Sarconesin II, a New Antimicrobial Peptide Isolated from Sarconesiopsis magellanica Excretions and Secretions.

Antibiotic resistance is at dangerous levels and increasing worldwide. The search for new antimicrobial drugs to counteract this problem is a priority for health institutions and organizations, both globally and in individual countries. Sarconesiopsis magellanica blowfly larval excretions and secretions (ES) are an important source for isolating antimicrobial peptides (AMPs). This study aims to identify and characterize a new S. magellanica AMP. RP-HPLC was used to fractionate ES, using C18 columns, and their antimicrobial activity was evaluated. The peptide sequence of the fraction collected at 43.7 min was determined by mass spectrometry (MS). Fluorescence and electronic microscopy were used to evaluate the mechanism of action. Toxicity was tested on HeLa cells and human erythrocytes; physicochemical properties were evaluated. The molecule in the ES was characterized as sarconesin II and it showed activity against Gram-negative (Escherichia coli MG1655, Pseudomonas aeruginosa ATCC 27853, P. aeruginosa PA14) and Gram-positive (Staphylococcus aureus ATCC 29213, Micrococcus luteus A270) bacteria. The lowest minimum inhibitory concentration obtained was 1.9 µM for M. luteus A270; the AMP had no toxicity in any cells tested here and its action in bacterial membrane and DNA was confirmed. Sarconesin II was documented as a conserved domain of the ATP synthase protein belonging to the Fli-1 superfamily. The data reported here indicated that peptides could be alternative therapeutic candidates for use in infections against Gram-negative and Gram-positive bacteria and eventually as a new resource of compounds for combating multidrug-resistant bacteria.

Sarconesin II, a new antimicrobial peptide isolated from Sarconesiopsis magellanica excretions and secretions

Antibiotic resistance is at dangerous levels and increasing worldwide. The search for new antimicrobial drugs to counteract this problem is a priority for health institutions and organizations, both globally and in individual countries. Sarconesiopsis magellanica blowfly larval excretions and secretions (ES) are an important source for isolating antimicrobial peptides (AMPs). This study aims to identify and characterize a new S. magellanica AMP. RP-HPLC was used to fractionate ES, using C18 columns, and their antimicrobial activity was evaluated. The peptide sequence of the fraction collected at 43.7 min was determined by mass spectrometry (MS). Fluorescence and electronic microscopy were used to evaluate the mechanism of action. Toxicity was tested on HeLa cells and human erythrocytes; physicochemical properties were evaluated. The molecule in the ES was characterized as sarconesin II and it showed activity against Gram-negative (Escherichia coli MG1655, Pseudomonas aeruginosa ATCC 27853, P. aeruginosa PA14) and Gram-positive (Staphylococcus aureus ATCC 29213, Micrococcus luteus A270) bacteria. The lowest minimum inhibitory concentration obtained was 1.9 µM for M. luteus A270; the AMP had no toxicity in any cells tested here and its action in bacterial membrane and DNA was confirmed. Sarconesin II was documented as a conserved domain of the ATP synthase protein belonging to the Fli-1 superfamily. The data reported here indicated that peptides could be alternative therapeutic candidates for use in infections against Gram-negative and Gram-positive bacteria and eventually as a new resource of compounds for combating multidrug-resistant bacteria.

NMR structures in different membrane environments of three ocellatin peptides isolated from Leptodactylus labyrinthicus.

The peptides ocellatin-LB1, -LB2 and -F1 have previously been isolated from anurans of the Leptodactylus genus and the sequences are identical from residue 1-22, which correspond to ocellatin-LB1 sequence (GVVDILKGAAKDIAGHLASKVM-NH2), whereas ocellatin-LB2 carries an extra N and ocellatin-F1 extra NKL residues at their C-termini. These peptides showed different spectra of activities and biophysical investigations indicated a direct correlation between membrane-disruptive properties and antimicrobial activities, i.e. ocellatin-F1 > ocellatin-LB1 > ocellatin-LB2. To better characterize their membrane interactions, we report here the detailed three-dimensional NMR structures of these peptides in TFE-d2:H2O (60:40) and in the presence of zwitterionic DPC-d38 and anionic SDS-d25 micellar solutions. Although the three peptides showed significant helical contents in the three mimetic environments, structural differences were noticed. When the structures of the three peptides in the presence of DPC-d38 micelles are compared to each other, a more pronounced curvature is observed for ocellatin-F1 and the bent helix, with the concave face composed mostly of hydrophobic residues, is consistent with the micellar curvature and the amphipathic nature of the molecule. Interestingly, an almost linear helical segment was observed for ocellatin-F1 in the presence of SDS-d25 micelles and the conformational differences in the two micellar environments are possibly related to the presence of the extra Lys residue near the peptide C-terminus, which increases the affinity of ocellatin-F1 to anionic membranes in comparison with ocellatin-LB1 and -LB2, as proved by isothermal titration calorimetry. To our knowledge, this work reports for the first time the three-dimensional structures of ocellatin peptides.

CARACTERIZACIÓN DE LA ESTRUCTURA SECUNDARIA DE SUBTIPOS DE LA HISTONA H1 POR DICROÍSMO CIRCULAR / CHARACTERIZATION OF THE SECONDARY STRUCTURE OF HISTONE H1 SUBTYPES BY CIRCULAR DICHROISM (CD)

Introducción: Las histonas H1 modulan la estructura y la función de la cromatina. Las células somáticas de mamífero contienen los subtipos H1º, H1a, H1b, H1c, H1d y H1e; en células germinales de testículo y en ovocito, se encuentran respectivamente H1t y H1oo. Su estructura está conformada por un dominio central globular flanqueado por los dominios N-Terminal (DNT) y C-Terminal (DCT). Objetivo: Caracterizar la estructura secundaria de subtipos de la histona H1 mediante dicroísmo circular (DC). Materiales y Métodos: La histona H1 total se extrajo de núcleos de cerebro de rata por cromatografía de intercambio catiónico; la H1º se purificó por filtración en gel y las H1a, H1b, H1c y H1e por cromatografía líquida de alta resolución de fase reversa (RF-HPLC). Los espectros de DC se realizaron en tampón fosfato 10 mM; tampón fosfato 10 mM, 20% TFE (trifluoroetanol); tampón fosfato 10 mM, 40% TFE; tampón fosfato 10 mM, 60% TFE; tampón fosfato 10 mM, 150 mM NaCl y tampón fosfato 10 mM, 1 M NaCl. El análisis de los espectros se realizó con el programa Standard Analysis. Resultados: El porcentaje de hélice-alfa se calculó por diferentes métodos matemáticos teniendo en cuenta elipticidad molar a 193 nm y a 222 nm; con programa de deconvolución K2D y con relaciones cualitativas R1 y R2. El TFE induce la estructura en hélice-alfa en cada uno de los subtipos, mientras que NaCl no induce ningún cambio importante. Conclusión: Los subtipos con mayor contenido de hélice-alfa son H1a y H1c. Las diferencias observadas en el porcentaje de hélice-alfa entre los diferentes subtipos puede ser importante para su diferenciación funcional.

H1 histones modulate the structure and function of chromatin. Mammalian somatic cells contain H1º, H1a, H1b, H1c, H1d and H1e subtypes; H1t and H1oo are found in testicular germ cells and oocyte, respectively. Its structure consists of a globular core domain flanked by N-terminal (DNT) and C-terminal (DCT) domains. Objective: To characterize the secondary structure of histone H1 subtypes through circular dichroism (CD). Materials and Methods: Total histone H1 was extracted for rat brain nuclei by cation exchange chromatography; histone H1º was purified by gel filtration and the histones H1a, H1b, H1c and H1e were purified by reversed phase high performance liquid chromatography (RP-HPLC). CD spectra were performed in 10 mM phosphate buffer; 10 mM, 20% TFE phosphate buffer (trifluoroethanol); 10 mM, 40% TFE; phosphate buffer 10 mM, 60% TFE; phosphate buffer 10 mM, 150 mM NaCl and phosphate buffer 10 mm, 1 M NaCl. The analysis of the spectra was performed with JASCO Standard Analysis. Results: The percentage of alpha-helix was calculated using different mathematical methods, taking into account the molar ellipticity at 193 nm, and 222 nm, with K2D deconvolution program and the R1 and R2 qualitative relationships. The results indicate that TFE induced the alpha-helix structure in each of the subtypes, whereas NaCl did not induce any significant change. Conclusion: H1a and H1c are subtypes with highest content of alpha-helix. The observed differences in the percentage of alpha-helix between different subtypes may be important for their functional differentiation.

Recognition of alpha-helix transmembrane domains with an amphipathy scale generated by molecular dynamics using only the primary sequence of proteins

We recently developed an amphipathy scale, elaborated from molecular dynamics data that can be used for the identification of hydrophobic or hydrophilic regions in proteins. This amphipathy scale reflects side chain/water molecule interaction energies. We have now used this amphipathy scale to find candidates for transmembrane segments, by examining a large sample of membrane proteins with alpha-helix segments. The candidates were selected based on an amphipathy coefficient value range and the minimum number of residues in a segment. We compared our results with the transmembrane segments previously identified in the PDB_TM database by the TMDET algorithm. We expected that the hydrophobic segments would be identified using only the primary structures of the proteins and the amphipathy scale. However, some of these hydrophobic segments may pertain to hydrophobic pockets not included in transmembrane regions. We found that our amphipathy scale could identify alpha-helix transmembrane regions with a probability of success of 76% when all segments were included and 90% when all membrane proteins were included.