Endothelial leakiness elicited by amyloid protein aggregation.

Alzheimer's disease (AD) is a major cause of dementia debilitating the global ageing population. Current understanding of the AD pathophysiology implicates the aggregation of amyloid beta (Aß) as causative to neurodegeneration, with tauopathies, apolipoprotein E and neuroinflammation considered as other major culprits. Curiously, vascular endothelial barrier dysfunction is strongly associated with Aß deposition and 80-90% AD subjects also experience cerebral amyloid angiopathy. Here we show amyloid protein-induced endothelial leakiness (APEL) in human microvascular endothelial monolayers as well as in mouse cerebral vasculature. Using signaling pathway assays and discrete molecular dynamics, we revealed that the angiopathy first arose from a disruption to vascular endothelial (VE)-cadherin junctions exposed to the nanoparticulates of Aß oligomers and seeds, preceding the earlier implicated proinflammatory and pro-oxidative stressors to endothelial leakiness. These findings were analogous to nanomaterials-induced endothelial leakiness (NanoEL), a major phenomenon in nanomedicine depicting the paracellular transport of anionic inorganic nanoparticles in the vasculature. As APEL also occurred in vitro with the oligomers and seeds of alpha synuclein, this study proposes a paradigm for elucidating the vascular permeation, systemic spread, and cross-seeding of amyloid proteins that underlie the pathogeneses of AD and Parkinson's disease.

Molecular Basis for Actin Polymerization Kinetics Modulated by Solution Crowding.

Actin polymerization drives cell movement and provides cells with structural integrity. Intracellular environments contain high concentrations of solutes, including organic compounds, macromolecules, and proteins. Macromolecular crowding has been shown to affect actin filament stability and bulk polymerization kinetics. However, the molecular mechanisms behind how crowding influences individual actin filament assembly are not well understood. In this study, we investigated how crowding modulates filament assembly kinetics using total internal reflection fluorescence (TIRF) microscopy imaging and pyrene fluorescence assays. The elongation rates of individual actin filaments analyzed from TIRF imaging depended on the type of crowding agent (polyethylene glycol, bovine serum albumin, and sucrose) as well as their concentrations. Further, we utilized all-atom molecular dynamics (MD) simulations to evaluate the effects of crowding molecules on the diffusion of actin monomers during filament assembly. Taken together, our data suggest that solution crowding can regulate actin assembly kinetics at the molecular level.

Distinct Conformations of SARS-CoV-2 Omicron Spike Protein and Its Interaction with ACE2 and Antibody.

Since November 2021, Omicron has been the dominant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant that causes the coronavirus disease 2019 (COVID-19) and has continuously impacted human health. Omicron sublineages are still increasing and cause increased transmission and infection rates. The additional 15 mutations on the receptor binding domain (RBD) of Omicron spike proteins change the protein conformation, enabling the Omicron variant to evade neutralizing antibodies. For this reason, many efforts have been made to design new antigenic variants to induce effective antibodies in SARS-CoV-2 vaccine development. However, understanding the different states of Omicron spike proteins with and without external molecules has not yet been addressed. In this review, we analyze the structures of the spike protein in the presence and absence of angiotensin-converting enzyme 2 (ACE2) and antibodies. Compared to previously determined structures for the wildtype spike protein and other variants such as alpha, beta, delta, and gamma, the Omicron spike protein adopts a partially open form. The open-form spike protein with one RBD up is dominant, followed by the open-form spike protein with two RBD up, and the closed-form spike protein with the RBD down. It is suggested that the competition between antibodies and ACE2 induces interactions between adjacent RBDs of the spike protein, which lead to a partially open form of the Omicron spike protein. The comprehensive structural information of Omicron spike proteins could be helpful for the efficient design of vaccines against the Omicron variant.

Anionic nanoplastic exposure induces endothelial leakiness.

The global-scale production of plastics has been instrumental in advancing modern society, while the rising accumulation of plastics in landfills, oceans, and anything in between has become a major stressor on environmental sustainability, climate, and, potentially, human health. While mechanical and chemical forces of man and nature can eventually break down or recycle plastics, our understanding of the biological fingerprints of plastics, especially of nanoplastics, remains poor. Here we report on a phenomenon associated with the nanoplastic forms of anionic polystyrene and poly(methyl methacrylate), where their introduction disrupted the vascular endothelial cadherin junctions in a dose-dependent manner, as revealed by confocal fluorescence microscopy, signaling pathways, molecular dynamics simulations, as well as ex vivo and in vivo assays with animal model systems. Collectively, our results implicated nanoplastics-induced vasculature permeability as primarily biophysical-biochemical in nature, uncorrelated with cytotoxic events such as reactive oxygen species production, autophagy, and apoptosis. This uncovered route of paracellular transport has opened up vast avenues for investigating the behaviour and biological effects of nanoplastics, which may offer crucial insights for guiding innovations towards a sustainable plastics industry and environmental remediation.

A Framework of Paracellular Transport via Nanoparticles-Induced Endothelial Leakiness.

Nanomaterial-induced endothelial leakiness (NanoEL) is an interfacial phenomenon denoting the paracellular transport of nanoparticles that is pertinent to nanotoxicology, nanomedicine and biomedical engineering. While the NanoEL phenomenon is complementary to the enhanced permeability and retention effect in terms of their common applicability to delineating the permeability and behavior of nanoparticles in tumoral environments, these two effects significantly differ in scope, origin, and manifestation. In the current study, the descriptors are fully examined of the NanoEL phenomenon elicited by generic citrate-coated gold nanoparticles (AuNPs) of changing size and concentration, from microscopic gap formation and actin reorganization down to molecular signaling pathways and nanoscale interactions of AuNPs with VE-cadherin and its intra/extracellular cofactors. Employing synergistic in silico methodologies, for the first time the molecular and statistical mechanics of cadherin pair disruption, especially in response to AuNPs of the smallest size and highest concentration are revealed. This study marks a major advancement toward establishing a comprehensive NanoEL framework for complementing the understanding of the transcytotic pathway and for guiding the design and application of future nanomedicines harnessing the myriad functions of the mammalian vasculature.

Crowding tunes the organization and mechanics of actin bundles formed by crosslinking proteins.

Fascin and α-actinin form higher-ordered actin bundles that mediate numerous cellular processes including cell morphogenesis and movement. While it is understood crosslinked bundle formation occurs in crowded cytoplasm, how crowding affects the bundling activities of the two crosslinking proteins is not known. Here, we demonstrate how solution crowding modulates the organization and mechanical properties of fascin- and α-actinin-induced bundles, utilizing total internal reflection fluorescence and atomic force microscopy imaging. Molecular dynamics simulations support the inference that crowding reduces binding interaction between actin filaments and fascin or the calponin homology 1 domain of α-actinin evidenced by interaction energy and hydrogen bonding analysis. Based on our findings, we suggest a mechanism of crosslinked actin bundle assembly and mechanics in crowded intracellular environments.

Molecular dynamics study of interactions between polymorphic actin filaments and gelsolin segment-1.

The assembly of protein actin into double-helical filaments promotes many eukaryotic cellular processes that are regulated by actin-binding proteins (ABPs). Actin filaments can adopt multiple conformations, known as structural polymorphism, which possibly influences the interaction between filaments and ABPs. Gelsolin is a Ca2+ -regulated ABP that severs and caps actin filaments. Gelsolin binding modulates filament structure; however, it is not known how polymorphic actin filament structures influence an interaction of gelsolin S1 with the barbed-end of filament. Herein, we investigated how polymorphic structures of actin filaments affect the interactions near interfaces between the gelsolin segment 1 (S1) domain and the filament barbed-end. Using all-atom molecular dynamics simulations, we demonstrate that different tilted states of subunits modulate gelsolin S1 interactions with the barbed-end of polymorphic filaments. Hydrogen bonding and interaction energy at the filament-gelsolin S1 interface indicate distinct conformations of filament barbed ends, resulting in different interactions of gelsolin S1. This study demonstrates that filament's structural multiplicity plays important roles in the interactions of actin with ABPs.

Experimental Realization of Few Layer Two-Dimensional MoS2 Membranes of Near Atomic Thickness for High Efficiency Water Desalination.

A globally imminent shortage of freshwater has been demanding viable strategies for improving desalination efficiencies with the adoption of cost- and energy-efficient membrane materials. The recently explored 2D transition metal dichalcogenides (2D TMDs) of near atomic thickness have been envisioned to offer notable advantages as high-efficiency membranes owing to their structural uniqueness; that is, extremely small thickness and intrinsic atomic porosity. Despite theoretically projected advantages, experimental realization of near atom-thickness 2D TMD-based membranes and their desalination efficiency assessments have remained largely unexplored mainly due to the technical difficulty associated with their seamless large-scale integration. Herein, we report the experimental demonstration of high-efficiency water desalination membranes based on few-layer 2D molybdenum disulfide (MoS2) of only ∼7 nm thickness. Chemical vapor deposition (CVD)-grown centimeter-scale 2D MoS2 layers were integrated onto porous polymeric supports with well-preserved structural integrity enabled by a water-assisted 2D layer transfer method. These 2D MoS2 membranes of near atomic thickness exhibit an excellent combination of high water permeability (>322 L m-2 h-1 bar-1) and high ionic sieving capability (>99%) for various seawater salts including Na+, K+, Ca2+, and Mg2+ with a range of concentrations. Moreover, they present near 100% salt ion rejection rates for actual seawater obtained from the Atlantic coast, significantly outperforming the previously developed 2D MoS2 layer membranes of micrometer thickness as well as conventional reverse osmosis (RO) membranes. Underlying principles behind such remarkably excellent desalination performances are attributed to the intrinsic atomic vacancies inherent to the CVD-grown 2D MoS2 layers as verified by aberration-corrected electron microscopy characterization.

Actin Filament Mechanics and Structure in Crowded Environments.

The cellular environment is crowded with high concentrations of macromolecules that significantly reduce accessible volume for biomolecular interactions. Reductions in cellular volume can generate depletion forces that affect protein assembly and stability. The mechanical and structural properties of actin filaments play critical roles in various cellular functions, including structural support, cell movement, division, and intracellular transport. Although the effects of molecular crowding on actin polymerization have been shown, how crowded environments affect filament mechanics and structure is unknown. In this study, we investigate the effects of solution crowding on the modulations of actin filament bending stiffness and conformations both in vitro and in silico. Direct visualization of thermally fluctuating filaments in the presence of crowding agents is achieved by fluorescence microscopy imaging. Biophysical analysis indicates that molecular crowding enhances filament's effective bending stiffness and reduces average filament lengths. Utilizing the all-atom molecular dynamics simulations, we demonstrate that molecular crowding alters filament conformations and intersubunit contacts that are directly coupled to the mechanical properties of filaments. Taken together, our study suggests that the interplay between excluded volume effects and nonspecific interactions raised from molecular crowding may modulate actin filament mechanics and structure.

Investigation of the role hydrophobin monomer loops using hybrid models via molecular dynamics simulation.

Hydrophobins are small amphiphilic fungal proteins that are highly surface-active and are used in various industrial applications such as dispersion, immobilization, and antifouling. At hydrophobic-hydrophilic interfaces, hydrophobins tend to self-assemble as rodlets or monolayers, depending on whether they are class I or II. Several studies have determined the three-dimensional structure and investigated the self-assembly formation mechanism of the class I EAS from Neurospora crassa and the class II HFBII from Trichoderma reesei. Although some studies have examined the performance of chimeric hydrophobins, they have not been investigated at the atomic scale. Here, we designed chimeric hydrophobins by grafting the L1 loop of Vmh2 and the L3 loop of EAS onto the class II hydrophobin HFBII by homology modeling and performed vacuum-water interface molecular simulations to determine their structural behaviors. We found that the chimeric hydrophobin grafted with the L3 of EAS became unstable under standard conditions, whereas that grafted with the L1 of Vmh2 became unstable in the presence of calcium ions. Moreover, when both the EAS L3 and Vmh2 L1 were grafted together, the structure became disordered and lost its amphiphilic characteristics in standard conditions. In the presence of calcium, however, its structural stability was restored. However, an additional external perturbation is required to trigger the conformational transition. Although our chimeric hydrophobin models were designed through homology modeling, our results provide detailed information regarding hydrophobin self-assembly and their surface-interactive behavior that may serve as a template for designing hydrophobins for future industrial applications.

Mechanically inferior constituents in spider silk result in mechanically superior fibres by adaptation to harsh hydration conditions: a molecular dynamics study.

Spider silk exhibits mechanical properties such as high strength and toughness that are superior to those of any man-made fibre (Bourzac 2015 Nature519, S4-S6 (doi:10.1038/519S4a)). This high strength and toughness originates from a combination of the crystalline (exhibiting robust strength) and amorphous (exhibiting superb extensibility) regions present in the silk (Asakura et al 2015 Macromolecules48, 2345-2357 (doi:10.1021/acs.macromol.5b00160)). The crystalline regions comprise a mixture of poly-alanine and poly-glycine-alanine. Poly-alanine is expected to be stronger than poly-glycine-alanine, because alanine exhibits greater interactions between the strands than glycine (Tokareva et al 2014 Acta Biomater.10, 1612-1626 (doi:10.1016/j.actbio.2013.08.020)). We connect this characteristic sequence to the interactions observed upon the hydration of spider silk. Like most proteinaceous materials, spider silks become highly brittle upon dehydration, and thus water collection is crucial to maintaining its toughness (Gosline et al 1986 Endeavour10, 37-43 (doi:10.1016/0160-9327(86)90049-9)). We report on the molecular dynamic simulations of spider silk structures with different sequences for the crystalline region of the silk structures, of wild-type (WT), poly-alanine, and poly-glycine-alanine. We reveal that the characteristic sequence of spider silk results in the ß-sheets being maintained as the degree of hydration changes and that the high water collection capabilities of WT spider silk sequence prevent the silk from becoming brittle and weak in dry conditions. The characteristic crystalline sequence of spider dragline silk is therefore relevant not for maximizing the interactions between the strands but for adaption to changing hydration conditions to maintain an optimal performance even in harsh conditions.

Metal ions affect the formation and stability of amyloid ß aggregates at multiple length scales.

Amyloid ß (Aß) aggregates, which are a hallmark for neurodegenerative disease, are formed through a self-assembly process such as aggregation of Aß peptide chains. This aggregation process depends on the solvent conditions under which the proteins are aggregated. Nevertheless, the underlying mechanism of the ionic effect on the formation and stability of amyloid aggregates has not been fully understood. Here, we report how metal ions play a role in the formation and stability of Aß aggregates at different length scales, i.e. oligomers and fibrils. It is shown that the metal (i.e. zinc or copper) ion increases the stability of Aß oligomers, whereas the metal ion reduces the stability of Aß fibrils. In addition, we found that zinc ions are able to more effectively destabilize fibril structures than copper ions. Metal ion-mediated (de)stabilization of Aß oligomers (or fibrils) is attributed to the critical effect of the metal ion on the ß-sheet rich crystalline structure of the amyloid aggregate and the status of hydrogen bonds within the aggregate. Our study sheds light on the role of the metal ion in stabilizing the amyloid oligomers known as a toxic agent (to functional cells), which is consistent with clinical observation that high concentrations of metal ions are found in patients suffering from neurodegenerative diseases.

Loading-device effects on the protein-unfolding mechanisms using molecular-dynamic simulations.

Experimental force spectroscopy has been effectively utilized for measuring structural characterization of biomolecules and mechanical properties of biomaterials. Specifically, atomic force microscopy (AFM) has been widely used to portray biomolecular characterization in single-molecule experiment by observing the unfolding behavior of the proteins. Not only the experimental techniques enable us to characterize globular protein, but computational methods like molecular dynamics (MD) also gives insight into understanding biomolecular structures. To better comprehend the behavior of biomolecules, conditions such as pulling velocities and loading rates are put to the test, yet there are still limitations in understanding the unfolding behavior of biomolecules with the effect of different loading devices. In this study, we performed an all-atom MD and steered molecular dynamics (SMD) simulations considering different loading device effects such as "soft" and "stiff" to characterize the anisotropic unfolding behavior of ubiquitin protein. We found out the anisotropic unfolding pathways of the protein through the broken number of hydrogen bonds and geometric secondary structures of the biomolecule. Our study provides the importance for usage of various loading-devices on biomolecules when analyzing the structural compositions and the characteristics of globular biomolecules.

Mechanical features of various silkworm crystalline considering hydration effect via molecular dynamics simulations.

Silk materials are receiving significant attention as base materials for various functional nanomaterials and nanodevices, due to its exceptionally high mechanical properties, biocompatibility, and degradable characteristics. Although crystalline silk regions are composed of various repetitive motifs with differing amino acid sequences, how the effect of humidity works differently on each of the motifs and their structural characteristics remains unclear. We report molecular dynamics (MD) simulations on various silkworm fibroins composed of major motifs (i.e. (GAGAGS)n, (GAGAGA)n, and (GAGAGY)n) at varying degrees of hydration, and reveal how each major motifs of silk fibroins change at each degrees of hydration using MD simulations and their structural properties in mechanical perspective via steered molecular dynamics simulations. Our results explain what effects humidity can have on nanoscale materials and devices consisting of crystalline silk materials.

Capping effects on polymorphic Aß16-21 amyloids depend on their size: A molecular dynamics simulation study.

Understanding Aß amyloid oligomers associated with neuro-degenerative diseases is needed due to their toxic characteristics and mediation of amyloid fibril growth. Depending on various physiological circumstances such as ionic strength, metal ion, and point-residue mutation, oligomeric amyloids exhibit polymorphic behavior and structural stabilities, i.e. showing different conformation and stabilities. Specifically, experimental and computational researchers have found that the capping modulates the physical and chemical properties of amyloids by preserving electrostatic energy interactions, which is one of the dominant factors for amyloid stability. Still, there is no detailed knowledge for the polymorphic amyloids with reflecting the terminal capping effects. In the present study, we investigated the role of terminal capping (i.e. N-terminal acetylation and C-terminal amidation) on polymorphic Aß16-21 amyloid oligomer and protofibrils via molecular dynamics (MD) simulations. We found that the capping effects have differently altered the conformation of polymorphic antiparallel-homo and -hetero Aß16-21 amyloid oligomer, but not Aß16-21 amyloid protofibrils. However, regardless of polymorphic composition of the amyloids, the capping induces the thermodynamic instabilities of Aß16-21 amyloid oligomers, but does not show any distinct affect on Aß16-21 amyloid protofibrils. Specifically, among the molecular mechanic factors, electrostatic energy dominantly contributes the thermodynamic stability of the Aß16-21 amyloids. We hope that our computation study about the role of the capping effects on the polymorphic amyloids will facilitate additional efforts to enhance degradation of amyloids and to design a selective drug in the future.

Mechanical and vibrational characterization of amyloid-like HET-s nanosheets based on the skewed plate theory.

Pathological amyloidogenic prion proteins have a toxic effect on functional cells in the human cerebrum because of poor degradability and the tendency to accumulate in an uncontrolled manner under physiological conditions. HET-s, a fungal prion protein, is known to undergo conformational variations from fibrillar to nanosheet structures during a change from low to high pH conditions. It has been said that this conformational change can lead to self-propagation by nucleating on the lateral surface of singlet fibrils. Efforts have been made toward the mechanical characterization of fibrillar amyloids, but a global understanding of amyloid-like HET-s nanosheet structures is lacking. In this study, we analyzed the mechanical and vibrational characteristics of the skewed HET-s nanosheet structures that developed under neutral pH conditions by performing various molecular dynamics simulations. By applying the skewed plate theory to HET-s nanosheets for various length scales with numerous pores inside the structures, we found that the skewed HET-s nanosheet structure has mechanical properties comparable to those of previously reported biological film materials and nanomaterials. Considering the inherent characteristics of structural stability, our observation provides valuable and detailed structural information on skewed amyloid-like HET-s nanosheets.

Characterizing Structural Stability of Amyloid Motif Fibrils Mediated by Water Molecules.

In biological systems, structural confinements of amyloid fibrils can be mediated by the role of water molecules. However, the underlying effect of the dynamic behavior of water molecules on structural stabilities of amyloid fibrils is still unclear. By performing molecular dynamics simulations, we investigate the dynamic features and the effect of interior water molecules on conformations and mechanical characteristics of various amyloid fibrils. We find that a specific mechanism induced by the dynamic properties of interior water molecules can affect diffusion of water molecules inside amyloid fibrils, inducing their different structural stabilities. The conformation of amyloid fibrils induced by interior water molecules show the fibrils' different mechanical features. We elucidate the role of confined and movable interior water molecules in structural stabilities of various amyloid fibrils. Our results offer insights not only in further understanding of mechanical features of amyloids as mediated by water molecules, but also in the fine-tuning of the functional abilities of amyloid fibrils for applications.

Structural analysis of oligomeric and protofibrillar Aß amyloid pair structures considering F20L mutation effects using molecular dynamics simulations.

Aß amyloid proteins are involved in neuro-degenerative diseases such as Alzheimer's, Parkinson's, and so forth. Because of its structurally stable feature under physiological conditions, Aß amyloid protein disrupts the normal cell function. Because of these concerns, understanding the structural feature of Aß amyloid protein in detail is crucial. There have been some efforts on lowering the structural stabilities of Aß amyloid fibrils by decreasing the aromatic residues characteristic and hydrophobic effect. Yet, there is a lack of understanding of Aß amyloid pair structures considering those effects. In this study, we provide the structural characteristics of wildtype (WT) and phenylalanine residue mutation to leucine (F20L) Aß amyloid pair structures using molecular dynamics simulation in detail. We also considered the polymorphic feature of F20L and WT Aß pair amyloids based on the facing ß-strand directions between the amyloid pairs. As a result, we were able to observe the varying effects of mutation, polymorphism, and protofibril lengths on the structural stability of pair amyloids. Furthermore, we have also found that opposite structural stability exists on a certain polymorphic Aß pair amyloids depending on its oligomeric or protofibrillar state, which can be helpful for understanding the amyloid growth mechanism via repetitive fragmentation and elongation mechanism. Proteins 2017; 85:580-592. © 2016 Wiley Periodicals, Inc.

Sodium chloride's effect on self-assembly of diphenylalanine bilayer.

Understanding self-assembling peptides becomes essential in nanotechnology, thereby providing a bottom-up method for fabrication of nanostructures. Diphenylalanine constitutes an outstanding building block that can be assembled into various nanostructures, including two-dimensional bilayers or nanotubes, exhibiting superb mechanical properties. It is known that the effect of the ions is critical in conformational and chemical interactions of bilayers or membranes. In this study, we analyzed the effect of sodium chloride on diphenylalanine bilayer using coarse-grained molecular dynamics simulations, and calculated the bending Young's modulus and the torsional modulus by applying normal modal analysis using an elastic network model. The results showed that sodium chloride dramatically increases the assembling efficiency and stability, thereby promising to allow the precise design and control of the fabrication process and properties of bio-inspired materials. © 2016 Wiley Periodicals, Inc.

Conformational changes of Aß (1-42) monomers in different solvents.

Amyloid proteins are known to be the main cause of numerous degenerative and neurodegenerative diseases. In general, amyloids are misfolded from monomers and they tend to have ß-strand formations. These misfolded monomers are then transformed into oligomers, fibrils, and plaques. It is important to understand the forming mechanism of amyloids in order to prevent degenerative diseases to occur. Aß protein is a highly noticeable protein which causes Alzheimer's disease. It is reported that solvents affect the forming mechanism of Aß amyloids. In this research, Aß1-42 was analyzed using an all-atom MD simulation with the consideration of effects induced by two disparate solvents: water and DMSO. As a result, two different conformation changes of Aß1-42 were exhibited in each solvent. It was found that salt-bridge of Asp23 and Lys28 in Aß1-42 was the key for amyloid folding based on the various analysis including hydrogen bond, electrostatic interaction energy and salt-bridge distance. Since this salt-bridge region plays a crucial role in initiating the misfolding of Aß1-42, this research may shed a light for studies related in amyloid folding and misfolding.