Exposure to Aldehyde Cherry e-Liquid Flavoring and Its Vaping Byproduct Disrupt Pulmonary Surfactant Biophysical Function.

Over the past decade, there has been a significant rise in the use of vaping devices, particularly among adolescents, raising concerns for effects on respiratory health. Pressingly, many recent vaping-related lung injuries are unexplained by current knowledge, and the overall implications of vaping for respiratory health are poorly understood. This study investigates the effect of hydrophobic vaping liquid chemicals on the pulmonary surfactant biophysical function. We focus on the commonly used flavoring benzaldehyde and its vaping byproduct, benzaldehyde propylene glycol acetal. The study involves rigorous testing of the surfactant biophysical function in Langmuir trough and constrained sessile drop surfactometer experiments with both protein-free synthetic surfactant and hydrophobic protein-containing clinical surfactant models. The study reveals that exposure to these vaping chemicals significantly interferes with the synthetic and clinical surfactant biophysical function. Further atomistic simulations reveal preferential interactions with SP-B and SP-C surfactant proteins. Additionally, data show surfactant lipid-vaping chemical interactions and suggest significant transfer of vaping chemicals to the experimental subphase, indicating a toxicological mechanism for the alveolar epithelium. Our study, therefore, reveals novel mechanisms for the inhalational toxicity of vaping. This highlights the need to reassess the safety of vaping liquids for respiratory health, particularly the use of aldehyde chemicals as vaping flavorings.

Why do polyarginines adsorb at neutral phospholipid bilayers and polylysines do not? An insight from density functional theory calculations and molecular dynamics simulations.

Adsorption of cell-penetrating peptides (CPPs) at cellular membranes is the first and necessary step for their subsequent translocation across cellular membranes into the cytosol. It has been experimentally shown that CPPs rich in arginine (Arg) amino acid penetrate across phospholipid bilayers more effectively than their lysine (Lys) rich counterparts. In this work, we aim to understand the differences in the first translocation step, adsorption of Arg9 and Lys9 peptides at fully hydrated neutral phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipid bilayers and evaluate in detail the energetics of the process using molecular dynamics (MD) simulations and free energy calculations of adsorption of the single peptide. We show that the adsorption of Arg9 is energetically feasible, with the free energy of adsorption being ∼-5.0 kcal mol-1 at PC and ∼-5.5 kcal mol-1 at PE bilayers. In contrast, adsorption of Lys9 is not observed at PC bilayers, and their adsorption at PE bilayers is very weak, being ∼-0.5 kcal mol-1. We show by energy decomposition and analysis of peptide hydration along the membrane that significantly stronger electrostatic interactions of Arg9 with lipid phosphate groups, together with the greater loss of peptide hydration (and in turn stronger hydrophobic interactions) along the membrane translocation path, are the main driving factors governing the adsorption of Arg-rich peptides at neutral lipid bilayers in contrast to Lys-rich peptides. Finally, we also compare the energetics in lipid/bilayer systems with the density functional theory (DFT) calculations of the corresponding model systems in the continuum water model and reveal the energetic differences in different environments.

Stealthy Player in Lipid Experiments? EDTA Binding to Phosphatidylcholine Membranes Probed by Simulations and Monolayer Experiments.

Ethylenediaminetetraacetic acid (EDTA) is frequently used in lipid experiments to remove redundant ions, such as Ca2+, from the sample solution. In this work, combining molecular dynamics (MD) simulations and Langmuir monolayer experiments, we show that on top of the expected Ca2+ depletion, EDTA anions themselves bind to phosphatidylcholine (PC) monolayers. This binding, originating from EDTA interaction with choline groups of PC lipids, leads to the adsorption of EDTA anions at the monolayer surface and concentration-dependent changes in surface pressure as measured by monolayer experiments and explained by MD simulations. This surprising observation emphasizes that lipid experiments carried out using EDTA-containing solutions, especially of high concentrations, must be interpreted very carefully due to potential interfering interactions of EDTA with lipids and other biomolecules involved in the experiment, e.g., cationic peptides, that may alter membrane-binding affinities of studied compounds.

Effects of Water Deuteration on Thermodynamic and Structural Properties of Proteins and Biomembranes.

Light and heavy water are often used interchangeably in spectroscopic experiments with the tacit assumption that the structure of the investigated biomolecule does not depend too much on employing one or the other solvent. While this may often be a good approximation, we demonstrate here using molecular dynamics simulations incorporating nuclear quantum effects via modification of the interaction potential that there are small but significant differences. Namely, as quantified and discussed in the present study, both proteins and biomembranes tend to be slightly more compact and rigid in D2O than in H2O, which reflects the stronger hydrogen bonding in the former solvent.

Ionic Strength and Solution Composition Dictate the Adsorption of Cell-Penetrating Peptides onto Phosphatidylcholine Membranes.

Adsorption of arginine-rich positively charged peptides onto neutral zwitterionic phosphocholine (PC) bilayers is a key step in the translocation of those potent cell-penetrating peptides into the cell interior. In the past, we have shown both theoretically and experimentally that polyarginines adsorb to the neutral PC-supported lipid bilayers in contrast to polylysines. However, comparing our results with previous studies showed that the results often do not match even at the qualitative level. The adsorption of arginine-rich peptides onto 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) may qualitatively depend on the actual experimental conditions where binding experiments have been performed. In this work, we systematically studied the adsorption of R9 and K9 peptides onto the POPC bilayer, aided by molecular dynamics (MD) simulations and fluorescence cross-correlation spectroscopy (FCCS) experiments. Using MD simulations, we tested a series of increasing peptide concentrations, in parallel with increasing Na+ and Ca2+ salt concentrations, showing that the apparent strength of adsorption of R9 decreases upon the increase of peptide or salt concentration in the system. The key result from the simulations is that the salt concentrations used experimentally can alter the picture of peptide adsorption qualitatively. Using FCCS experiments with fluorescently labeled R9 and K9, we first demonstrated that the binding of R9 to POPC is tighter by almost 2 orders of magnitude compared to that of K9. Finally, upon the addition of an excess of either Na+ or Ca2+ ions with R9, the total fluorescence correlation signal is lost, which implies the unbinding of R9 from the PC bilayer, in agreement with our predictions from MD simulations.

The "good," the "bad," and the "hidden" in neutron scattering and molecular dynamics of ionic aqueous solutions.

We characterize a concentrated 7.3 m CaCl2 solution, combining neutron diffraction with chloride isotopic substitution (Cl-NDIS) in null water and molecular dynamics (MD) simulations. We elucidate the solution structure, thermodynamic properties, and extent of ion pairing previously suggested as concentration-dependent and often not observed at lower concentrations. Our Cl-NDIS measurements designate the solvent-shared ion pairing as dominant and the contact ion pairing (CIP) as insignificant even under conditions close to the solubility limit. The MD models parameterized against neutron diffraction with calcium isotopic substitution (Ca-NDIS) overestimate CIP despite successfully reproducing most of the Cl-NDIS signal. This drawback originates from the fact that Ca2+-Cl- interactions were primarily "hidden" in the Ca-NDIS signal due to overlapping with Ca2+-Ow and Ca2+-Hw contributions to the total scattering. Contrary, MD models with moderate CIP and possessing generally good performance at high concentrations fail to reproduce the NDIS measurements accurately. Therefore, the electronic polarization, introduced in most of the recent MD models via scaling ionic charges, resolves some but not all parameterization drawbacks. We conclude that despite improving the quality of MD models "on average," the question "which model is the best" has not been answered but replaced by the question "which model is better for a given research." An overall "good" model can still be inappropriate or, in some instances, "bad" and, unfortunately, produce erroneous results. The accurate interpretation of several NDIS datasets, complemented by MD simulations, can prevent such mistakes and help identify the strengths, weaknesses, and convenient applications for corresponding computational models.

A unifying framework for amyloid-mediated membrane damage: The lipid-chaperone hypothesis.

Over the past thirty years, researchers have highlighted the role played by a class of proteins or polypeptides that forms pathogenic amyloid aggregates in vivo, including i) the amyloid Aß peptide, which is known to form senile plaques in Alzheimer's disease; ii) α-synuclein, responsible for Lewy body formation in Parkinson's disease and iii) IAPP, which is the protein component of type 2 diabetes-associated islet amyloids. These proteins, known as intrinsically disordered proteins (IDPs), are present as highly dynamic conformational ensembles. IDPs can partially (mis) fold into (dys) functional conformations and accumulate as amyloid aggregates upon interaction with other cytosolic partners such as proteins or lipid membranes. In addition, an increasing number of reports link the toxicity of amyloid proteins to their harmful effects on membrane integrity. Still, the molecular mechanism underlying the amyloidogenic proteins transfer from the aqueous environment to the hydrocarbon core of the membrane is poorly understood. This review starts with a historical overview of the toxicity models of amyloidogenic proteins to contextualize the more recent lipid-chaperone hypothesis. Then, we report the early molecular-level events in the aggregation and ion-channel pore formation of Aß, IAPP, and α-synuclein interacting with model membranes, emphasizing the complexity of these processes due to their different spatial-temporal resolutions. Next, we underline the need for a combined experimental and computational approach, focusing on the strengths and weaknesses of the most commonly used techniques. Finally, the last two chapters highlight the crucial role of lipid-protein complexes as molecular switches among ion-channel-like formation, detergent-like, and fibril formation mechanisms and their implication in fighting amyloidogenic diseases.

Accurate Simulations of Lipid Monolayers Require a Water Model with Correct Surface Tension.

Lipid monolayers provide our lungs and eyes their functionality and serve as proxy systems in biomembrane research. Therefore, lipid monolayers have been studied intensively including using molecular dynamics simulations, which are able to probe their lateral structure and interactions with, e.g., pharmaceuticals or nanoparticles. However, such simulations have struggled in describing the forces at the air-water interface. Particularly, the surface tension of water and long-range van der Waals interactions have been considered critical, but their importance in monolayer simulations has been evaluated only separately. Here, we combine the recent C36/LJ-PME lipid force field that includes long-range van der Waals forces with water models that reproduce experimental surface tensions to elucidate the importance of these contributions in monolayer simulations. Our results suggest that a water model with correct surface tension is necessary to reproduce experimental surface pressure-area isotherms and monolayer phase behavior. The latter includes the liquid expanded and liquid condensed phases, their coexistence, and the opening of pores at the correct area per lipid upon expansion. Despite these improvements of the C36/LJ-PME with certain water models, the standard cutoff-based CHARMM36 lipid model with the 4-point OPC water model still provides the best agreement with experiments. Our results emphasize the importance of using high-quality water models in applications and parameter development in molecular dynamics simulations of biomolecules.

Heavy Water Models for Classical Molecular Dynamics: Effective Inclusion of Nuclear Quantum Effects.

Small differences in physical and chemical properties of H2O and D2O, such as melting and boiling points or pKa, can be traced back to a slightly stronger hydrogen bonding in heavy versus normal water. In particular, deuteration reduces zero-point vibrational energies as a demonstration of nuclear quantum effects. In principle, computationally demanding quantum molecular dynamics is required to model such effects. However, as already demonstrated by Feynmann and Hibbs, zero-point vibrations can be effectively accounted for by modifying the interaction potential within classical dynamics. In the spirit of the Feymann-Hibbs approach, we develop here two water models for classical molecular dynamics by fitting experimental differences between H2O and D2O. We show that a three-site SPCE-based model accurately reproduces differences between properties of the two water isotopes, with a four-site TIP4P-2005/based model in addition capturing also the absolute values of key properties of heavy water. The present models are computationally simple enough to allow for extensive simulations of biomolecules in heavy water relevant, for example, for experimental techniques such as NMR or neutron scattering.

Sweet taste of heavy water.

Hydrogen to deuterium isotopic substitution has only a minor effect on physical and chemical properties of water and, as such, is not supposed to influence its neutral taste. Here we conclusively demonstrate that humans are, nevertheless, able to distinguish D2O from H2O by taste. Indeed, highly purified heavy water has a distinctly sweeter taste than same-purity normal water and can add to perceived sweetness of sweeteners. In contrast, mice do not prefer D2O over H2O, indicating that they are not likely to perceive heavy water as sweet. HEK 293T cells transfected with the TAS1R2/TAS1R3 heterodimer and chimeric G-proteins are activated by D2O but not by H2O. Lactisole, which is a known sweetness inhibitor acting via the TAS1R3 monomer of the TAS1R2/TAS1R3, suppresses the sweetness of D2O in human sensory tests, as well as the calcium release elicited by D2O in sweet taste receptor-expressing cells. The present multifaceted experimental study, complemented by homology modelling and molecular dynamics simulations, resolves a long-standing controversy about the taste of heavy water, shows that its sweet taste is mediated by the human TAS1R2/TAS1R3 taste receptor, and opens way to future studies of the detailed mechanism of action.

The role of alpha-helix on the structure-targeting drug design of amyloidogenic proteins.

The most accredited hypothesis links the toxicity of amyloid proteins to their harmful effects on membrane integrity through the formation of prefibrillar-transient oligomers able to disrupt cell membranes. However, damage mechanisms necessarily assume a first step in which the amyloidogenic protein transfers from the aqueous phase to the membrane hydrophobic core. This determinant step is still poorly understood. However, according to our lipid-chaperon hypothesis, free lipids in solution play a crucial role in facilitating this footfall. Free phospholipid concentration in the aqueous phase acts as a switch between ion channel-like pore and fibril formation, so that high free lipid concentration in solution promotes pore and repress fibril formation. Conversely, low free lipids in the solution favor fibril and repress pore formation. This behavior is due to the formation of stable lipid-protein complexes. Here, we hypothesize that the helix propensity is a fundamental requirement to fulfill the lipid-chaperon model. The alpha-helix region seems to be responsible for the binding with amphiphilic molecules fostering the proposed mechanism. Indeed, our results show the dependency of protein-lipid binding from the helical structure presence. When the helix content is substantially lower than the wild type, the contact probability decreases. Instead, if the helix is broadening, the contact probability increases. Our findings open a new perspective for in silico screening of secondary structure-targeting drugs of amyloidogenic proteins.

Lipid-Chaperone Hypothesis: A Common Molecular Mechanism of Membrane Disruption by Intrinsically Disordered Proteins.

An increasing number of human diseases has been shown to be linked to aggregation and amyloid formation by intrinsically disordered proteins (IDPs). Amylin, amyloid-ß, and α-synuclein are, indeed, involved in type-II diabetes, Alzheimer's, and Parkinson's, respectively. Despite the correlation of the toxicity of these proteins at early aggregation stages with membrane damage, the molecular events underlying the process is quite complex to understand. In this study, we demonstrate the crucial role of free lipids in the formation of lipid-protein complex, which enables an easy membrane insertion for amylin, amyloid-ß, and α-synuclein. Experimental results from a variety of biophysical methods and molecular dynamics results reveal that this common molecular pathway in membrane poration is shared by amyloidogenic (amylin, amyloid-ß, and α-synuclein) and nonamyloidogenic (rat IAPP, ß-synuclein) proteins. Based on these results, we propose a "lipid-chaperone" hypothesis as a unifying framework for protein-membrane poration.

Modulating Aß aggregation by tyrosol-based ligands: The crucial role of the catechol moiety.

The abnormal deposition of Aß amyloid deposits in the brain is a hallmark of Alzheimer's disease (AD). Based on this evidence, many current therapeutic approaches focus on the development of small molecules halting Aß aggregation. However, due to the temporary and elusive structures of amyloid assemblies, the rational design of aggregation inhibitors remains a challenging task. Here we combine ThT assays and MD simulations to study Aß aggregation in the presence of the natural compounds tyrosol (TY), 3-hydroxytyrosol (HDT), and 3-methoxytyrosol (homovanillyl alcohol - HVA). We show that albeit HDT is a potent inhibitor of amyloid growth, TY and HVA catalyze fibril formation. An inspection of MD simulations trajectories revealed that the different effects of these three molecules on Aß1-40 aggregation are ascribable to their capacity to arrange H-bonds network between the ligand (position C-3) and the peptide (Glu22). We believe that our results may contribute to the design of more effective and safe small molecules able to contrast pathogenic amyloid aggregation.

Phospholipids Critical Micellar Concentrations Trigger Different Mechanisms of Intrinsically Disordered Proteins Interaction with Model Membranes.

Amyloidogenic proteins are involved in many diseases, including Alzheimer's, Parkinson's, and type II diabetes. These proteins are thought to be toxic for cells because of their abnormal interaction with the cell membrane. Simpler model membranes (LUVs) have been used to study the early steps of membrane-protein interactions and their subsequent evolution. Phospholipid LUVs formed in water solution establish a chemical equilibrium between self-assembled LUVs and a small amount of phospholipids in water solution (CMC). Here, using both experimental and molecular dynamics simulations approach we demonstrate that the insertion of IAPP, an amyloidogenic peptide involved in diabetes, in membranes is driven by free lipids in solution in dynamic equilibrium with the self-assembled lipids of the bilayer. It is suggested that this could be a general mechanism lying at the root of membrane insertion processes of self-assembling peptides.

Detection and characterization at nM concentration of oligomers formed by hIAPP, Aß(1-40) and their equimolar mixture using SERS and MD simulations.

We report a structural investigation on IAPP, Aß(1-40) and their equi-molar mixture aggregation pathway at nano-molar concentration using the Surface Enhanced Raman Spectroscopy (SERS) effect induced by silver metal colloids prepared by laser processes in solution and molecular dynamics simulations. Our data show the ability of silver NPs coupled with SERS to detect secondary structures of IAPP, Aß(1-40) and their 1 : 1 molar ratio mixture in the oligomeric state. The preparation of silver colloids shows superior performance with respect to chemically prepared nano-particles. SERS spectroscopy shows both selectivity and sensitivity in detecting the secondary structures of hIAPP and Aß(1-40) and to recognize both proteins in their mixture. On the other hand, molecular dynamics simulations confirm SERS structural data and the given atomistic details about the structural organization of IAPP and Aß(1-40) oligomers. Our study shows an inhomogeneity in the chemical composition of IAPP/Aß(1-40) oligomer aggregates.

Amyloid growth and membrane damage: Current themes and emerging perspectives from theory and experiments on Aß and hIAPP.

Alzheimer's Disease (AD) and Type 2 diabetes mellitus (T2DM) are two incurable diseases both hallmarked by an abnormal deposition of the amyloidogenic peptides Aß and Islet Amyloid Polypeptide (IAPP) in affected tissues. Epidemiological data demonstrate that patients suffering from diabetes are at high risk of developing AD, thus making the search for factors common to the two pathologies of special interest for the design of new therapies. Accumulating evidence suggests that the toxic properties of both Aß or IAPP are ascribable to their ability to damage the cell membrane. However, the molecular details describing Aß or IAPP interaction with membranes are poorly understood. This review focuses on biophysical and in silico studies addressing these topics. Effects of calcium, cholesterol and membrane lipid composition in driving aberrant Aß or IAPP interaction with the membrane will be specifically considered. The cross correlation of all these factors appears to be a key issue not only to shed light in the countless and often controversial reports relative to this area but also to gain valuable insights into the central events leading to membrane damage caused by amyloidogenic peptides. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.