The Peptide Salamandrin-I Modulates Components Involved in Pyroptosis and Induces Cell Death in Human Leukemia Cell Line HL-60.

Amphibian secretions have been extensively investigated for the production of bioactive molecules. Salamandrin-I is an antioxidant peptide, isolated from the skin secretion of the fire salamander, that has induced no toxicity in microglia or erythrocytes. Importantly, the administration of antioxidants may constitute an adequate therapeutic approach to cancer treatment. Here, with the purpose of better characterizing the therapeutic potential of salamandrin-I, we investigated whether this antioxidant peptide also exerts anticancer activity, using the human leukemia cell line HL-60 as a cancer model. Salamandrin-I treatment induced a significant reduction in HL-60 proliferation, which was accompanied by cell cycle arrest. Furthermore, the peptide-induced cell death showed a significant increase in the LDH release in HL-60 cells. The cellular toxicity exerted by salamandrin-I is possibly related to pyroptosis, since the HL-60 cells showed loss of mitochondrial membrane potential and hyperexpression of inflammasome components following the peptide treatment. This is the first demonstration of the anticancer potential of the salamandrin-I peptide. Such results are important, as they offer relevant insights into the field of cancer therapy and allow the design of future bioactive molecules using salamandrin-I as a template.

Purification and Biological Properties of Raniseptins-3 and -6, Two Antimicrobial Peptides from Boana raniceps (Cope, 1862) Skin Secretion.

The number of multidrug-resistant pathogenic microorganisms has been growing in recent years, most of which is due to the inappropriate use of the commercial antibiotics that are currently available. The dissemination of antimicrobial resistance represents a serious global public health problem. Thus, it is necessary to search for and develop new drugs that can act as antimicrobial agents. Antimicrobial peptides are a promising alternative for the development of new therapeutic drugs. Anurans' skin glands are a rich source of broad-spectrum antimicrobial compounds and hylids, a large and diverse family of tree frogs, are known as an important source of antimicrobial peptides. In the present study, two novel antimicrobial peptides, named Raniseptins-3 and -6, were isolated from Boana raniceps skin secretion and their structural and biological properties were evaluated. Raniseptins-3 and -6 are cationic, rich in hydrophobic residues, and adopt an α-helix conformation in the presence of SDS (35 mM). Both peptides are active against Gram-negative bacteria and Gram-positive pathogens, with low hemolytic activity at therapeutic concentrations. No activity was observed for yeasts, but the peptides are highly cytotoxic against B16F10 murine melanoma cells and NIH3T3 mouse fibroblast cells. None of the tested compounds showed improvement trends in the MTT and LDH parameters of MHV-3 infected cells at the concentrations tested.

Release of immunomodulatory peptides at bacterial membrane interfaces as a novel strategy to fight microorganisms.

Cationic and amphiphilic peptides can be used as homing devices to accumulate conjugated antibiotics to bacteria-enriched sites and promote efficient microbial killing. However, just as important as tackling bacterial infections, is the modulation of the immune response in this complex microenvironment. In the present report, we designed a peptide chimaera called Chim2, formed by a membrane-active module, an enzyme hydrolysis site and a formyl peptide receptor 2 (FPR2) agonist. This molecule was designed to adsorb onto bacterial membranes, promote their lysis, and upon hydrolysis by local enzymes, release the FPR2 agonist sequence for activation and recruitment of immune cells. We synthesized the isolated peptide modules of Chim2 and characterized their biological activities independently and as a single polypeptide chain. We conducted antimicrobial assays, along with other tests aiming at the analyses of the cellular and immunological responses. In addition, assays using vesicles as models of eukaryotic and prokaryotic membranes were conducted and solution structures of Chim2 were generated by 1H NMR. Chim2 is antimicrobial, adsorbs preferentially to negatively charged vesicles while adopting an α-helix structure and exposes its disorganized tail to the solvent, which facilitates hydrolysis by tryptase-like enzymes, allowing the release of the FPR2 agonist fragment. This fragment was shown to induce accumulation of the cellular activation marker, lipid bodies, in mouse macrophages and the release of immunomodulatory interleukins. In conclusion, these data demonstrate that peptides with antimicrobial and immunomodulatory activities can be considered for further development as drugs.

A first look at the N- and O-glycosylation landscape in anuran skin secretions.

Amphibians secrete a complex array of molecules that shape their interactions with coinhabiting microorganisms and macroscopic predators. Glycans are a rapidly evolving and complex class of biomolecules implicated in intrinsic and extrinsic recognition events. Despite the numerous studies aiming at the biochemical characterization of anuran skin secretions, little is known about protein-linked oligosaccharides, their synthesis pathways, and their homing secreted glycoproteins. In the present report, LC-MS/MS was used to investigate the diversity of N- and O-linked oligosaccharides in the skin secretion of two South American frogs, Pithecopus azureus and Boana raniceps. Additionally, the enzymes responsible for glycan synthesis pathways were evaluated based on their skin tissue transcriptome. Our analyses allowed the annotation of various N- and O-glycan structures commonly found in vertebrate proteins. Paucimannosidic glycans were abundant in the skin secretion of both amphibians; however, hybrid and complex N-glycan structures were detected only in B. raniceps. A good correlation between the structures discovered in glycomic analyses and transcripts encoding enzymes necessary for their synthesis was obtained. Some transcripts such as those of MAN1A2, FUT8, and ST6GALNAC were found solely in B. raniceps. Finally, secreted N- and O- linked glycoproteins were predicted from the transcriptomic data, indicating that proteases and protease inhibitors are putative sources of the glycans described herein. Overall, our results show the presence of oligosaccharides in amphibians skin secretions and suggest that their diversity is species-specific, paving the way for novel perspectives involving amphibian evolution and ecology.

Isolation and Sequencing of Cu-, Fe-, and Zn-Binding Whey Peptides for Potential Neuroprotective Applications as Multitargeted Compounds.

This study aims to isolate metal-binding peptides and synthesize promising amino acid sequences to potentially act as neuroprotective compounds in the future, targeting different mechanisms. Fractions of whey metal-binding peptides (Cu, Fe, and Zn) isolated by immobilized metal affinity chromatography showed different amino acid profiles according to the metal. The Cu-binding peptides presented roughly twofold increase in the in vitro antioxidant, as assessed by oxygen radical absorbance capacity and anticholinesterase activities over the hydrolysate. This is probably because of the higher concentration of aromatic and basic residues, the latter being crucial for binding to the anionic sites of acetylcholinesterase. Six peptide sequences were synthesized based on the metal-binding sites, molecular mass, hydrophobicity, and bioactivity probability. Among the synthetic peptides, the VF dipeptide stood out both for its in vitro antioxidant and anticholinesterase activities. This peptide, as well as the fraction of Cu-binding peptides, should be further studied because it may act through different mechanisms related to neurodegenerative diseases, in addition to the chelation of the excess of metals in the central nervous system.

Diagnosing lysosomal storage diseases in a Brazilian non-newborn population by tandem mass spectrometry.

OBJECTIVES: High-throughput mass spectrometry methods have been developed to screen newborns for lysosomal storage disorders, allowing the implementation of newborn screening pilot studies in North America and Europe. It is currently feasible to diagnose Pompe, Fabry, Gaucher, Krabbe, and Niemann-Pick A/B diseases, as well as mucopolysaccharidosis I, by tandem mass spectrometry in dried blood spots, which offers considerable technical advantages compared with standard methodologies. We aimed to investigate whether the mass spectrometry methodology for lysosomal storage disease screening, originally developed for newborns, can also discriminate between affected patients and controls of various ages. METHODS: A total of 205 control individuals were grouped according to age and subjected to mass spectrometry quantification of lysosomal α-glucosidase, ß-glucocerebrosidase, α-galactosidase, acid sphingomyelinase, galactocerebrosidase, and α-L-iduronidase activities. Additionally, 13 affected patients were analyzed. RESULTS: The median activities for each enzyme and each age group were determined. Enzyme activities were significantly lower in individuals aged older than 18 years compared with those in newborns. Affected patients presented enzymatic activities corresponding to less than 20% of the age-matched controls. CONCLUSIONS: Our data indicate that the mass spectrometry methodology can be used for the screening of lysosomal storage diseases in non-newborn patients. However, for some diseases, such as Fabry and mucopolysaccharidosis I, a combination of biochemical and clinical data may be necessary to achieve accurate diagnoses.

Diagnosing lysosomal storage diseases in a Brazilian non-newborn population by tandem mass spectrometry

OBJECTIVES: High-throughput mass spectrometry methods have been developed to screen newborns for lysosomal storage disorders, allowing the implementation of newborn screening pilot studies in North America and Europe. It is currently feasible to diagnose Pompe, Fabry, Gaucher, Krabbe, and Niemann-Pick A/B diseases, as well as mucopolysaccharidosis I, by tandem mass spectrometry in dried blood spots, which offers considerable technical advantages compared with standard methodologies. We aimed to investigate whether the mass spectrometry methodology for lysosomal storage disease screening, originally developed for newborns, can also discriminate between affected patients and controls of various ages. METHODS: A total of 205 control individuals were grouped according to age and subjected to mass spectrometry quantification of lysosomal α-glucosidase, β-glucocerebrosidase, α-galactosidase, acid sphingomyelinase, galactocerebrosidase, and α−L-iduronidase activities. Additionally, 13 affected patients were analyzed. RESULTS: The median activities for each enzyme and each age group were determined. Enzyme activities were significantly lower in individuals aged older than 18 years compared with those in newborns. Affected patients presented enzymatic activities corresponding to less than 20% of the age-matched controls. CONCLUSIONS: Our data indicate that the mass spectrometry methodology can be used for the screening of lysosomal storage diseases in non-newborn patients. However, for some diseases, such as Fabry and mucopolysaccharidosis I, a combination of biochemical and clinical data may be necessary to achieve accurate diagnoses. .

Analysis of novel ARG1 mutations causing hyperargininemia and correlation with arginase I activity in erythrocytes.

Hyperargininemia (HA) is an autosomal recessive disease that typically has a clinical presentation that is distinct from other urea cycle disorders. It is caused by the deficient activity of the enzyme arginase I, encoded by the gene ARG1. We screened for ARG1 mutations and measured erythrocyte enzyme activity in a series of 16 Brazilian HA patients. Novel mutations, in addition to previously described missense mutations, were analysed for their effect on the structure, stability and/or function of arginase I (ARG1) using bioinformatics tools. Three previously reported mutations were found (p.R21X; p.I11T and p.W122X), and five novel mutations were identified (p.G27D; p.G74V; p.T134I; p.R308Q; p.I174fs179). The p.T134I mutation was the most frequent in the Brazilian population. Patients carrying the p.R308Q mutation had higher residual ARG1 decreased activity, but presented no distinguishable phenotype compared to the other patients. Bioinformatics analyses revealed that missense mutations (1) affect the ARG1 active site, (2) interfere with the stability of the ARG1 folded conformation or (3) alter the quaternary structure of the ARG1. Our study reinforced the role of Arg308 residue for assembly of the ARG1 homotrimer. The panel of heterogeneous ARG1 mutations that cause HA was expanded, nevertheless a clear genotype-phenotype correlation was not observed in our series.