A dual role of ERGIC-localized Rabs in TMED10-mediated unconventional protein secretion.

Cargo translocation across membranes is a crucial aspect of secretion. In conventional secretion signal peptide-equipped proteins enter the endoplasmic reticulum (ER), whereas a subset of cargo lacking signal peptides translocate into the ER-Golgi intermediate compartment (ERGIC) in a process called unconventional protein secretion (UcPS). The regulatory events at the ERGIC in UcPS are unclear. Here we reveal the involvement of ERGIC-localized small GTPases, Rab1 (Rab1A and Rab1B) and Rab2A, in regulating UcPS cargo transport via TMED10 on the ERGIC. Rab1 enhances TMED10 translocator activity, promoting cargo translocation into the ERGIC, whereas Rab2A, in collaboration with KIF5B, regulates ERGIC compartmentalization, establishing a UcPS-specific compartment. This study highlights the pivotal role of ERGIC-localized Rabs in governing cargo translocation and specifying the ERGIC's function in UcPS.

Neuroinflammation catching nanobubbles for microglia-neuron unit modulation against epilepsy.

Epilepsy is a common neurological disease caused by synchronous firing of hyperexcitable neurons. Currently, patients with epilepsy are typically treated with antiseizure medicines that work by interrupting the hyperexcitability or hypersynchrony of localized neurons or by inhibiting excitatory neurotransmission. However, these drugs do not treat the underlying causes of epilepsy, and nearly one-third of patients have seizures that cannot be controlled by these medications. Animal and clinical evidence suggests that inflammation caused by neuronal and non-neuronal cells within the epilepsy lesion could play a central role in seizure disorders. Here we report a gas-filled nanobubble (NB) conjugated with diammonium glycyrrhizinate (DG) drugs and sphingosine-1-phosphate (S1P) molecules (S1P@DG-NBs) on the lipid shell for targeted therapy and real-time ultrasound visualization applications against neuroinflammatory injury. Affinity of S1P@DG-NBs for the S1P receptor endows these NBs with enhanced targeting capability to the neuroinflammatory microenvironment of epilepsy, where the DG drugs modulate endothelium-microglia-neuron inflammation by inhibiting high-mobility group box 1 molecules and downregulating the Toll-like receptor 4 signaling pathway, resulting in anti-inflammatory M2 microglia that exert anti-epilepsy effects. Our results show that this technology can enhance visualization of epileptic brain and deliver drugs with anti-inflammatory and immunomodulatory properties to ameliorate seizures symptoms.

Membrane Domain Anti-Registration Induces an Intrinsic Transmembrane Potential.

Plasma membrane segregation into various nanoscale membrane domains is driven by distinct interactions between diverse lipids and proteins. Among them, liquid-ordered (Lo) membrane domains are defined as "lipid rafts" and liquid-disordered (Ld) ones as "lipid non-rafts". Using model membrane systems, both intra-leaflet and inter-leaflet dynamics of these membrane domains are widely studied. Nevertheless, the biological impact of the latter, which is accompanied by membrane domain registration/anti-registration, is far from clear. Hence, in this work, we studied the biological relevance of the membrane domain anti-registration using both all-atom molecular dynamics (MD) simulations and confocal fluorescence microscopy. All-atom MD simulations suggested an intrinsic transmembrane potential for the case of the membrane anti-registration (Lo/Ld). Meanwhile, confocal fluorescence microscopy experiments of HeLa and 293T cell lines indicated that membrane cholesterol depletion could significantly alter the transmembrane potential of cells. Considering differences in the cholesterol content between Lo and Ld membrane domains, our confocal fluorescence microscopy experiments are consistent with our all-atom MD simulations. In short, membrane domain anti-registration induces local membrane asymmetry and, thus, an intrinsic transmembrane potential.

Bio-inspired peptide-conjugated liposomes for enhanced planktonic bacteria killing and biofilm eradication.

Developing new antimicrobial agents has become an urgent task to address the increasing prevalence of multidrug-resistant pathogens and the emergence of biofilms. Cationic antimicrobial peptides (AMPs) have been regarded as promising candidates due to their unique non-specific membrane rupture mechanism. However, a series of problems with the peptides hindered their practical application due to their high toxicity and low bioactivity and stability. Here, inspired by broadening the application of cell-penetrating peptides (CPPs), we selected five different sequences of cationic peptides which are considered as both CPPs and AMPs, and developed a biomimetic strategy to construct cationic peptide-conjugated liposomes with the virus-like structure for both enhancements of antibacterial efficacy and biosafety. The correlation between available peptide density/peptide variety and antimicrobial capabilities was evaluated from quantitative perspectives. Computational simulation and experimental investigations assisted to identify the optimal peptide-conjugated liposomes and revealed that the designed system provides high charge density for enhanced anionic bacterial membrane binding capability without compromised cytotoxicity, being capable of enhanced antibacterial efficacy of bacteria/biofilm of clinically important pathogens. The bio-inspired design has shown enhanced therapeutic efficiency of peptides and may promote the development of next-generation antimicrobials.

The stiffness-dependent tumor cell internalization of liquid metal nanoparticles.

The properties of nanoparticle (NP) carriers, such as size, shape and surface state, have been proven to dramatically affect their uptake by tumor cells, thereby influencing and determining the effect of nanomedicine on tumor theranostics. However, the effect of the stiffness of NPs on their cellular internalization remains unclear, especially for circumstances involving active or passive NP targeting. In this work, we constructed eutectic gallium indium liquid metal NPs with the same particle size, shape and surface charge properties but distinct stiffness via tailoring the surface oxidation and silica coating. It has been found that the softer NPs would be endocytosed much slower than their stiffer counterparts in the presence of specific ligand-receptor interaction. Interestingly, once the interaction is eliminated, softer NPs are internalized faster than the stiffer ones. Based on experimental observations and theoretical verification, we demonstrate that this phenomenon is mainly caused by varying degrees of deformation of soft NPs induced by ligand-receptor interactions. Such a finding of the stiffness effect of NPs implies great potential for fundamental biomedical applications, such as the rational design of nanomedicines.

Molecular dynamics simulation insights into the cellular uptake of elastic nanoparticles through human pulmonary surfactant.

The interaction between inhaled nanoparticles (NPs) and the pulmonary surfactant (PS) monolayer has drawn significant attention due to its potential in drug delivery design and application for respiratory therapeutics in active and passive cellular uptake pathways. Even though much attention has been given to explore the interaction between NPs and the PS monolayer, the effects of the NP elasticity on the translocation across the PS monolayer have not been thoroughly studied. Here, we performed a series of coarse-grained (CG) molecular dynamics simulations to study active or passive cellular uptake pathways of three NPs with different elasticities through a PS monolayer. The differences between active and passive pathways underly the enhanced targeting ability by ligand-receptor interaction (L-R interaction). In the active or passive cellular uptake pathways, it is found that the increase in stiffness level leads to a higher penetrability of NPs at the same time range. The soft NP has always been withheld inside the PS monolayer due to the lowest level of elasticity, while the other two types of NPs penetrate through the PS monolayer as the simulation progresses toward the end. The NPs in the active cellular uptake pathways take a longer time to penetrate the PS monolayer, resulting in a longer average penetration distance of approximately 40.55% and a higher average number of contacts, approximately 36.11%, than passive cellular uptake pathways, due to the L-R interaction. Moreover, it demonstrates that NPs in active cellular uptake pathways have a significantly higher targeting ability with the PS monolayer. We conclude that the level of NP elasticities has a substantial link to the penetrability in active or passive cellular uptake pathways. These results provide valuable insights into drug delivery and nanoprobe design for inhaled NPs within the lungs.

Molecular View on the Impact of DHA Molecules on the Physical Properties of a Model Cell Membrane.

Docosahexaenoic acid (DHA) is a ω-3 polyunsaturated fatty acid, which can be uptaken by cells and is essential for proper neuronal and retinal function. However, the detailed physical impact of DHA molecules on the plasma membrane is still unclear. Hence, in this work, we carried out µs-scale coarse-grained molecular dynamics (MD) simulations to reveal the interactions between DHA molecules and a model cell membrane. As is known, the cell membrane can segregate into liquid-ordered (Lo) and liquid-disordered (Ld) membrane domains due to the differential interactions between lipids and proteins. In order to capture this feature, we adopted the three-component phase-separated lipid membranes and considered both anionic and neutral DHA molecules in the current work. Our results showed that DHA molecules can spontaneously self-assemble into nanoclusters, fuse with lipid membranes, and localize preferably in Ld membrane domains. During the membrane fusion process, DHA molecules can change the intrinsic transmembrane potential of the lipid membrane, and the effects of anionic DHA molecules are much more significant. Besides, the presence of DHA molecules mainly in the Ld membrane domains could regulate the differences in the lipid chain order, membrane thickness, cholesterol preference, and cholesterol flip-flop basically in a concentration-dependent manner, which further promote the stability of the intraleaflet dynamics and inhibit the interleaflet dynamics (or promote membrane domain registration) of the membrane domains. In short, the impact of DHA molecules on the physical properties of a model cell membrane on the molecular level revealed in our work will provide useful insights for understanding the biological functions of DHA molecules.

Evaluation of Interactions between SARS-CoV-2 RBD and Full-Length ACE2 with Coarse-Grained Molecular Dynamics Simulations.

Compared to all-atom models, coarse-grained models enable the investigation of the dynamics of simulation systems on a much larger length scale and a longer time scale, which makes them suitable for studying macromolecular systems. Hence, in this work, we performed multiple µs-scale Martini coarse-grained molecular dynamics simulations to reveal the interaction details between SARS-CoV-2 RBD and full-length human ACE2. Besides, the key coarse-grained systems were backmapped into the corresponding all-atom system for the display of structural details. Our results indicated that the plier structure in two ends of the binding interface plays a key role in the binding process of SARS-CoV-2 RBD with ACE2. Furthermore, we also found that when there is no B0AT1 in the simulation system, the N-terminus of ACE2 is more likely to approach the cell membrane, which has a strong correlation with the subsequent fusion of the virus with the cell membrane. These binding details of SARS-CoV-2 RBD and the ACE2 protease domain (PD) as well as the membrane orientation thermodynamics can promote the development of therapeutic drugs and preventive vaccines against SARS-CoV-2.

Ultra-Stretchable and Fast Self-Healing Ionic Hydrogel in Cryogenic Environments for Artificial Nerve Fiber.

Self-healing materials behave with irreplaceable advantages in biomimetic intelligent robots (BIR) for avoiding or reducing safety hazards and economic losses from accidental damage during service. However, the self-healing ability is unreservedly lost and even becomes rigid and fragile in the cryogenic environment where BIR are precisely needed. Here, the authors report a versatile ionic hydrogel with fast self-healing ability, ultra-stretchability, and stable conductivity, even at -80 °C. The hydrogel is systematically optimized to improve a hydrogen-bonded network nanostructure, coordinated achieving a quick self-healing ability within 10 min, large deformation tolerance of over 7000%, superior conductivity of 11.76 S cm-1 and anti-freezing ability, which is difficult to obtain simultaneously. Such a hydrogel provides new opportunities for artificial electronic devices in harsh environments. As a prospective application, they fabricate an artificial nerve fiber by mimicking the structure and functions of the myelinated axon, exhibiting the property of fast and potential-gated signal transmission. This artificial nerve fiber is integrated into a robot for demonstrating a real-time high fidelity and high throughput information interaction under big deformation and cryogenic temperature. The hydrogel and bionic device will bring pioneering functions for robots and open a broad application scenario in extreme conditions.

Designing amphiphilic Janus nanoparticles with tunable lipid raft affinity via molecular dynamics simulation.

Due to the differential interactions among lipids and proteins, the plasma membrane can segregate into a series of functional nanoscale membrane domains ("lipid rafts"), which are essential in multiple biological processes such as signaling transduction, protein trafficking and endocytosis. On the other hand, Janus nanoparticles (NPs) have shown great promise in various biomedical applications due to their asymmetric characteristics and can integrate different surface properties and thus synergetic functions. Hence, in this work, we aim to design an amphiphilic Janus NP to target and regulate lipid rafts via tuning its surface ligand amphiphilicity using coarse-grained molecular dynamics (MD) simulations. Our µs-scale free coarse-grained MD simulations as well as umbrella sampling free energy calculations indicated that the hydrophobicity of the hydrophobic surface ligands not only determined the lateral membrane partitioning thermodynamics of Janus NPs in phase-separated lipid membranes, but also the difficulty in their insertion into different membrane domains of the lipid membrane. These two factors jointly regulated the lipid raft affinity of Janus NPs. Meanwhile, the hydrophilicity of the hydrophilic surface ligands could affect the insertion ability of Janus NPs. Besides, the ultra-small size could ensure the membrane-bound behavior of Janus NPs without disrupting the overall structure and phase separation kinetics of the lipid membrane. These results may provide valuable insights into the design of functional NPs targeting and controllably regulating lipid rafts.

Surface ligand rigidity modulates lipid raft affinity of ultra-small hydrophobic nanoparticles: insights from molecular dynamics simulations.

Differential preferences between lipids and proteins drive the formation of dynamical nanoscale membrane domains (lipid rafts), which play key roles in the proper functioning of cells. On the other hand, due to the potent physicochemical properties of nanoparticles (NPs), they have been widely used in drug delivery, bio-imaging and regulating various essential biological processes of the cells. Hence, in this work, we aim to design ultra-small hydrophobic NPs with tunable raft affinity, which is supposed to partition into the hydrophobic region of lipid membranes and be able to regulate the dynamics of the lipid raft domains. A series of µs-scale coarse-grained molecular dynamics simulations and umbrella sampling free energy calculations were performed to investigate the role of surface ligand rigidity of ultra-small hydrophobicNPs in their raft affinity. Our results indicated that the preferred localization of NPs can be tuned by adjusting their surface ligand rigidity. Generally, rigid NPs tended to target the raft domain, while soft NPs preferred the interface of the raft and non-raft domains. The free energy analysis further indicated that the surface ligand rigidity of NPs can enhance their targeting to lipid raft domains. Besides, we found that these ultra-small NPs had no significant effects on the phase separation of the lipid membrane although they might cause some local interference to surrounding lipids. These results indicate that the targeting to the lipid raft domain can be achieved by the surface ligand rigidity of NPs, which provides helpful insights for further regulations of lipid raft-mediated biological processes.

Cell membrane-biomimetic coating via click-mediated liposome fusion for mitigating the foreign-body reaction.

The foreign-body reaction (FBR) caused by the implantation of synthetic polymer scaffolds seriously affects tissue-biomaterial integration and tissue repair. To address this issue, we developed a cell membrane-biomimetic coating formed by "click"-mediated liposome immobilization and fusion on the surface of electrospun fibers to mitigate the FBR. Utilization of electrospun polystyrene microfibrous scaffold as a model matrix, we deposited azide-incorporated silk fibroin on the surface of the fibers by the layer-by-layer assembly, finally, covalently modified with clickable liposomes via copper-free SPAAC click reaction. Compared with physical adsorption, liposomes click covalently binding can quickly fuse to form lipid film and maintain fluidity, which also improved liposome stability in vitro and in vivo. Molecular dynamics simulation proved that "click" improves the binding rate and strength of liposome to silk substrate. Importantly, histological observation and in vivo fluorescent probes imaging showed that liposome-functionalized electrospun fibers had negligible characteristics of the FBR and were accompanied by many infiltrated host cells and new blood vessels. We believe that the promotion of macrophage polarization toward a pro-regenerative phenotype plays an important role in vascularization. This bioinspired strategy paves the way for utilizing cell membrane biomimetic coating to resist the FBR and promote tissue-scaffold integration.

Transmembrane potential of physiologically relevant model membranes: Effects of membrane asymmetry.

Transmembrane potential difference (Vm) plays important roles in regulating various biological processes. At the macro level, Vm can be experimentally measured or calculated using the Nernst or Goldman-Hodgkin-Katz equation. However, the atomic details responsible for its generation and impact on protein and lipid dynamics still need to be further elucidated. In this work, we performed a series of all-atom molecular dynamics (MD) simulations of symmetric model membranes of various lipid compositions and cation contents to evaluate the relationship between membrane asymmetry and Vm. Specifically, we studied the impact of the asymmetric distribution of POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine), PIP2 (phosphatidylinositol 4,5-bisphosphate), as well as Na+ and K+ on Vm using atomically detailed MD simulations of symmetric model membranes. The results suggest that, for an asymmetric POPC-POPC/POPS bilayer in the presence of NaCl, the presence of the monovalent anionic lipid POPS in the inner leaflet polarizes the membrane (ΔVm < 0). Intriguingly, replacing a third of the POPS lipids by the polyvalent anionic signaling lipid PIP2 counteracts this effect, resulting in a smaller negative membrane potential. We also found that replacing Na+ ions in the inner region by K+ depolarizes the membrane (ΔVm > 0). These divergent effects arise from variations in the strength of cation-lipid interactions and are correlated with changes in lipid chain order and head-group orientation.

Correction: Optimization of hydrophobic nanoparticles to better target lipid rafts with molecular dynamics simulations.

Correction for 'Optimization of hydrophobic nanoparticles to better target lipid rafts with molecular dynamics simulations' by Xiaoqian Lin et al., Nanoscale, 2020, 12, 4101-4109, DOI: 10.1039/C9NR09226A.

Single-Molecule Study of Peptides with the Same Amino Acid Composition but Different Sequences by Using an Aerolysin Nanopore.

Nanopores are original sensors employed for highly sensitive peptides/proteins detection. Herein, we describe the use of an aerolysin nanopore to identify two similar model peptides, YEQYEQQDDDRQQQ (YEQ2Q3) and QDDDRQQQYEQYEQ (Q3YEQ2), with the same amino acid composition but different sequences. All-atom molecular dynamics (MD) simulations reveal that YEQ2Q3 possesses fewer hydrogen bonds and a more extended conformation than Q3YEQ2. These two peptides, which fold differently, exhibit obviously distinct mass-independent current blockades with characteristic dwell times when entering the aerolysin nanopore. Typically, at +60 mV, the statistical dwell time of 0.630±0.018 ms for peptide Q3YEQ2 is four times longer than the value of 0.160±0.001 ms for peptide YEQ2Q3, and yet peptide YEQ2Q3 induces ∼1.9 % larger blockade current amplitude than peptide Q3YEQ2. The obtained results show the remarkable potential of aerolysin nanopore for peptides/proteins identification, characterization, sequencing and also demonstrate that the mass identification of nonuniformly charged peptides/proteins by using the nanopore technique could be complicated by their folded structure and complex analyte-pore interaction.

A Translocation Pathway for Vesicle-Mediated Unconventional Protein Secretion.

Many cytosolic proteins lacking a signal peptide, called leaderless cargoes, are secreted through unconventional secretion. Vesicle trafficking is a major pathway involved. It is unclear how leaderless cargoes enter into the vesicle. Here, we find a translocation pathway regulating vesicle entry and secretion of leaderless cargoes. We identify TMED10 as a protein channel for the vesicle entry and secretion of many leaderless cargoes. The interaction of TMED10 C-terminal region with a motif in the cargo accounts for the selective release of the cargoes. In an in vitro reconstitution assay, TMED10 directly mediates the membrane translocation of leaderless cargoes into the liposome, which is dependent on protein unfolding and enhanced by HSP90s. In the cell, TMED10 localizes on the endoplasmic reticulum (ER)-Golgi intermediate compartment and directs the entry of cargoes into this compartment. Furthermore, cargo induces the formation of TMED10 homo-oligomers which may act as a protein channel for cargo translocation.

A Review on Applications of Computational Methods in Drug Screening and Design.

Drug development is one of the most significant processes in the pharmaceutical industry. Various computational methods have dramatically reduced the time and cost of drug discovery. In this review, we firstly discussed roles of multiscale biomolecular simulations in identifying drug binding sites on the target macromolecule and elucidating drug action mechanisms. Then, virtual screening methods (e.g., molecular docking, pharmacophore modeling, and QSAR) as well as structure- and ligand-based classical/de novo drug design were introduced and discussed. Last, we explored the development of machine learning methods and their applications in aforementioned computational methods to speed up the drug discovery process. Also, several application examples of combining various methods was discussed. A combination of different methods to jointly solve the tough problem at different scales and dimensions will be an inevitable trend in drug screening and design.

Optimization of hydrophobic nanoparticles to better target lipid rafts with molecular dynamics simulations.

Due to different interactions between lipids and proteins, a plasma membrane can segregate into different membrane domains. Among them, ordered functional membrane domains are defined as "lipid rafts", which play key roles in many biological processes (e.g., signal transduction, endocytosis, etc.) in the cell. Hence, it will be of much biological significance to monitor and even regulate the dynamics of lipid rafts. In this work, we designed a ligand-modified spherical nanoparticle with coarse-grained molecular dynamics simulations, which can be encapsulated into the hydrophobic region of the lipid membrane and specifically target either raft or non-raft membrane domains. The preferred localization of the nanoparticle can be tuned by adjusting ligand hydrophobicity, length and density. Generally, more hydrophobic nanoparticles tend to target the raft domain, while less hydrophobic nanoparticles prefer the non-raft domain. Besides, ligand length and density jointly determine the exposure of nanoparticle cores and thus affect the roles of ligands in nanoparticles' final localization. Our results may provide insights into the experimental design of functional nanoparticles, targeting the lipid raft and regulating its dynamics.

Lipid Acyl Chain cis Double Bond Position Modulates Membrane Domain Registration/Anti-Registration.

Inter-leaflet coupling is critical to control the dynamics of membrane domain registration/anti-registration, which is important in maintaining proper biological functions. Factors such as lipid acyl chain inter-digitation and membrane remodeling have been found to be able to regulate the inter-leaflet coupling. However, detailed molecular mechanisms that dominate the inter-leaflet coupling are still far from clear. Here, we revealed that lipid acyl chain cis double bond position can regulate the inter-leaflet coupling according to our coarse-grained and all-atom molecular dynamics simulations. The farther the double bond is away from the lipid tail terminal, the weaker the inter-leaflet attractive interactions between unsaturated lipids. Therefore, the relative motions of membrane domains in two membrane leaflets become more obvious (membrane domain anti-registration). Generally, our simulations validated a direct indicator for the inter-leaflet coupling strength, which provides physical insights into the molecular mechanisms of membrane domain registration/anti-registration.