluta dilata, which highlights the importance of the female preservation for conservation of species that depend on the production of foam nests in the early stages of development. Overall, our results showed the foam nest of L. vastus is composed of a great diversity of proteins and that besides Lv-RSN-1, the main protein in the foam, other proteins must have a coadjuvant role in building and stability of the nest.

Aims According to the WHO, 20–25% of people worldwide are affected by skin infections caused by dermatophytes, such as those of the Trichophyton genus. Additionally, several dermatophytes have developed resistance to drugs such as griseofulvin and itraconazole. This study tested 2S albumins-derived antimicrobial peptides (AMPs) as alternative antidermatophytic molecules. Main methods Membrane pore formation assays, tests to detect overproduction of ROS, scanning electron microscopy (SEM) and fluorescence microscopy (FM) were carried out to provide insight into the mechanisms of antidermatophytic action. Key findings All AMPs (at 50 μg mL−1) tested reduced the mycelial growth of T. mentagrophytes and T. rubrum by up to 95%. In contrast, using a concentration 20-fold higher, griseofulvin only inhibited T. mentagrophytes by 35%, while itraconazole was not active against both dermatophytes. Scanning electron and fluorescence microscopies revealed that the six AMPs caused severe damage to hyphal morphology by inducing cell wall rupture, hyphal content leakage, and death. Peptides also induced membrane pore formation and oxidative stress by overproduction of ROS. Based on the stronger activity of peptides than the commercial drugs and the mechanism of action, all six peptides have the potential to be either employed as models to develop new antidermatophytic drugs or as adjuvants to existing ones. Significance The synthetic peptides are more efficient than conventional drug to treat infection caused by dermatophytes being potential molecules to develop new drugs.

A partial cDNA sequence from Anacardium occidentale CCP 76 was obtained, encoding a GH19 chitinase (AoChi) belonging to class VI. AoChi exhibits distinct structural features in relation to previously characterized plant GH19 chitinases from classes I, II, IV and VII. For example, a conserved Glu residue at the catalytic center of typical GH19 chitinases, which acts as the proton donor during catalysis, is replaced by a Lys residue in AoChi. To verify if AoChi is a genuine chitinase or is a chitinase-like protein that has lost its ability to degrade chitin and inhibit the growth of fungal pathogens, the recombinant protein was expressed in Pichia pastoris, purified and biochemically characterized. Purified AoChi (45 kDa apparent molecular mass) was able to degrade colloidal chitin, with optimum activity at pH 6.0 and at temperatures from 30 °C to 50 °C. AoChi activity was completely lost when the protein was heated at 70 °C for 1 h or incubated at pH values of 2.0 or 10.0. Several cation ions (Al3+, Cd2+, Ca2+, Pb2+, Cu2+, Fe3+, Mn2+, Rb+, Zn2+ and Hg2+), chelating (EDTA) and reducing agents (DTT, β-mercaptoethanol) and the denaturant SDS, drastically reduced AoChi enzymatic activity. AoChi chitinase activity fitted the classical Michaelis-Menten kinetics, although turnover number and catalytic efficiency were much lower in comparison to typical GH19 plant chitinases. Moreover, AoChi inhibited in vitro the mycelial growth of Lasiodiplodia theobromae, causing several alterations in hyphae morphology. Molecular docking of a chito-oligosaccharide in the substrate-binding cleft of AoChi revealed that the Lys residue (theoretical pKa = 6.01) that replaces the catalytic Glu could act as the proton donor during catalysis.

Peruvianin-I is a cysteine peptidase (EC 3.4.22) purified from Thevetia peruviana. Previous studies have shown that it is the only germin-like protein (GLP) with proteolytic activity described so far. In this work, the X-ray crystal structure of peruvianin-I was determined to a resolution of 2.15 A (PDB accession number: 6ORM) and its specific location was evaluated by different assays. Its overall structure shows an arrangement composed of a homohexamer (a trimer of dimers) where each monomer exhibits a typical β-barrel fold and two glycosylation sites (Asn55 and Asn144). Analysis of its active site confirmed the absence of essential amino acids for typical oxalate oxidase activity of GLPs. Details of the active site and molecular docking results, using a specific cysteine peptidase inhibitor (iodoacetamide), were used to discuss a plausible mechanism for proteolytic activity of peruvianin-I. Histological analyses showed that T. peruviana has articulated anastomosing laticifers, i.e., rows of cells which merge to form continuous tubes throughout its green organs. Moreover, peruvianin-I was detected exclusively in the latex. Because latex peptidases have been described as defensive molecules against insects, we hypothesize that peruvianin-I contributes to protect T. peruviana plants against herbivory.

The present study evaluated four laticifer fluids as a novel source of peptidases capable of hydrolyzing proteins in cow’s milk. The latex peptidases from Calotropis procera (CpLP), Cryptostegia grandiflora (CgLP), and Carica papaya (CapLP) were able to perform total hydrolysis of caseins after 30 min at pH 6.5, as confirmed by a significant reduction in the residual antigenicity. Casein hydrolysis by Plumeria rubra latex peptidases (PrLP) was negligible. Moreover, whey proteins were more resistant to proteolysis by latex peptidases; however, heat pretreatment of the whey proteins enhanced the degree of hydrolysis and reduced the residual antigenicity of the hydrolysates. The in vivo assays show that the cow’s milk proteins hydrolysed by CgLP and CapLP exhibited no immune reactions in mice allergic to cow’s milk, similar to a commercial partially hydrolysed formula. Thus, these peptidases are promising enzymes for the development of novel hypoallergenic formulas for children with a milk allergy., Instituto de Estudios Inmunológicos y Fisiopatológicos

The germin-like protein (GLP) purified from Thevetia peruviana, Peruvianin-I, is the only one described as having proteolytic activity. Therefore, the goal of this study was to investigate the structural features responsible for its enzymatic activity. Although the amino acid sequence of Peruvianin-I showed high identity with other GLPs, it exhibited punctual mutations, which were responsible for the absence of oxalate oxidase activity. The phylogenetic analysis showed that Peruvianin-I does not belong to any classification of GLP subfamilies. Moreover, Peruvianin-I contains a catalytic triad found in all plant cysteine peptidases. Molecular docking simulations confirmed the role of the catalytic triad in its proteolytic activity. Synchrotron radiation circular dichroism assays confirmed that Peruvianin-I was stable at pH ranging from 5.0 to 8.0 and that it presented significant structural changes only above 60 °C. The addition of iodoacetamide caused changes in its native conformation, but only a slight effect was observed after adding a reducing agent. This study reports an unusual protein with germin-like structure, lacking typical oxalate oxidase activity. Instead, the proteolytic activity observed suggests that the protein is a cysteine peptidase. These structural peculiarities make Peruvianin‑I an interesting model for further understanding of the action of laticifer fluids in plant defense.

Osmotin- and thaumatin-like proteins (OLPs and TLPs) have been associated with plant defense responses to different biotic stresses. In the present work, several in silico sequences from OLPs and TLPs were investigated by means of bioinformatics tools aiming to prospect for antimicrobial peptides. The peptide sequences chosen were further synthesized and characterized, and their activities and action mechanisms were assayed against some phytopathogenic fungi, bacteria and yeasts of clinical importance. From this survey approach, four peptide sequences (GDCKATSC, CPRALKVPGGCN, IVGQCPAKLKA, and CAADIVGQCPAKLK) were selected considering some chemical parameters commonly attributed to antimicrobial peptides. Antimicrobial assays showed that these peptides were unable to inhibit mycelial growth of phytopathogenic fungi and they did not affect bacterial cell growth. Nevertheless, significant inhibitory activity was found for CPRALKVPGGCN and CAADIVGQCPAKLK against Candida albicans and Saccharomyces cerevisiae. Fluorescence and scanning electron microscopy assays suggested that CAADIVGQCPAKLK did not damage the overall cell structure, or its activity was negligible on yeast membrane and cell wall integrity. However, it induced the production of reactive oxygen species (ROS) and apoptosis. Molecular docking analysis showed that CAADIVGQCPAKLK had strong affinity to interact with specific plasma membrane receptors of C. albicans and S. cerevisiae, which have been described as promoting the induction of apoptosis. The results indicate that CAADIVGQCPAKLK can be a valuable target for the development of a desired antimicrobial agent against the pathogen C. albicans.

The latex from Thevetia peruviana is rich in plant defense proteins, including a 120 kDa cysteine peptidase with structural characteristics similar to germin-like proteins. More than 20,000 plant species produce latex, including Apocynaceae, Sapotaceae, Papaveraceae and Euphorbiaceae. To better understand the physiological role played by latex fluids, a proteomic analysis of Thevetia peruviana (Pers.) Schum latex was performed using two-dimensional gel electrophoresis and mass spectrometry. A total of 33 proteins (86 %) were identified, including storage proteins, a peptidase inhibitor, cysteine peptidases, peroxidases and osmotins. An unusual cysteine peptidase, termed peruvianin-I, was purified from the latex by a single chromatographic step involving gel filtration. The enzyme (glycoprotein) was inhibited by E-64 and iodoacetamide and exhibited high specific activity towards azocasein (K m 17.6 µM), with an optimal pH and temperature of 5.0–6.0 and 25–37 °C, respectively. Gel filtration chromatography, two-dimensional gel electrophoresis, and mass spectrometry revealed that peruvianin-I possesses 120 kDa, pI 4.0, and six subunits (20 kDa). A unique N-terminal amino acid sequence was obtained to oligomer and monomers of peruvianin-I (1ADPGPLQDFCLADLNSPLFINGYPCRNPALAISDDF36). High-resolution images from atomic force microscopy showed the homohexameric structure of peruvianin-I may be organized as a trimer of dimers that form a central channel similar to germin-like proteins. Peruvianin-I exhibited no oxalate oxidase and superoxide dismutase activity or antifungal effects. Peruvianin-I represents the first germin-like protein (GLP) with cysteine peptidase activity, an activity unknown in the GLP family so far.

Proteins that share similar primary sequences to the protein originally described in salt-stressed tobacco cells have been named osmotins. So far, only two osmotin-like proteins were purified and characterized of latex fluids. Osmotin from Carica papaya latex is an inducible protein lacking antifungal activity, whereas the Calotropis procera latex osmotin is a constitutive antifungal protein. To get additional insights into this subject, we investigated osmotins in latex fluids of five species. Two potential osmotin-like proteins in Cryptostegia grandiflora and Plumeria rubra latex were detected by immunological cross-reactivity with polyclonal antibodies produced against the C. procera latex osmotin (CpOsm) by ELISA, Dot Blot and Western Blot assays. Osmotin-like proteins were not detected in the latex of Thevetia peruviana, Himatanthus drasticus and healthy Carica papaya fruits. Later, the two new osmotin-like proteins were purified through immunoaffinity chromatography with anti-CpOsm immobilized antibodies. Worth noting the chromatographic efficiency allowed for the purification of the osmotin-like protein belonging to H. drasticus latex, which was not detectable by immunoassays. The identification of the purified proteins was confirmed after MS/MS analyses of their tryptic digests. It is concluded that the constitutive osmotin-like proteins reported here share structural similarities to CpOsm. However, unlike CpOsm, they did not exhibit antifungal activity against Fusarium solani and Colletotrichum gloeosporioides. These results suggest that osmotins of different latex sources may be involved in distinct physiological or defensive events.

Seeds of native species from the rain forest (Amazon) are source of chitinases and their protein extracts exhibited strong and broad antifungal activity. Numerous plant species native to the Amazon have not yet been chemically studied. Studies of seeds are scarcer, since adversities in accessing study areas and seasonality pose constant hurdles to systematic research. In this study, proteins were extracted from seeds belonging to endemic Amazon species and were investigated for the first time. Proteolytic activity, peptidase inhibitors, and chitinases were identified, but chitinolytic activity predominated. Four proteins were purified through chromatography and identified as lectin and chitinases by MS/MS analyses. The proteins were examined for inhibition of a phytopathogen (Fusarium oxysporum). Analyses by fluorescence microscopy suggested binding of propidium iodide to DNA of fungal spores, revealing that spore integrity was lost when accessed by the proteins. Further structural and functional analyses of defensive proteins belonging to species facing highly complex ecosystems such as Amazonia should be conducted, since these could provide new insights into specificity and synergism involving defense proteins of plants submitted to a very complex ecosystem.