Lipid Affinity Research Articles

Biocompatible Hydrogel Coating on Silicone Rubber with Improved Antifouling and Durable Lubricious Properties.

Silicone rubber is widely used in various medical applications. However, silicone rubber is prone to biofouling due to their affinity for lipids and has a high friction coefficient, which can significantly impact their efficacy and performance used as medical devices. Thus, the development of hydrogels with antifouling and lubricious abilities for the modification of silicone rubber is in high demand. We herein prepared a variety of hydrogel coatings mainly based on polyvinylpyrrolidone (PVP) and poly (ethylene glycol) diacrylate (PEGDA). We modified the silicone rubber using the prepared hydrogel coatings and cured it using a heating method. Then, we characterized its surface and evaluated the antifouling property, lubricious property, cytotoxicity, sensitization, and vaginal irritation. The results of water contact angle (WCA), protein adsorption, and friction coefficient indicated the success of the modification of the silicone rubber, leading to a significant decrease in the corresponding test values. Meanwhile, the results of cytotoxicity, sensitization, and vaginal irritation tests showed that the hydrogel coating-modified silicone rubbers have an excellent biocompatibility. This study describes how the silicone rubber could be modified with a biocompatible hydrogel coating. The hydrogel coating-modified silicone rubbers have improved antifouling and durable lubricious properties.

S-acylation of Ca2+ transport proteins in cancer.

Alterations in cellular calcium (Ca2+) signals have been causally associated with the development and progression of human cancers. Cellular Ca2+ signals are generated by channels, pumps, and exchangers that move Ca2+ ions across membranes and are decoded by effector proteins in the cytosol or in organelles. S-acylation, the reversible addition of 16-carbon fatty acids to proteins, modulates the activity of Ca2+ transporters by altering their affinity for lipids, and enzymes mediating this reversible post-translational modification have also been linked to several types of cancers. Here, we compile studies reporting an association between Ca2+ transporters or S-acylation enzymes with specific cancers, as well as studies reporting or predicting the S-acylation of Ca2+ transporters. We then discuss the potential role of S-acylation in the oncogenic potential of a subset of Ca2+ transport proteins involved in cancer.

TREK2 Lipid Binding Preferences Revealed by Native Mass Spectrometry.

TREK2, a two-pore domain potassium channel, is recognized for its regulation by various stimuli, including lipids. While previous members of the TREK subfamily, TREK1 and TRAAK, have been investigated to elucidate their lipid affinity and selectivity, TREK2 has not been similarly studied in this regard. Our findings indicate that while TRAAK and TREK2 exhibit similarities in terms of electrostatics and share an overall structural resemblance, there are notable distinctions in their interaction with lipids. Specifically, SAPI(4,5)P2,1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-(1'-myo-inositol-4',5'-bisphosphate) exhibits a strong affinity for TREK2, surpassing that of dOPI(4,5)P2,1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-4',5'-bisphosphate), which differs in its acyl chains. TREK2 displays lipid binding preferences not only for the headgroup of lipids but also toward the acyl chains. Functional studies draw a correlation for lipid binding affinity and activity of the channel. These findings provide important insight into elucidating the molecular prerequisites for specific lipid binding to TREK2 important for function.

Optimisation of analytical methods for tuberculosis drug detection in wastewater: A multinational study

Wastewater-based epidemiology (WBE) is a robust tool for disease surveillance and monitoring of pharmaceutical consumption. However, monitoring tuberculosis (TB) drug consumption faces challenges due to limited data availability. This study aimed to optimise methods for detecting TB drugs in treated and untreated wastewater from four African countries: South Africa, Nigeria, Kenya, and Cameroon. The limit of detection (LOD) for these drugs ranged from a minimum of 2.20 (±1.02) for rifampicin to a maximum of 2.95 (±0.79) for pyrazinamide. A parallel trend was observed concerning the limit of quantification (LOQ), with rifampicin reporting the lowest average LOQ of 7.33 (±3.44) and pyrazinamide showing the highest average LOQ of 9.81 (±2.64). The variance in LOD and LOQ values could be attributed to factors such as drug polarity. Erythromycin and rifampicin exhibited moderately polar LogP values (2.6 and 2.95), indicating higher lipid affinity and lower water affinity.Conversely, ethambutol, pyrazinamide, and isoniazid displayed polar LogP values (−0.059, −0.6, and −0.7), suggesting lower lipid affinity and greater water affinity. The study revealed that storing wastewater samples for up to 5 days did not result in significant drug concentration loss, with concentration reduction remaining below 1 log throughout the storage period. Application of the optimised method for drug detection and quantification in both treated and untreated wastewater unveiled varied results. Detection frequencies varied among drugs, with ethambutol consistently most detected, while pyrazinamide and isoniazid were least detected in wastewater from only two countries. Most untreated wastewater samples had undetectable drug concentrations, ranging from 1.21 ng/mL for erythromycin to 54.61 ng/mL for isoniazid. This variability may suggest differences in drug consumption within connected communities. In treated wastewater samples, detectable drug concentrations ranged from 1.27 ng/mL for isoniazid to 10.20 ng/mL for ethambutol. Wastewater treatment plants exhibited variable removal efficiencies for different drugs, emphasising the need for further optimisation. Detecting these drugs in treated wastewater suggests potential surface water contamination and subsequent risks of human exposure, underscoring continued research's importance.

Sequestration of membrane cholesterol by cholesterol-binding proteins inhibits SARS-CoV-2 entry into Vero E6 cells

Membrane lipids and proteins form dynamic domains crucial for physiological and pathophysiological processes, including viral infection. Many plasma membrane proteins, residing within membrane domains enriched with cholesterol (CHOL) and sphingomyelin (SM), serve as receptors for attachment and entry of viruses into the host cell. Among these, human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use proteins associated with membrane domains for initial binding and internalization. We hypothesized that the interaction of lipid-binding proteins with CHOL in plasma membrane could sequestrate lipids and thus affect the efficiency of virus entry into host cells, preventing the initial steps of viral infection. We have prepared CHOL-binding proteins with high affinities for lipids in the plasma membrane of mammalian cells. Binding of the perfringolysin O domain four (D4) and its variant D4E458L to membrane CHOL impaired the internalization of the receptor-binding domain of the SARS-CoV-2 spike protein and the pseudovirus complemented with the SARS-CoV-2 spike protein. SARS-CoV-2 replication in Vero E6 cells was also decreased. Overall, our results demonstrate that the integrity of CHOL-rich membrane domains and the accessibility of CHOL in the membrane play an essential role in SARS-CoV-2 cell entry.

Pyroptosis in health and disease.

The field of cell death has witnessed significant advancements since the initial discovery of apoptosis in the 1970s. This review delves into the intricacies of pyroptosis, a more recently identified form of regulated, lytic cell death, and explores the roles of pyroptotic effector molecules, with a strong emphasis on their mechanisms and relevance in various diseases. Pyroptosis, characterized by its proinflammatory nature, is driven by the accumulation of large plasma membrane pores comprised of gasdermin family protein subunits. In different contexts of cellular homeostatic perturbations, infections, and tissue damage, proteases, such as caspase-1 and caspase-4/5, play pivotal roles in pyroptosis by cleaving gasdermins. Gasdermin-D (GSDMD), the most extensively studied member of the gasdermin protein family, is expressed in various immune cells and certain epithelial cells. Upon cleavage by caspases, GSDMD oligomerizes and forms transmembrane pores in the cell membrane, leading to the release of proinflammatory cytokines. GSDMD-N, the NH2-terminal fragment, displays an affinity for specific lipids, contributing to its role in pore formation in pyroptosis. While GSDMD is the primary focus, other gasdermin family members are also discussed in detail. These proteins exhibit distinct tissue-specific functions and contribute to different facets of cell death regulation. Additionally, genetic variations in some gasdermins have been linked to diseases, underscoring their clinical relevance. Furthermore, the interplay between GSDM pores and the activation of other effectors, such as ninjurin-1, is elucidated, providing insights into the complexity of pyroptosis regulation. The findings underscore the molecular mechanisms that govern pyroptosis and its implications for various physiological and pathological processes.

Gold nanoparticle-powered screening of membrane protein-specific lipids from complex lipid mixtures

Membrane proteins (MPs) are affected by binding of specific lipids. We previously developed a methodology for systematically analyzing MP-lipid interactions leveraging surface plasmon resonance (SPR). In this method, the gold sensor chip surface was modified with a self-assembled monolayer (SAM), which allowed for a larger amount of MP-immobilization. However, the laborious lipid purification step remained a bottleneck. To address this issue, a new strategy has been developed utilizing gold nanoparticles (AuNPs) instead of the gold sensor chip. AuNPs were coated with SAM, on which MP was covalently anchored. The MP-immobilized AuNPs were mixed with a lipid mixture, and the recovered lipids were quantified by LC–MS. Bacteriorhodopsin (bR) was used as an MP to demonstrate this concept. We optimized immobilization conditions and confirmed the efficient immobilization of bR by dynamic light scattering and electron micrographs. Washing conditions for pulldown experiments were optimized to efficiently remove non-specific lipids. A new binding index was introduced to qualitatively reproduce the known affinity of lipids for bR. Consequently, the low-abundant and least-studied lipid S-TeGD was identified as a candidate for bR-specific lipids. This technique can skip the laborious lipid purification process, accelerating the screening of MP-specific lipids from complex lipid mixtures.

Identifying Membrane Protein-Lipid Interactions with Lipidomic Lipid Exchange-Mass Spectrometry.

Lipids can play important roles in modulating membrane protein structure and function. However, it is challenging to identify natural lipids bound to membrane proteins in complex bilayers. Here, we developed lipidomic lipid exchange-mass spectrometry (LX-MS) to study the lipid affinity for membrane proteins on a lipidomic scale. We first mix membrane protein nanodiscs with empty nanodiscs that have no embedded membrane proteins. After allowing lipids to passively exchange between the two populations, we separate the two types of nanodiscs and perform lipidomic analysis on each with liquid chromatography and MS. Enrichment of lipids in the membrane protein nanodiscs reveals the affinity of individual lipids for binding the target membrane protein. We apply this approach to study three membrane proteins. With the Escherichia coli ammonium transporter AmtB and aquaporin AqpZ in nanodiscs with E. coli polar lipid extracts, we detected binding of cardiolipin and phosphatidyl-glycerol lipids to the proteins. With the acetylcholine receptor in nanodiscs with brain polar lipid extracts, we discovered a complex set of lipid interactions that depended on the head group and tail composition. Overall, lipidomic LX-MS provides a detailed understanding of the lipid-binding affinity and thermodynamics for membrane proteins in complex bilayers and provides a unique perspective on the chemical environment surrounding membrane proteins.

Independent Membrane Binding Properties of the Caspase Generated Fragments of the Beaded Filament Structural Protein 1 (BFSP1) Involves an Amphipathic Helix.

BFSP1 (beaded filament structural protein 1) is a plasma membrane, Aquaporin 0 (AQP0/MIP)-associated intermediate filament protein expressed in the eye lens. BFSP1 is myristoylated, a post-translation modification that requires caspase cleavage at D433. Bioinformatic analyses suggested that the sequences 434-452 were α-helical and amphipathic. By CD spectroscopy, we show that the addition of trifluoroethanol induced a switch from an intrinsically disordered to a more α-helical conformation for the residues 434-467. Recombinantly produced BFSP1 fragments containing this amphipathic helix bind to lens lipid bilayers as determined by surface plasmon resonance (SPR). Lastly, we demonstrate by transient transfection of non-lens MCF7 cells that these same BFSP1 C-terminal sequences localise to plasma membranes and to cytoplasmic vesicles. These can be co-labelled with the vital dye, lysotracker, but other cell compartments, such as the nuclear and mitochondrial membranes, were negative. The N-terminal myristoylation of the amphipathic helix appeared not to change either the lipid affinity or membrane localisation of the BFSP1 polypeptides or fragments we assessed by SPR and transient transfection, but it did appear to enhance its helical content. These data support the conclusion that C-terminal sequences of human BFSP1 distal to the caspase site at G433 have independent membrane binding properties via an adjacent amphipathic helix.

VDAC interactions with lipids and their effect on isoform specificity.

The Voltage-Dependent Anion Channel (VDAC) is a β-barrel, the most abundant protein of the mitochondrial outer membrane, allowing passage of ions and metabolites (such as ATP, ADP, NADH⋯) in and out of the mitochondria. Besides its transport function, VDAC is also implicated in mitochondrial regulation with roles in apoptosis, calcium homeostasis or neurodegeneration. However, its role in those different pathways remains to be understood at the molecular level. VDAC possesses 3 isoforms in mammals (VDAC1, 2 and 3), sharing a high sequence identity, the same architecture and an overlapping tissue distribution. They display very similar transport properties such as conductance, selectivity and voltage-induced closure while their isoform specificity originates from their different interaction with partner proteins. This may determine their unique role in mitochondrial regulation, where each isoform seems to have different functions and a different set of partners: VDAC1 is implicated in neurodegeneration (Alzheimer and Parkinson disease), VDAC2 in apoptosis and calcium homeostasis, and VDAC3 probably in spermatogenesis and oxidative stress. Using a combination of biochemistry, molecular dynamics and electrophysiology, we investigated the lipid organization around each VDAC isoform and their impact on VDAC function. Our data suggest that the three isoforms have different affinities for lipids, and that could contribute to the selection of interacting partners.

Structure–activity relationship of gallic acid from Paeonia lactiflora and its synthetic analogs against human breast cancer cells

AbstractHigh‐performance liquid chromatography analysis of the ethyl acetate fraction of Paeonia lactiflora roots led to the detection of five compounds: gallic acid (GA) (1), methyl gallate (2), albiflorin (4), paeoniflorin (5), and pentagalloylglucose (6). Among them, pentagalloylglucose (6) showed cytotoxicity against human breast cancer cells MCF‐7 and MDA‐MB‐231 in vitro with IC50 values of 20.1 and 14.4 μM, respectively. Through the structure–activity relationship of GA, which is an important backbone of pentagalloylglucose, we found that its three hydroxy groups were important for its cytotoxicity, and that the 3‐O‐methylgallic acid structure was only effective against a triple‐negative breast cancer cell line. Furthermore, drug efficacy was confirmed by increasing its lipid affinity through the synthesis of various ester derivatives of gallic acid.

Mutational Insight into Allosteric Regulation of Kir Channel Activity.

Potassium (K+) channels are regulated in part by allosteric communication between the helical bundle crossing, or inner gate, and the selectivity filter, or outer gate. This network is triggered by gating stimuli. In concert, there is an allosteric network which is a conjugated set of interactions which correlate long-range structural rearrangements necessary for channel function. Inward-rectifier K+ (Kir) channels favor inward K+ conductance, are ligand-gated, and help establish resting membrane potentials. KirBac1.1 is a bacterial Kir (KirBac) channel homologous to human Kir (hKir) channels. Additionally, KirBac1.1 is gated by the anionic phospholipid ligand phosphatidylglycerol (PG). In this study, we use site-directed mutagenesis to investigate residues involved in the KirBac1.1 gating mechanism and allosteric network we previously proposed using detailed solid-state NMR (SSNMR) measurements. Using fluorescence-based K+ and sodium (Na+) flux assays, we identified channel mutants with impaired function that do not alter selectivity of the channel. In tandem, we performed coarse grain molecular dynamics simulations, observing changes in PG-KirBac1.1 interactions correlated with mutant channel activity and contacts between the two transmembrane helices and pore helix tied to this behavior. Lipid affinity is closely tied to the proximity of two tryptophan residues on neighboring subunits which lure anionic lipids to a cationic pocket formed by a cluster of arginine residues. Thus, these simulations establish a structural and functional basis for the role of each mutated site in the proposed allosteric network. The experimental and simulated data provide insight into key functional residues involved in gating and lipid allostery of K+ channels. Our findings also have direct implications on the physiology of hKir channels due to conservation of many of the residues identified in this work from KirBac1.1.

Exploring lipid affinities of persistent organic pollutants and MeO-PBDEs in blubber of marine mammals

Although lipophilic compounds have been the focus of numerous studies in marine mammals, their association with lipids is widely accepted, but rarely scrutinized. This pilot study aimed to investigate potential relationships between individual lipids from different lipid classes identified through a non-targeted Nuclear Magnetic Resonance (NMR) based lipidomics approach and legacy POPs in the blubber of long-finned pilot whales, sperm whales, common bottlenose dolphins, and Indo-Pacific bottlenose dolphins. Concentrations of selected POPs such as HCB and HCHs in sperm whales from Tasmania were found to differ from those in long-finned pilot whales and common bottlenose dolphins from the same location. Profiles of NMR spectra measured in blubber of sperm whales were also distinctly different compared to the pilot whales and common bottlenose dolphins. Two groups of Indo-Pacific bottlenose dolphins from South Australia that were 20 years apart showed highly comparable profiles of NMR signals despite having higher concentrations of several POP classes in the more recent group. More specific correlations were investigated between selected POPs (n = 12) and all detected NMR signals (n = 63) in all species. Outcomes were species-specific, but difficult to interpret due to the lack of available literature for marine mammals and the small sample sizes per species. Because of the key role of lipids in the bioaccumulation of POPs and in the incidence of diseases, more attention should be given to the identification and characterization of lipid species in future toxicological studies. However, future studies should focus on one marine mammal species to increase sample sizes and limit the number of confounding factors, such as diet, that can influence POP and lipid levels and profiles.

Antibacterial and Anti-Inflammatory Effects of Apolipoprotein E.

Apolipoprotein E (APOE) is a lipid-transport protein that functions as a key mediator of lipid transport and cholesterol metabolism. Recent studies have shown that peptides derived from human APOE display anti-inflammatory and antimicrobial effects. Here, we applied in vitro assays and fluorescent microscopy to investigate the anti-bacterial effects of full-length APOE. The interaction of APOE with endotoxins from Escherichia coli was explored using surface plasmon resonance, binding assays, transmission electron microscopy and all-atom molecular dynamics (MD) simulations. We also studied the immunomodulatory activity of APOE using in vitro cell assays and an in vivo mouse model in combination with advanced imaging techniques. We observed that APOE exhibits anti-bacterial activity against several Gram-negative bacterial strains of Pseudomonas aeruginosa and Escherichia coli. In addition, we showed that APOE exhibits a significant binding affinity for lipopolysaccharide (LPS) and lipid A as well as heparin. MD simulations identified the low-density lipoprotein receptor (LDLR) binding region in helix 4 of APOE as a primary binding site for these molecules via electrostatic interactions. Together, our data suggest that APOE may have an important role in controlling inflammation during Gram-negative bacterial infection.

Interactions between Hydrolysable Tannins and Lipid Vesicles from Escherichia coli with Isothermal Titration Calorimetry.

Isothermal titration calorimetry (ITC) was used to study the interactions between hydrolysable tannins (HTs) and lipid vesicles prepared from a phospholipid extract of Escherichia coli (E. coli). A group of 24 structurally different HTs was selected, and structural differences affecting their affinities to interact with lipid vesicles in aqueous buffered media were identified. In general, the interactions between HTs and lipid vesicles were exothermic in nature, and ITC as a technique functioned well in the screening of HTs for their affinity for lipids. Most notably, the galloyl moiety, the structural flexibility of the entire tannin structure, the hydrophobicity of the tannin, and higher molecular weight were observed to be important for the stronger interactions with the lipids. The strongest interactions with lipids were observed for rugosins D and G. It was also observed that some HTs with moderate hydrophobicities, such as geraniin, chebulagic acid, and chebulinic acid, did not have any detectable interactions with the lipid vesicles, suggesting that a hydrophobic structure alone does not guarantee an affinity for lipids.

Albumin Lipidomics Reveals Meaningful Compositional Changes in Advanced Cirrhosis and Its Potential to Promote Inflammation Resolution.

Albumin infusions are therapeutically used to revert hypoalbuminemia and to replace the extensively oxidized albumin molecule circulating in patients with acutely decompensated (AD) cirrhosis. Because albumin has high affinity for lipids, here we characterized the albumin lipidome in patients with AD and explored the albumin effects on the release of fatty acid (FA)–derived lipid mediators by peripheral leukocytes. Lipids and lipid mediators were measured by liquid chromatography–tandem mass spectrometry in albumin‐enriched and albumin‐depleted plasma fractions separated by affinity chromatography and in leukocyte incubations from 18 patients with AD and 10 healthy subjects (HS). Lipid mediators were also measured in 41 patients with AD included in an albumin therapy trial. The plasma lipidome associated with AD cirrhosis was characterized by generalized suppression of all lipid classes except FAs. In contrast to HS, albumin from patients with AD had lower content of polyunsaturated FAs (PUFAs), especially of the omega‐3‐PUFA docosahexaenoic acid. Consistent with this, the PUFA‐derived lipid mediator landscape of albumin from patients with AD was dominated by lower content of monohydroxy FA precursors of anti‐inflammatory/pro‐resolving lipid mediators (i.e., 15‐hydroxyeicosatetraenoic acid [15‐HETE]). In addition, albumin from patients with AD was depleted in prostaglandin (PG) E2, suggesting that this proinflammatory PG primarily travels disassociated to albumin in these patients. Incubation of leukocytes with exogenous albumin reduced PG production while inducing 15‐lipoxygenase expression and 15‐HETE release. Similar effects were seen under lipopolysaccharide plus N‐formylmethionyl‐leucyl‐phenylalanine‐stimulated conditions. Finally, PG levels were lower in patients with AD receiving albumin therapy, whereas 15‐HETE was increased after albumin treatment compared with baseline. Conclusion: Our findings indicate that the albumin lipid composition is severely disorganized in AD cirrhosis and that administration of exogenous albumin has the potential to redirect leukocyte biosynthesis from pro‐inflammatory to pro‐resolving lipid mediators.

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions.

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Protein dynamics and lipid affinity of monomeric, zeaxanthin-binding LHCII in thylakoid membranes

The xanthophyll cycle in the antenna of photosynthetic organisms under light stress is one of the most well-known processes in photosynthesis, but its role is not well understood. In the xanthophyll cycle, violaxanthin (Vio) is reversibly transformed to zeaxanthin (Zea) that occupies Vio binding sites of light-harvesting antenna proteins. Higher monomer/trimer ratios of the most abundant light-harvesting protein, the light-harvesting complex II (LHCII), usually occur in Zea accumulating membranes and have been observed in plants after prolonged illumination and during high-light acclimation. We present a combined NMR and coarse-grained simulation study on monomeric LHCII from the npq2 mutant that constitutively binds Zea in the Vio binding pocket. LHCII was isolated from 13C-enriched npq2 Chlamydomonas reinhardtii (Cr) cells and reconstituted in thylakoid lipid membranes. NMR results reveal selective changes in the fold and dynamics of npq2 LHCII compared with the trimeric, wild-type and show that npq2 LHCII contains multiple mono- or digalactosyl diacylglycerol lipids (MGDG and DGDG) that are strongly protein bound. Coarse-grained simulations on npq2 LHCII embedded in a thylakoid lipid membrane agree with these observations. The simulations show that LHCII monomers have more extensive lipid contacts than LHCII trimers and that protein-lipid contacts are influenced by Zea. We propose that both monomerization and Zea binding could have a functional role in modulating membrane fluidity and influence the aggregation and conformational dynamics of LHCII with a likely impact on photoprotection ability.

Chemistry of Lipid Nanoparticles for RNA Delivery.

Lipid nanoparticles (LNPs) are a type of lipid vesicles that possess a homogeneous lipid core. These vesicles are widely used in small-molecule drug and nucleic acid delivery and recently gained much attention because of their remarkable success as a delivery platform for COVID-19 mRNA vaccines. Nonetheless, the utility of transient protein expression induced by mRNA extends far beyond vaccines against infectious diseases─they also hold promise as cancer vaccines, protein replacement therapies, and gene editing components for rare genetic diseases. However, naked mRNA is inherently unstable and prone to rapid degradation by nucleases and self-hydrolysis. Encapsulation of mRNA within LNPs protects mRNA from extracellular ribonucleases and assists with intracellular mRNA delivery.In this Account, we discuss the core features of LNPs for RNA delivery. We focus our attention on LNPs designed to deliver mRNA; however, we also include examples of siRNA-LNP delivery where appropriate to highlight the commonalities and the dissimilarities due to the nucleic acid structure. First, we introduce the concept of LNPs, the advantages and disadvantages of utilizing nucleic acids as therapeutic agents, and the general reasoning behind the molecular makeup of LNPs. We also briefly highlight the most recent clinical successes of LNP-based nucleic acid therapies. Second, we describe the theory and methods of LNP self-assembly. The common idea behind all of the preparation methods is inducing electrostatic interactions between the nucleic acid and charged lipids and promoting nanoparticle growth via hydrophobic interactions. Third, we break down the LNP composition with special attention to the fundamental properties and purposes of each component. This includes the identified molecular design criteria, commercial sourcing, impact on intracellular trafficking, and contribution to the properties of LNPs. One of the key components of LNPs is ionizable lipids, which initiate electrostatic binding with endosomal membranes and facilitate cytosolic release; however, the roles of other lipid components should not be disregarded, as they are associated with stability, clearance, and distribution of LNPs. Fourth, we review the attributes of LNP constructs as a whole that can heavily influence RNA delivery. These attributes are LNP size, charge, internal structure, lipid packing, lipid membrane hydration, stability, and affinity toward biomacromolecules. We also discuss the specific techniques used to examine these attributes and how they can be adjusted. Finally, we offer our perspective on the future of RNA therapies and some questions that remain in the realm of LNP formulation and optimization.

Nanodisc-Based Proteomics Identify Caj1 as an Hsp40 with Affinity for Phosphatidic Acid Lipids.

Many soluble proteins interact with membranes to perform important biological functions, including signal transduction, regulation, transport, trafficking, and biogenesis. Despite their importance, these protein-membrane interactions are difficult to characterize due to their often-transient nature as well as phospholipids' poor solubility in aqueous solution. Here, we employ nanodiscs-small, water-soluble patches of a lipid bilayer encircled with amphipathic scaffold proteins-along with quantitative proteomics to identify lipid-binding proteins in Saccharomyces cerevisiae. Using nanodiscs reconstituted with yeast total lipid extracts or only phosphatidylethanolamine (PE-nanodiscs), we capture several known membrane-interacting proteins, including the Rab GTPases Sec4 and Ypt1, which play key roles in vesicle trafficking. Utilizing PE-nanodiscs enriched with phosphatidic acid (PEPA-nanodiscs), we specifically capture a member of the Hsp40/J-protein family, Caj1, whose function has recently been linked to membrane protein quality control. We show that the Caj1 interaction with liposomes containing PA is modulated by pH and PE lipids and depends on two patches of positively charged residues near the C-terminus of the protein. The protein Caj1 is the first example of an Hsp40/J-domain protein with affinity for membranes and phosphatidic acid lipid specificity. These findings highlight the utility of combining proteomics with lipid nanodiscs to identify and characterize protein-lipid interactions that may not be evident using other methods. Data are available via ProteomeXchange with the identifier PXD027992.