Azucena Gonzalez-Horta

Effects of Palmitoylation on Dynamics and Phospholipid-Bilayer-Perturbing Properties of the N-Terminal Segment of Pulmonary Surfactant Protein SP-C as Shown by 2H-NMR

It has been proposed that palmitoylation of the N-terminal segment of surfactant protein SP-C is important for maintaining association of pulmonary surfactant complexes with interfacial films compressed to high pressures at the end of expiration. In this study, we examined surfactant membrane models containing palmitoylated and nonpalmitoylated synthetic peptides, based on the N-terminal SP-C sequence, in dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylglycerol (7:3, w/w) by (2)H-NMR. Perturbations of lipid properties by the peptide versions were compared in samples containing chain- and headgroup-deuterated lipid (DPPC-d(62) and DPPC-d(4) respectively). Also, deuterated peptide palmitate chains were compared with those of DPPC in otherwise identical lipid-protein mixtures. Palmitoylated peptide increased average DPPC-d(62) chain orientational order slightly, particularly for temperatures spanning gel and liquid crystalline coexistence, implying penetration of palmitoylated peptide into ordered membrane. In contrast, the nonpalmitoylated peptide had a small disordering effect in this temperature range. Both peptide versions perturbed DPPC-d(4) headgroup orientation similarly, suggesting little effect of palmitoylation on the largely electrostatic peptide-headgroup interaction. Deuterated acyl chains attached to the SP-C N-terminal segment displayed a qualitatively different distribution of chain order, and lower average order, than DPPC-d(62) in the same membranes. This likely reflects local perturbation of lipid headgroup spacing by the peptide portion interacting with the bilayer near the peptide palmitate chains. This study suggests that SP-C-attached acyl chains could be important for coupling of lipid and protein motions in surfactant bilayers and monolayers, especially in the context of ordered phospholipid structures such as those potentially formed during exhalation, when stabilization of the respiratory surface by surfactant is the most crucial.

Interfacial Behavior of Recombinant Forms of Human Pulmonary Surfactant Protein SP-C

The behavior at air-liquid interfaces of two recombinant versions of human surfactant protein SP-C has been characterized in comparison with that of native palmitoylated SP-C purified from porcine lungs. Both native and recombinant proteins promoted interfacial adsorption of dipalmitoylphosphatidylcholine bilayers to a limited extent, but catalyzed very rapid formation of films from different lipid mixtures containing both zwitterionic and anionic phospholipids. Once at the interface, the recombinant variants exhibited compression-driven structural transitions, consistent with changes in the orientation of the deacylated N-terminal segment, which were not observed in the native protein. Compression isotherms of lipid/protein films suggest that the recombinant SP-C forms promote expulsion at high pressures of a higher number of lipid molecules per mole of protein than does native SP-C. A more dynamic conformation of the N-terminal segment in recombinant SP-C forms is likely also responsible for facilitating compression-driven condensation of domains in anionic phospholipid films as observed by epifluorescence microscopy. Finally, both native palmitoylated SP-C and the phenylalanine-containing recombinant versions facilitate similarly the repetitive compression-expansion dynamics of lipid/protein films, which were able to reach maximal surface pressures with practically no hysteresis along multiple quasi-static or dynamic cycles.

Synthetic peptides representing the N-terminal segment of surfactant protein C modulate LPS-stimulated TNF-α production by macrophages

Surfactant protein C (SP-C) consists of a hydrophobic α-helix inserted in pulmonary surfactant membranes, and a more polar N-terminal palmitoylated segment exposed to the aqueous phase. Previously, we showed that SP-C inserted in lipid vesicles interacts with bacterial lipopolysaccharide (LPS) and reduces LPS-elicited responses. As the N-terminal segment of SP-C was the most likely region responsible for these effects, a set of synthetic analogs of this stretch (SPC(1-13) ) were studied. Binding studies showed that SPC(1-13) binds LPS to the same extent as porcine SP-C under lipid-free conditions. In the absence of serum, both, palmitoylated and non-palmitoylated analogs enhanced the binding of tritiated LPS to macrophages as well as the LPS-induced production of TNF-α by these cells. These effects were reversed in the presence of serum; the analogs reduced the production of TNF-α in LPS-stimulated macrophages, probably by interfering with the formation of LPS/CD14/LBP complexes as suggested by analysis of the fluorescence emitted by a FITC derivative of Re-LPS. Our data indicate that water-soluble analogs of the N-terminal segment of SP-C can reduce LPS effects in the presence of serum, and thus might help in the design of new derivatives to fight endotoxic shock and pro-inflammatory events.

New temperature-assisted ionic liquid-based dispersive liquid–liquid microextraction method for the determination of glyphosate and aminomethylphosphonic acid in water samples

ABSTRACT A new analytical temperature-assisted ionic liquid-based dispersive liquid–liquid microextraction (TA-IL-DLLME) method was developed for glyphosate and aminomethylphosphonic acid determination in water samples. Extracted analytes were derivatized using 9-fluoroenylmethylchloroformate and quantified by liquid chromatography with fluorescence detection. For the TA-IL-DLLME method, two strategies for phase solubilization were evaluated; in approach 1, the ionic liquid and aqueous matrix sample were mixed and then heated, while in approach 2, the aqueous sample was first heated and then the ionic liquid was injected. For both approaches, optimization included parameters that significantly affect extraction efficiency: ionic liquid type and volume, solubilization temperature and time, cooling and centrifugation time. Among the evaluated ionic liquids, 1-decyl-3-methylimidazolium tetrafluoroborate showed the best performance for TA-IL-DLLME and was selected for the two solubilization approaches; with approach 2, slightly better results were obtained. Thus, sample analyses were performed using a procedure based on approach 2. An important matrix effect, attributed to the presence of salts and metals in real water samples was observed. Sample acidification before derivatization allowed this problem to diminish, with recoveries ranging from 75 and 99%, and enrichment factors between 57 and 76 for target analytes. GRAPHICAL ABSTRACT

Biodegradable nanoparticles loaded with tetrameric melittin: preparation and membrane disruption evaluation

Melittin is the main component of bee venom consisting of 26 amino acids that has multiple effects, including antibacterial, antiviral and anti-inflammatory in various cell types. This peptide forms pores in biological membranes and triggers cell death. Therefore it has potential as an anti-cancer therapy. However, the therapeutic application of melittin is limited due to its main side effect, hemolysis, which is especially pronounced following intravenous administration. In the present study, we formulated tetrameric melittin-carrying poly-D,L-lactic-co-glycolic acid nanoparticles (PLGA-NPs) and analyzed the lytic activity of this system on liposomes that resembles breast cancer cells. Tetrameric melittin binds avidly to PLGA-NPs with an encapsulation efficiency of 97% and retains its lytic activity demonstrating the effectiveness of PLGA-NPs as nanocarriers for this cytolytic peptide.

Developments of solid-phase microextraction fiber coatings for environmental pharmaceutical and personal care products analysis

Abstract Solid-phase microextraction (SPME) is a sample preparation technique with many applications that is being continuously developed. In this technique, the type of fiber coating plays a crucial role for extraction efficiency. Currently available commercial coatings have certain drawbacks that have been overcome by the development of new coatings based on novel materials; these have improved the efficiency of extraction, selectivity and stability of commercial coatings. Pharmaceutical and personal care products (PPCPs) are one of the most important groups of emerging contaminants; however, some studies suggest that these compounds can cause adverse health effects. No official monitoring protocols for these compounds are currently available, so the establishment of analytical methods that allow their determination in environmental samples is required. The complexity of environmental samples together with the low concentration levels of these compounds makes necessary the use of sample preparation techniques capable of removing interferences, as well as preconcentrated analytes, and SPME is a very promising alternative to achieve this. This review describes the recent developments in SPME with classical and novel coatings and its applications for PPCP determination in environmental samples.

Differential interaction of α-synuclein N-terminal segment with mitochondrial model membranes

Alpha-synuclein (α-syn) is an intrinsically-disordered protein that has been associated with Parkinsons disease through its deposition in an amyloid fibril form within Lewy Body. Several lines of evidence suggest that the physical association of α-syn with the mitochondrial membranes may cause membrane damage and mitochondrial dysfunction, playing an important role in disease progression. Although there is strong evidence that the N-terminus part of α-syn is essential for membrane affinity, cooperative formation of helical domains and regulation of mitochondria membrane permeability, the amino acids involve in this membrane binding is still controversial. Fluorescence spectroscopy, circular dichroism and Langmuir monolayer technique were used to elucidate this recognition process of mitochondrial membrane system by synthetic peptides derived from α-syn N-terminal segment. The results obtained in this work show that the first 15 amino acid of the α-syn N-terminal segment mainly participate in the anchoring, perturbing the membrane hydrophobic region, while the peptide corresponding to 16-30 residues interacts only with the phospholipid polar headgroup, confirming that the binding affinity of the N-terminus is nonuniform.