Structural Characterization and Rat Aortic Vascular Reactivity of Bradykinin-Potentiating Peptides (BPPs) from the Snake Venom of Bothrops moojeni from Delta do Parnaíba Region, Brazil.

Snake venoms contain various bradykinin-potentiating peptides (BPPs). First studied for their vasorelaxant properties due to angiotensin converting enzyme (ACE) inhibition, these molecules present a range of binding partners, among them the argininosuccinate synthase (AsS) enzyme. This has renewed interest in their characterization from biological sources and the evaluation of their pharmacological activities. In the present work, the low molecular weight fraction of Bothrops moojeni venom was obtained and BPPs were characterized by mass spectrometry. Eleven BPPs or related peptides were sequenced, and one of them, BPP-Bm01, was new. Interestingly, some oxidized BPPs were detected. The three most abundant peptides were BPP-Bm01, BPP-Bax12, and BPP-13a, and their putative interactions with the AsS enzyme were investigated in silico. A binding cavity for these molecules was predicted, and docking studies allowed their ranking. Three peptides were synthesized and submitted to vasorelaxation assays using rat aortic rings. While all BPPs were active, BPP-Bm01 showed the highest potency in this assay. This work adds further diversity to BPPs from snake venoms and suggests, for the first time, a putative binding pocket for these molecules in the AsS enzyme. This can guide the design of new and more potent AsS activators.

Pseudobombax parvifolium Hydroalcoholic Bark Extract: Chemical Characterisation and Cytotoxic, Mutagenic, and Preclinical Aspects Associated with a Protective Effect on Oxidative Stress.

Plants have long been used in traditional medicine to treat illnesses. Nevertheless, their chemical diversity requires studies to establish the extract dosage and its safe use. Pseudobombax parvifolium, an endemic species of the Brazilian Caatinga biome, is commonly used in folk medicine, due to its anti-inflammatory properties related to cellular oxidative stress; however, its biological properties have scarcely been studied. In this study, we chemically characterized the P. parvifolium hydroalcoholic bark extract (EBHE) and evaluated its cytotoxic, mutagenic, and preclinical aspects, as well as its antioxidant effect. Our phytochemical analysis revealed a significative total polyphenol content and identified loliolide for the first time in this species. Cytotoxicity, mutagenicity, and acute oral and repeated dose indicated no toxic effects on cell culture, Drosophila melanogaster, and Wistar rat exposure to different EBHE concentrations, respectively. Furthermore, we observed a significant decrease in lipid peroxidation and a mild hypoglycemic and hypolipidemic effect with repeated oral dosing of EBHE. Although there were no significant changes in glutathione content, we did observe a significant increase in superoxide dismutase at a dose of 400 mg/kg and in glutathione peroxidase at doses of 100, 200, and 400 mg/kg. These findings suggest that EBHE has potential as a source of bioactive molecules, and it can be used safely in traditional medicine and in the development of herbal medicines for application in the public health system.

Chemical Characterization of Flowers and Leaf Extracts Obtained from Turnera subulata and Their Immunomodulatory Effect on LPS-Activated RAW 264.7 Macrophages.

The anti-inflammatory properties of Turnera subulata have been evaluated as an alternative drug approach to treating several inflammatory processes. Accordingly, in this study, aqueous and hydroalcoholic extracts of T. subulata flowers and leaves were analyzed regarding their phytocomposition by ultrafast liquid chromatography coupled to mass spectrometry, and their anti-inflammatory properties were assessed by an in vitro inflammation model, using LPS-stimulated RAW-264.7 macrophages. The phytochemical profile indicated vitexin-2-O-rhamnoside as an important constituent in both extracts, while methoxyisoflavones, some bulky amino acids (e.g., tryptophan, tyrosine, phenylalanine), pheophorbides, and octadecatrienoic, stearidonic, and ferulic acids were detected in hydroalcoholic extracts. The extracts displayed the ability to modulate the in vitro inflammatory response by altering the secretion of proinflammatory (TNF-α, IL-1ß, and IL-6) and anti-inflammatory (IL-10) cytokines and inhibiting the PGE-2 and NO production. Overall, for the first time, putative compounds from T. subulata flowers and leaves were characterized, which can modulate the inflammatory process. Therefore, the data highlight this plant as an option to obtain extracts for phytotherapic formulations to treat and/or prevent chronic diseases.

Novel disintegrin-like peptides derived from an amphibian skin cDNA sequence of Hypsiboas punctatus.

Disintegrins comprise a family of small proteins that bind to and alter the physiological function of integrins, especially integrins that mediate platelet aggregation in blood. Here, we report a lysine-glycine-aspartic acid (KGD) disintegrin-like motif present in a 15-amino acid residue peptide identified in a cDNA library of the amphibian Hypsiboas punctatus skin. The original peptide sequence was used as a template from which five new analogs were designed, chemically synthesized by solid phase, and tested for disintegrin activity and tridimensional structural studies using NMR spectroscopy. The original amphibian peptide had no effect on integrin-mediated responses. Nevertheless, derived peptide analogs inhibited integrin-mediated platelet function, including platelet spreading on fibrinogen.

Identification of Differential N-Glycan Compositions in the Serum and Tissue of Colon Cancer Patients by Mass Spectrometry.

Colorectal cancer (CRC) ranks second as the leading cause of cancer-related deaths worldwide. N-glycosylation is one of the most common posttranslational protein modifications. Therefore, we studied the total serum N-glycome (TSNG) of 13 colon cancer patients compared to healthy controls using MALDI-TOF/MS and LC-MS. N-glycosylation of cancer tumor samples from the same cohort were further quantified using a similar methodology. In total, 23 N-glycan compositions were down-regulated in the serum of colon cancer patients, mostly galactosylated forms whilst the mannose-rich HexNAc2Hex7, the fucosylated bi-antennary glycan HexNAc4Hex5Fuc1NeuAc2, and the tetra-antennary HexNAc6Hex7NeuAc3 were up-regulated in serum. Hierarchical clustering analysis of TSNG correctly singled out 85% of the patients from controls. Albeit heterogenous, N-glycosylation of tumor samples showed overrepresented oligomannosidic, bi-antennary hypogalactosylated, and branched compositions related to normal colonic tissue, in both MALDI-TOF/MS and LC-MS analysis. Moreover, compositions found upregulated in tumor tissue were mostly uncorrelated to compositions in serum of cancer patients. Mass spectrometry-based N-glycan profiling in serum shows potential in the discrimination of patients from healthy controls. However, the compositions profile in serum showed no parallel with N-glycans in tumor microenvironment, which suggests a different origin of compositions found in serum of cancer patients.

The Antioxidant Peptide Salamandrin-I: First Bioactive Peptide Identified from Skin Secretion of Salamandra Genus (Salamandra salamandra).

Amphibian skin is a multifunctional organ that plays key roles in defense, breathing, and water balance. In this study, skin secretion samples of the fire salamander (Salamandra salamandra) were separated using RP-HPLC and de novo sequenced using MALDI-TOF MS/MS. Next, we used an in silico platform to screen antioxidant molecules in the framework of density functional theory. One of the identified peptides, salamandrin-I, [M + H]+ = 1406.6 Da, was selected for solid-phase synthesis; it showed free radical scavenging activity against DPPH and ABTS radicals. Salamandrin-I did not show antimicrobial activity against Gram-positive and -negative bacteria. In vitro assays using human microglia and red blood cells showed that salamandrin-I has no cytotoxicity up to the concentration of 100 µM. In addition, in vivo toxicity tests on Galleria mellonella larvae resulted in no mortality at 20 and 40 mg/kg. Antioxidant peptides derived from natural sources are increasingly attracting interest. Among several applications, these peptides, such as salamandrin-I, can be used as templates in the design of novel antioxidant molecules that may contribute to devising strategies for more effective control of neurological disease.

Identification and characterization of phospholipases A2 from the skin secretion of Pithecopus azureus anuran.

The present work reports the isolation, characterization and the complete sequence of a phospholipase A2 (PLA2) present in the skin secretion of Pithecopus azureus. Among several peptides and small proteins previously described by our group from some species belonging to this amphibian genus (formerly named Phyllomedusa), a 15 kDa N-glycosylated protein showing PLA2 activity was purified, assayed, sequenced and named Pa-PLA2. The Pithecopus azureus skin phospholipase A2 polypeptide chain is composed by 125 amino acid residues linked by seven disulfide bonds and two N-glycosylated sites (N67 and N108). The Pa-PLA2 enzymatic activity was qualitatively evaluated and compared to classical viperid PLA2 showing that both, native and deglycosylated Pa-PLA2 forms, are catalytically functional. The tridimensional molecular model of Pa-PLA2 indicates that the observed glycan moieties are suggestively placed far from the active site of that enzyme and therefore having little or no significant role on the direct interaction of the Pa-PLA2 catalytic pocket and its substrates.

Lipid transfer proteins in coffee: isolation of Coffea orthologs, Coffea arabica homeologs, expression during coffee fruit development and promoter analysis in transgenic tobacco plants.

The aim of the present study was to perform a genomic analysis of non-specific lipid-transfer proteins (nsLTPs) in coffee. Several nsLTPs-encoding cDNA and gene sequences were cloned from Coffea arabica and Coffea canephora species. In this work, their analyses revealed that coffee nsLTPs belong to Type II LTP characterized under their mature forms by a molecular weight of around 7.3 kDa, a basic isoelectric points of 8.5 and the presence of typical CXC pattern, with X being an hydrophobic residue facing towards the hydrophobic cavity. Even if several single nucleotide polymorphisms were identified in these nsLTP-coding sequences, 3D predictions showed that they do not have a significant impact on protein functions. Northern blot and RT-qPCR experiments revealed specific expression of Type II nsLTPs-encoding genes in coffee fruits, mainly during the early development of endosperm of both C. arabica and C. canephora. As part of our search for tissue-specific promoters in coffee, an nsLTP promoter region of around 1.2 kb was isolated. It contained several DNA repeats including boxes identified as essential for grain specific expression in other plants. The whole fragment, and a series of 5' deletions, were fused to the reporter gene ß-glucuronidase (uidA) and analyzed in transgenic Nicotiana tabacum plants. Histochemical and fluorimetric GUS assays showed that the shorter (345 bp) and medium (827 bp) fragments of nsLTP promoter function as grain-specific promoters in transgenic tobacco plants.

Conformational and functional effects induced by D- and L-amino acid epimerization on a single gene encoded peptide from the skin secretion of Hypsiboas punctatus.

Skin secretion of Hypsiboas punctatus is the source of a complex mixture of bioactive compounds where peptides and small proteins prevail, similarly to many other amphibians. Among dozens of molecules isolated from H. punctatus in a proteomic based approach, we report here the structural and functional studies of a novel peptide named Phenylseptin (FFFDTLKNLAGKVIGALT-NH2) that was purified as two naturally occurring D- and L-Phes configurations. The amino acid epimerization and C-terminal amidation for both molecules were confirmed by a combination of techniques including reverse-phase UFLC, ion mobility mass spectrometry, high resolution MS/MS experiments, Edman degradation, cDNA sequencing and solid-phase peptide synthesis. RMSD analysis of the twenty lowest-energy (1)H NMR structures of each peptide revealed a major 90° difference between the two backbones at the first four N-terminal residues and substantial orientation changes of their respective side chains. These structural divergences were considered to be the primary cause of the in vitro quantitative differences in antimicrobial activities between the two molecules. Finally, both molecules elicited equally aversive reactions in mice when delivered orally, an effect that depended entirely on peripheral gustatory pathways.

Characterization of a novel peptide toxin from Acanthoscurria paulensis spider venom: a distinct cysteine assignment to the HWTX-II family.

Spider venom toxins have raised interest in prospecting new drugs and pesticides. Nevertheless, few studies are conducted with tarantula toxins, especially with species found in Brazil. This study aims to characterize chemically and biologically the first toxin isolated from Acanthoscurria paulensis venom. Ap1a consists of 48 amino acid residues and has a molecular mass of 5457.79 Da. The cloned gene encodes a putative sequence of 23 amino acid residues for the signal peptide and 27 for the pro-peptide. The sequence of the mature peptide is 60-84% identical with those of toxins of the HWTX-II family. Different from the structural pattern proposed for these toxins, the disulfide pairing of Ap1a is of the ICK type motif, which is also shared by the U1-TRTX-Bs1a toxin. Ap1a induced a dose-dependent and reversible paralytic effect in Spodoptera frugiperda caterpillars, with an ED50 of 13.0 ± 4.2 µg/g 8 h after injections. In the Drosophila melanogaster Giant Fiber circuit, Ap1a (1.14-22.82 µg/g) reduces both the amplitude and frequency of responses from GF-TTM and GF-DLM pathways, suggesting an action at the neuromuscular junction, which is mediated by glutamatergic receptors. It is also lethal to mice (1.67 µg/g, intracranial route), inducing effects similar to those reported with intracerebroventricular administration of NMDA. Ap1a (1 µM) does not alter the response induced by acetylcholine on the rhabdomyosarcoma cell preparation and shows no significant effects on hNav1.2, hNav1.4, hNav1.5, and hNav1.6 channels. Because of its unique sequence and cysteine assignment to the HWTX-II family, Ap1a is a significant contribution to the structure-function study of this family of toxins.

Post-secretory events alter the peptide content of the skin secretion of Hypsiboas raniceps.

A novel family of antimicrobial peptides, named raniseptins, has been characterized from the skin secretion of the anuran Hypsiboas raniceps. Nine cDNA molecules have been successfully cloned, sequenced, and their respective polypeptides were characterized by mass spectrometry and Edman degradation. The encoded precursors share structural similarities with the dermaseptin prepropeptides from the Phyllomedusinae subfamily and the mature 28-29 residue long peptides undergo further proteolytic cleavage in the crude secretion yielding consistent fragments of 14-15 residues. The biological assays performed demonstrated that the Rsp-1 peptide has antimicrobial activity against different bacterial strains without significant lytic effect against human erythrocytes, whereas the peptide fragments generated by endoproteolysis show limited antibiotic potency. MALDI imaging mass spectrometry in situ studies have demonstrated that the mature raniseptin peptides are in fact secreted as intact molecules within a defined glandular domain of the dorsal skin, challenging the physiological role of the observed raniseptin fragments, identified only as part of the crude secretion. In this sense, stored and secreted antimicrobial peptides may confer distinct protective roles to the frog.

Novel dermaseptins from Phyllomedusa hypochondrialis (Amphibia).

Six new antimicrobial peptides structurally related to the dermaseptin family have been isolated from the skin secretion of the amphibian Phyllomedusa hypochondrialis. The primary structures of these molecules named as DShypo 01, 02, 03, 04, 06, and 07 were determined by de novo MS/MS experiments, Edman degradation, and cDNA sequencing. The fifth peptide was found to be precisely the same DS 01 from Phyllomedusa oreades previously described by our group. The majority of the peptides purified from the crude skin secretion could be directly localized and mapped onto a freshly dissected dorsal skin fragment using mass spectrometry-imaging techniques. Comparisons between peptides and commercial drugs on their antibacterial and anti-Leishmania amazonensis efficiencies, associated with peptide lytic effects on mammalian blood cells and surface plasmon resonance interaction studies on immobilized DMPC vesicles, were also performed.