Discoidal High-density Lipoprotein Research Articles

Abstract 114: Inhibition Of Lecithin Cholesterol Acyl Transferase By A Novel Anti-apolipoprotein A1 Antibody Fragment

Apolipoprotein A1 (APOA1), the most abundant protein in high-density lipoproteins (HDL), is the primary activator of lecithin cholesterol acyl transferase (LCAT). LCAT’s cholesteryl esterification activity is key for maturing nascent HDL discs into the spherical forms commonly found in plasma. However, the molecular mechanism for APOA1 activation of LCAT is poorly understood. Using phage display selection against nanodiscs (lipid complexes stabilized by an APOA1 variant commonly used in transmembrane protein structural biology), we have identified, isolated, and expressed a novel Fab fragment (Fab16). Size exclusion chromatography elution shift experiments showed that Fab16 readily bound reconstituted HDL (rHDL) particles containing human purified plasma APOA1 with nearly quantitative binding at a 1:1 molar ratio of Fab16 to APOA1. Negative stain electron microscopy (NSEM), confirmed that 1-2 Fab16 bound to each rHDL particle in a specific pattern around the disc periphery. Additionally, Fab16 effectively bound both large and small spherical HDL particles isolated from human plasma. Chemical cross-linking / mass spectrometry experiments indicated that Fab16 binds an APOA1 epitope between residues 94 and 118 (i.e., within amphipathic helix 4). As our previous work has shown that APOA1 helix 4 is important for LCAT activation, we hypothesized that Fab16 would inhibit LCAT activity. Using a novel gas chromatography cholesterol esterification assay, we found that Fab16 applied at a 1.5:1 molar ratio to APOA1 inhibited LCAT cholesterol esterification activity by 56±7%. The symmetrical nature of both discoidal and spherical HDL particles has hampered the application of cryogenic electron microscopy (cryo EM) for understanding HDL structure. Fab16 may provide a valuable ‘landmark’ to assist in deriving a high-resolution structure of the complex. Work to optimize conditions for collecting high quality cryo EM datasets of discoidal and spherical HDL in the presence and absence of LCAT is ongoing. Deriving a high-resolution understanding of HDL structure and its interaction with remodeling factors such LCAT has been a long-standing problem but is crucial for deriving new strategies to manipulate HDL function for therapeutic benefit.

Nasal delivery of polymeric nanoDisc mobilizes a synergy of central and peripheral amyloid-β clearance to treat Alzheimer's disease.

The disequilibrium of amyloid β-peptide (Aβ) between the central and peripheral pools has been claimed as an initiating event in Alzheimer's disease (AD). In this study, we employ discoidal high-density lipoproteins (HDL-Disc) mimicking Aβ antibody for directional flux of Aβ from central to peripheral catabolism, with desirable safety and translation potential. Structurally, HDL-Disc assembly (polyDisc) is prepared with aid of chitosan derivative polymerization. After intranasal administration and response to slightly acidic nasal microenvironment, polyDisc depolymerizes into carrier-free HDL-Disc with chitosan derivatives that adhere to the mucosal layer to reversibly open tight junctions, helping HDL-Disc penetrate the olfactory pathway into brain. Thereafter, HDL-Disc captures Aβ into microglia for central clearance or ferries Aβ out of the brain for liver-mediated compensatory catabolism. For synergy therapy, intranasal administration of polyDisc can effectively reduce intracerebral Aβ burden by 97.3% and vascular Aβ burden by 73.5%, ameliorate neurologic damage, and rescue memory deficits in APPswe/PS1dE9 transgenic AD mice with improved safety, especially vascular safety. Collectively, this design provides a proof of concept for developing Aβ antibody mimics to mobilize a synergy of central and peripheral Aβ clearance for AD treatment.

A Nanodisc-Paved Biobridge Facilitates Stem Cell Membrane Fusogenicity for Intracerebral Shuttling and Bystander Effects.

Mesenchymal stem cell (MSC) therapies experience steadfast clinical advances but are still hindered by inefficient site-specific migration. An adaptable MSC membrane fusogenicity technology is conceptualized for lipid raft-mediated targeting ligand embedding by using toolkits of discoidal high-density lipoprotein (HDL)-containing biomimicking 4F peptides. According to the pathological clues of brain diseases, the vascular cell adhesion molecule 1 specialized VBP peptide is fused with 4F to assemble 4F-VBP (HDL), which acts as a biobridge and transfers VBP onto the living cell membrane via lipid rafts for surface engineering of MSCs in suspension. When compared with the membrane-modifying strategies of PEGylated phospholipids, 4F-VBP (HDL) provides a 3.86 higher linkage efficiency to obtain MSCs4F-VBP(HDL) , which can recognize and adhere to the inflammatory endothelium for efficient blood-brain barrier crossing and brain accumulation. In APPswe/PSEN1dE9 mice with Alzheimer's disease (AD), the transcriptomic analysis reveals that systemic administration of MSCs4F-VBP(HDL) can activate pathways associated with neuronal activity and diminish neuroinflammation for rewiring AD brains. This customizable HDL-mediated membrane fusogenicity platform primes MSC inflammatory brain delivery, which can be expanded to other disease treatments by simply fusing 4F with relevant ligands for living cell engineering.

Lutein nanodisks protect human retinal pigment epithelial cells from UV light-induced damage.

The hydrophobic carotenoid, lutein, was conferred with aqueous solubility upon formulation into reconstituted discoidal high density lipoprotein particles, termed lutein nanodisks (ND). When formulated with phosphatidylcholine (PC), apolipoprotein (apo) A-I and lutein (formulation ratio = 5 mg PC/2 mg apoA-I/1 mg lutein), lutein solubilization efficiency in phosphate buffered saline (PBS) was ~90%. The UV/Vis absorbance maxima for lutein ND in PBS were red shifted by 6-13 nm versus the corresponding lutein absorbance maxima in ethanol. FPLC gel filtration chromatography gave rise to a single major absorbance peak in the size range of ND. Incubation of cultured ARPE-19 cells with lutein ND resulted in lutein uptake, as determined by HPLC analysis of cell extracts. Compared to control incubations, ARPE-19 cells incubated with lutein ND were protected from UV light-induced loss of cell viability. Consistent with this, reactive oxygen species generation, induced by exposure to UV irradiation, was lower in lutein-enriched cells than in control cells. Thus, uptake of ND-associated lutein protects ARPE-19 cells from UV light-induced damage. Taken together, the data indicate ND provide an aqueous lutein delivery vehicle for biotechnological or therapeutic applications.

HDL maturation and remodelling

Cholesterol in the circulation is mostly transported in an esterified form as a component of lipoproteins. The majority of these cholesteryl esters are produced in nascent, discoidal high density lipoproteins (HDLs) by the enzyme, lecithin:cholesterol acyltransferase (LCAT). Discoidal HDLs are discrete populations of particles that consist of a phospholipid bilayer, the hydrophobic acyl chains of which are shielded from the aqueous environment by apolipoproteins that also confer water solubility on the particles. The progressive LCAT-mediated accumulation of cholesteryl esters in discoidal HDLs generates the spherical HDLs that predominate in normal human plasma. Spherical HDLs contain a core of water insoluble, neutral lipids (cholesteryl esters and triglycerides) that is surrounded by a surface monolayer of phospholipids with which apolipoproteins associate. Although spherical HDLs all have the same basic structure, they are extremely diverse in size, composition, and function. This review is concerned with how the biogenesis of discoidal and spherical HDLs is regulated and the mechanistic basis of their size and compositional heterogeneity. Current understanding of the impact of this heterogeneity on the therapeutic potential of HDLs of varying size and composition is also addressed in the context of several disease states.

Foam fractionation of a recombinant biosurfactant apolipoprotein

Locusta migratoria apolipophorin III (apoLp-III) possesses the ability to exist as a water soluble amphipathic α-helix bundle and a lipid surface seeking apolipoprotein. The intrinsic ability of apoLp-III to transform phospholipid vesicles into reconstituted discoidal high-density lipoproteins (rHDL) has led to myriad applications. To improve the yield of recombinant apoLp-III, studies were performed in a bioreactor. Induction of apoLp-III expression generated a protein product that is secreted from E. coli into the culture medium. Interaction of apoLp-III with gas and liquid components in media produced large quantities of thick foam. A continuous foam fractionation process yielded a foamate containing apoLp-III as the sole major protein component. The yield of recombinant apoLp-III was ~0.2 g / liter bacterial culture. Mass spectrometry analysis verified the identity of the target protein and indicated no modifications or changes to apoLp-III occurred as a result of foam fractionation. The functional ability of apoLp-III to induce rHDL formation was evaluated by incubating foam fractionated apoLp-III with phosphatidylcholine vesicles. FPLC size exclusion chromatography revealed a single major population of particles in the size range of rHDL. The results described offer a novel approach to bioreactor-based apoLp-III production that takes advantage of its intrinsic biosurfactant properties.

Current models of apolipoprotein A-I lipidation by adenosine triphosphate binding cassette transporter A1.

The primary cardioprotective function of high-density lipoprotein (HDL) is to remove excess cellular free cholesterol (FC) from peripheral tissues and deliver it to the liver. Here, we summarize recent research that examines apolipoprotein A-I (apoA-I) lipidation models by adenosine triphosphate binding cassette transporter A1 (ABCA1) and discuss its relevance in atherosclerotic cardiovascular disease (ASCVD). The first step in HDL formation involves the interaction between apoA-I and ABCA1, where ABCA1 mediates the removal of FC and phospholipids from lipid-laden macrophages to form discoidal nascent HDL (nHDL). However, there are currently no clear-cut systematic models that characterize HDL formation. A number of recent studies have investigated the importance of apoA-I C- and N-terminal domains required for optimal cholesterol efflux and nHDL production. Furthermore, functional ABCA1 is required for direct or indirect binding to apoA-I where ABCA1 dimer-monomer interconversion facilitates apoA-I lipidation from plasma membrane microdomains. Microparticles are also another lipid source for apoA-I solubilization into nHDL. ApoA-I and ABCA1 are key factors in macrophage-mediated cholesterol efflux and nHDL production. Understanding of the key steps in HDL formation may unlock the therapeutic potential of HDL and improve clinical management of ASCVD.

Natural discoidal lipoproteins with tiny modification for tumor extracellular dissociation in antitumor chemoimmunotherapy

Appealing cancer immunotherapy requires synchronous presentation of tumor antigens and immunoadjuvant. Herein, a “one-step” modification strategy is proposed to tinily remould endogenous discoidal high density lipoprotein (dHDL) for tumor-homing and site-specific chemoimmunotherapy. For molecular targeting therapy, lipophilic immunoadjuvant CpG oligodeoxynucleotides is conjugated to facilitate HDL-surface anchoring; and GC nucleotides provide enough reservoir for completion of doxorubicin (Dox) “sandwich”. After administration, the tiny size (~30 nm) of disc nanodrug can maneuver deeply into tumors for receptor binding and in situ structural collapse. The intracellular concentrated CpG-Dox induce potent immunogenic cell death from burst Dox liberation at acidic pH. In turn, the released antigens and CpG motifs are simultaneously recognized by dendritic cells for antigen presentation and antitumor T cell responses. Combination chemoimmunotherapy with discoidal nanodrugs performed highest tumor weight inhibitory of 93.2% and extend the median survival time at a safe level. Collectively, this study suggests that the minimalist revolution of natural dHDL particulates may provide a biomimicry nanoplatform for site-specific amplified chemoimmunotherapy.

Novel Cocrystallization of Apolipoprotein A‐I with Butyric Acid

Diseases of lipid metabolism, in particular cardiovascular atherosclerosis, remain the number one cause of morbidity and mortality in Western society. The well-documented anti-atherogenic role of high-density lipoprotein (HDL) is related to its involvement in reverse cholesterol transport. When the transporter ABCA1 initiates cholesterol efflux from peripheral and hepatic cells, apolipoprotein A-I (apoA-I) binds the free cholesterol together with phospholipid to form discoidal nascent HDL (“good cholesterol”). The enzyme lecithin:cholesterol acyltransferase (LCAT) catalyzes the esterification of the cholesterol carried by HDL. ApoA-I activates LCAT for reverse cholesterol transport, wherein LCAT esterifies the cholesterol within the nascent HDL2 and facilitates maturation of HDL to a mature spherical form. After esterification, cholesterol can no longer diffuse back to the peripheral and hepatic cells. The esterified cholesterol, or cholesteryl ester, molecules form the hydrophobic core of the mature HDL particle and leave the circulation upon uptake by a hepatic scavenger receptor for excretion by the liver. Research in the Atkinson laboratory has implicated the arginine 123 residue of apoA-I in the function of LCAT. Based on the X-ray crystal structure, the Arg123 residues of each apoA-I monomer enclose a hydrophobic tunnel into which the lipid substrates of LCAT may be sequestered. ApoA-I has a closed conformation without lipid bound that transforms to a “double belt” encircling the nascent HDL disc. When apoA-I activates LCAT, the tunnel should widen in order to present the substrates to LCAT for esterification. However, the mechanism by which apoA-I interfaces with LCAT substrates remains unknown. Cocrystallization of apoA-I with the lipid component, butyric acid, will enable us to assess how the “double belt” of apoA-I opens in three-dimensional space.

Paraoxonase-1 and -3 Protein Expression in the Brain of the Tg2576 Mouse Model of Alzheimer's Disease.

Background: Brain oxidative lipid damage and inflammation are common in neurodegenerative diseases such as Alzheimer’s disease (AD). Paraoxonase-1 and -3 (PON1 and PON3) protein expression was demonstrated in tissue with no PON1 or PON3 gene expression. In the present study, we examine differences in PON1 and PON3 protein expression in the brain of a mouse model of AD. Methods: we used peroxidase- and fluorescence-based immunohistochemistry in five brain regions (olfactory bulb, forebrain, posterior midbrain, hindbrain and cerebellum) of transgenic (Tg2576) mice with the Swedish mutation (KM670/671NL) responsible for a familial form of AD and corresponding wild-type mice. Results: We found intense PON1 and PON3-positive staining in star-shaped cells surrounding Aβ plaques in all the studied Tg2576 mouse-brain regions. Although we could not colocalize PON1 and PON3 with astrocytes (star-shaped cells in the brain), we found some PON3 colocalization with microglia. Conclusions: These results suggest that (1) PON1 and PON3 cross the blood–brain barrier in discoidal high-density lipoproteins (HDLs) and are transferred to specific brain-cell types; and (2) PON1 and PON3 play an important role in preventing oxidative stress and lipid peroxidation in particular brain-cell types (likely to be glial cells) in AD pathology and potentially in other neurodegenerative diseases as well.

Urea-Assisted Reconstitution of Discoidal High-Density Lipoprotein.

High-density lipoprotein (HDL) is a naturally occurring composite of lipids and lipid-binding proteins. The cholate dialysis method, first reported by Jonas in 1969, is the most widely used approach for reconstituting discoidal HDL (dHDL) in test tubes with phospholipids and the most dominant protein, apolipoprotein A-1 (apoA-I). Here, we show that a dHDL-relevant complex can also be prepared by gently mixing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and apoA-I or its mutants in ethanol/H2O solutions containing urea at a concentration of a few molar and then incubating the mixture at the gel-liquid crystalline phase transition temperature in test tubes. Subsequent purification steps involve quick dialysis following size exclusion chromatography. The yields (73 ± 3% and 70 ± 1% protein and DMPC, respectively) of the resulting HDL-like nanoparticles, designated as uHDL, were comparable to the values of 68 ± 9% and 71 ± 12% obtained in the cholate dialysis method. Using apoA-I and two mutants, the key factor in this method was found to be urea at the folded and unfolded transition midpoint concentration. By using this urea-assisted method in the presence of a hydrophobic drug, all-trans-retinoic acid (ATRA), one-step preparation of ATRA-loaded uHDL was also possible. The loading efficiency was comparable to that in the mixing of ATRA and uHDL or dHDL reconstituted by the cholate dialysis method. Atomic force microscopy analysis revealed that uHDL and ATRA-loaded uHDL were discoidal. Our urea-assisted method is an easy and efficient method for reconstituting dHDL and can be utilized to prepare various drug-dHDL complexes.

Structural analysis of lecithin:cholesterol acyltransferase bound to high density lipoprotein particles

Lecithin:cholesterol acyltransferase (LCAT) catalyzes a critical step of reverse cholesterol transport by esterifying cholesterol in high density lipoprotein (HDL) particles. LCAT is activated by apolipoprotein A-I (ApoA-I), which forms a double belt around HDL, however the manner in which LCAT engages its lipidic substrates and ApoA-I in HDL is poorly understood. Here, we used negative stain electron microscopy, crosslinking, and hydrogen-deuterium exchange studies to refine the molecular details of the LCAT–HDL complex. Our data are consistent with LCAT preferentially binding to the edge of discoidal HDL near the boundary between helix 5 and 6 of ApoA-I in a manner that creates a path from the lipid bilayer to the active site of LCAT. Our results provide not only an explanation why LCAT activity diminishes as HDL particles mature, but also direct support for the anti-parallel double belt model of HDL, with LCAT binding preferentially to the helix 4/6 region.

Protein Backbone and Average Particle Dynamics in Reconstituted Discoidal and Spherical HDL Probed by Hydrogen Deuterium Exchange and Elastic Incoherent Neutron Scattering

Lipoproteins are supramolecular assemblies of proteins and lipids with dynamic characteristics critically linked to their biological functions as plasma lipid transporters and lipid exchangers. Among them, spherical high-density lipoproteins are the most abundant forms of high-density lipoprotein (HDL) in human plasma, active participants in reverse cholesterol transport, and associated with reduced development of atherosclerosis. Here, we employed elastic incoherent neutron scattering (EINS) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to determine the average particle dynamics and protein backbone local mobility of physiologically competent discoidal and spherical HDL particles reconstituted with human apolipoprotein A-I (apoA-I). Our EINS measurements indicated that discoidal HDL was more dynamic than spherical HDL at ambient temperatures, in agreement with their lipid-protein composition. Combining small-angle neutron scattering (SANS) with contrast variation and MS cross-linking, we showed earlier that the most likely organization of the three apolipoprotein A-I (apoA-I) chains in spherical HDL is a combination of a hairpin monomer and a helical antiparallel dimer. Here, we corroborated those findings with kinetic studies, employing hydrogen-deuterium exchange mass spectrometry (HDX-MS). Many overlapping apoA-I digested peptides exhibited bimodal HDX kinetics behavior, suggesting that apoA-I regions with the same amino acid composition located on different apoA-I chains had different conformations and/or interaction environments.

The Extent of Human Apolipoprotein A-I Lipidation Strongly Affects the β-Amyloid Efflux Across the Blood-Brain Barrier in vitro.

Much evidence suggests a protective role of high-density lipoprotein (HDL) and its major apolipoprotein apoA-I, in Alzheimer’s disease (AD). The biogenesis of nascent HDL derived from a first lipidation of apoA-I, which is synthesized by the liver and intestine but not in the brain, in a process mediated by ABCA1. The maturation of nascent HDL in mature spherical HDL is due to a subsequent lipidation step, LCAT-mediated cholesterol esterification, and the change of apoA-I conformation. Therefore, different subclasses of apoA-I-HDL simultaneously exist in the blood circulation. Here, we investigated if and how the lipidation state affects the ability of apoA-I-HDL to target and modulate the cerebral β-amyloid (Aβ) content from the periphery, that is thus far unclear. In particular, different subclasses of HDL, each with different apoA-I lipidation state, were purified from human plasma and their ability to cross the blood-brain barrier (BBB), to interact with Aβ aggregates, and to affect Aβ efflux across the BBB was assessed in vitro using a transwell system. The results showed that discoidal HDL displayed a superior capability to promote Aβ efflux in vitro (9 × 10-5 cm/min), when compared to apoA-I in other lipidation states. In particular, no effect on Aβ efflux was detected when apoA-I was in mature spherical HDL, suggesting that apoA-I conformation, and lipidation could play a role in Aβ clearance from the brain. Finally, when apoA-I folded its structure in discoidal HDL, rather than in spherical ones, it was able to cross the BBB in vitro and strongly destabilize the conformation of Aβ fibrils by decreasing the order of the fibril structure (-24%) and the β-sheet content (-14%). These data suggest that the extent of apoA-I lipidation, and consequently its conformation, may represent crucial features that could exert their protective role in AD pathogenesis.

High-density lipoproteins induce miR-223–3p biogenesis and export from myeloid cells: Role of scavenger receptor BI-mediated lipid transfer

Background and aimsWe recently showed that miR-223–3p on high-density lipoproteins (HDL) is exported to endothelial cells, where it inhibits inflammation. However, the origin of miR-223–3p on HDL is unknown. We hypothesize that HDL-associated miR-223–3p originates in myeloid cells and is exported to HDL in a scavenger receptor BI (SR-BI)-dependent manner. MethodsPolymorphonuclear neutrophils (PMNs) and human monocyte derived macrophages (HMDMs) were incubated with native HDL (nHDL) or discoidal reconstituted HDL (rHDL). Total RNA was isolated before and after incubation. Mature and primary miR-223–3p (pri-mir-223–3p) levels were quantified by real-time PCR. ResultsIncubation with nHDL and rHDL increased miR-223–3p export from PMNs and HMDMs. In PMNs, nHDL but not rHDL, increased mature and pri-mir-223–3p. Incubation with HDL also increased Dicer mRNA, a critical regulator of miRNA biogenesis. Incubation of HMDMs with nHDL did not increase cellular levels of mature miR-223–3p, but significantly increased pri-mir-223 levels. Incubation with rHDL had no effect on either mature or pri-mir-223–3p levels.Activated PMNs increased miR-223–3p export to HDL and the production of reactive oxygen species and activated protein kinase C. Blocking HDL binding to SR-BI increased miR-223–3p export to HDL in both PMNs and HMDMs, but did not affect mature and primary miR-223–3p levels. Chemical inhibition of cholesterol flux by Block Lipid Transport (BLT)-1 inhibited HDL-induced pri-mir-223 expression in PMNs. ConclusionsHDL-associated miR-223–3p originates in PMNs and macrophages. HDL stimulates miR-223–3p biogenesis in PMNs in a process that is regulated by SR-BI-mediated lipid flux.

Apolipoprotein A1 Forms 5/5 and 5/4 Antiparallel Dimers in Human High-density Lipoprotein

Apolipoprotein A1 (APOA1), the major protein of high-density lipoprotein (HDL), contains 10 helical repeats that play key roles in protein-protein and protein-lipid interactions. The current structural model for HDL proposes that APOA1 forms an antiparallel dimer in which helix 5 in monomer 1 associates with helix 5 in monomer 2 along a left-left (LL5/5) interface, forming a protein complex with a 2-fold axis of symmetry centered on helix 5. However, computational studies suggest that other orientations are possible. To test this idea, we used a zero-length chemical cross-linking reagent that forms covalent bonds between closely apposed basic and acidic residues. Using proteolytic digestion and tandem mass spectrometry, we identified amino acids in the central region of the antiparallel APOA1 dimer of HDL that were in close contact. As predicted by the current model, we found six intermolecular cross-links that were consistent with the antiparallel LL5/5 registry. However, we also identified three intermolecular cross-links that were consistent with the antiparallel LL5/4 registry. The LL5/5 is the major structural conformation of the two complexes in both reconstituted discoidal HDL particles and in spherical HDL from human plasma. Molecular dynamic simulations suggest that that LL5/5 and LL5/4 APOA1 dimers possess similar free energies of dimerization, with LL5/5 having the lowest free energy. Our observations indicate that phospholipidated APOA1 in HDL forms different antiparallel dimers that could play distinct roles in enzyme regulation, assembly of specific protein complexes, and the functional properties of HDL in humans.

Dynamic Analysis of Discoidal Nascent HDL Structure by IPET

Although circulating high density lipoproteins (HDL) are considered protective from cardiovascular disease, we have a remarkably limited understanding of their structure. In the recent decades, several models of HDL are built and debated each other, but the exact structure of HDL can't be confirmed even now. In our previous work, we used cross‐linking chemistry and mass spectrometry to generate detailed models of apoA‐I in reconstituted particles as well as “real” HDL from human plasma. Despite substantial differences in size and shape, these structures all shared the theme of an antiparallel belt‐like arrangement. Thus, direct visible observation of the conformation of discoidal HDL with the help of electron microscopy is a possible way to solve the debate. Here we report a study of structure of HDL by using negative‐staining electron microscopy and cryo‐electron microscopy as well as electron tomography. The 3D structures of dynamical individual HDL molecules are reconstructed by the individual‐particle electron tomography reconstruction (IPET). The reconstructed density maps at ~ 2 nm resolution display the size heterogeneity and conformation variety of HDL particles, and reveal a fusion/release mechanism of discoidal HDL in lipid transfer. These results revealed that apoA‐I makes highly specific contacts with itself to form a molecular scaffold that stabilizes HDL and facilitates, through specific protein: protein interactions, the association of HDL partner proteins to define particle function. The notion is crucial for us to further understand how the major HDL protein, apolipoprotein (apo)A‐I are involved in cholesterol transfer and lipid metabolism and cardiovascular disease.Support or Funding InformationThis work was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health (2R01 HL115153‐06, R01GM104427, and P01HL030086). Work conducted at the Molecular Foundry was supported by the Office of Basic Energy Sciences of the Office of Science at the U.S. Department of Energy under Contract No. DE‐AC02‐05CH11231.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reconstituted Discoidal High-Density Lipoproteins: Bioinspired Nanodiscs with Many Unexpected Applications.

Summarize the initial discovery of discoidal high-density lipoprotein (HDL) in human plasma and review more recent innovations that span the use of reconstituted nanodisc HDL for membrane protein characterization to its use as a drug carrier and a novel therapeutic agent for cardiovascular disease. Using a wide variety of biophysical techniques, the structure and composition of endogenous discoidal HDL have now largely been solved. This has led to the development of new methods for the in vitro reconstitution of nanodisc HDL, which have proven to have a wide variety of biomedical applications. Nanodisc HDL has been used as a platform for mimicking the plasma membrane for the reconstitution and investigation of the structures of several plasma membrane proteins, such as cytochrome P450s and ABC transporters. Nanodisc HDL has also been designed as drug carriers to transport amphipathic, as well as hydrophobic small molecules, and has potential therapeutic applications for several diseases. Finally, nanodisc HDL itself like native discoidal HDL can mediate cholesterol efflux from cells and are currently being tested in late-stage clinical trials for cardiovascular disease. The discovery of the characterization of native discoidal HDL has inspired a new field of synthetic nanodisc HDL, which has offered a growing number of unanticipated biomedical applications.

Abstract 026: Apolipoprotein A-I Helical Registry Modulates Lecithin:cholesterol Acyl Transferase Activity

The structure of apolipoprotein (APOA1), the major protein of high density lipoproteins (HDL), can modulate protein interactions affecting the cardioprotective functions of HDL particles. APOA1 on nascent discoidal HDL is composed of ten, tandem, α-helical repeats arranged in an anti-parallel, stacked ring-structure that encapsulates a lipid bilayer. Previous chemical cross-linking studies suggested that these APOA1 rings may adopt two different orientations, or registries, with respect to each other. These orientations include one where the bulk of APOA1 orients with the 5 th helix of one molecule opposing the 5 th helix of the other (5/5 helical registry), and another orientation adopting a 5/2 helical registry. Engineered HDL particles locked in both 5/5 and 5/2 registries, or rotamers, equally promoted cholesterol efflux from macrophages, indicating functional particles. However, the ability of lecithin:cholesterol acyl transferase (LCAT) to esterify cholesterol was increased over WT in the 5/5 rotamer, and dramatically impaired in the 5/2 rotamer ( p<0.001 ), despite surface plasmon resonance studies showing that LCAT bound equally to both. Cross-linking experiments indicated that LCAT activity requires a hybrid epitope composed of helices 5-7 on one APOA1 molecule and 3-4 on the other. Thus, the reciprocating nature of APOA1’s structure may represent a thumbwheel mechanism that influences the activity of HDL remodeling factors in plasma.

Macrophages Shed Excess Cholesterol in Unique Extracellular Structures Containing Cholesterol Microdomains.

Cells use various mechanisms to maintain cellular cholesterol homeostasis including efflux of cholesterol from the cellular plasma membrane to cholesterol acceptors such as HDLs (high-density lipoproteins). Little is known about the transfer of cholesterol from cells into the extracellular matrix. Using a unique monoclonal antibody that detects ordered cholesterol arrays (ie, cholesterol micro[or nano]-domains), we previously identified that particles containing these cholesterol domains accumulate in the extracellular matrix during cholesterol enrichment of human monocyte-derived macrophages and are found in atherosclerotic lesions. In this study, we further investigate these deposited particles containing cholesterol microdomains and discover their unexpected morphology. Although appearing spherical at the resolution of the conventional fluorescence microscope, super-resolution immunofluorescence and atomic force microscopy of in situ cholesterol microdomains, and immunoelectron microscopy of isolated cholesterol microdomains revealed that the microdomains are not vesicles or 3-dimensional crystals but rather appear as branching irregularly shaped deposits of varying size. These cholesterol microdomain-containing deposits are shed from the plasma membrane into the extracellular matrix. To date, research on cellular excretion of excess cholesterol has demonstrated cellular cholesterol efflux in the form of membranous vesicles and discoidal HDL particles released into the fluid-phase medium. Shedding of plasma membrane cholesterol microdomains provides an additional mechanism for cells such as macrophages to maintain plasma membrane cholesterol homeostasis. Furthermore, recognition that macrophages shed cholesterol microdomains into the extracellular matrix is important to our understanding of extracellular buildup of cholesterol in atherosclerosis.