Apolipoprotein A-I Assumes a “Looped Belt” Conformation on Reconstituted High Density Lipoprotein

Self Cite

High density lipoproteins (HDL) represent a heterogeneous population of particles with apoA-I as the major protein (1). Whether apoA-I/HDL 2 plays a direct role in cardiovascular disease prevention (e.g. removal of cholesterol from clogged arteries) or an indirect one (e.g. acts as a platform for the clustering of protective molecules, such as anti-inflammatory or antioxidant proteins), detailed knowledge of HDL structure is a key to understanding the molecular mechanism underlying these processes. Because the conformation of apoA-I on HDL is highly plastic (1), understanding apoA-I/HDL structure and dynamics is not straightforward.Since the early 1970s, the standard model for HDL particles reconstituted from apolipoprotein A-I (apoA-I) and phospholipid (apoA-I/HDL) has been that of a discoidal particle on the order of 100 Å in diameter and the thickness of a phospholipid bilayer. The initial observation of discoidal HDL particles was made by Forte et al. (2) when they examined HDL from patients with familial lecithin:cholesterol acyltransferase deficiency by negative stain EM. Reconstituted apoA-I/HDL particles (3) and nascent HDL from lymph (4) were then observed by negative stain EM to also have a discoidal shape. X-ray and neutron scattering studies (5, 6) provided further support for the standard discoidal model. The standard discoidal model has been used to interpret the results of a large number of experimental studies of the structure of reconstituted apoA-I/HDL particles. For review, see however, Wu et al. (11) used small angle neutron scattering to develop a model for apoA-I/HDL particles that is dramatically different from the standard model. In their model, termed a double superhelix (DSH), apoA-I possesses an open helical shape that twists around a central prolate ellipsoidal particle resembling a partial type I hexagonal lyotropic liquid crystalline phase.The DSH model is interesting. It is similar to MD simulation-based models (12) in that the proposed double superhelix forms a left-handed spiral as it wraps around the prolate ellip-

Section: Experimental Studiesmentioning

confidence: 99%

Section: Experimental Studiesmentioning

confidence: 99%

Assessment of the Validity of the Double Superhelix Model for Reconstituted High Density Lipoproteins

Jones

Zhang

Catte

et al. 2010

Self Cite

“…Fluorescent Labeling of Proteins-AEDANS and ALEXA350 fluorophores were covalently linked to Cys-136 as described (51). Briefly, the cysteine disulfide bond was reduced by overnight incubation in the presence of tris(2-carboxyethyl)phosphine at a final molar ratio of 1:10 apoA-I:tris(2-carboxyethyl)phosphine.…”

Section: Production Of Recombinant Apoa-i Proteins-mutationsmentioning

confidence: 99%

Exchange of Apolipoprotein A-I between Lipid-associated and Lipid-free States

Cavigiolio¹,

Geier²,

Shao

et al. 2010

Self Cite

An important event in cholesterol metabolism is the efflux of cellular cholesterol by apolipoprotein A-I (apoA-I), the major protein of high density lipoproteins (HDL). Lipid-free apoA-I is the preferred substrate for ATP-binding cassette A1, which promotes cholesterol efflux from macrophage foam cells in the arterial wall. However, the vast majority of apoA-I in plasma is associated with HDL, and the mechanisms for the generation of lipid-free apoA-I remain poorly understood. In the current study, we used fluorescently labeled apoA-I that exhibits a distinct fluorescence emission spectrum when in different states of lipid association to establish the kinetics of apoA-I transition between the lipid-associated and lipid-free states. This approach characterized the spontaneous and rapid exchange of apoA-I between the lipid-associated and lipid-free states. In contrast, the kinetics of apoA-I exchange were significantly reduced when apoA-I on HDL was cross-linked with a bi-functional reagent or oxidized by myeloperoxidase. Our observations support the hypothesis that oxidative damage to apoA-I by myeloperoxidase limits the ability of apoA-I to be liberated in a lipid-free form from HDL. This impairment of apoA-I exchange reaction may be a trait of dysfunctional HDL contributing to reduced ATP-binding cassette A1-mediated cholesterol efflux and atherosclerosis.Apolipoprotein A-I (apoA-I), 4 the primary protein constituent of HDL, inhibits atherosclerosis in hypercholesterolemic mice (1-3). Moreover, increasing evidence indicates that HDL is cardioprotective in humans (4). The ability of HDL to promote cholesterol efflux from cholesteryl ester-laden macrophage foam cells is of central importance to the initiation and progression of atherosclerosis (5-7).In plasma, the vast majority of apoA-I associates with spherical HDL, a complex of apolipoproteins, phospholipids, triglycerides, free cholesterol, and cholesterol esters (8). However, the primary acceptor of cholesterol and phospholipids from macrophages is lipid-free or lipid-poor apoA-I (containing up to four phospholipid molecules (9)), which is the preferred substrate of the plasma membrane transporter ATP-binding cassette A1 (ABCA1) (10 -15). Circulating HDL is remodeled by the action of proteins and enzymes (16) such as cholesteryl ester transfer protein (17), lechitin:cholesterol acyltransferase (LCAT) (18, 19), phospholipid transfer protein (20, 21), and hepatic lipase (22). Plasma HDL remodeling can result in the destabilization of HDL and the release of lipid-free/lipid-poor apoA-I. Furthermore, the selective uptake of lipids from HDL through scavenger receptor B, type 1 can yield lipid-poor apoA-I (23).We hypothesized that the production of lipid-free/lipid-poor apoA-I from mature HDL and relipidation by ABCA1 is a dynamic process in the arterial wall, which is critical in protecting macrophages from cholesteryl ester accumulation. Previously, we showed that lipid-free/lipid-poor apoA-I is released from reconstituted HDL (rHDL) by long term (3-7 days) incu...

“…By electron paramagnetic resonance (EPR) spectroscopy analysis, we have determined the location of this "hinge domain" as a 12-amino acid-long segment centered on residue 139. Because this portion of apoA-I aligns with its counterpart in a paired apoA-I on HDL, we hypothesized this stretch of residues forms a pore-like structure, described in the "looped belt" model (14), wherein this pore provides lecithin:cholesterol acyltransferase access to cholesterol and the acyl chain of POPC. Recently, this has been supported by molecular dynamic simulation computational analysis, wherein Jones et al (23) determined that this region could form an amphipathic presentation tunnel for the acyl chains of POPC.…”

mentioning

confidence: 99%

Structure of Apolipoprotein A-I N Terminus on Nascent High Density Lipoproteins

Lagerstedt

Cavigiolio²,

Budamagunta

et al. 2011

Self Cite

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.