Structure of apolipoprotein A-I N terminus on nascent high density lipoproteins.
ABSTRACT: Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and a critical element of cholesterol metabolism. To better elucidate the role of the apoA-I structure-function in cholesterol metabolism, the conformation of the apoA-I N terminus (residues 6-98) on nascent HDL was examined by electron paramagnetic resonance (EPR) spectroscopic analysis. A series of 93 apoA-I variants bearing single nitroxide spin label at positions 6-98 was reconstituted onto 9.6-nm HDL particles (rHDL). These particles were subjected to EPR spectral analysis, measuring regional flexibility and side chain solvent accessibility. Secondary structure was elucidated from side-chain mobility and molecular accessibility, wherein two major ?-helical domains were localized to residues 6-34 and 50-98. We identified an unstructured segment (residues 35-39) and a ?-strand (residues 40-49) between the two helices. Residues 14, 19, 34, 37, 41, and 58 were examined by EPR on 7.8, 8.4, and 9.6 nm rHDL to assess the effect of particle size on the N-terminal structure. Residues 14, 19, and 58 showed no significant rHDL size-dependent spectral or accessibility differences, whereas residues 34, 37, and 41 displayed moderate spectral changes along with substantial rHDL size-dependent differences in molecular accessibility. We have elucidated the secondary structure of the N-terminal domain of apoA-I on 9.6 nm rHDL (residues 6-98) and identified residues in this region that are affected by particle size. We conclude that the inter-helical segment (residues 35-49) plays a role in the adaptation of apoA-I to the particle size of HDL.
Project description:The antiatherogenic properties of apolipoprotein A-I (apoA-I) are derived, in part, from lipidation-state-dependent structural elements that manifest at different stages of apoA-I's progression from lipid-free protein to spherical high-density lipoprotein (HDL). Previously, we reported the structure of apoA-I's N-terminus on reconstituted HDLs (rHDLs) of different sizes. We have now investigated at the single-residue level the conformational adaptations of three regions in the central domain of apoA-I (residues 119-124, 139-144, and 164-170) upon apoA-I lipid binding and HDL formation. An important function associated with these residues of apoA-I is the activation of lecithin:cholesterol acyltransferase (LCAT), the enzyme responsible for catalyzing HDL maturation. Structural examination was performed by site-directed tryptophan fluorescence and spin-label electron paramagnetic resonance spectroscopies for both the lipid-free protein and rHDL particles 7.8, 8.4, and 9.6 nm in diameter. The two methods provide complementary information about residue side chain mobility and molecular accessibility, as well as the polarity of the local environment at the targeted positions. The modulation of these biophysical parameters yielded new insight into the importance of structural elements in the central domain of apoA-I. In particular, we determined that the loosely lipid-associated structure of residues 134-145 is conserved in all rHDL particles. Truncation of this region completely abolished LCAT activation but did not significantly affect rHDL size, reaffirming the important role of this structural element in HDL function.
Project description:Apolipoprotein A-I (ApoA-I) is the major protein component of high-density lipoprotein (HDL), and is critical for maintenance of cholesterol homeostasis. During reverse cholesterol transport, HDL transitions between an array of subclasses, differing in size and composition. This process requires ApoA-I to adapt to changes in the shape of the HDL particle, transiting from an apolipoprotein to a myriad of HDL subclass-specific conformations. Changes in ApoA-I structure cause alterations in HDL-specific enzyme and receptor-binding properties, and thereby direct the HDL particle through the reverse cholesterol transport pathway. In this study, we used site-directed spin label spectroscopy to examine the conformational details of the ApoA-I central domain on HDL. The motional dynamics and accessibility to hydrophobic/hydrophilic relaxation agents of ApoA-I residues 99-163 on 9.6-nm reconstituted HDL was analyzed by EPR. In previous analyses, we examined residues 6-98 and 164-238 (of ApoA-I's 243 residues), and combining these findings with the current results, we have generated a full-length map of the backbone structure of reconstituted HDL-associated ApoA-I. Remarkably, given that the majority of ApoA-I's length is composed of amphipathic helices, we have identified nonhelical residues, specifically the presence of a ?-strand (residues 149-157). The significance of these nonhelical residues is discussed, along with the other features, in the context of ApoA-I function in contrast to recent models derived by other methods.
Project description:Apolipoprotein A-I and A-IV are protein constituents of high-density lipoproteins although their functional difference in lipoprotein metabolism is still unclear. To compare anti-atherogenic properties between apoA-I and apoA-4, we characterized both proteins in lipid-free and lipid-bound state. In lipid-free state, apoA4 showed two distinct bands, around 78 and 67 Å on native gel electrophoresis, while apoA-I showed scattered band pattern less than 71 Å. In reconstituted HDL (rHDL) state, apoA-4 showed three major bands around 101 Å and 113 Å, while apoA-I-rHDL showed almost single band around 98 Å size. Lipid-free apoA-I showed 2.9-fold higher phospholipid binding ability than apoA-4. In lipid-free state, BS3-crosslinking revealed that apoA-4 showed less multimerization tendency upto dimer, while apoA-I showed pentamerization. In rHDL state (95:1), apoA-4 was existed as dimer as like as apoA-I. With higher phospholipid content (255:1), five apoA-I and three apoA-4 were required to the bigger rHDL formation. Regardless of particle size, apoA-I-rHDL showed superior LCAT activation ability than apoA-4-rHDL. Uptake of acetylated LDL was inhibited by apoA-I in both lipid-free and lipid-bound state, while apoA-4 inhibited it only lipid-free state. ApoA-4 showed less anti-atherogenic activity with more sensitivity to glycation. In conclusion, apoA-4 showed inferior physiological functions in lipid-bound state, compared with those of apoA-I, to induce more pro-atherosclerotic properties.
Project description:Specific mutations in Apolipoprotein A-I (ApoA-I) of high-density lipoprotein (HDL) are responsible for a late-onset systemic amyloidosis. Carriers do not exhibit increased cardiovascular disease risk despite reduced levels of ApoA-I/ HDL-cholesterol. To explain this paradox, we show that the HDL particle profile of L75P and L174S patients presents a higher relative abundance of the 8.4 nm vs 9.6 nm particles, and that serum from patients, as well as reconstituted 8.4 and 9.6 nm HDL particles (rHDL), possess increased capacity to catalyze cholesterol efflux from macrophages. Synchrotron radiation circular dichroism and hydrogen-deuterium exchange revealed that the variants in 8.4 nm rHDL have altered secondary structure composition and display a more flexible binding to lipids compared to their native counterpart. The reduced HDL-cholesterol levels of patients carrying ApoA-I amyloidogenic variants are thus balanced by higher proportion of small, dense HDL particles and better cholesterol efflux due to altered, region-specific protein structure dynamics.
Project description:1. Phospholipid transfer protein (PLTP) mediates conversion of high-density lipoprotein (HDL3) to large particles, with concomitant release of apolipoprotein A-I (apoA-I). To study the mechanisms involved in this conversion, reconstituted HDL (rHDL) particles containing either fluorescent pyrenylacyl cholesterol ester (PyrCE) in their core (PyrCE-rHDL) or pyrenylacyl phosphatidylcholine (PysPC) in their surface lipid layer (PyrPC-rHDL) were prepared. Upon incubation with PLTP they behaved as native HDL3, in that their size increased considerably. 2. When PyrPC-rHDL was incubated with HDL3 in the presence of PLTP, a rapid decline of the pyrene excimer/monomer fluorescence ratio (E/M) occurred, demonstrating that PLTP induced mixing of the surface lipids of PyrPC-rHDL and HDL3. As this mixing was almost complete before any significant increase in HDL particle size was observed, it represents PLTP-mediated phospholipid transfer or exchange that is not directly coupled to the formation of large HDL particles. 3. When core-labelled PyrCE-rHDL was incubated in the presence of PLTP, a much slower, time-dependent decrease of E/M was observed, demonstrating that PLTP also promotes mixing of the core lipids. The rate and extent of mixing of core lipids correlated with the amount of PLTP added and with the increase in particle size. The enlarged particles formed could be visualized as discrete, non-aggregated particles by electron microscopy. Concomitantly with the appearance of enlarged particles, lipid-poor apoA-I molecules were released. These data, together with the fact that PLTP has been shown not to mediate transfer of cholesterol esters, strongly suggest that particle fusion rather than (net) lipid transfer or particle aggregation is responsible for the enlargement of HDL particles observed upon incubation with PLTP.4.ApoA-I rHDL, but not apoA-II rHDL, were converted into large particles, suggesting that the presence of apoA-I is required for PLTP-mediated HDL fusion. A model for PLTP-mediated enlargement of HDL particles is presented.
Project description:Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and plays a central role in cholesterol metabolism. The lipid-free/lipid-poor form of apoA-I is the preferred substrate for the ATP-binding cassette transporter A1 (ABCA1). The interaction of apoA-I with ABCA1 leads to the formation of cholesterol laden high density lipoprotein (HDL) particles, a key step in reverse cholesterol transport and the maintenance of cholesterol homeostasis. Knowledge of the structure of lipid-free apoA-I is essential to understanding its critical interaction with ABCA1 and the molecular mechanisms underlying HDL biogenesis. We therefore examined the structure of lipid-free apoA-I by electron paramagnetic resonance spectroscopy (EPR). Through site directed spin label EPR, we mapped the secondary structure of apoA-I and identified sites of spin coupling as residues 26, 44, 64, 167, 217 and 226. We capitalize on the fact that lipid-free apoA-I self-associates in an anti-parallel manner in solution. We employed these sites of spin coupling to define the central plane in the dimeric apoA-I complex. Applying both the constraints of dipolar coupling with the EPR-derived pattern of solvent accessibility, we assembled the secondary structure into a tertiary context, providing a solution structure for lipid-free apoA-I. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).
Project description:Apolipoprotein (apo) A-I is the major protein component of high-density lipoprotein (HDL) and plays key roles in the Reverse Cholesterol Transport pathway. In the past decade, reconstituted HDL (rHDL) has been employed as a therapeutic agent for treatment of atherosclerosis. The ability of rHDL to promote cholesterol efflux from peripheral cells has been documented to reduce the size of atherosclerotic plaque lesions. However, development of apoA-I rHDL-based therapeutics for human use requires a cost effective process to generate an apoA-I product that meets "Good Manufacturing Practice" standards. Methods available for production and isolation of unmodified recombinant human apoA-I at scale are cumbersome, laborious and complex. To overcome this obstacle, a streamlined two-step procedure has been devised for isolation of recombinant untagged human apoA-I from E. coli that takes advantage of its ability to re-fold to a native conformation following denaturation. Heat treatment of a sonicated E. coli supernatant fraction induced precipitation of a large proportion of host cell proteins (HCP), yielding apoA-I as the major soluble protein. Reversed-phase HPLC of this material permitted recovery of apoA-I largely free of HCP and endotoxin. Purified apoA-I possessed ?-helix secondary structure, formed rHDL upon incubation with phospholipid and efficiently promoted cholesterol efflux from cholesterol loaded J774 macrophages.
Project description:Atherosclerosis affects millions of people worldwide. However, the wide variety of limitations in the current therapeutic options leaves much to be desired in future lipid-lowering therapies. For example, although statins, which are the first-line treatment for coronary heart disease (CHD), reduce the risk of cardiovascular events in a large percentage of patients, they lead to optimal levels of low density lipoprotein-cholesterol (LDL-C) in only about one-third of patients. A new promising research direction against atherosclerosis aims to improve lipoprotein metabolism. Novel therapeutic approaches are being developed to increase the levels of functional high density lipoprotein (HDL) particles. This review aims to highlight the atheroprotective potential of the in vitro synthesized reconstituted HDL particles containing apolipoprotein E (apoE) as their sole apolipoprotein component (rHDL-apoE). For this purpose, we provide: (1) a summary of the atheroprotective properties of native plasma HDL and its apolipoprotein components, apolipoprotein A-I (apoA-I) and apoE; (2) an overview of the anti-atherogenic functions of rHDL-apoA-I and apoA-I-containing HDL, i.e., natural HDL isolated from transgenic Apoa1-/- × Apoe-/- mice overexpressing human apoA-I (HDL-apoA-I); and (3) the latest developments and therapeutic potential of HDL-apoE and rHDL-apoE. Novel rHDL formulations containing apoE could possibly present enhanced biological functions, leading to improved therapeutic efficacy against atherosclerosis.
Project description:Reconstituted high-density lipoprotein particles (rHDL) are powerful platforms used as a model phospholipid bilayer system to study membrane proteins. They consist of a discoidal-shaped planar bilayer of phospholipids that is surrounded by a dimer of apolipoprotein A-I (apoA-I). The amphipathic nature of apoA-1 shields the hydrophobic acyl chains of the lipids from solvent and keeps the particles soluble in aqueous environments. These monodispersed, nanoscale discoidal HDL particles are approximately 10-11 nm in diameter with a thickness that is dependent on the length of the phospholipid acyl chain. Reconstituted HDL particles can be assembled in vitro using purified apoA-1 and purified lipids. Investigators have utilized this model bilayer system to co-reconstitute membrane proteins, and take advantage of the small size and its monodispersion. Our laboratory and others have utilized the rHDL approach to study the behavior of G protein-coupled receptors. In this chapter, we describe strategies for the preparation of rHDL particles containing GPCRs in their monomeric form and discuss various methodologies used to analyze the reconstituted receptor function.
Project description:Mechanisms to increase plasma high-density lipoprotein (HDL) or to promote egress of cholesterol from cholesterol-loaded cells (e.g., foam cells from atherosclerotic lesions) remain an important target to regress heart disease. Reconstituted HDL (rHDL) serves as a valuable vehicle to promote cellular cholesterol efflux in vitro and in vivo. rHDL were prepared with wild type apolipoprotein (apo) A-I and the rare variant, apoA-I Milano (M), and each apolipoprotein was reconstituted with phosphatidylcholine (PC) or sphingomyelin (SM). The four distinct rHDL generated were incubated with CHO cells, J774 macrophages, and BHK cells in cellular cholesterol efflux assays. In each cell type, apoA-I(M) SM-rHDL promoted the greatest cholesterol efflux. In BHK cells, the cholesterol efflux capacities of all four distinct rHDL were greatly enhanced by increased expression of ABCG1. Efflux to PC-containing rHDL was stimulated by transfection of a nonfunctional ABCA1 mutant (W590S), suggesting that binding to ABCA1 represents a competing interaction. This interpretation was confirmed by binding experiments. The data show that cholesterol efflux activity is dependent upon the apoA-I protein employed, as well as the phospholipid constituent of the rHDL. Future studies designed to optimize the efflux capacity of therapeutic rHDL may improve the value of this emerging intervention strategy.