The Effect of Selected Flavonoids and Lipoic Acid on Natural and Model Cell Membranes: Langmuir and Microelectrophoretic Methods.

The influence of kaempferol (K), myricetin (M) and lipoic acid (LA) on the properties of natural erythrocytes, isolated from animal blood and biological membrane models (monolayers and liposomes) made of phosphatidylcholine (PC), cholesterol (CHOL), and sphingomyelin (SM), CHOL in a ratio of 10:9, was investigated. The Langmuir method, Brewster angle microscopy (BAM) and microelectrophoresis were used. The presented results showed that modification of liposomes with kaempferol, myricetin and lipoic acid caused changes in the surface charge density and the isoelectric point value. Comparing the tested systems, several conclusions were made. (1) The isoelectric point for the DPPC:Chol:M (~2.2) had lower pH values compared to lipoic acid (pH~2.5) and kaempferol (pH~2.6). (2) The isoelectric point for the SM-Chol with myricetin (~3.0) had lower pH values compared to kaempferol (pH~3.4) and lipoic acid (pH~4.7). (3) The surface charge density values for the DPPC:Chol:M system in the range of pH 2-9 showed values from 0.2 to -2.5 × 10-2 C m-2. Meanwhile, for the DPPC:Chol:K and DPPC:Chol:LA systems, these values were higher at pH~2 (0.7 × 10-2 C m-2 and 0.8 × 10-2 C m-2) and lower at pH~9 (-2.1 × 10-2 C m-2 and -1.8 × 10-2 C m-2), respectively. (4) The surface charge density values for the SM:Chol:M system in the range of pH 2-9 showed values from 0.5 to -2.3 × 10-2 C m-2. Meanwhile, for the DPPC:Chol:K and DPPC:Chol:LA systems, these values were higher at pH~2 (0.8 × 10-2 C m-2), and lower at pH~9 (-1.0 × 10-2 C m-2 and -1.8 × 10-2 C m-2), respectively. (5) The surface charge density values for the erythrocytes with myricetin in the range of pH 2-9 showed values from 1.0 to -1.8 × 10-2 C m-2. Meanwhile, for the erythrocytes:K and erythrocytes:LA systems, these values, at pH~2, were 1.3 × 10-2 C m-2 and 0.8 × 10-2 C m-2 and, at pH~9, -1.7 × 10-2 C m-2 and -1.0 × 10-2 C m-2, respectively.

Cholesterol Binds the Amphipathic Helix of IFITM3 and Regulates Antiviral Activity.

The interferon-induced transmembrane (IFITM) proteins broadly inhibit the entry of diverse pathogenic viruses, including Influenza A virus (IAV), Zika virus, HIV-1, and SARS coronaviruses by inhibiting virus-cell membrane fusion. IFITM3 was previously shown to disrupt cholesterol trafficking, but the functional relationship between IFITM3 and cholesterol remains unclear. We previously showed that inhibition of IAV entry by IFITM3 is associated with its ability to promote cellular membrane rigidity, and these activities are functionally linked by a shared requirement for the amphipathic helix (AH) found in the intramembrane domain (IMD) of IFITM3. Furthermore, it has been shown that the AH of IFITM3 alters lipid membranes in vitro in a cholesterol-dependent manner. Therefore, we aimed to elucidate the relationship between IFITM3 and cholesterol in more detail. Using a fluorescence-based in vitro binding assay, we found that a peptide derived from the AH of IFITM3 directly interacted with the cholesterol analog, NBD-cholesterol, while other regions of the IFITM3 IMD did not, and native cholesterol competed with this interaction. In addition, recombinant full-length IFITM3 protein also exhibited NBD-cholesterol binding activity. Importantly, previously characterized mutations within the AH of IFITM3 that strongly inhibit antiviral function (F63Q and F67Q) disrupted AH structure in solution, inhibited cholesterol binding in vitro, and restricted bilayer insertion in silico. Our data suggest that direct interactions with cholesterol may contribute to the inhibition of membrane fusion pore formation by IFITM3. These findings may facilitate the design of therapeutic peptides for use in broad-spectrum antiviral therapy.

Strikingly Different Roles of SARS-CoV-2 Fusion Peptides Uncovered by Neutron Scattering.

Coronavirus disease-2019 (COVID-19), a potentially lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of selected SARS-CoV-2 Spike fusion peptides, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin-echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium, however, reorients the peptide back to the lipid leaflet closest to the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain, which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed.

Microscopic Interactions of Melatonin, Serotonin and Tryptophan with Zwitterionic Phospholipid Membranes.

The interactions at the atomic level between small molecules and the main components of cellular plasma membranes are crucial for elucidating the mechanisms allowing for the entrance of such small species inside the cell. We have performed molecular dynamics and metadynamics simulations of tryptophan, serotonin, and melatonin at the interface of zwitterionic phospholipid bilayers. In this work, we will review recent computer simulation developments and report microscopic properties, such as the area per lipid and thickness of the membranes, atomic radial distribution functions, angular orientations, and free energy landscapes of small molecule binding to the membrane. Cholesterol affects the behaviour of the small molecules, which are mainly buried in the interfacial regions. We have observed a competition between the binding of small molecules to phospholipids and cholesterol through lipidic hydrogen-bonds. Free energy barriers that are associated to translational and orientational changes of melatonin have been found to be between 10-20 kJ/mol for distances of 1 nm between melatonin and the center of the membrane. Corresponding barriers for tryptophan and serotonin that are obtained from reversible work methods are of the order of 10 kJ/mol and reveal strong hydrogen bonding between such species and specific phospholipid sites. The diffusion of tryptophan and melatonin is of the order of 10-7 cm2/s for the cholesterol-free and cholesterol-rich setups.