Structure–activity relationships of the antimicrobial peptide gramicidin S and its analogs: Aqueous solubility,self-association,conformation, antimicrobial activity and interaction with model lipid membranes

GS10 [cyclo-(VKLdYPVKLdYP)] is a synthetic analog of the naturally occurring antimicrobial peptide gramicidin (GS) in which the two positively charged ornithine (Orn) residues are replaced by two positively charged lysine (Lys) residues and the two less polar aromatic phenylalanine (Phe) residues are replaced by the more polar tyrosine (Tyr) residues. In this study, we examine the effects of these seemingly conservative modifications to the parent GS molecule on the physical properties of the peptide, and on its interactions with lipid bilayer model and biological membranes, by a variety of biophysical techniques. We show that although GS10 retains the largely β-sheet conformation characteristic of GS, it is less structured in both water and membrane-mimetic solvents. GS10 is also more water soluble and less hydrophobic than GS, as predicted, and also exhibits a reduced tendency for self-association in aqueous solution. Surprisingly, GS10 associates more strongly with zwitterionic and anionic phospholipid bilayer model membranes than does GS, despite its greater water solubility, and the presence of anionic phospholipids and cholesterol (Chol) modestly reduces the association of both GS10 and GS to these model membranes. The strong partitioning of both peptides into lipid bilayers is driven by a large favorable entropy change opposed by a much smaller unfavorable enthalpy change. However, GS10 is also less potent than GS at inducing inverted cubic phases in phospholipid bilayer model membranes and at inhibiting the growth of the cell wall-less bacterium Acholeplasma laidlawii B. These results are discussed in terms of the comparative antibiotic and hemolytic activities of these peptides.     

Role of the EF-hand-like Motif in the 14-3-3 Protein-mediated Activation of Yeast Neutral Trehalase Nth1

Trehalases hydrolyze the non-reducing disaccharide trehalose amassed by cells as a universal protectant and storage carbohydrate. Recently, it has been shown that the activity of neutral trehalase Nth1 from Saccharomyces cerevisiae is mediated by the 14-3-3 protein binding that modulates the structure of both the catalytic domain and the region containing the EF-hand-like motif, whose role in the activation of Nth1 is unclear. In this work, the structure of the Nth1·14-3-3 complex and the importance of the EF-hand-like motif were investigated using site-directed mutagenesis, hydrogen/deuterium exchange coupled to mass spectrometry, chemical cross-linking, and small angle x-ray scattering. The low resolution structural views of Nth1 alone and the Nth1·14-3-3 complex show that the 14-3-3 protein binding induces a significant structural rearrangement of the whole Nth1 molecule. The EF-hand-like motif-containing region forms a separate domain that interacts with both the 14-3-3 protein and the catalytic trehalase domain. The structural integrity of the EF-hand like motif is essential for the 14-3-3 protein-mediated activation of Nth1, and calcium binding, although not required for the activation, facilitates this process by affecting its structure. Our data suggest that the EF-hand like motif-containing domain functions as the intermediary through which the 14-3-3 protein modulates the function of the catalytic domain of Nth1.     

Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells

Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism.     

Genetic signature of differential sensitivity to stevioside in the Italian population

The demand for diet products is continuously increasing, together with that for natural food ingredients. Stevioside and other steviol glycosides extracted from the leaves of the plant Stevia rebaudiana Bertoni are the first natural high-potency sweeteners to be approved for consumption in the United States and the European Union. However, the sweetness of these compounds is generally accompanied by aversive sensations, such as bitter and off-tastes, which may constitute a limit to their consumption. Moreover, consumers’ differences in sensitivity to high-potency sweeteners are well known, as well as difficulties in characterizing their aftertaste. Recently, TAS2R4 and TAS2R14 have been identified as the receptors that mediate the bitter off-taste of steviol glycosides in vitro. In the present study, we demonstrate that TAS2R4 gene polymorphism rs2234001 and TAS2R14 gene polymorphism rs3741843 are functional for stevioside bitterness perception.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0401-y) contains supplementary material, which is available to authorized users.     

Labeled chemical biology tools for investigating sphingolipid metabolism,trafficking and interaction with lipids and proteins

The unraveling of sphingolipid metabolism and function in the last 40 years relied on the extensive study of inherited human disease and specifically-tailored mouse models. However, only few of the achievements made so far would have been possible without chemical biology tools, such as fluorescent and/or radio-labeled and other artificial substrates, (mechanism-based) enzyme inhibitors, cross-linking probes or artificial membrane models. In this review we provide an overview over chemical biology tools that have been used to gain more insight into the molecular basis of sphingolipid-related biology. Many of these tools are still of high relevance for the investigation of current sphingolipid-related questions, others may stimulate the tailoring of novel probes suitable to address recent and future issues in the field. This article is part of a Special Issue entitled Tools to study lipid functions.     

Pre-treatment with H<Subscript>2</Subscript>O<Subscript>2</Subscript> induces salt tolerance in Barley seedlings

Barley seedlings were pre-treated with 1 and 5 μM H₂O₂ for 2 d and then supplied with water or 150 mM NaCl for 4 and 7 d. Exogenous H₂O₂ alone had no effect on the proline, malondialdehyde (MDA) and H₂O₂ contents, decreased catalase (CAT) activity and had no effect on peroxidase (POX) activity. Three new superoxide dismutase (SOD) isoenzymes appeared in the leaves as a result of 1 μM H₂O₂ treatment. NaCl enhanced CAT and POX activity. SOD activity and isoenzyme patterns were changed due to H₂O₂ pre-treatment, NaCl stress and leaf ageing. In pre-treated seedlings the rate of ¹⁴CO₂ fixation was higher and MDA, H₂O₂ and proline contents were lower in comparison to the seedlings subjected directly to NaCl stress. Cl⁻ content in the leaves 4 and 7 d after NaCl supply increased considerably, but less in pre-treated plants. It was suggested that H₂O₂ metabolism is involved as a signal in the processes of barley salt tolerance.     

NMR spectral mapping of Lipid A molecular patterns affected by interaction with the innate immune receptor CD14

Soluble CD14 (sCD14) is a serum glycoprotein that binds to the Lipid A moiety of lipopolysaccharides (LPS) with high affinity as part of the innate immune response to bacterial endotoxins. In order to investigate structural interactions of Lipid A with sCD14, we have prepared an isotopically labeled form of a fully active and chemically defined endotoxin, Kdo₂-Lipid A, which allowed us to carry out detailed NMR spectral mapping of this agonist ligand bound to sCD14 and identify for the first time structural regions that are strongly affected during complex formation with sCD14. These map to two adjacent areas comprising the lower portions of the sugar headgroup and upper half of the acyl chains I, III, and V, which are spatially proximal to the 1- and 4′-phosphate ends. Additionally, we have detected for the first time, presence of differential dynamic behavior for the affected resonances, suggesting a likely role for dynamics in the mechanism of Lipid A pattern recognition by sCD14.     

Elevated Proton Leak of the Intermediate OH in Cytochrome c Oxidase

The kinetics of the formation and relaxation of transmembrane electric potential (Δψ) during the complete single turnover of CcO was studied in the bovine heart mitochondrial and the aa₃-type Paracoccus denitrificans enzymes incorporated into proteoliposome membrane. The real-time Δψ kinetics was followed by the direct electrometry technique. The prompt oxidation of CcO and formation of the activated, oxidized (O_H) state of the enzyme leaves the enzyme trapped in the open state that provides an internal leak for protons and thus facilitates dissipation of Δψ (τ^app ≤ 0.5-0.8 s). By contrast, when the enzyme in the O_H state is rapidly re-reduced by sequential electron delivery, Δψ dissipates much slower (τ^app > 3 s). In P. denitrificans CcO proteoliposomes the accelerated Δψ dissipation is slowed down by a mutational block of the proton conductance through the D-, but not K-channel. We concluded that in contrast to the other intermediates the O_H state of CcO is vulnerable to the elevated internal proton leak that proceeds via the D-channel.     

An Integrated Model of Eicosanoid Metabolism and Signaling Based on Lipidomics Flux Analysis

There is increasing evidence for a major and critical involvement of lipids in signal transduction and cellular trafficking, and this has motivated large-scale studies on lipid pathways. The Lipid Metabolites and Pathways Strategy consortium is actively investigating lipid metabolism in mammalian cells and has made available time-course data on various lipids in response to treatment with KDO₂-lipid A (a lipopolysaccharide analog) of macrophage RAW 264.7 cells. The lipids known as eicosanoids play an important role in inflammation. We have reconstructed an integrated network of eicosanoid metabolism and signaling based on the KEGG pathway database and the literature and have developed a kinetic model. A matrix-based approach was used to estimate the rate constants from experimental data and these were further refined using generalized constrained nonlinear optimization. The resulting model fits the experimental data well for all species, and simulated enzyme activities were similar to their literature values. The quantitative model for eicosanoid metabolism that we have developed can be used to design experimental studies utilizing genetic and pharmacological perturbations to probe fluxes in lipid pathways.