Bound Phospholipids Assist Cholesteryl Ester Transfer in the Cholesteryl Ester Transfer Protein.

Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that assists the transfer of cholesteryl esters (CEs) from antiatherogenic high-density lipoproteins (HDLs) to proatherogenic low-density lipoproteins (LDLs), initiating cholesterol plaques in the arteries. Consequently, inhibiting the activity of CETP is therefore being pursued as a novel strategy to reduce the risk of cardiovascular diseases (CVDs). The crystal structure of CETP has revealed the presence of two CEs running in the hydrophobic tunnel and two plugged-in phospholipids (PLs) near the concave surface. Other than previous animal models that rule out the PL transfer by CETP and PLs in providing the structural stability, the functional importance of bound phospholipids in CETP is not fully explored. Here, we employ a series of molecular dynamics (MD) simulations, steered molecular dynamics (SMD) simulations, and free energy calculations to unravel the effect of PLs on the functionality of the protein. Our results suggest that PLs play an important role in the transfer of neutral lipids by transforming the unfavorable bent conformation of CEs into a favorable linear conformation to facilitate the smooth transfer. The results also suggest that the making and breaking interactions of the hydrophobic tunnel residues with CEs with a combined effort from PLs are responsible for the transfer of CEs. Further, the findings demonstrate that the N-PL has a more pronounced effort on CE transfer than C-PL but efforts from both PLs are essential in the transfer. Thus, we propose that the functionally important PLs can be considered with potential research interest in targeting cardiovascular diseases.

Looking in Depth at Oxidized Cholesteryl Esters by LC-MS/MS: Reporting Specific Fragmentation Fingerprints and Isomer Discrimination.

Cholesteryl esters (CE) are prone to oxidation under increased oxidative stress conditions, but little is known about oxidized CE species (oxCE). To date, only a few oxCE have been identified, however, mainly based on the detection of molecular ions by mass spectrometry (MS) or target approaches for specific oxCE. The study of oxCE occurring from radical oxidation is still scarcely addressed. In this work, we made a comprehensive assessment of oxCE derivatives and their specific fragmentation patterns to identify detailed structural features and isomer differentiation using high-resolution C18 HPLC-MS- and MS/MS-based lipidomic approaches. The LC-MS/MS analysis allowed us to pinpoint oxCE structural isomers of long-chain and short-chain species, eluting at different retention times (tR). Data analysis revealed that oxCE can be modified either in the fatty acyl moiety or in the cholesterol ring. The location of the hydroxy/hydroperoxy group originates characteristic fragment ions, namely the unmodified cholestenyl cation (m/z 369) for the isomer with oxidation in the fatty acyl chain or ions at m/z 367 and m/z 385 (369 + 16) when oxygenation occurs in the cholesterol ring. Additionally, we identified CE 18:2 and 20:4 aldehydic and carboxylic short-chain products that showed a clear fragmentation pattern that confirmed the modification in the fatty acyl chain. Specific fragmentation fingerprinting allowed discrimination of the isobaric short-chain species, namely carboxylic short-chain products, from hydroxy aldehyde short-chain products, with a hydroxycholesterol moiety. This new information is important to identify different oxCE in biological samples and will contribute to unraveling their role in biological conditions and diseases such as cardiovascular disease.

On the importance of chain branching in tear film lipid layer wax and cholesteryl esters.

The tear film lipid layer (TFLL) is important to the maintenance of ocular surface health. Surprisingly, information on the individual roles of the myriad of unique lipids found therein is limited. The most abundant lipid species are the wax esters (WE) and cholesteryl esters (CE), and, especially their branched analogs. The isolation of these lipid species from the TFLL has proved to be tedious, and as a result, insights on their biophysical profiles and role in the TFLL is currently lacking. Herein, we circumvent these issues by a total synthesis of the most abundant iso-methyl branched WEs and CEs found in the TFLL. Through a detailed characterization of the biophysical properties, by the use of Langmuir monolayer and wide-angle X-ray scattering techniques, we demonstrate that chain branching alters the behavior of these lipid species on multiple levels. Taken together, our results fill an important knowledge gap concerning the structure and function of the TFLL on the whole.

Cholesterol Binds in a Reversed Orientation to TCRß-TM in Which Its OH Group is Localized to the Center of the Lipid Bilayer.

T cell receptor (TCR) signaling in response to antigen recognition is essential for the adaptive immune response. Cholesterol keeps TCRs in the resting conformation and mediates TCR clustering by directly binding to the transmembrane domain of the TCRß subunit (TCRß-TM), while cholesterol sulfate (CS) displaces cholesterol from TCRß. However, the atomic interaction of cholesterol or CS with TCRß remains elusive. Here, we determined the cholesterol and CS binding site of TCRß-TM in phospholipid bilayers using solution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulation. Cholesterol binds to the transmembrane residues within a CARC-like cholesterol recognition motif. Surprisingly, the polar OH group of cholesterol is placed in the hydrophobic center of the lipid bilayer stabilized by its polar interaction with K154 of TCRß-TM. An aromatic interaction with Y158 and hydrophobic interactions with V160 and L161 stabilize this reverse orientation. CS binds to the same site, explaining how it competes with cholesterol. Site-directed mutagenesis of the CARC-like motif disrupted the cholesterol/CS binding to TCRß-TM, validating the NMR and MD results.

Allosteric modulators enhance agonist efficacy by increasing the residence time of a GPCR in the active state.

Much hope in drug development comes from the discovery of positive allosteric modulators (PAM) that display target subtype selectivity and act by increasing agonist potency and efficacy. How such compounds can allosterically influence agonist action remains unclear. Metabotropic glutamate receptors (mGlu) are G protein-coupled receptors that represent promising targets for brain diseases, and for which PAMs acting in the transmembrane domain have been developed. Here, we explore the effect of a PAM on the structural dynamics of mGlu2 in optimized detergent micelles using single molecule FRET at submillisecond timescales. We show that glutamate only partially stabilizes the extracellular domains in the active state. Full activation is only observed in the presence of a PAM or the Gi protein. Our results provide important insights on the role of allosteric modulators in mGlu activation, by stabilizing the active state of a receptor that is otherwise rapidly oscillating between active and inactive states.

Mechanistic Insight into How PEGylation Reduces the Efficacy of pH-Sensitive Liposomes from Molecular Dynamics Simulations.

Liposome-based drug delivery systems composed of DOPE stabilized with cholesteryl hemisuccinate (CHMS) have been proposed as a drug delivery mechanism with pH-triggered release as the anionic form (CHSa) is protonated (CHS) at reduced pH; PEGylation is known to decrease this pH sensitivity. In this manuscript, we set out to use molecular dynamics (MD) simulations with a model with all-atom resolution to provide insight into why incorporation of poly(ethyleneglycol) (PEG) into DOPE-CHMS liposomes reduces their pH sensitivity; we also address two additional questions: (1) How CHSa stabilizes DOPE bilayers into a lamellar conformation at a physiological pH of 7.4? and (2) how the change from CHSa to CHS at acidic pH triggers the destabilization of DOPE bilayers? We found that (A) CHSa stabilizes the DOPE lipid membrane by increasing the hydrophilicity of the bilayer surface, (B) when CHSa changes to CHS by pH reduction, DOPE bilayers are destabilized due to a reduction in bilayer hydrophilicity and a reduction in the area per lipid, and (C) PEG stabilizes DOPE bilayers into the lamellar phase, thus reducing the pH sensitivity of the liposomes by increasing the area per lipid through penetration into the bilayer, which is our main focus.

Single-cell lipidomics with high structural specificity by mass spectrometry.

Single-cell analysis is critical to revealing cell-to-cell heterogeneity that would otherwise be lost in ensemble analysis. Detailed lipidome characterization for single cells is still far from mature, especially when considering the highly complex structural diversity of lipids and the limited sample amounts available from a single cell. We report the development of a general strategy enabling single-cell lipidomic analysis with high structural specificity. Cell fixation is applied to retain lipids in the cell during batch treatments prior to single-cell analysis. In addition to tandem mass spectrometry analysis revealing the class and fatty acyl-chain for lipids, batch photochemical derivatization and single-cell droplet treatment are performed to identify the C=C locations and sn-positions of lipids, respectively. Electro-migration combined with droplet-assisted electrospray ionization enables single-cell mass spectrometry analysis with easy operation but high efficiency in sample usage. Four subtypes of human breast cancer cells are correctly classified through quantitative analysis of lipid C=C location or sn-position isomers in ~160 cells. Most importantly, the single-cell deep lipidomics strategy successfully discriminates gefitinib-resistant cells from a population of wild-type human lung cancer cells (HCC827), highlighting its unique capability to promote precision medicine.

Cervidins A-D: Novel Glycine Conjugated Fatty Acids from the Tarsal Gland of Male Whitetail Deer, Odocoileus virginianus.

Sexually mature male deer are known to rub-urinate, a process where urine is deposited on the tarsal gland. The resulting mixture of compounds from urine and secretions from the tarsal gland are used to signal sex, age, maturation status, and other information at close distance. We examined the difference in metabolites of tarsal gland extracts from male and female whitetail deer, Odocoileus virginianus, harvested during the mating season. Using NMR spectroscopy and high-pressure liquid chromatography linked to high resolution mass spectrometry (HPLC/HR-MS) we identified a homologous series of four male-specific compounds. The compounds are novel glycine conjugates of 10-hydroxy-6,9-oxido fatty acids, which we term cervidins A-D. Cervidins were deemed to possess the absolute configuration 6S,9R,10R through comparison of their spectroscopic data with those of known compounds. In addition, cholesterol 3-sulfate and 3-(3-hydroxyphenyl)-propanoic acid were found to be present in the extracts. Our results clearly demonstrate the diversity of potential semiochemicals contained in the mammalian integument.

From Inert Storage to Biological Activity-In Search of Identity for Oxidized Cholesteryl Esters.

Esterification of cholesterol is a universal mechanism to store and transport large quantities of cholesterol between organs and tissues and to avoid toxicity of the excess of cellular cholesterol. Intended for transport and storage and thus to be inert, cholesteryl esters (CEs) reside in hydrophobic cores of circulating lipoproteins and intracellular lipid droplets. However, the inert identity of CEs is dramatically changed if cholesterol is esterified to a polyunsaturated fatty acid and subjected to oxidative modification. Post-synthetic, or epilipidomic, oxidative modifications of CEs are mediated by specialized enzymes, chief among them are lipoxygenases, and by free radical oxidation. The complex repertoire of oxidized CE (OxCE) products exhibit various, context-dependent biological activities, surveyed in this review. Oxidized fatty acyl chains in OxCE can be hydrolyzed and re-esterified, thus seeding oxidized moieties into phospholipids (PLs), with OxPLs having different from OxCEs biological activities. Technological advances in mass spectrometry and the development of new anti-OxCE antibodies make it possible to validate the presence and quantify the levels of OxCEs in human atherosclerotic lesions and plasma. The article discusses the prospects of measuring OxCE levels in plasma as a novel biomarker assay to evaluate risk of developing cardiovascular disease and efficacy of treatment.

Transmembrane Polar Relay Drives the Allosteric Regulation for ABCG5/G8 Sterol Transporter.

The heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the biliary and transintestinal secretion of cholesterol and dietary plant sterols. Missense mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis. A new molecular framework was recently established by a crystal structure of human ABCG5/G8 and reveals a network of polar and charged amino acids in the core of the transmembrane domains, namely, a polar relay. In this study, we utilize genetic variants to dissect the mechanistic role of this transmembrane polar relay in controlling ABCG5/G8 function. We demonstrated a sterol-coupled ATPase activity of ABCG5/G8 by cholesteryl hemisuccinate (CHS), a relatively water-soluble cholesterol memetic, and characterized CHS-coupled ATPase activity of three loss-of-function missense variants, R543S, E146Q, and A540F, which are respectively within, in contact with, and distant from the polar relay. The results established an in vitro phenotype of the loss-of-function and missense mutations of ABCG5/G8, showing significantly impaired ATPase activity and loss of energy sufficient to weaken the signal transmission from the transmembrane domains. Our data provide a biochemical evidence underlying the importance of the polar relay and its network in regulating the catalytic activity of ABCG5/G8 sterol transporter.

Sequential purification and characterization of Torpedo californica nAChR-DC supplemented with CHS for high-resolution crystallization studies.

Over the past 10 years we have been developing a multi-attribute analytical platform that allows for the preparation of milligram amounts of functional, high-pure, and stable Torpedo (muscle-type) nAChR detergent complexes for crystallization purpose. In the present work, we have been able to significantly improve and optimize the purity and yield of nicotinic acetylcholine receptors in detergent complexes (nAChR-DC) without compromising stability and functionality. We implemented new methods in the process, such as analysis and rapid production of samples for future crystallization preparations. Native nAChR was extracted from the electric organ of Torpedo californica using the lipid-like detergent LysoFos Choline 16 (LFC-16), followed by three consecutive steps of chromatography purification. We evaluated the effect of cholesteryl hemisuccinate (CHS) supplementation during the affinity purification steps of nAChR-LFC-16 in terms of receptor secondary structure, stability and functionality. CHS produced significant changes in the degree of ß-secondary structure, these changes compromise the diffusion of the nAChR-LFC-16 in lipid cubic phase. The behavior was reversed by Methyl-ß-Cyclodextrin treatment. Also, CHS decreased acetylcholine evoked currents of Xenopus leavis oocyte injected with nAChR-LFC-16 in a concentration-dependent manner. Methyl-ß-Cyclodextrin treatment do not reverse functionality, however column delipidation produced a functional protein similar to nAChR-LFC-16 without CHS treatment.

Machine-learning assisted confocal imaging of intracellular sites of triglycerides and cholesteryl esters formation and storage.

All living systems are maintained by a constant flux of metabolic energy and, among the different reactions, the process of lipids storage and lipolysis is of fundamental importance. Current research has focused on the investigation of lipid droplets (LD) as a powerful biomarker for the early detection of metabolic and neurological disorders. Efforts in this field aim at increasing selectivity for LD detection by exploiting existing or newly synthesized probes. However, LD constitute only the final product of a complex series of reactions during which fatty acids are transformed into triglycerides and cholesterol is transformed in cholesteryl esters. These final products can be accumulated in intracellular organelles or deposits other than LD. A complete spatial mapping of the intracellular sites of triglycerides and cholesteryl esters formation and storage is, therefore, crucial to highlight any potential metabolic imbalance, thus predicting and counteracting its progression. Here, we present a machine learning assisted, polarity-driven segmentation which enables to localize and quantify triglycerides and cholesteryl esters biosynthesis sites in all intracellular organelles, thus allowing to monitor in real-time the overall process of the turnover of these non-polar lipids in living cells. This technique is applied to normal and differentiated PC12 cells to test how the level of activation of biosynthetic pathways changes in response to the differentiation process.

Self-assembled Angelica sinensis polysaccharide nanoparticles with an instinctive liver-targeting ability as a drug carrier for acute alcoholic liver damage protection.

The present work aimed to study the feasibility of Angelica sinensis polysaccharide (ASP) as an instinctive liver-targeting drug delivery carrier with applications in acute alcoholic liver damage (ALD). Amphipathic cholesteryl hemisuccinate-ASP (ASP-CHEMS) conjugate was synthesized by an esterification reaction and characterized by conventional methods. ASP-CHEMS self-assembled nanoparticles (ACNPs) and Curcumin-loaded ACNPs (Cur/ACNPs) were fabricated with a roughly spherical shape, and their sizes were ranged from 200 to 260 nm in aqueous solution. Compared with free Cur, Cur/ACNPs displayed enhanced solubility, good photostability, and a sustained release of Cur over 72 h. In the in vivo cellular uptake behavior study and in vivo bioimaging experiments, the ACNPs showed excellent liver-targeting capability because of the specific recognition by the asialoglycoprotein receptor (ASGPR) overexpressed on the hepatocyte membrane. The tissue distribution of Cur/ACNPs in mice further demonstrated that ACNPs could distinctly enhance the distribution of Cur into the liver. Furthermore, Cur/ACNPs protected the liver from acute ALD by attenuating oxidative stress and were superior to the protective effects of free Cur and the Cur-loaded CHEMS modified-dextran derivative. According to the results, ACNPs may serve as a promising liver-targeting drug delivery carrier for liver disease prevention.

A new 5α,8α-epidioxysterol with immunosuppressive activity from the South China Sea soft coral Sinularia sp.

A new 5α,8α-epidioxysterol, namely yalongsterol A (1), along with two known related steroids 5α,8α-epidioxy-24-methyl-cholesta-6,24(28)-dien-3ß-ol (2) and (22E,24S)-5α,8α-epidioxy-24-methyl-cholesta-6,22-dien-3ß-ol (3), were isolated from the South China Sea soft coral Sinularia sp. Their structures were established by extensive spectroscopic analyses and comparisons of the spectral data with those reported in the literature. In bioassay, compounds 1-3 showed moderate immunosuppressive activities against T and/or B lymphocyte cells with IC50 values ranging from 19.30 to 59.49 µM, and low cytotoxicity on murine splenocytes with CC50 values ranging from 40.88 to 62.29 µM.[Formula: see text].

Effects of disulfide bond and cholesterol derivatives on human calcitonin amyloid formation.

Human calcitonin (hCT) is a 32-residue peptide that aggregates to form amyloid fibrils under appropriate conditions. In this study, we investigated the effect of the intramolecular disulfide bond formed at the N-terminal region of the peptide in the aggregation kinetics of hCT. Our results indicate that the presence of the disulfide bond in hCT plays a crucial role in forming the critical nucleus needed for fibril formation, facilitating the rate of hCT amyloidogenesis. Furthermore, we reported for the first time the effects of cholesterol, cholesterol sulfate, and 3ß-[N-(dimethylaminoethane)carbamoyl]-cholesterol (DC-cholesterol) on the amyloid formation of oxidized hCT. Our results show that while cholesterol does not affect amyloidogenesis of oxidized hCT, high concentrations of cholesterol sulfate exhibits a moderate inhibiting activity on hCT amyloid formation. In particular, our results show that DC-cholesterol strongly inhibits amyloidogenesis of oxidized hCT in a dose-dependent manner. Further studies at different pH conditions imply the crucial impact of electrostatic and hydrogen bonding interactions in mediating the interplay of hCT and the surface of DC-cholesterol vesicles and the inhibiting function of DC-cholesterol on hCT fibrillization.

Different metabolism of EPA, DPA and DHA in humans: A double-blind cross-over study.

This study aimed to compare eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) incorporated into red blood cells (RBC) phospholipids (PL), plasma PL, plasma triglyceride (TAG), and plasma cholesteryl ester (CE) fractions, and the metabolomics profiles in a double-blind cross-over study. Twelve female healthy subjects randomly consumed 1 g per day for 6 days of pure EPA, DPA, or DHA. The placebo treatment was olive oil. The fasting venous blood was taken at days 0, 3 and 6, and the RBC PL and plasma lipid fractions were separated for fatty acid determination using thin layer chromatography followed by gas chromatography. Plasma metabolites were analyzed by UHPLC-Q-Exactive Orbitrap/MS. Supplemental EPA significantly increased the concentrations of EPA in RBC PL (days 3 and 6). For subjects consuming the DPA supplement, the concentrations of both DPA and EPA were significantly increased in RBC PL over a 6-day period, respectively. For plasma PL fraction, EPA and DPA supplementation significantly increased the concentrations of EPA and DPA at both days 3 and 6, respectively. Supplemental DHA significantly increased the concentrations of DHA in plasma PL at day 6. For plasma TAG fraction, supplementation with EPA and DPA significantly increased the concentrations of EPA and DPA at both days 3 and 6, respectively. After DHA supplementation, significant increases in the concentrations of DHA were found relative to baseline at both days 3 and 6. For plasma CE fraction, EPA supplementation significantly increased the concentrations of EPA (days 3 and 6) and DPA (days 6), respectively. Supplemental DPA significantly increased the concentrations of EPA at day 6. Meanwhile, the concentrations of DHA were significantly increased over a 6-day period of intervention after subjects consuming the DHA supplements. There were a total of 922 plasma metabolites identified using metabolomics analyses. Supplementation with DPA and DHA significantly increased the levels of sphingosine 1-phosphate (P for DPA = 0.025, P for DHA = 0.029) and 15-deoxy-Δ12,14-prostaglandin A1 (P for DPA = 0.034; P for DHA = 0.021) in comparison with olive oil group. Additionally, supplementation with EPA (P = 0.007) and DHA (P = 0.005) significantly reduced the levels of linoleyl carnitine, compared with olive oil group. This study shows that DPA might act as a reservoir of n-3 LCP incorporated into blood lipid fractions, metabolized into DHA, and retro-converted back to EPA. Metabolomics analyses indicate that supplemental EPA, DPA and DHA have shared and differentiated metabolites. The differences of these metabolic biomarkers should be investigated in additional studies.

Universal phase behaviors of intracellular lipid droplets.

Lipid droplets are cytoplasmic microscale organelles involved in energy homeostasis and handling of cellular lipids and proteins. The core structure is mainly composed of two kinds of neutral lipids, triglycerides and cholesteryl esters, which are coated by a phospholipid monolayer and proteins. Despite the liquid crystalline nature of cholesteryl esters, the connection between the lipid composition and physical states is poorly understood. Here, we present a universal intracellular phase diagram of lipid droplets, semiquantitatively consistent with the in vitro phase diagram, and reveal that cholesterol esters cause the liquid-liquid crystal phase transition under near-physiological conditions. We moreover combine in vivo and in vitro studies, together with the theory of confined liquid crystals, to suggest that the radial molecular alignments in the liquid crystallized lipid droplets are caused by an anchoring force at the droplet surface. Our findings on the phase transition of lipid droplets and resulting molecular organization contribute to a better understanding of their biological functions and diseases.

Cholesteryl Hemisuccinate Is Not a Good Replacement for Cholesterol in Lipid Nanodiscs.

Nanodiscs are suitable tools for studies of membrane proteins (MPs) due to their ability to mimic native biological membranes, and several MP structures are solved in nanodiscs. Among the various cell membrane components, cholesterol (CHL) is known to regulate protein function and its concentration can reach up to 50 mol %. However, studies comprising cholesterol are challenging due to its hydrophobic nature, hence, nanodiscs with only a low cholesterol concentration have been studied. To overcome the problem, cholesterol analogs with high solubility in polar solutions are often used, and one of them is cholesteryl hemisuccinate (CHS). Nevertheless, in molecular dynamics (MD) simulation, this is not an obstacle. In this study, we performed MD simulations of nanodiscs containing neutral phosphatidylcholine (POPC) lipids, negatively charged phosphatidylglycerol (POPG) lipids, CHL, or negatively charged cholesterol analog, CHS. Our simulations show that CHS increases the order of lipids in nanodiscs; the effect is, however, weaker than CHL and even smaller in nanodiscs. Furthermore, CHS gathered around scaffold proteins while cholesterol was uniformly distributed in the nanodiscs. Thus, nanodiscs with CHS are heterogeneous and not equivalent to nanodiscs with CHL. Finally, we also observed the increased concentration of POPG near the scaffold proteins, driven by electrostatic interactions. The MD results are experimentally validated using electron paramagnetic resonance spectroscopy. These results show that nanodiscs are, in fact, complex structures not easily comparable with planar lipid bilayers.

Development and characterization of an improved formulation of cholesteryl oleate-loaded cationic solid-lipid nanoparticles as an efficient non-viral gene delivery system.

Nanoparticle-mediated plasmid delivery is considered a useful tool to introduce foreign DNA into the cells for the purpose of DNA vaccination and/or gene therapy. Cationic solid-lipid nanoparticles (cSLNs) are considered one of the most promising non-viral vectors for nucleic acid delivery. Based on the idea that the optimization of the components is required to improve transfection efficiency, the present study aimed to formulate and characterize cholesteryl oleate-containing solid-lipid nanoparticles (CO-SLNs) incorporating protamine (P) to condense DNA to produce P:DNA:CO-SLN complexes as non-viral vectors for gene delivery with reduced cytotoxicity and high cellular uptake efficiency. For this purpose, CO-SLNs were used to prepare DNA complexes with and without protamine as DNA condenser and nuclear transfer enhancer. The main physicochemical characteristics, binding capabilities, cytotoxicity and cellular uptake of these novel CO-SLNs were analyzed. Positively charged spherical P:DNA:CO-SLN complexes with a particle size ranging from 330.1 ± 14.8 nm to 347.0 ± 18.5 nm were obtained. Positive results were obtained in the DNase I protection assay with a protective effect of the genetic material and 100% loading efficiency was achieved at a P:DNA:CO-SLN ratio of 2:1:7. Transfection studies in human embryonic kidney (HEK293T) cells showed the versatility of adding protamine to efficiently transfect cells, widening the potential applications of CO-SLN-based vectors, since the incorporation of protamine induced almost a 200-fold increase in the transfection capacity of CO-SLNs without toxicity. These results indicate that CO-SLNs with protamine are a safe and effective platform for non-viral nucleic acid delivery.

Steering the Lipid Transfer To Unravel the Mechanism of Cholesteryl Ester Transfer Protein Inhibition.

Human plasma cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids from antiatherogenic high-density lipoproteins (HDLs) to proatherogenic low-density lipoproteins (LDLs). Recent cryo-electron microscopy studies have suggested that CETP penetrates its N- and C-terminal domains in HDL and LDL to form a ternary complex, which facilitates the lipid transfer between different lipoproteins. Inhibition of CETP lipid transfer activity has been shown to increase the plasma HDL-C levels and, therefore, became an effective strategy for combating cardiovascular diseases. Thus, understanding the molecular mechanism of inhibition of lipid transfer through CETP is of paramount importance. Recently reported inhibitors, torcetrapib and anacetrapib, exhibited low potency in addition to severe side effects, which essentially demanded a thorough knowledge of the inhibition mechanism. Here, we employ steered molecular dynamics simulations to understand how inhibitors interfere with the neutral lipid transfer mechanism of CETP. Our study revealed that inhibitors physically occlude the tunnel posing a high energy barrier for lipid transfer. In addition, inhibitors bring about the conformational changes in CETP that hamper CE passage and expose protein residues that disrupt the optimal hydrophobicity of the CE transfer path. The atomic level details presented here could accelerate the designing of safe and efficacious CETP inhibitors.