Interaction between Alzheimer's Aβ(25–35) peptide and phospholipid bilayers: The role of cholesterol

AbstractThere is mounting evidence that the lipid matrix of neuronal cell membranes plays an important role in the accumulation of β-amyloid peptides into senile plaques, one of the hallmarks of Alzheimer's disease (AD). With the aim to clarify the molecular basis of the interaction between amyloid peptides and cellular membranes, we investigated the interaction between a cytotoxic fragment of Aβ(1–42), i.e., Aβ(25–35), and phospholipid bilayer membranes. These systems were studied by Electron Paramagnetic Resonance (EPR) spectroscopy, using phospholipids spin-labeled on the acyl chain. The effect of inclusion of charged phospholipids or/and cholesterol in the bilayer composition was considered in relation to the peptide/membrane interaction. The results show that Aβ(25–35) inserts in bilayers formed by the zwitterionic phospholipid dilauroyl phosphatidylcholine (DLPC), positioning between the outer part of the hydrophobic core and the external hydrophilic layer. This process is not significantly influenced by the inclusion of the anionic phospholipid phosphatidylglycerol (DLPG) in the bilayer, indicating the peptide insertion to be driven by hydrophobic rather than electrostatic interactions. Cholesterol plays a fundamental role in regulating the peptide/membrane association, inducing a membrane transition from a fluid-disordered to a fluid-ordered phase. At low cholesterol content, in the fluid-disordered phase, the insertion of the peptide in the membrane causes a displacement of cholesterol towards the more external part of the membrane. The crowding of cholesterol enhances its rigidifying effect on this region of the bilayer. Finally, the cholesterol-rich fluid-ordered membrane looses the ability to include Aβ(25–35)

Flavonoid microparticles by spray-drying: Influence of enhancersof the dissolution rate on properties and stability

Naringenin (Nn) and Quercetin (Q) have numerous health benefits particularly due to their antioxidant properties. However, their low solubility, bioavailability and stability limit their use as components for functional foods, nutraceuticals and pharmaceutical agents. In this research, Nn- and Q-microparticles were produced by a spray-drying process using a combination of cellulose acetate phthalate (CAP) as coating gastroresistant polymer and swelling or surfactant agents as enhancers of dissolution rate. Raw materials and microparticles produced were all characterized by particle size analysis, differential scanning calorimetry, X-ray diffraction, and imaged by electron and fluorescence microscopy. During 12 months, storage stability was evaluated by analyzing drug content, HPLC and DSC profiles, as well as antioxidant activity (DPPH test). In vitro dissolution tests, using a pH-change method, were carried out to investigate the influence of formulative parameters on flavonoid release from the microparticles. Presence of a combination of CAP and surfactants or swelling agents in the formulations produced microparticles with good resistance at low pH of the gastric fluid and complete flavonoid release in the intestinal environment. The spray-drying technique and the process conditions selected have given satisfying encapsulation efficiency and product yield. The microencapsulation have improved the technological characteristics of the powders such as morphology and size, have given long-lasting storage stability and have preserved the antioxidant properties

Structural analysis of a simplified model reproducing SARS-CoV-2 S RBD/ACE2 binding site

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus identified as the cause of the coronavirus outbreak in December 2019 (COVID-19). Like all the RNA viruses, SARS-CoV-2 constantly evolves through mutations in its genome, accumulating 1–2 nucleotide changes every month, giving the virus a selective advantage through enhanced transmissibility, greater pathogenicity, and the possibility of circumventing immunity previously acquired by an individual either by natural infection or by vaccination. Several SARS-CoV-2 variants of concern (VoC) have been identified, among which we find Alpha (Lineage B.1.1.7), Beta (Lineage B.1.351), and Gamma (Lineage P.1) variants. Most of the mutations occur in the spike (S) protein, a surface glycoprotein that plays a crucial role in viral infection; the S protein binds the host cell receptor, the angiotensin-converting enzyme of type 2 (ACE2) via the receptor binding domain (RBD) and catalyzes the fusion of the viral membrane with the host cell. In this work, we present the development of a simplified system that would afford to study the change in the SARS-CoV-2 S RBD/ACE2 binding related to the frequent mutations. In particular, we synthesized and studied the structure of short amino acid sequences, mimicking the two proteins’ critical portions. Variations in the residues were easily managed through the one-point alteration of the sequences. Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopies provide insights into ACE2 and SARS-CoV-2 S RBD structure with its related three variants (Alpha, Beta, and Gamma). Spectroscopy data supported by molecular dynamics lead to the description of an ACE2/RBD binding model in which the effect of a single amino acid mutation in changing the binding of S protein to the ACE2 receptor is predictable

A thermodynamic signature of lipid segregation in biomembranes induced by a short peptide derived from glycoprotein gp36 of feline immunodeficiency virus.

The interactions between proteins/peptides and lipid bilayers are fundamental in a variety of key biological processes, and among these, the membrane fusion process operated by viral glycoproteins is one of the most important, being a fundamental step of the infectious event. In the case of the feline immunodeficiency virus (FIV), a small region of the membrane proximal external region (MPER) of the glycoprotein gp36 has been demonstrated to be necessary for the infection to occur, being able to destabilize the membranes to be fused. In this study, we report a physicochemical characterization of the interaction process between an eight-residue peptide, named C8, modeled on that gp36 region and some biological membrane models (liposomes) by using calorimetric and spectroscopic measurements. CD studies have shown that the peptide conformation changes upon binding to the liposomes. Interestingly, the peptide folds from a disordered structure (in the absence of liposomes) to a more ordered structure with a low but significant helix content. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) results show that C8 binds with high affinity the lipid bilayers and induces a significant perturbation/reorganization of the lipid membrane structure. The type and the extent of such membrane reorganization depend on the membrane composition. These findings provide interesting insights into the role of this short peptide fragment in the mechanism of virus-cell fusion, demonstrating its ability to induce lipid segregation in biomembranes

Nusinersen Induces Disease-Severity-Specific Neurometabolic Effects in Spinal Muscular Atrophy

Intrathecal delivery of Nusinersen-an antisense oligonucleotide that promotes survival motor neuron (SMN) protein induction-is an approved therapy for spinal muscular atrophy (SMA). Here, we employed nuclear magnetic resonance (NMR) spectroscopy to longitudinally characterize the unknown metabolic effects of Nusinersen in the cerebrospinal fluid (CSF) of SMA patients across disease severity. Modulation of amino acid metabolism is a common denominator of biochemical changes induced by Nusinersen, with distinct downstream metabolic effects according to disease severity. In severe SMA1 patients, Nusinersen stimulates energy-related glucose metabolism. In intermediate SMA2 patients, Nusinersen effects are also related to energy homeostasis but involve ketone body and fatty acid biosynthesis. In milder SMA3 patients, Nusinersen mainly modulates amino acid metabolism. Moreover, Nusinersen modifies the CSF metabolome of a more severe clinical group towards the profile of untreated SMA patients with milder disease. These findings reveal disease severity-specific neurometabolic signatures of Nusinersen treatment, suggesting a selective modulation of peripheral organ metabolism by this CNS-directed therapy in severe SMA patients

Analytical characterization of an inulin-type fructooligosaccharide from root-tubers of Asphodelus ramosus L

Plant-based systems continue to play a pivotal role in healthcare, and their use has been extensively documented. Asphodelus L. is a genus comprising various herbaceous species, known by the trivial name Asphodelus. These plants have been known since antiquity for both food and therapeutic uses, especially for treating several diseases associated with inflammatory and infectious skin disorders. Phytochemical studies revealed the presence of different constituents, mainly anthraquinones, triterpenoids, phenolic acids, and flavonoids. Although extensive literature has been published on these constituents, a paucity of information has been reported regarding the carbohydrate composition, such as fructans and fructan-like derivatives. The extraction of watersoluble neutral polysaccharides is commonly performed using water extraction, at times assisted by microwaves and ultrasounds. Herein, we reported the investigation of the alkaline extraction of roottubers of Asphodelus ramosus L., analyzing the water-soluble polysaccharides obtained by precipitation from the alkaline extract and its subsequent purification by chromatography. A polysaccharide was isolated by alkaline extraction; the HPTLC study to determine its composition showed fructose as the main monosaccharide. FT-IR analysis showed the presence of an inulin-type structure, and NMR analyses allowed us to conclude that A. ramosus roots contain polysaccharide with an inulin-type fructooligosaccharide with a degree of polymerization of 7-8

Environmental constraints in the study of flexible segments of proteins

The structural problem posed by ill-defined segments in protein structures is similar to those encountered in the study of most peptide hormones, with terminal tracts resembling linear peptides and loops resembling cyclic peptides, The conformational preferences of short linear peptides in solution can be influenced by the use of solvent mixtures of viscosity higher than that of pure water but comparable to that of cytoplasm. In order to check whether it is possible to use these media in the structural study of proteins, we undertook an exploratory study on BPTI in a mixture of dimethylsulfoxide and water. The complete assignment of BPTI in an 80:20 (by volume) DMSO-d(6)/water cryomixture at two temperatures showed that all resonances parallel those in water, hinting at the persistence of the correct protein architecture, which is also confirmed by NOESY experiments. In addition to the NOEs present in the aqueous solution it was possible to detect numerous new cross peaks, in particular from residues belonging to the less-defined regions. The new cross peaks do not originate from spin diffusion and are consistent with the best NMR structure and with the X-ray structures of BPTI