Diego O. Nolasco

In silico identification of novel hevein-like peptide precursors.

Lectins are proteins with ability to bind reversibly and non-enzymatically to a specific carbohydrate. They are involved in numerous biological processes and show enormous biotechnological potential. Among plant lectins, the hevein domain is extremely common, being observed in several kinds of lectins. Moreover, this domain is also observed in an important class of antimicrobial peptides named hevein-like peptides. Due to higher cysteine residues conservation, hevein-like peptides could be mined among the sequence databases. By using the pattern CX(4,5)CC[GS]X(2)GXCGX[GST]X(2,3)[FWY]C[GS]X[AGS] novel hevein-like peptide precursors were found from three different plants: Oryza sativa, Vitis vinifera and Selaginella moellendorffii. In addition, an hevein-like peptide precursor from the phytopathogenic fungus Phaeosphaeria nodorum was also identified. The molecular models indicate that they have the same scaffold as others, composed of an antiparallel β-sheet and short helices. Nonetheless, the fungal hevein-like peptide probably has a different disulfide bond pattern. Despite this difference, the complexes between peptide and N,N,N-triacetylglucosamine are stable, according to molecular dynamics simulations. This is the first report of an hevein-like peptide from an organism outside the plant kingdom. The exact role of an hevein-like peptide in the fungal biology must be clarified, while in plants they are clearly involved in plant defense. In summary, data here reported clear shows that an in silico strategy could lead to the identification of novel hevein-like peptides that could be used as biotechnological tools in the fields of health and agribusiness.

Structural and Functional Characterization of a Multifunctional Alanine-Rich Peptide Analogue from Pleuronectes americanus

Recently, defense peptides that are able to act against several targets have been characterized. The present work focuses on structural and functional evaluation of the peptide analogue Pa-MAP, previously isolated as an antifreeze peptide from Pleuronectes americanus. Pa-MAP showed activities against different targets such as tumoral cells in culture (CACO-2, MCF-7 and HCT-116), bacteria (Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 25923), viruses (HSV-1 and HSV-2) and fungi (Candida parapsilosis ATCC 22019, Trichophyton mentagrophytes (28d&E) and T. rubrum (327)). This peptide did not show toxicity against mammalian cells such as erythrocytes, Vero and RAW 264.7 cells. Molecular mechanism of action was related to hydrophobic residues, since only the terminal amino group is charged at pH 7 as confirmed by potentiometric titration. In order to shed some light on its structure-function relations, in vitro and in silico assays were carried out using circular dichroism and molecular dynamics. Furthermore, Pa-MAP showed partial unfolding of the peptide changes in a wide pH (3 to 11) and temperature (25 to 95°C) ranges, although it might not reach complete unfolding at 95°C, suggesting a high conformational stability. This peptide also showed a conformational transition with a partial α-helical fold in water and a full α-helical core in SDS and TFE environments. These results were corroborated by spectral data measured at 222 nm and by 50 ns dynamic simulation. In conclusion, data reported here show that Pa-MAP is a potential candidate for drug design against pathogenic microorganisms due to its structural stability and wide activity against a range of targets.

A polyalanine peptide derived from polar fish with anti-infectious activities

Due to the growing concern about antibiotic-resistant microbial infections, increasing support has been given to new drug discovery programs. A promising alternative to counter bacterial infections includes the antimicrobial peptides (AMPs), which have emerged as model molecules for rational design strategies. Here we focused on the study of Pa-MAP 1.9, a rationally designed AMP derived from the polar fish Pleuronectes americanus. Pa-MAP 1.9 was active against Gram-negative planktonic bacteria and biofilms, without being cytotoxic to mammalian cells. By using AFM, leakage assays, CD spectroscopy and in silico tools, we found that Pa-MAP 1.9 may be acting both on intracellular targets and on the bacterial surface, also being more efficient at interacting with anionic LUVs mimicking Gram-negative bacterial surface, where this peptide adopts α-helical conformations, than cholesterol-enriched LUVs mimicking mammalian cells. Thus, as bacteria present varied physiological features that favor antibiotic-resistance, Pa-MAP 1.9 could be a promising candidate in the development of tools against infections caused by pathogenic bacteria.

Functional and structural insights on self-assembled nanofiber-based novel antibacterial ointment from antimicrobial peptides, bacitracin and gramicidin S

A novel antibacterial ointment using bacitracin, specific for Gram-positive bacteria, and gramicidin S, a highly toxic antibacterial peptide, was here developed showing broad-spectrum antibacterial activities against pathogenic strains with less toxicity after self-assembly into nanofiber structures. Such structures were confirmed with scanning electron microscopy and CD analyses. In addition, in silico studies using docking associated with molecular dynamics were carried out to obtain information about fiber structural oligomerization. Thus, the bacitracin and gramicidin S-based self-assembled nanopeptide ribbon may be a successful ointment formulation for bacterial infection control.

Shedding some light over the floral metabolism by arum lily (Zantedeschia aethiopica) spathe de novo transcriptome assembly.

Zantedeschia aethiopica is an evergreen perennial plant cultivated worldwide and commonly used for ornamental and medicinal purposes including the treatment of bacterial infections. However, the current understanding of molecular and physiological mechanisms in this plant is limited, in comparison to other non-model plants. In order to improve understanding of the biology of this botanical species, RNA-Seq technology was used for transcriptome assembly and characterization. Following Z. aethiopica spathe tissue RNA extraction, high-throughput RNA sequencing was performed with the aim of obtaining both abundant and rare transcript data. Functional profiling based on KEGG Orthology (KO) analysis highlighted contigs that were involved predominantly in genetic information (37%) and metabolism (34%) processes. Predicted proteins involved in the plant circadian system, hormone signal transduction, secondary metabolism and basal immunity are described here. In silico screening of the transcriptome data set for antimicrobial peptide (AMP) –encoding sequences was also carried out and three lipid transfer proteins (LTP) were identified as potential AMPs involved in plant defense. Spathe predicted protein maps were drawn, and suggested that major plant efforts are expended in guaranteeing the maintenance of cell homeostasis, characterized by high investment in carbohydrate, amino acid and energy metabolism as well as in genetic information.

Structural and functional evaluation of the palindromic alanine-rich antimicrobial peptide Pa-MAP2.

Recently, several peptides have been studied regarding the defence process against pathogenic microorganisms, which are able to act against different targets, with the purpose of developing novel bioactive compounds. The present work focuses on the structural and functional evaluation of the palindromic antimicrobial peptide Pa-MAP2, designed based on the peptide Pa-MAP from Pleuronectes americanus. For a better structural understanding, molecular modelling analyses were carried out, together with molecular dynamics and circular dichroism, in different media. Antibacterial activity against Gram-negative and positive bacteria was evaluated, as well as cytotoxicity against human erythrocytes, RAW 264.7, Vero and L6 cells. In silico docking experiments, lipid vesicle studies, and atomic force microscopy (AFM) imaging were carried out to explore the activity of the peptide. In vivo studies on infected mice were also done. The palindromic primary sequence favoured an α-helix structure that was pH dependent, only present on alkaline environment, with dynamic N- and C-terminals that are stabilized in anionic media. Pa-MAP2 only showed activity against Gram-negative bacteria, with a MIC of 3.2 μM, and without any cytotoxic effect. In silico, lipid vesicles and AFM studies confirm the preference for anionic lipids (POPG, POPS, DPPE, DPPG and LPS), with the positively charged lysine residues being essential for the initial electrostatic interaction. In vivo studies showed that Pa-MAP2 increases to 100% the survival rate of mice infected with Escherichia coli. Data here reported indicated that palindromic Pa-MAP2 could be an alternative candidate for use in therapeutics against Gram-negative bacterial infections.

Cm-p5: an antifungal hydrophilic peptide derived from the coastal mollusk Cenchritis muricatus (Gastropoda: Littorinidae)

Antimicrobial peptides form part of the first line of defense against pathogens formany organisms. Current treatments for fungal infections are limited by drug toxicity and pathogen resistance. Cm‐p5 (SRSE‐LIVHQRLF), a peptide derived from the marine mollusk Cenchritis muricatus peptide Cm‐p1, has a significantly increased fungistatic activity against pathogenic Candida albicans (minimal inhibitory concentration, 10 μg/ml; EC50, 1.146 μg/ml) while exhibiting low toxic effects against a cultured mammalian cell line. Cm‐p5 as characterized by circular dichroism and nuclear magnetic resonance revealed an α‐helical structure in membrane‐mimetic conditions and a tendency to random coil folding in aqueous solutions. Additional studies modeling Cm‐p5 binding to a phosphatidylserine bilayer in silico and isothermal titration calorimetry using lipid monophases demonstrated that Cm‐p5 has a high affinity for the phospholipids of fungal membranes (phosphatidylserine and phosphatidylethanolamine), only moderate interactions with a mammalian membrane phospholipid, low interaction with ergosterol, and no interaction with chitin. Adhesion of Cm‐p5 to living C. albicans cells was confirmed by fluorescence microscopy with FITC‐labeled peptide. In a systemic candidiasis model in mice, intraperitoneal administration of Cm‐p5 was unable to control the fungal kidney burden, although its low amphiphaticity could be modified to generate new derivatives with improved fungicidal activity and stability.—López‐Abarrategui, C., McBeth, C., Mandai, S. M., Sun, Z. J., Heffron, G., Alba‐Menéndez, A., Migliolo, L., Reyes‐Acosta, O., Garcia‐Villarino, M., Nolasco, D. O., Falcão, R., Cherobim, M. D., Dias, S. C., Brandt, W., Wessjohann, L., Starnbach, M., Franco, O. L., Otero‐González, A. J. Cm‐p5: an antifungal hydrophilic peptide derived from the coastal mollusk Cenchritis muricatus (Gastropoda: Littorinidae). FASEB J. 29, 3315‐3325 (2015). www.fasebj.org

Impact of kinesin Eg5 inhibition by 3,4-dihydropyrimidin-2(1H)-one derivatives on various breast cancer cell features

BackgroundBreast cancer is a complex heterogeneous disease and is one of the leading causes of death among women. In addressing the need for treatments of this life-threatening illness, we studied 3,4-dihydropyrimidin-2(1H)-one (or thione) derivatives (DHPMs), a class of inhibitor molecules of the Eg5 motor spindle protein that shows pronounced antitumor activity against several cancer cell lines.MethodsAn in vitro screening was performed for identification of DHPMs with potent antitumor effects on MCF-7 and MDA-MB-231 cells and the selected DHPMs were evaluated for their inhibitory activity on Eg5 both in silico, using Molecular dynamics, and in vitro Eg5 inhibition assays. Analysis of cell death induction, proliferation, cell cycle and cancer stem cells (CSC) profile were performed by flow cytometry to assess the influence of the selected DPHMs on these important tumor features. Finally, the effects of DHPM treatment on tube formation were evaluated in vitro using HUVEC cells, and in vivo using a model on chorioallantoic membrane (CAM) of fertilized eggs.ResultsWe identified five DHPMs with pronounced inhibitory activity on Eg5 motor protein interfering with the proper mitotic spindle assembly during cell division. These compounds impair the correct conclusion of cell cycle of the breast cancer cells and showed to be selective for tumor cells. Moreover, DHPMs modulate the CD44+/CD24− phenotype leading to a decrease in the CSC population in MDA-MB-231 cells, an important effect since CSC are resistant to many conventional cancer therapies and play a pivotal role in tumor initiation and maintenance. This observation was confirmed by the results which demonstrated that DHPM treated cells had impaired proliferation and were unable to sustain angiogenesis events. Finally, the DHMP treated cells were induced to apoptosis, which is one of the most pursued goals in drug development.ConclusionsThe results of our study strongly suggest that DHPMs inhibit important tumorigenic features of breast cancer cells leading them to death by apoptosis. These findings firmly point to DHPM molecular architecture as a promising alternative against breast cancer.

Prediction of the Impact of Coding Missense and Nonsense Single Nucleotide Polymorphisms on HD5 and HBD1 Antibacterial Activity against Escherichia coli

Defensins confer host defense against microorganisms and are important for human health. Single nucleotide polymorphisms (SNPs) in defensin gene‐coding regions could lead to less active variants. Using SNP data available at the dbSNP database and frequency information from the 1000 Genomes Project, two DEFA5 (L26I and R13H) and eight DEFB1 (C35S, K31T, K33R, R29G, V06I, C12Y, Y28* and C05*) missense and nonsense SNPs that are located within mature regions of the coded defensins were retrieved. Such SNPs are rare and population restricted. In order to assess their antibacterial activity against Escherichia coli, two linear regression models were used from a previous work, which models the antibacterial activity as a function of solvation potential energy, using molecular dynamics data. Regarding only the antibacterial predictions, for HD5, no biological differences between wild‐type and its variants were observed; while for HBD1, the results suggest that the R29G, K31T, Y28* and C05* variants could be less active than the wild‐type one. The data here reported could lead to a substantial improvement in knowledge about the impact of missense SNPs in human defensins and their world distribution.

Comparative transcriptomic analysis indicates genes associated with local and systemic resistance to Colletotrichum graminicola in maize

The hemibiotrophic fungus Colletotrichum graminicola may cause severe damage to maize, affecting normal development of the plant and decreasing grain yield. In this context, understanding plant defense pathways at the inoculation site and systemically in uninoculated tissues can help in the development of genetic engineering of resistance against this pathogen. Previous work has discussed the molecular basis of maize - C. graminicola interaction. However, many genes involved in defense have not yet been exploited for lack of annotation in public databases. Here, changes in global gene expression were studied in root, male and female inflorescences of maize under local and systemic fungal infection treatments, respectively. RNA-Seq with qPCR was used to indicate genes involved in plant defense. We found that systemic acquired resistance induction in female inflorescences mainly involves accumulation of salicylic acid (SA)-inducible defense genes (ZmNAC, ZmHSF, ZmWRKY, ZmbZIP and PR1) and potential genes involved in chromatin modification. Furthermore, transcripts involved in jasmonic acid (JA) and ethylene (ET) signaling pathways were also accumulated and may participate in plant immunity. Moreover, several genes were functionally re-annotated based on domain signature, indicating novel candidates to be tested in strategies involving gene knockout and overexpression in plants.