Visualization of specific intracellular biological activities plays a key role in the understanding of cells behavior; therefore the development of new generations of probes and sensors remains an important task. Despite the growing utility of small-molecule fluorophores and a wide variety of fluorescent probes, only few of them are capable of penetrating spontaneously into the cell; thus cell sensors are usually composed of a recognition unit, a cell penetrating unit and a fluorescent unit.
In our laboratory we developed a new versatile methodology for the fast synthesis of fluorescently labelled peptides. The particularity of the strategy is that the fluorescent probe, generated on the peptide backbone, functions both as fluorescent sensor as well as cell penetrating element. Our fluorophores are derivatives of natural products having hexa-substituted benzene obtained by MCR4. Their synthesis is based on the new multicomponent reaction recently developed in our laboratory: the reaction of substituted chiral tetramic acids which together with an aldehyde, an iso-cyanide, a dienophile and a Lewis acid; produce 3-substituted iso-indolinones in one pot.¹ We applied this strategy to synthesize a targeted drug model consisting of a permeable fluorophore and D-[KLAKLAK]₂ peptide with mitochondrial localization and toxicity that mediates apoptosis. The drug model was tested for biological activity using XTT, flow cytometry and confocal fluorescent microscopy. Together, these experiments proved the ability of the fluorescent molecule to transport a non-permeable cell toxic peptide into the cell with the concomitant recovery of toxicity. Results indicate that the new fluorescent probes are potentially applicable as cell sensors as well as drug delivery carriers.

• Gelman, M; Massarano, T.; Lavi, R.; Byk, G. A New Multicomponent Reaction MCR4 for the Synthesis of Analogs of Staurosporine. Curr. Org. Chem. 2018, 22, 505-517.
• Massarano, T.; Mazir, A.; Lavi, R.; Byk, G. Solid-Phase Multicomponent Synthesis of 3-Substituted Isoindolinones Generates New Cell-Penetrating Probes as Drug Carriers. ChemMedChem 2020, 15(10), 833-838.

There has been a long-felt need for sanitizer hydrogel, which has a high degree of antimicrobial efficacy while posing no toxicity and environmental incompatibility. This study aims to develop and characterize functional hydrogels from biocompatible and biodegradable natural ingredients, with broad antimicrobial activity against pathogenic and spoilage organisms. Four natural antimicrobial compounds, namely reuterin, pediocin, microcin J25, and lactic acid, were produced, purified, and their antimicrobial activity was evaluated (alone or in combination) using agar well diffusion and microtitration assays. The combined effect among these compounds was investigated by checkerboard assay based on predetermined MIC values against the indicator strains S. Newport ATCC 6962 and L. ivanovii HPB28. A consortium of microcin J25, reuterin, and lactic acid was selected due to its high synergistic interaction. Hydrogel formulas containing the selected consortium were developed from carbohydrate-based biopolymer chitosan and Charboxymethylcellulose (CMC). They were characterized in terms of viscosity, antimicrobial activity, and stability during storage at room temperature. Hydrogels made from chitosan (1.5% and 2.5%) and CMC (3% and 5%) were selected based on their required viscosity and inhibition activity against indicator strains. Both hydrogels were shown to remain stable and active during four-week storage at room temperature. This antimicrobial hydrogel formula represents therefore a promising and safer alternative to commercial chemical products for hands and food surface disinfection.

Cathelicidins are host defense peptides (HDPs) widely recognized for their multifaceted functions in the innate and adaptive immune response. Among them, the only human cathelicidin is represented by the 37-residue peptide LL-37, which has pleiotropic activity ranging from immunological to antineoplastic. Some pathological conditions are characterized by the imbalance of the cathelicidin expression and consequent activation of inflammatory pathways and apoptosis. Therefore, the development of strategies aimed to reduce the side-effect of LL-37 dysregulated expression is highly desired. It might be achieved by inducing the rearrangement of the peptide structure and consequent impairment of its binding to the cell surface. In this study, we demonstrated a remarkable attenuation of LL-37 cytotoxicity towards colon and monocytic cell lines in the presence of the drug suramin. Additionally, to unravel the molecular mechanism underlying the alteration of its activity, a detailed analysis of the peptide structure was performed. In this respect, an LL-37 fragment peptide, FK-16, which retains its antibacterial activity, was also used. The study of the molecular mechanisms of peptide-drug interaction revealed the ability of suramin to induce alteration in the peptide structure, aggregation, and formation of complexes. This latter case might hinder the interaction of LL-37 with the cell membrane. Moreover, a comparison with other therapeutic agents having common features revealed the peculiar ability of suramin to optimize the interaction with the studied peptides. The newly discovered suramin action on LL-37 cytotoxicity can contribute to the development of novel repurposing strategies aimed to prevent inflammatory responses triggered by overexpression of host defense peptides.

Acknowledgments: This work was supported through grants provided by the Momentum Program (LP2016‐2), the National Competitiveness and Excellence Program (NVKP_16‐1‐2016‐0007), and GINOP (BIONANO_GINOP‐2.3.2‐15‐2016‐00017).

Extracellular vesicles (EVs) are lipid bilayer enclosed nanoparticles secreted by most cells and found in body fluids. They have the advantage of displaying native membrane proteins on their surface and with the ability to interact with other cells play important role in many biological processes like cellular uptake, immune response, and cancer progression. Furthermore, their size distribution makes them promising candidates to expand biophysical understanding of membrane active peptides (MAPs). To study the action mechanism of MAPs we used red blood cell-derived extracellular vesicles (REV) as a model membrane system. We selected well-known MAPs: KLA, PNC28, CM15 and Melittin, and their interaction was investigated using several biophysical techniques such as polarized light spectroscopy (Szigyártó et al. 2018), microfluidic resistive pulse sensing and freeze-fracture transmission electron microscopy.

We observed that CM15 and Melittin efficiently remove the proteins from REV membrane surfaces even at lower 5 µM concentrations, resulting in smoothed membrane surface while leaving the vesicle intact (Singh et al. 2020). Whereas a similar effect was not detected in the case of KLA and PNC28 even at a higher concentration of 160 µM. The overall results indicate that REV as a complex model membrane provides an excellent platform to understand better their interactions with MAPs and reveals novel peptide functions. This further highlights their role in possible future applications for nanoparticle and surface engineering of EV-based therapeutics.

Conventional cancer treatments have side effects, decreased quality of life of patients and may contribute to multidrug resistance. An alternative to the current anticancer treatments is the use of antitumor peptides, such as Melittin, because they are molecules that inhibit the proliferation of tumor cells and are selective to tumor targets. However, they have low specificity and high toxicity relative to normal cells, leading to the need to work with bioconjugated antitumor aptamers peptides, which are more selective and lead to greater efficacy in killing tumor cells. In this work, three peptides (sequences: FNLPLPSRPLLR, GGVSCMQTSPVCENNL, NPGTCKDKWIECLLNG) were synthesized by Solid Phase Peptide Synthesis and purified by High Performance Liquid Chromatography. The strains worked on were MCF-7, MRC-5 and A549. Membrane labeling of the aptamers peptides was performed by In Cell Analyzer and flow cytometry. Cell viability was performed by MTT assays. The results obtained demonstrate that the aptamers peptides did not cause the death of the cell lines and that they present more evident markings on membrane proteins of the MCF-7 strain. Melittin showed cytotoxic action in all cell lines. Aptamers peptides can bring a greater specificity and selectivity for the anticancer molecules, as Melittin, considering its binding to target proteins in tumor cells. Next steps include conjugation of aptamers with Melittin, evaluation of the antitumor potential and mechanism of action of the bioconjugates.

Peptides with the ability to self-assemble into defined nanoparticles have gained increase interest for the design of antigen delivery platform for subunit vaccines. By modulating the primary sequences and the self-assembly conditions, the shape, size and surface chemistry of the final supramolecular structures can be precisely modulated, opening to a diversity of immunological functionalities. We recently reported that nanofilaments with a cross-β quaternary structure and assembled from a short 10-mer amyloidogenic peptide derived from the islet amyloid polypeptide (I10), constitute promising self-adjuvanted assemblies suitable for anchoring antigenic determinants and increasing their immunogenicity. In the present study, we took advantage of non-covalent molecular self-assembly to integrate different densities of antigens on the fibril in a controlled manner by adjusting the stoichiometry of the different monomer building blocks. The M2e epitope derived from the matrix 2 protein of the influenza virus was conjugated to the I10 self-assembling sequence by a flexible short linker on solid support. Chimeric M2e-I10 peptides were assembled in presence of different molar ratio of naked I10 under continuous rotary agitation. Structural conversion of soluble peptides into cross-β filaments was followed by thioflavin T fluorescence, circular dichroism spectroscopy and atomic force microscopy. By ELISA, we observed that the density of the M2e epitope accessible on the fibril surface could be finely modulated by controlling the stoichiometry of the building blocks. Finally, the capacity of the assemblies to activate the Toll-like receptor 2 (TLR2) was evaluated using HEK-Blue-hTLR6/TLR2 cells that have a NFκB-inducible reporter gene SEAP (secreted embryonic alkaline phosphatase). Overall, this study indicates that the density of a given epitope on a nanofibril can be precisely controlled through molecular co-assembly, ultimately fine regulating the amplitude of the antigen-specific immune response.

The resistance of microorganisms to antibiotics is occurring worldwide. An alternative against resistant bacteria has been antimicrobial peptides. Cell penetrating peptides are a family of peptides that are able to cross the biological membrane. In the present study, the peptide desCys¹¹/Lys¹²/Lys¹³(p-BthTX-I)₂K was coupled to the cell penetrating peptides PFVYLI, HIV-TAT (47-57) and AIP-6 to improve its antimicrobial action. The peptides were synthesized by SPPS. Circular dichroism spectroscopy was used to evaluated structure of conjugates. The biological activity was determined by broth microdilution tests and the minimum inhibitory concentration and the minimum bactericidal concentration were obtained. The hemolytic activity was performed to measure the toxicity against erythrocytes. The results obtained demonstrate that the peptides do not have a defined structure, which is consistent with the structure of desCys¹¹/Lys¹²/Lys¹³(p-BthTX-I)₂K. Conjugates showed activity against Gram-negative E. coli similar to the wild type peptide. However, the MIC decreased from 128 µM to values ​​between 16 µM and 8 µM in Gram-positive S. aureus. Except to PFVYLI conjugate, hemolytic activity was not found. These results showed that the addition of the fusion peptides to the original peptide, improves the activity of the peptide against Gram-positive bacteria, without decrease its activity in Gram-negative bacteria.

Nearly all the vegetation in our planet earth is susceptible to invading pathogens, which significantly affect the crop and its yield. The chemical bio-control agents are the wide spread tool to get rid of plant pathogens. However, the excessive use of such chemical bio-control agents has the potential to wipe-out entire humanity. In this situation, advanced research in the development of eco-friendly bio-control agents is an absolute necessity. The present study put forward a novel strain Bacillus tequilensis PP1 (accession number MK648314) from the gills of an estuarine water fish Chelon parsia with the potential to counterfeit most relevant phyto-pathogenic fungi. An unknown molecule with m/z 1018.54 was found as the key molecule of antifungal activity, primary structural investigation revealed that the molecule comes under lipopeptide class. The isolated lipopeptide is efficient enough to inhibit the growth of phytopathogens such as Aspergillus niger, Trichoderma viride, Mucor racemosus and Fusarium oxysporum with minimum inhibitory concentration range (MIC) of 6 to 12 µg/ml. The derived lipopeptide exhibits tremendous surfactant property with an emulsification index of 32.21 %, which opens up extended application of the molecule in various fields. It is expected that, with further research, the molecule may serve as a replacement for chemical-biocontrol agents

Background

Australia annually produces around 3.5 million metric tons of rapeseed/Canola, and about half of this remains as de-oiled by-product, and largely consists of seed proteins. This rich source of proteins is primarily streamlined for animal feed as these proteins cannot be utilised for human consumption due to potential for triggering allergic reactions. When ingested these proteins are not digested in the stomach and therefore remain intact in the intestine where they trigger allergic reactions by binding with immunoglobulin E antibodies. Epitopes, the regions on the rapeseed seed proteins that bind these antibodies are yet to be characterised.

Methods

Rapeseed major seed proteins were probed by aligning the reported immunodominant linear epitopes of known allergens with respect to the sequences of rapeseed proteins to detect the corresponding epitope sites. Sequence alignment, evolutionary relations, allergen database surveys, and modelling of three-dimensional structure were performed to characterise the epitopes.

Results

The molecular surface epitope mapping identified epitopes in rapeseed proteins with high sequence coverage and identity to known food epitopes. This indicated conservation of allergenic epitope motifs, while the three-dimensional structure modelling allowed the prediction of ligand binding sites on human H1 Histamine receptors.

Conclusion

The epitopes identified in this study could be used in the development of recombinant proteins for diagnosis, to develop synthetic and recombinant vaccines for immunotherapy and therapeutic purposes against rapeseed allergy. This work enriches our existing knowledge on immunogenesis to seed proteins and provides a robust foundation and rational basis for protein bioengineering of seed storage proteins for human food.

Mitochondrial dysfunction has been implicated in many diseases including cancer, cardiovascular and neurodegenerative diseases^1,2. The voltage-dependant anion channel 1 (VDAC1) is the most abundant protein found in the outer mitochondrial membrane. VDAC1 functions as a gatekeeper and is considered as a multi-functional porin involved in cell survival and cell death³. VDAC1 mediates energy production as well as ions and metabolic exchanges between the mitochondria and other cellular compartments, thus modulating mitochondrial permeability. VDAC1 is also involved in the cellular Ca²⁺ homeostasis by its capacity to transport Ca²⁺ in and out of mitochondria⁴. In addition, VDAC1 interacts with multiple proteins implicated in neurodegeneration process⁵ as well as with pro-apoptotic and anti-apoptotic regulating proteins, and in particular Hexokinase isoforms I and II, its main ligand.

Hexokinase I (HK I) is found in brain tissues and is known as the “guardian of the mitochondria”. HK possess a hydrophobic N-terminal structured in α-helix that is necessary for the binding of HK to VDAC⁶. The interaction between HK and VDAC1 has attracted much interest, and several studies have proposed different models of binding to VDAC1^7,8. However, the precise mechanism of binding remains undefined up to now.

In order to give some insights to the nature of HK binding to VDAC1, we have developed new peptide analogs of HK I based on Hexokinase I N-terminal sequence. In this study, the in-vitro activity of the peptides was studied, the peptide helical content was investigated by circular dichroism and NMR, and peptide proteolytic stability was assessed.

(1) Shoshan-Barmatz, V.; Shteinfer-Kuzmine, A.; Verma, A. VDAC1 at the Intersection of Cell Metabolism, Apoptosis, and Diseases. Biomolecules 2020, 10 (11). https://doi.org/10.3390/biom10111485.
(2) Abramov, A. Y.; Berezhnov, A. V.; Fedotova, E. I.; Zinchenko, V. P.; Dolgacheva, L. P. Interaction of Misfolded Proteins and Mitochondria in Neurodegenerative Disorders. Biochem Soc Trans 2017, 45 (4), 1025–1033. https://doi.org/10.1042/BST20170024.
(3) Shoshan-Barmatz, V.; Ben-Hail, D. VDAC, a Multi-Functional Mitochondrial Protein as a Pharmacological Target. Mitochondrion 2012, 12 (1), 24–34. https://doi.org/10.1016/j.mito.2011.04.001.
(4) Shoshan-Barmatz, V.; Krelin, Y.; Shteinfer-Kuzmine, A. VDAC1 Functions in Ca2+ Homeostasis and Cell Life and Death in Health and Disease. Cell Calcium 2018, 69, 81–100. https://doi.org/10.1016/j.ceca.2017.06.007.
(5) Magri, A.; Messina, A. Interactions of VDAC with Proteins Involved in Neurodegenerative Aggregation: An Opportunity for Advancement on Therapeutic Molecules. Curr Med Chem 2017, 24 (40), 4470–4487. https://doi.org/10.2174/0929867324666170601073920.
(6) Xie, G. C.; Wilson, J. E. Rat Brain Hexokinase: The Hydrophobic N-Terminus of the Mitochondrially Bound Enzyme Is Inserted in the Lipid Bilayer. Arch Biochem Biophys 1988, 267 (2), 803–810. https://doi.org/10.1016/0003-9861(88)90090-2.
(7) Rosano, C. Molecular Model of Hexokinase Binding to the Outer Mitochondrial Membrane Porin (VDAC1): Implication for the Design of New Cancer Therapies. Mitochondrion 2011, 11 (3), 513–519. https://doi.org/10.1016/j.mito.2011.01.012.
(8) Abu-Hamad, S.; Zaid, H.; Israelson, A.; Nahon, E.; Shoshan-Barmatz, V. Hexokinase-I Protection against Apoptotic Cell Death Is Mediated via Interaction with the Voltage-Dependent Anion Channel-1: MAPPING THE SITE OF BINDING. J. Biol. Chem. 2008, 283 (19), 13482–13490. https://doi.org/10.1074/jbc.M708216200.