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Cholesteryl ester transfer protein (CETP) shuttles various lipids between lipoproteins, resulting in the net transfer of cholesteryl esters from atheroprotective, high-density lipoproteins (HDL) to atherogenic, lower-density species. Inhibition of CETP raises HDL cholesterol and may potentially be used to treat cardiovascular disease. Here we describe the structure of CETP at 2.2-Å resolution, revealing a 60-Å-long tunnel filled with two hydrophobic cholesteryl esters and plugged by an amphiphilic phosphatidylcholine at each end. The two tunnel openings are large enough to allow lipid access, which is aided by a flexible helix and possibly also by a mobile flap. The curvature of the concave surface of CETP matches the radius of curvature of HDL particles, and potential conformational changes may occur to accommodate larger lipoprotein particles. Point mutations blocking the middle of the tunnel abolish lipid-transfer activities, suggesting that neutral lipids pass through this continuous tunnel.

CETP transfers cholesteryl esters (CEs) and triglycerides (TGs) between different lipoproteins and came in limelight as a drug-target against CVD. In the search for detailed mechanism of lipid transfer through CETP, enormous effort is devoted employing crystallographic, cryo-EM, and Molecular Dynamics (MD) studies. However, these studies primarily focused on CE-bound CETP structure and CE transfer mechanism. With the reported correlation that CETP looses significant CE transfer activity upon inhibiting TG transfer, it is of tremendous importance to understand the structure and dynamics of TG-bound CETP. Our results from large-scale all-atom and coarse-grained MD simulations show that CETP can accommodate two TG molecules in parallel N-N orientation with TG oleate chains majorly attaining the tuning-fork conformation. In TG-bound form, CETP not only maintained its secondary structures but also exhibited similar bending-twisting motions as reported for CE-CETP crystal structure. Obtained structural information are further validated by correlating to available functional data of 2–8 fold slower transfer rate of TG through CETP, where we show that TGs make 20% additional contacts with CETP compared to CEs. Identified CETP residues facilitating TG binding also match very well with reported mutagenesis data. The study could accelerate the drug-designing processes to combat CETP functionality and CVD.

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. Analyzing the lipidomes of single cells remains a challenge. Here, the authors present a strategy to identify class, fatty acyl-chain, C=C locations and sn -positions of lipids in single cells, and use their method to identify individual gefitinib-resistant cells in a wild-type lung cancer cell population.

Export of LDL-derived cholesterol from lysosomes requires the cooperation of the integral membrane protein Niemann–Pick C1 (NPC1) and a soluble protein, Niemann–Pick C2 (NPC2). Mutations in the genes encoding these proteins lead to Niemann–Pick disease type C (NPC). NPC2 binds to NPC1’s second (middle), lumenally oriented domain (MLD) and transfers cholesterol to NPC1’s N-terminal domain (NTD). Here, we report the 2.4-Å resolution crystal structure of a complex of human NPC1–MLD and NPC2 bearing bound cholesterol-3-O-sulfate. NPC1–MLD uses two protruding loops to bind NPC2, analogous to its interaction with the primed Ebola virus glycoprotein. Docking of the NPC1–NPC2 complex onto the full-length NPC1 structure reveals a direct cholesterol transfer tunnel between NPC2 and NTD cholesterol binding pockets, supporting the “hydrophobic hand-off” cholesterol transfer model.

The steroid cholesterol is an essential component of eukaryotic membranes, and it functionally modulates membrane proteins, including G protein-coupled receptors. To reveal insight into how cholesterol modulates G protein-coupled receptors, we have used dynamic single-molecule force spectroscopy to quantify the mechanical strength and flexibility, conformational variability, and kinetic and energetic stability of structural segments stabilizing the human β 2 -adrenergic receptor (β 2 AR) in the absence and presence of the cholesterol analog cholesteryl hemisuccinate (CHS). CHS considerably increased the kinetic, energetic, and mechanical stability of almost every structural segment at sufficient magnitude to alter the structure and functional relationship of β 2 AR. One exception was the structural core segment of β 2 AR, which establishes multiple ligand binding sites, and its properties were not significantly influenced by CHS.

Apolipoprotein A1 (ApoA1) is a main protein moiety in high-density lipoprotein (HDL) particles. Generally, ApoA1 and HDL are considered as atheroprotective. In prooxidant and inflammatory microenvironment in the vicinity to the atherosclerotic lesion, ApoA1/HDL are subjected to modification. The chemical modifications such as oxidation, nitration, etc result in altering native architecture of ApoA1 toward dysfunctionality and abnormality. Neutrophil myeloperoxidase has a prominent role in this mechanism. Neo-epitopes could be formed and then exposed that makes them immunogenic. Indeed, these epitopes may be recognized by immune cells and induce production of proatherogenic ApoA1-specific IgG antibodies. These antibodies are biologically relevant because they are able to react with Toll-like receptor (TLR)-2 and TLR4 in target cells and induce a variety of pro-inflammatory responses. Epidemiological and functional studies underline a prognostic value of ApoA1 self-antibodies for several cardiovascular diseases, including myocardial infarction, acute coronary syndrome, and severe carotid stenosis.

PurposeTo fabricate an acid-cleavable PEG polymer for the development of PEG-cleavable pH-sensitive liposomes (CL-pPSL), and to investigate their ability for endosomal escape and long circulation.MethodsPEG-benzaldehyde-hydrazone-cholesteryl hemisuccinate (PEGB-Hz-CHEMS) containing hydrazone and ester bonds was synthesised and used to fabricate a dual pH-sensitive CL-pPSL. Non-cleavable PEGylated pH-sensitive liposome (pPSL) was used as a reference and gemcitabine as a model drug. The cell uptake and endosomal escape were investigated in pancreatic cancer Mia PaCa-2 cells and pharmacokinetics were studied in rats.ResultsThe CL-pPSL showed accelerated drug release at endosomal pH 5.0 compared to pPSL. Compared to pPSL, CL-pPSL released their fluorescent payload to cytosol more efficiently and showed a 1.4-fold increase in intracellular gemcitabine concentration and higher cytotoxicity. In rats, injection of gemcitabine loaded CL-pPSL resulted in a slightly smaller Vd (149 ± 27 ml/kg; 170 ± 30 ml/kg) and shorter terminal T1/2 (5.4 ± 0.3 h; 5.8 ± 0.6 h) (both p > 0.05) but a significantly lower AUC (p < 0.01), than pPSL, due to the lower PEGylation degree (1.7 mol%) which means a ‘mushroom’ configuration of PEG. A five-time increase in the dose with CL-pPSL resulted in a 11-fold increase in AUC and a longer T1/2 (8.2 ± 0.5 h).ConclusionThe PEG-detachment from the CL-pPSL enhanced endosome escape efficiency compared with pPSL, without significantly compromising their stealth abilities.

The lipophilization of polyphenols (phenolipids) may increase their affinity for membranes, leading to better antioxidant protection. Cholesteryl esters of caffeic, dihydrocaffeic, homoprotocatechuic and protocatechuic acids were synthetized in a one-step procedure with good to excellent yields of ~50–95%. After evaluation of their radical scavenging capacity by the DPPH method and establishing the anodic peak potential by cyclic voltammetry, their antioxidant capacity against AAPH-induced oxidative stress in soybean PC liposomes was determined. Their interaction with the liposomal membrane was studied with the aid of three fluorescence probes located at different depths in the membrane. The cholesteryl esters showed a better or similar radical scavenging capacity to that of α-tocopherol and a lower anodic peak potential than the corresponding parental phenolic acids. Cholesteryl esters were able to protect liposomes to a similar or greater extent than α-tocopherol. However, despite their antiradical capacity and being able to penetrate and orientate in the membrane in a parallel position to phospholipids, the antioxidant efficiency of cholesteryl esters was deeply dependent on the phenolipid polyphenolic moiety structure. When incorporated during liposome preparation, cholesteryl protocatechuate and caffeate showed more than double the activity of α-tocopherol. Thus, cholesteryl phenolipids may protect biomembranes against oxidative stress to a greater extent than α-tocopherol.

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.

Monounsaturated fatty acids (MUFA) are emerging health biomarkers, and in particular the ratio between palmitoleic acid (9cis-16:1) and palmitic acid (16:0) affords the delta-9 desaturase index that is increased in obesity. Recently, other positional and geometrical MUFA isomers belonging to the hexadecenoic family (C16 MUFA) were found in circulating lipids, such as sapienic acid (6cis-16:1), palmitelaidic acid (9trans-16:1) and 6trans-16:1. In this work we report: i) the identification of sapienic acid as component of human erythrocyte membrane phospholipids with significant increase in morbidly obese patients (n = 50) compared with age-matched lean controls (n = 50); and ii) the first comparison of erythrocyte membrane phospholipids (PL) and plasma cholesteryl esters (CE) in morbidly obese patients highlighting that some of their fatty acid levels have opposite trends: increases of both palmitic and sapienic acids with the decrease of linoleic acid (9cis,12cis-18:2, omega-6) in red blood cell (RBC) membrane PL were reversed in plasma CE, whereas the increase of palmitoleic acid was similar in both lipid species. Consequentially, desaturase enzymatic indexes gave different results, depending on the lipid class used for the fatty acid content. The fatty acid profile of morbidly obese subjects also showed significant increases of stearic acid (C18:0) and C20 omega-6, as well as decreases of oleic acid (9cis-18:1) and docosahexaenoic acid (C22:6 omega-3) as compared with lean healthy controls. Trans monounsaturated and polyunsaturated fatty acids were also measured and found significantly increased in both lipid classes of morbidly obese subjects. These results highlight the C16 MUFA isomers as emerging metabolic marker provided that the assignment of the double bond position and geometry is correctly performed, thus identifying the corresponding lipidomic pathway. Since RBC membrane PL and plasma CE have different fatty acid trends, caution must also be used in the choice of lipid species for the interpretation of lipidomic profiles.