Helical structure, stability, and dynamics in human apolipoprotein E3 and E4 by hydrogen exchange and mass spectrometry.

Apolipoprotein E (apoE) plays a critical role in cholesterol transport in both peripheral circulation and brain. Human apoE is a polymorphic 299-residue protein in which the less common E4 isoform differs from the major E3 isoform only by a C112R substitution. ApoE4 interacts with lipoprotein particles and with the amyloid-ß peptide, and it is associated with increased incidence of cardiovascular and Alzheimer's disease. To understand the structural basis for the differences between apoE3 and E4 functionality, we used hydrogen-deuterium exchange coupled with a fragment separation method and mass spectrometric analysis to compare their secondary structures at near amino acid resolution. We determined the positions, dynamics, and stabilities of the helical segments in these two proteins, in their normal tetrameric state and in mutation-induced monomeric mutants. Consistent with prior X-ray crystallography and NMR results, the N-terminal domain contains four α-helices, 20 to 30 amino acids long. The C-terminal domain is relatively unstructured in the monomeric state but forms an α-helix ∼70 residues long in the self-associated tetrameric state. Helix stabilities are relatively low, 4 kcal/mol to 5 kcal/mol, consistent with flexibility and facile reversible unfolding. Secondary structure in the tetrameric apoE3 and E4 isoforms is similar except that some helical segments in apoE4 spanning residues 12 to 20 and 204 to 210 are unfolded. These conformational differences result from the C112R substitution in the N-terminal helix bundle and likely relate to a reduced ability of apoE4 to form tetramers, thereby increasing the concentration of functional apoE4 monomers, which gives rise to its higher lipid binding compared with apoE3.

Is ABCA1 a lipid transfer protein?

ABCA1 functions as a lipid transporter because it mediates the transfer of cellular phospholipid (PL) and free (unesterified) cholesterol (FC) to apoA-I and related proteins present in the extracellular medium. ABCA1 is a membrane PL translocase and its enzymatic activity leads to transfer of PL molecules from the cytoplasmic leaflet to the exofacial leaflet of a cell plasma membrane (PM). The presence of active ABCA1 in the PM promotes binding of apoA-I to the cell surface. About 10% of this bound apoA-I interacts directly with ABCA1 and stabilizes the transporter. Most of the pool of cell surface-associated apoA-I is bound to lipid domains in the PM that are created by the activity of ABCA1. The amphipathic α-helices in apoA-I confer detergent-like properties on the protein enabling it to solubilize PL and FC in these membrane domains to create a heterogeneous population of discoidal nascent HDL particles. This review focuses on current understanding of the structure-function relationships of human ABCA1 and the molecular mechanisms underlying HDL particle production.

Directional ABCA1-mediated cholesterol efflux and apoB-lipoprotein secretion in the retinal pigment epithelium.

Cholesterol-containing soft drusen and subretinal drusenoid deposits (SDDs) occur at the basolateral and apical side of the retinal pigment epithelium (RPE), respectively, in the chorioretina and are independent risk factors for late age-related macular degeneration (AMD). Cholesterol in these deposits could originate from the RPE as nascent HDL or apoB-lipoprotein. We characterized cholesterol efflux and apoB-lipoprotein secretion in RPE cells. Human RPE cells, ARPE-19, formed nascent HDL that was similar in physicochemical properties to nascent HDL formed by other cell types. In highly polarized primary human fetal RPE (phfRPE) monolayers grown in low-lipid conditions, cholesterol efflux to HDL was moderately directional to the apical side and much stronger than ABCA1-mediated efflux to apoA-I at both sides; ABCA1-mediated efflux was weak and equivalent between the two sides. Feeding phfRPE monolayers with oxidized or acetylated LDL increased intracellular levels of free and esterified cholesterol and substantially raised ABCA1-mediated cholesterol efflux at the apical side. phfRPE monolayers secreted apoB-lipoprotein preferentially to the apical side in low-lipid and oxidized LDL-feeding conditions. These findings together with evidence from human genetics and AMD pathology suggest that RPE-generated HDL may contribute lipid to SDDs.

CRISPR/Cas9-Mediated Gene Editing in Human iPSC-Derived Macrophage Reveals Lysosomal Acid Lipase Function in Human Macrophages-Brief Report.

OBJECTIVE: To gain mechanistic insights into the role of LIPA (lipase A), the gene encoding LAL (lysosomal acid lipase) protein, in human macrophages. APPROACH AND RESULTS: We used CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) technology to knock out LIPA in human induced pluripotent stem cells and then differentiate to macrophage (human-induced pluripotent stem cells-derived macrophage [IPSDM]) to explore the human macrophage LIPA loss-of-function phenotypes. LIPA was abundantly expressed in monocyte-derived macrophages and was markedly induced on IPSDM differentiation to comparable levels as in human monocyte-derived macrophage. IPSDM with knockout of LIPA (LIPA-/-) had barely detectable LAL enzymatic activity. Control and LIPA-/- IPSDM were loaded with [3H]-cholesteryl oleate-labeled AcLDL (acetylated low-density lipoprotein) followed by efflux to apolipoprotein A-I. Efflux of liberated [3H]-cholesterol to apolipoprotein A-I was abolished in LIPA-/- IPSDM, indicating deficiency in LAL-mediated lysosomal cholesteryl ester hydrolysis. In cells loaded with [3H]-cholesterol-labeled AcLDL, [3H]-cholesterol efflux was, however, not different between control and LIPA-/- IPSDM. ABCA1 (ATP-binding cassette, subfamily A, member 1) expression was upregulated by AcLDL loading but to a similar extent between control and LIPA-/- IPSDM. In nonlipid loaded state, LIPA-/- IPSDM had high levels of cholesteryl ester mass compared with minute amounts in control IPSDM. Yet, with AcLDL loading, overall cholesteryl ester mass was increased to similar levels in both control and LIPA-/- IPSDM. LIPA-/- did not impact lysosomal apolipoprotein-B degradation or expression of IL1B, IL6, and CCL5. CONCLUSIONS: LIPA-/- IPSDM reveals macrophage-specific hallmarks of LIPA deficiency. CRISPR/Cas9 and IPSDM provide important tools to study human macrophage biology and more broadly for future studies of disease-associated LIPA genetic variation in human macrophages.

Cell lipid metabolism modulators 2-bromopalmitate, D609, monensin, U18666A and probucol shift discoidal HDL formation to the smaller-sized particles: implications for the mechanism of HDL assembly.

ATP-binding cassette transporter A1 (ABCA1) mediates formation of disc-shaped high-density lipoprotein (HDL) from cell lipid and lipid-free apolipoprotein A-I (apo A-I). Discoidal HDL particles are heterogeneous in physicochemical characteristics for reasons that are understood incompletely. Discoidal lipoprotein particles similar in characteristics and heterogeneity to cell-formed discoidal HDL can be reconstituted from purified lipids and apo A-I by cell-free, physicochemical methods. The heterogeneity of reconstituted HDL (rHDL) is sensitive to the lipid composition of the starting lipid/apo A-I mixture. To determine whether the heterogeneity of cell-formed HDL is similarly sensitive to changes in cell lipids, we investigated four compounds that have well-established effects on cell lipid metabolism and ABCA1-mediated cell cholesterol efflux. 2-Bromopalmitate, D609, monensin and U18666A decreased formation of the larger-sized, but dramatically increased formation of the smaller-sized HDL. 2-Bromopalmitate did not appear to affect ABCA1 activity, subcellular localization or oligomerization, but induced dissolution of the cholesterol-phospholipid complexes in the plasma membrane. Arachidonic and linoleic acids shifted HDL formation to the smaller-sized species. Tangier disease mutations and inhibitors of ABCA1 activity wheat germ agglutinin and AG 490 reduced formation of both larger-sized and smaller-sized HDL. The effect of probucol was similar to the effect of 2-bromopalmitate. Taking rHDL formation as a paradigm, we propose that ABCA1 mutations and activity inhibitors reduce the amount of cell lipid available for HDL formation, and the compounds in the 2-bromopalmitate group and the polyunsaturated fatty acids change cell lipid composition from one that favors formation of the larger-sized HDL particles to one that favors formation of the smaller-sized species.

From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus.

Recent genome-wide association studies (GWASs) have identified a locus on chromosome 1p13 strongly associated with both plasma low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) in humans. Here we show through a series of studies in human cohorts and human-derived hepatocytes that a common noncoding polymorphism at the 1p13 locus, rs12740374, creates a C/EBP (CCAAT/enhancer binding protein) transcription factor binding site and alters the hepatic expression of the SORT1 gene. With small interfering RNA (siRNA) knockdown and viral overexpression in mouse liver, we demonstrate that Sort1 alters plasma LDL-C and very low-density lipoprotein (VLDL) particle levels by modulating hepatic VLDL secretion. Thus, we provide functional evidence for a novel regulatory pathway for lipoprotein metabolism and suggest that modulation of this pathway may alter risk for MI in humans. We also demonstrate that common noncoding DNA variants identified by GWASs can directly contribute to clinical phenotypes.

Kinetic and thermodynamic analyses of spontaneous exchange between high-density lipoprotein-bound and lipid-free apolipoprotein A-I.

It is thought that apolipoprotein A-I (apoA-I) spontaneously exchanges between high-density lipoprotein (HDL)-bound and lipid-free states, which is relevant to the occurrence of preß-HDL particles in plasma. To improve our understanding of the mechanistic basis for this phenomenon, we performed kinetic and thermodynamic analyses for apoA-I exchange between discoidal HDL-bound and lipid-free forms using fluorescence-labeled apoA-I variants. Gel filtration experiments demonstrated that addition of excess lipid-free apoA-I to discoidal HDL particles promotes exchange of apoA-I between HDL-associated and lipid-free pools without alteration of the steady-state HDL particle size. Kinetic analysis of time-dependent changes in NBD fluorescence upon the transition of NBD-labeled apoA-I from HDL-bound to lipid-free state indicates that the exchange kinetics are independent of the collision frequency between HDL-bound and lipid-free apoA-I, in which the lipid binding ability of apoA-I affects the rate of association of lipid-free apoA-I with the HDL particles and not the rate of dissociation of HDL-bound apoA-I. Thus, C-terminal truncations or mutations that reduce the lipid binding affinity of apoA-I strongly impair the transition of lipid-free apoA-I to the HDL-bound state. Thermodynamic analysis of the exchange kinetics demonstrated that the apoA-I exchange process is enthalpically unfavorable but entropically favorable. These results explain the thermodynamic basis of the spontaneous exchange reaction of apoA-I associated with HDL particles. The altered exchangeability of dysfunctional apoA-I would affect HDL particle rearrangement, leading to perturbed HDL metabolism.

Robust passive and active efflux of cellular cholesterol to a designer functional mimic of high density lipoprotein.

The ability of HDL to support macrophage cholesterol efflux is an integral part of its atheroprotective action. Augmenting this ability, especially when HDL cholesterol efflux capacity from macrophages is poor, represents a promising therapeutic strategy. One approach to enhancing macrophage cholesterol efflux is infusing blood with HDL mimics. Previously, we reported the synthesis of a functional mimic of HDL (fmHDL) that consists of a gold nanoparticle template, a phospholipid bilayer, and apo A-I. In this work, we characterize the ability of fmHDL to support the well-established pathways of cellular cholesterol efflux from model cell lines and primary macrophages. fmHDL received cell cholesterol by unmediated (aqueous) and ABCG1- and scavenger receptor class B type I (SR-BI)-mediated diffusion. Furthermore, the fmHDL holoparticle accepted cholesterol and phospholipid by the ABCA1 pathway. These results demonstrate that fmHDL supports all the cholesterol efflux pathways available to native HDL and thus, represents a promising infusible therapeutic for enhancing macrophage cholesterol efflux. fmHDL accepts cholesterol from cells by all known pathways of cholesterol efflux: unmediated, ABCG1- and SR-BI-mediated diffusion, and through ABCA1.

Molecular mechanisms of cellular cholesterol efflux.

Most types of cells in the body do not express the capability of catabolizing cholesterol, so cholesterol efflux is essential for homeostasis. For instance, macrophages possess four pathways for exporting free (unesterified) cholesterol to extracellular high density lipoprotein (HDL). The passive processes include simple diffusion via the aqueous phase and facilitated diffusion mediated by scavenger receptor class B, type 1 (SR-BI). Active pathways are mediated by the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, which are membrane lipid translocases. The efflux of cellular phospholipid and free cholesterol to apolipoprotein A-I promoted by ABCA1 is essential for HDL biogenesis. Current understanding of the molecular mechanisms involved in these four efflux pathways is presented in this minireview.

Fluorescence study of domain structure and lipid interaction of human apolipoproteins E3 and E4.

Human apolipoprotein E (apoE) isoforms exhibit different conformational stabilities and lipid-binding properties that give rise to altered cholesterol metabolism among the isoforms. Using Trp-substituted mutations and site- directed fluorescence labeling, we made a comprehensive comparison of the conformational organization of the N- and C-terminal domains and lipid interactions between the apoE3 and apoE4 isoforms. Trp fluorescence measurements for selectively Trp-substituted variants of apoE isoforms demonstrated that apoE4 adopts less stable conformations in both the N- and C-terminal domains compared to apoE3. Consistent with this, the conformational reorganization of the N-terminal helix bundle occurs at lower guanidine hydrochloride concentration in apoE4 than in apoE3 as monitored by fluorescence resonance energy transfer (FRET) from Trp residues to acrylodan attached at the N-terminal helix. Upon binding of apoE3 and apoE4 variants to egg phosphatidylcholine small unilamellar vesicles, similar changes in Trp fluorescence or FRET efficiency were observed for the isoforms, indi- cating that the opening of the N-terminal helix bundle occurs similarly in apoE3 and apoE4. Introduction of mutations into the C-terminal domain of the apoE isoforms to prevent self-association and maintain the monomeric state resulted in great increase in the rate of binding of the C-terminal helices to a lipid surface. Overall, our results demonstrate that the different conformational organizations of the N- and C-terminal domains have a minor effect on the steady-state lipid-binding behavior of apoE3 and apoE4: rather, self-association property is a critical determinant in the kinetics of lipid binding through the C-terminal helices of apoE isoforms.

The roles of C-terminal helices of human apolipoprotein A-I in formation of high-density lipoprotein particles.

Apolipoprotein A-I (apoA-I) accepts cholesterol and phospholipids from ATP-binding cassette transporter A1 (ABCA1)-expressing cells to form high-density lipoprotein (HDL). Human apoA-I has two tertiary structural domains and the C-terminal domain (approximately amino acids 190-243) plays a key role in lipid binding. Although the high lipid affinity region of the C-terminal domain of apoA-I (residues 223-243) is essential for the HDL formation, the function of low lipid affinity region (residues 191-220) remains unclear. To evaluate the role of residues 191-220, we analyzed the structure, lipid binding properties, and HDL formation activity of Δ191-220 apoA-I, in comparison to wild-type and Δ223-243 apoA-I. Although deletion of residues 191-220 has a slight effect on the tertiary structure of apoA-I, the Δ191-220 variant showed intermediate behavior between wild-type and Δ223-243 regarding the formation of hydrophobic sites and lipid interaction through the C-terminal domain. Physicochemical analysis demonstrated that defective lipid binding of Δ191-220 apoA-I is due to the decreased ability to form α-helix structure which provides the energetic source for lipid binding. In addition, the ability to form HDL particles in vitro and induce cholesterol efflux from ABCA1-expressing cells of Δ191-220 apoA-I was also intermediate between wild-type and Δ223-243 apoA-I. These results suggest that despite possessing low lipid affinity, residues 191-220 play a role in enhancing the ability of apoA-I to bind to and solubilize lipids by forming α-helix upon lipid interaction. Our results demonstrate that the combination of low lipid affinity region and high lipid affinity region of apoA-I is required for efficient ABCA1-dependent HDL formation.

Apolipoprotein A-I helical structure and stability in discoidal high-density lipoprotein (HDL) particles by hydrogen exchange and mass spectrometry.

To understand high-density lipoprotein (HDL) structure at the molecular level, the location and stability of α-helical segments in human apolipoprotein (apo) A-I in large (9.6 nm) and small (7.8 nm) discoidal HDL particles were determined by hydrogen-deuterium exchange (HX) and mass spectrometry methods. The measured HX kinetics of some 100 apoA-I peptides specify, at close to amino acid resolution, the structural condition of segments throughout the protein sequence and changes in structure and stability that occur on incorporation into lipoprotein particles. When incorporated into the large HDL particle, the nonhelical regions in lipid-free apoA-I (residues 45-53, 66-69, 116-146, and 179-236) change conformation from random coil to α-helix so that nearly the entire apoA-I molecule adopts helical structure (except for the terminal residues 1-6 and 237-243). The amphipathic α-helices have relatively low stability, in the range 3-5 kcal/mol, indicating high flexibility and dynamic unfolding and refolding in seconds or less. A segment encompassed by residues 125-158 exhibits bimodal HX labeling indicating co-existing helical and disordered loop conformations that interchange on a time scale of minutes. When incorporated around the edge of the smaller HDL particle, the increase in packing density of the two apoA-I molecules forces about 20% more residues out of direct contact with the phospholipid molecules to form disordered loops, and these are the same segments that form loops in the lipid-free state. The region of disc-associated apoA-I that binds the lecithin-cholesterol acyltransferase enzyme is well structured and not a protruding unstructured loop as reported by others.

Influence of domain stability on the properties of human apolipoprotein E3 and E4 and mouse apolipoprotein E.

The human apolipoprotein (apo) E4 isoform, which differs from wild-type apoE3 by the single amino acid substitution C112R, is associated with elevated risk of cardiovascular and Alzheimer's diseases, but the molecular basis for this variation between isoforms is not understood. Human apoE is a two-domain protein comprising an N-terminal helix bundle and a separately folded C-terminal region. Here, we examine the concept that the ability of the protein to bind to lipid surfaces is influenced by the stability (or readiness to unfold) of these domains. The lipid-free structures and abilities to bind to lipid and lipoprotein particles of a series of human and mouse apoE variants with varying domain stabilities and domain­domain interactions are compared. As assessed by urea denaturation, the two domains are more unstable in apoE4 than in apoE3. To distinguish the contributions of the destabilization of each domain to the greater lipid-binding ability of apoE4, the properties of the apoE4 R61T and E255A variants, which have the same helix bundle stabilities but altered C-terminal domain stabilities, are compared. In these cases, the effects on lipid-binding properties are relatively minor, indicating that the destabilization of the helix bundle domain is primarily responsible for the enhanced lipid-binding ability of apoE4. Unlike human apoE, mouse apoE behaves essentially as a single domain, and its lipid-binding characteristics are more similar to those of apoE4. Together, the results show that the overall stability of the entire apoE molecule exerts a major influence on its lipid- and lipoprotein-binding properties.

Interaction of thioflavin T with amyloid fibrils of apolipoprotein A-I N-terminal fragment: resonance energy transfer study.

Apolipoprotein A-I is amenable to a number of specific mutations associated with hereditary systemic amyloidoses. Amyloidogenic properties of apoA-I are determined mainly by its N-terminal fragment. In the present study Förster resonance energy transfer between tryptophan as a donor and Thioflavin T as an acceptor was employed to obtain structural information on the amyloid fibrils formed by apoA-I variant 1-83/G26R/W@8. Analysis of the dye-fibril binding data provided evidence for the presence of two types of ThT binding sites with similar stoichiometries (bound dye to monomeric protein molar ratio ∼10), but different association constants (∼6 and 0.1µM(-1)) and ThT quantum yields in fibril-associated state (0.08 and 0.05, respectively). A ß-strand-loop-ß-strand structural model of 1-83/G26R/W@8 apoA-I fibrils has been proposed, with potential ThT binding sites located in the solvent-exposed grooves of the N-terminal ß-sheet layer. Reasoning from the expanded FRET analysis allowing for heterogeneity of ThT binding centers and fibril polymorphism, the most probable locations of high- and low-affinity ThT binding sites were attributed to the grooves T16_Y18 and D20_L22, respectively.

Dual role of an N-terminal amyloidogenic mutation in apolipoprotein A-I: destabilization of helix bundle and enhancement of fibril formation.

A number of naturally occurring mutations of apolipoprotein (apo) A-I, the major protein of HDL, are known to be associated with hereditary amyloidosis and atherosclerosis. Here, we examined the effects of the G26R point mutation in apoA-I (apoA-I(Iowa)) on the structure, stability, and aggregation propensity to form amyloid fibril of full-length apoA-I and the N-terminal fragment of apoA-I. Circular dichroism and fluorescence measurements demonstrated that the G26R mutation destabilizes the N-terminal helix bundle domain of full-length protein, leading to increased hydrophobic surface exposure, whereas it has no effect on the initial structure of the N-terminal 1-83 fragment, which is predominantly a random coil structure. Upon incubation for extended periods at neutral pH, the N-terminal 1-83 variants undergo a conformational change to ß-sheet-rich structure with a great increase in thioflavin T fluorescence, whereas no structural change is observed in full-length proteins. Comparison of fibril-forming propensity among substituted mutants at Gly-26 position of 1-83 fragments demonstrated that the G26R mutation enhances the nucleation step of fibril formation, whereas G26K and G26E mutations have small or inhibiting effects on the formation of fibrils. These fibrils of the 1-83 variants have long and straight morphology as revealed by atomic force microscopy and exhibited significant toxicity with HEK293 cells. Our results indicate dual critical roles of the arginine residue at position 26 in apoA-I(Iowa): destabilization of the N-terminal helix bundle structure in full-length protein and enhancement of amyloid fibril formation by the N-terminal 1-83 fragment.

Mechanisms responsible for the compositional heterogeneity of nascent high density lipoprotein.

Apolipoprotein (apo) A-I-containing nascent HDL particles produced by the ATP binding cassette transporter A1 have different sizes and compositions. To understand the molecular basis for this heterogeneity, the HDL particles produced by apoA-I-mediated solubilization of phospholipid (PL)/free (unesterified) cholesterol (FC) bilayer membranes in cell and cell-free systems are compared. Incubation of apoA-I with ATP binding cassette transporter A1-expressing baby hamster kidney cells leads to formation of two populations of FC-containing discoidal nascent HDL particles. The larger 11-nm diameter particles are highly FC-enriched (FC/PL = 1.2/1 mol/mol) relative to the smaller 8 nm particles and the cell plasma membrane (FC/PL = 0.4/1). ApoA-I-mediated spontaneous solubilization of either multilamellar or unilamellar vesicles made of a membrane-PL mixture and FC yields discoidal HDL particles with diameters in the range 9-17 nm and, as found with the cell system, the larger particles are relatively enriched in FC despite the fact that all particles are created by solubilization of a common FC/PL membrane domain. The size-dependent distribution of FC among HDL particles is due to varying amounts of PL being sequestered in a boundary layer by interaction with apoA-I at the disc edge. The presence of a relatively large boundary layer in smaller discoidal HDL promotes preferential distribution of phosphatidylserine to such particles. However, phosphatidylcholine and sphingomyelin which are the primary PL constituents of nascent HDL do not exhibit selective incorporation into HDL discs of different sizes. This understanding of the mechanisms responsible for the heterogeneity in lipid composition of nascent HDL particles may provide a basis for selecting subspecies with preferred cardio-protective properties.

Apolipoprotein E isoforms and lipoprotein metabolism.

Apolipoprotein (apo) E is a 299-residue protein which functions as a key regulator of plasma lipid levels. Human apoE exists as three common isoforms and the parent form, apoE3, operates optimally in promoting clearance of triglyceride (TG)-rich lipoproteins and is associated with normal plasma lipid levels. This result occurs because apoE3 possesses both the requisite lipid-binding ability and affinity for the low density lipoprotein receptor (LDLR) to mediate appropriate lipolytic processing and endocytosis of TG-rich lipoprotein remnant particles. ApoE2 which differs from apoE3 by the single amino acid substitution Arg158Cys located near the LDLR recognition site exhibits impaired binding to the receptor and an inability to promote clearance of TG-rich lipoprotein remnant particles; this isoform is associated with Type-III hyperlipoproteinemia. ApoE4 which differs from apoE3 by the single amino acid substitution Cys112Arg is also associated with dyslipidemia although binding of this isoform to the LDLR is unaffected. The amino acid substitution affects the organization and stability of both the N-terminal helix bundle domain and separately folded C-terminal domain so that apoE4 has enhanced lipid binding ability. As a consequence, apoE4 binds better than apoE3 to the surface of very low density lipoprotein (VLDL) particles and impairs their lipolytic processing in the circulation so that apoE4 is associated with a more pro-atherogenic lipoprotein-cholesterol distribution (higher VLDL-cholesterol/high density lipoprotein-cholesterol ratio). This review summarizes current understanding of the structural differences between apoE2, apoE3, and apoE4, and the molecular mechanisms responsible for the alterations in lipoprotein metabolism resulting from this polymorphism of apoE. Detailed knowledge of how expression of structurally distinct apoE variants modifies lipoprotein metabolism provides a basis for developing ways to manipulate the functionality of apoE in vivo.

Factors controlling nascent high-density lipoprotein particle heterogeneity: ATP-binding cassette transporter A1 activity and cell lipid and apolipoprotein AI availability.

Nascent high-density lipoprotein (HDL) particles arise in different sizes. We have sought to uncover factors that control this size heterogeneity. Gel filtration, native PAGE, and protein cross-linking were used to analyze the size heterogeneity of nascent HDL produced by BHK-ABCA1, RAW 264.7, J774, and HepG2 cells under different levels of two factors considered as a ratio, the availability of apolipoprotein AI (apoAI) -accessible cell lipid, and concentration of extracellular lipid-free apoAI. Increases in the available cell lipid:apoAI ratio due to either elevated ATP-binding cassette transporter A1 (ABCA1) expression and activity or raised cell density (i.e., increasing numerator) shifted the production of nascent HDL from smaller particles with fewer apoAI molecules per particle and fewer molecules of choline-phospholipid and cholesterol per apoAI molecule to larger particles that contained more apoAI and more lipid per molecule of apoAI. A further shift to larger particles was observed in BHK-ABCA1 cells when the available cell lipid:apoAI ratio was raised still higher by decreasing the apoAI concentration (i.e., the denominator). These changes in nascent HDL biogenesis were reminiscent of the transition that occurs in the size composition of reconstituted HDL in response to an increasing initial lipid:apoAI molar ratio. Thus, the ratio of available cell lipid:apoAI is a fundamental cause of nascent HDL size heterogeneity, and rHDL formation is a good model of nascent HDL biogenesis.

Molecular mechanisms responsible for the differential effects of apoE3 and apoE4 on plasma lipoprotein-cholesterol levels.

OBJECTIVE: The goal of this study was to understand the molecular basis of how the amino acid substitution C112R that distinguishes human apolipoprotein (apo) E4 from apoE3 causes the more proatherogenic plasma lipoprotein-cholesterol distribution that is known to be associated with the expression of apoE4. APPROACH AND RESULTS: Adeno-associated viruses, serotype 8 (AAV8), were used to express different levels of human apoE3, apoE4, and several C-terminal truncation and internal deletion variants in C57BL/6 apoE-null mice, which exhibit marked dysbetalipoproteinemia. Plasma obtained from these mice 2 weeks after the AAV8 treatment was analyzed for cholesterol and triglyceride levels, as well as for the distribution of cholesterol between the lipoprotein fractions. Hepatic expression of apoE3 and apoE4 induced similar dose-dependent decreases in plasma cholesterol and triglyceride to the levels seen in control C57BL/6 mice. Importantly, at the same reduction in plasma total cholesterol, expression of apoE4 gave rise to higher very low-density lipoprotein-cholesterol (VLDL-C) and lower high-density lipoprotein-cholesterol levels relative to the apoE3 situation. The C-terminal domain and residues 261 to 272 in particular play a critical role, because deleting them markedly affected the performance of both isoforms. CONCLUSIONS: ApoE4 possesses enhanced lipid and VLDL-binding ability relative to apoE3, which gives rise to impaired lipolytic processing of VLDL in apoE4-expressing mice. These effects reduce VLDL remnant clearance from the plasma compartment and decrease the amount of VLDL surface components available for incorporation into the high-density lipoprotein pool, accounting for the more proatherogenic lipoprotein profile (higher VLDL-C/high-density lipoprotein-cholesterol ratio) occurring in apoE4-expressing animals compared with their apoE3 counterparts.

Outcomes after Surgical Microwave Ablation for the Treatment of Colorectal Liver Metastasis.

BACKGROUND: Colorectal cancer (CRC) is the third most common cause of cancer mortality worldwide. Of these, approximately 25% will have liver metastasis. We performed 394 microwave ablations (MWA) and analyzed outcomes for survival and ablation failure. STUDY DESIGN: Retrospective review of patients who underwent a surgical microwave ablation at a single center high-volume institution from October 2006 through September 2022 using a prospectively maintained database. Primary outcome was overall survival. RESULTS: A total of 394 operations were performed on 328 patients with 842 tumors undergoing MWA. Median tumor size was 1.5 cm (range 0.4-7.0 cm), with the median number of tumors ablated per operation being 1 (range 1-11). A laparoscopic approach was used 77.9% of the time. Concomitant procedures were performed 63% of the time, most commonly hepatectomy (22.3%), cholecystectomy (17.5%), and colectomy (6.6%). Clavien-Dindo Grade III or IV complications occurred in 12 patients (3.6%), and all of these patients had undergone concomitant procedures. Mortality within 30 days occurred in 4 patients (1.2%). The rate of incomplete ablation (IA) was 1.5% per tumor. Local recurrence (LR) occurred at a rate of 6.3% per tumor. African Americans were found to have a higher incidence of IA and LR. One year survival probability was 91% [95% CI: 87.9 -94.3], with a mean overall survival of 57.6 months [95% CI: 49.9-65.4 months]. CONCLUSION: Surgical MWA offers a low-morbidity approach to treatment of colorectal liver metastasis (CRLM), with low rates of failure. This large series reviews the outcomes of MWA as definitive treatment for CRLM.