Aza- and Mixed Thia/Aza-Macrocyclic Receptors with Quinoline-Bearing Pendant Arms for Optical Discrimination of Zinc(II) or Cadmium(II) Ions.

The synthesis and coordination properties of two fluorescent chemosensors, featuring [9]aneN3 (1,4,7-triazacyclononane; L1) and [12]aneNS3 (1-aza-4,7,10-trithiacyclododecane; L2) as receptor units, and a quinoline pendant arm with an amide group as a functional group spacer are described. The optical responses of L1 and L2 in the presence of several metal ions were analysed in MeCN/H2 O (1 : 4 v/v) solutions. A selective chelation enhancement of fluorescence (CHEF) effect was observed in the presence of Zn2+ in the case of L1, and in the presence of Cd2+ in the case of L2, following the formation of a 1 : 1 and a 1 : 2 metal/ligand complex, respectively, which was also confirmed by potentiometric measurements. 1 H and 13 C NMR measurements in CD3 CN/CDCl3 in combination with molecular mechanics calculations show that for both complexes of L1 and L2 with Zn2+ and Cd2+ , respectively, the coordination of the carbonyl group from the pendant arm could be the origin of the observed optical selectivity.

Folded Structure and Membrane Affinity of the N-Terminal Domain of the Three Human Isoforms of the Mitochondrial Voltage-Dependent Anion-Selective Channel.

Voltage-dependent anion-selective channels (VDACs) are primarily located in the mitochondrial outer membrane (MOM). They are essential for the regulation of ion and metabolite exchanges. In particular, their role in energy-related nucleotide exchange has many implications in apoptosis, cancer, and neurodegenerative diseases. It has been proposed that VDACs' functions are regulated by mobility of the N-terminal helical domain, which is bound to the inner wall of the main ß-barrel domain but exists in equilibrium between the bound-folded and the unbound-unfolded state. When the N-terminal domain detaches from the channel's wall and eventually leaves the lumen, it can either stay exposed to the cytosolic environment or interact with the outer leaflet of the MOM; then, it may also interact with other protein partners. In humans, three different VDAC isoforms are expressed at different tissue-specific levels with evidence of distinct roles. Although the N-terminal domains share high sequence similarity, important differences do exist, with the functionality of the entire protein mostly attributed to them. In this work, the three-dimensional structure and membrane affinity of the three isolated hVDAC N-terminal peptides have been compared through Fourier-transform infrared and NMR spectroscopy in combination with molecular dynamics simulations, and measurement of the surface pressure of lipid monolayers. Although peptides were studied as isolated from the ß-barrel domain, the observed differences are relevant for those whole protein's functions in which a protein-protein interaction is mediated by the N-terminal domain.

The singular behavior of a ß-type semi-synthetic two branched polypeptide: three-dimensional structure and mode of action.

Dendrimeric peptides make a versatile group of bioactive peptidomimetics and a potential new class of antimicrobial agents to tackle the pressing threat of multi-drug resistant pathogens. These are branched supramolecular assemblies where multiple copies of the bioactive unit are linked to a central core. Beyond their antimicrobial activity, dendrimeric peptides could also be designed to functionalize the surface of nanoparticles or materials for other medical uses. Despite these properties, however, little is known about the structure-function relationship of such compounds, which is key to unveil the fundamental physico-chemical parameters and design analogues with desired attributes. To close this gap, we focused on a semi-synthetic, two-branched peptide, SB056, endowed with remarkable activity against both Gram-positive and Gram-negative bacteria and limited cytotoxicity. SB056 can be considered the smallest prototypical dendrimeric peptide, with the core restricted to a single lysine residue and only two copies of the same highly cationic 10-mer polypeptide; an octanamide tail is present at the C-terminus. Combining NMR and Molecular Dynamics simulations, we have determined the 3D structure of two analogues. Fluorescence spectroscopy was applied to investigate the water-bilayer partition in the presence of vesicles of variable charge. Vesicle leakage assays were also performed and the experimental data were analyzed by applying an iterative Monte Carlo scheme to estimate the minimum number of bound peptides needed to achieve the release. We unveiled a singular beta hairpin-type structure determined by the peptide chains only, with the octanamide tail available for further functionalization to add new potential properties without affecting the structure.

VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications.

Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes.

A computational study of ion current modulation in hVDAC3 induced by disulfide bonds.

The human VDAC channel exists in three isoforms characterized by high sequence homology and structural similarity. Yet the function and mode of action of hVDAC3 are still elusive. The presence of six surface cysteines exposed to the oxidizing environment of the mitochondrial inter-membrane space suggests the possible establishment of intramolecular disulfide bonds. Two natural candidates for disulfide bridge formation are Cys2 and Cys8 that, located on the flexible N-terminal domain, can easily come in contact. A third potentially important residue is Cys122 that is close to Cys2 in the homology model of VDAC3. Here we analyzed the impact of SS bonds through molecular dynamics simulations of derivatives of hVDAC3 (dubbed SS-2-8, SS-2-122, SS-8-122) including a single disulfide bond. Simulations showed that in SS-8-122, the fragment 1-7 crosses the top part of the barrel partially occluding the pore and causing a 20% drop of conductance. In order to identify other potential channel-occluding disulfide bonds, we used a set of neural networks and structural bioinformatics algorithms, after filtering with the steric constraints imposed by the 3D-structure. We identified other three species, namely SS-8-65, SS-2-36 and SS-8-36. While the conductance of SS-8-65 and SS-2-36 is about 30% lower than that of the species without disulfide bonds, the conductance of SS-8-36 was 40-50% lower. The results show how VDAC3 is able to modulate its pore size and current by exploiting the mobility of the N-terminal and forming, upon external stimuli, disulfide bridges with cysteine residues located on the barrel and exposed to the inter-membrane space.

Conformational Analysis of the Host-Defense Peptides Pseudhymenochirin-1Pb and -2Pa and Design of Analogues with Insulin-Releasing Activities and Reduced Toxicities.

Pseudhymenochirin-1Pb (Ps-1Pb; IKIPSFFRNILKKVGKEAVSLIAGALKQS) and pseudhymenochirin-2Pa (Ps-2Pa; GIFPIFAKLLGKVIKVASSLISKGRTE) are amphibian peptides with broad spectrum antimicrobial activities and cytotoxicity against mammalian cells. In the membrane-mimetic solvent 50% (v/v) trifluoroethanol-H2O, both peptides adopt a well-defined α-helical conformation that extends over almost all the sequence and incorporates a flexible bend. Both peptides significantly (p < 0.05) stimulate the rate of release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥ 0.1 nM but produce loss of integrity of the plasma membrane at concentrations ≥ 1 µM. Increasing cationicity by the substitution Glu(17) → l-Lys in Ps-1Pb and Glu(27) → l-Lys in Ps-2Pa generates analogues with increased cytotoxicity and reduced insulin-releasing potency. In contrast, the analogues [R8r]Ps-1Pb and [K8k,K19k]Ps-2Pa, incorporating d-amino acid residues to destabilize the α-helical domains, retain potent insulin-releasing activity but are nontoxic to BRIN-BD11 cells at concentrations of 3 µM. [R8r]Ps-1Pb produces a significant increase in insulin release rate at 0.3 nM and [K8k,K19k]Ps-2Pa at 0.01 nM. Both analogues show low hemolytic activity (IC50 > 100 µM) but retain broad-spectrum antimicrobial activity and remain cytotoxic to a range of human tumor cell lines, albeit with lower potency than the naturally occurring peptides. These analogues show potential for development into agents for type 2 diabetes therapy.

The N-Terminal Peptides of the Three Human Isoforms of the Mitochondrial Voltage-Dependent Anion Channel Have Different Helical Propensities.

The voltage-dependent anion channel (VDAC) is the main mitochondrial porin allowing the exchange of ions and metabolites between the cytosol and the mitochondrion. In addition, VDAC was found to actively interact with proteins playing a fundamental role in the regulation of apoptosis and being of central interest in cancer research. VDAC is a large transmembrane ß-barrel channel, whose N-terminal helical fragment adheres to the channel interior, partially closing the pore. This fragment is considered to play a key role in protein stability and function as well as in the interaction with apoptosis-related proteins. Three VDAC isoforms are differently expressed in higher eukaryotes, for which distinct and complementary roles are proposed. In this work, the folding propensity of their N-terminal fragments has been compared. By using multiple spectroscopic techniques, and complementing the experimental results with theoretical computer-assisted approaches, we have characterized their conformational equilibrium. Significant differences were found in the intrinsic helical propensity of the three peptides, decreasing in the following order: hVDAC2 > hVDAC3 > hVDAC1. In light of the models proposed in the literature to explain voltage gating, selectivity, and permeability, as well as interactions with functionally related proteins, our results suggest that the different chemicophysical properties of the N-terminal domain are possibly correlated to different functions for the three isoforms. The overall emerging picture is that a similar transmembrane water accessible conduit has been equipped with not identical domains, whose differences can modulate the functional roles of the three VDAC isoforms.

Synthesis, characterization, antimicrobial activity and LPS-interaction properties of SB041, a novel dendrimeric peptide with antimicrobial properties.

Multimeric peptides offer several advantages with respect to their monomeric counterparts, as increased activity and greater stability to peptidases and proteases. SB041 is a novel antimicrobial peptide with dendrimeric structure; it is a tetramer of pyrEKKIRVRLSA linked by a lysine core, with an amino valeric acid chain. Here, we report on its synthesis, NMR characterization, antimicrobial activity, and LPS-interaction properties. The peptide was especially active against Gram-negative strains, with a potency comparable (on molar basis) to that of lipopeptides colistin and polymixin B, but it also displayed some activity against selected Gram-positive strains. Following these indications, we investigated the efficacy of SB041 in binding Escherichia coli and Pseudomonas aeruginosa LPS in vitro and counteracting its biological effects in RAW-Blue cells, derived from RAW 264.7 macrophages. SB041 strongly bound purified LPS, especially that of E. coli, as proved by fluorescent displacement assay, and readily penetrated into LPS monolayers. However, the killing activity of SB041 against E. coli was not inhibited by increasing concentrations of LPS added to the medium. Checking the SB041 effect on LPS-induced activation of pattern recognition receptors (PRRs) in Raw-Blue cells revealed that while the peptide gave a statistically significant decrease in PRRs stimulation when RAW-Blue cells were challenged with P. aeruginosa LPS, the same was not seen when E. coli LPS was used to activate innate immune defense-like responses. Thus, as previously seen for other antimicrobial peptides, also for SB041 binding to LPS did not translate necessarily into LPS-neutralizing activity, suggesting that SB041-LPS interactions must be of complex nature.