Biomedical subjects

Michael C Phillips

Publications and source records attributed to Michael C Phillips.

At least 19 recordsLinked to original sources

PubMed2006

Contributions of the N- and C-terminal helical segments to the lipid-free structure and lipid interaction of apolipoprotein A-I.

The tertiary structure of lipid-free apolipoprotein (apo) A-I in the monomeric state comprises two domains: a N-terminal alpha-helix bundle and a less organized C-terminal domain. This study examined how the N- and C-terminal segments of apoA-I (residues 1-43 and 223-243), which contain the most hydrophobic regions in the molecule and are located in opposite structural domains, contribute to the lipid-free conformation and lipid interaction. Measurements of circular dichroism in conjunction with tryptophan and 8-anilino-1-naphthalenesulfonic acid fluorescence data demonstrated that single (L230P) or triple (L230P/L233P/Y236P) proline insertions into the C-terminal alpha helix disrupted the organization of the C-terminal domain without affecting the stability of the N-terminal helix bundle. In contrast, proline insertion into the N terminus (Y18P) disrupted the bundle structure in the N-terminal domain, indicating that the alpha-helical segment in this region is part of the helix bundle. Calorimetric and gel-filtration measurements showed that disruption of the C-terminal alpha helix significantly reduced the enthalpy and free energy of binding of apoA-I to lipids, whereas disruption of the N-terminal alpha helix had only a small effect on lipid binding. Significantly, the presence of the Y18P mutation offset the negative effects of disruption/removal of the C-terminal helical domain on lipid binding, suggesting that the alpha helix around Y18 concealed a potential lipid-binding region in the N-terminal domain, which was exposed by the disruption of the helix-bundle structure. When these results are taken together, they indicate that the alpha-helical segment in the N terminus of apoA-I modulates the lipid-free structure and lipid interaction in concert with the C-terminal domain.

Amino Acid Substitution↗

PubMed2006

Effect of carboxyl-terminal truncation on structure and lipid interaction of human apolipoprotein E4.

Apolipoprotein (apo) E4 has been identified as a major risk factor for Alzheimer's disease. Recently, apoE4 was found to undergo proteolytic cleavage in Alzheimer's disease brains, resulting in neurotoxic C-terminal-truncated fragments. In this study, we examined the effect of progressive truncation of the C-terminal domain in apoE4 on its lipid-free structure and lipid binding properties. Circular dichroism measurements demonstrated that deletion of residues 273-299 or 261-299 significantly decreased the number of helical residues, suggesting that the C-terminal residues 261-299 have alpha-helical structure. Although the progressive deletions in the C-terminal domain appear to somewhat increase thermal stability, apoE4 (delta273-299) and apoE4 (delta261-299) showed stability similar to that of the apoE4 22-kDa fragment (residues 1-191) when denatured with guanidine-HCl, indicating that residues 192-272 have a negligible effect on the stability of the C-terminal-truncated apoE4. Comparison of Trp-264 fluorescence in single Trp mutants of full-length and C-terminal-truncated apoE4 (delta273-299) indicated that the C-terminal domain structure in the latter is both less organized and cooperative. In addition, comparison of the binding of the C-terminal-truncated mutants to a hydrophobic fluorescent dye and to lipid emulsions revealed that residues 261-272 create a hydrophobic site which is critical for lipid binding. These results suggest that removal of a hydrophobic C-terminal alpha-helical segment (residues 273-299) to create C-terminal-truncated apoE4 forms found in brain leads to less organized C-terminal structure while still retaining a second alpha-helical lipid-binding region (residues 261-272) that is available for interaction with cell membranes and other proteins such as amyloid beta peptide.

Amino Acid Sequence↗

PubMed2006

Characterization of nascent HDL particles and microparticles formed by ABCA1-mediated efflux of cellular lipids to apoA-I.

The nascent HDL created by ABCA1-mediated efflux of cellular phospholipid (PL) and free (unesterified) cholesterol (FC) to apolipoprotein A-I (apoA-I) has not been defined. To address this issue, we characterized the lipid particles released when J774 mouse macrophages and human skin fibroblasts in which ABCA1 is activated are incubated with human apoA-I. In both cases, three types of nascent HDL containing two, three, or four molecules of apoA-I per particle are formed. With J774 cells, the predominant species have hydrodynamic diameters of approximately 9 and 12 nm. These discoidal HDL particles have different FC contents and PL compositions, and the presence of acidic PL causes them to exhibit alpha-electrophoretic mobility. These results are consistent with ABCA1 located in more than one membrane microenvironment being responsible for the production of the heterogeneous HDL. Activation of ABCA1 also leads to the release of apoA-I-free plasma membrane vesicles (microparticles). These larger, spherical particles released from J774 cells have the same PL composition as the 12 nm HDL and contain CD14 and ganglioside, consistent with their origin being plasma membrane raft domains. The various HDL particles and microparticles are created concurrently, and there is no precursor-product relationship between them. Importantly, a large fraction of the cellular FC effluxed from these cells by ABCA1 is located in microparticles. Collectively, these results show that the products of the apoA-I/ABCA1 interaction include discoidal HDL particles containing different numbers of apoA-I molecules. The cellular PLs and cholesterol incorporated into these nascent HDL particles originate from different cell membrane domains.

ATP Binding Cassette Transporter 1↗

PubMed2005

Structural analysis of lipoprotein E particles.

Apolipoprotein E (apoE) is a key regulator of cholesterol homeostasis. Human apoE has three common isoforms, each with different risk implications for cardiovascular and neurodegenerative disease. Neither the structure of lipoprotein E particles nor the structural consequences of the isoform differences are known. In this investigation, synthetic lipoprotein particles were prepared by complexing phospholipids with full-length apoE isoforms, or with truncated N-terminal and C-terminal domains of apoE. These particles were examined with calorimetry, electron microscopy, circular dichroism spectroscopy, and internal reflection infrared spectroscopy. Results indicate that particles made with the three full-length apoE isoforms are discoidal in shape, and structurally indistinguishable. Thus, differences in their pathological consequences are not due to gross differences in particle structure. Although apoE is predominantly helical, and the axes of the helices are parallel to the flat surfaces of the particles, the orientational order of lipid acyl chains is low and inconsistent with the belt model of lipoprotein A-I structure. Instead, the data suggest that there are at least two different types of apoE-lipid interactions within lipoprotein E particles. One type occurs between apoE helices and the edge of the lipid bilayer as in the belt model, while a second type involves apoE helices that situate in the plane of the membrane and disturb acyl chain order. These interactions allow LpE particles to form with different protein/lipid ratios, and they account for the structure of LpE particles made with only the truncated domains.

Apolipoproteins E↗

PubMed2005

Effects of the core lipid on the energetics of binding of ApoA-I to model lipoprotein particles of different sizes.

Interaction of apolipoproteins (apo) with lipid surfaces plays crucial roles in lipoprotein metabolism and cholesterol homeostasis. To elucidate the thermodynamics of binding of apoA-I to lipid, we used lipid emulsions composed of triolein (TO) and egg phosphatidylcholine (PC) as lipoprotein models. Determination of the level of binding of wild-type (WT) apoA-I and some deletion mutants to large (120 nm diameter; LEM) and small (35 nm diameter; SEM) emulsions indicated that N-terminal (residues 44-65) and C-terminal (residues 190-243 and 223-243) deletions have large effects on lipid interaction, whereas deletion of the central region (residues 123-166) has little effect. Substitution of amino acids at either L230 or L230, L233, and Y236 with proline residues also decreases the level of binding, indicating that an alpha-helix conformation in this C-terminal region is required for efficient lipid binding. Calorimetry showed that binding of WT apoA-I to SEM generates endothermic heat (DeltaH approximately 30 kcal/mol) in contrast to the exothermic heat (ca. -85 kcal/mol) generated upon binding to LEM and egg PC small unilamellar vesicles (SUV). This exothermic heat arises from an approximately 25% increase in alpha-helix content, and it drives the binding of apoA-I to LEM and SUV. There is a similar increase in alpha-helix content of apoA-I upon binding to either SEM or SUV, but the binding of apoA-I to SEM is an entropy-driven process. These results suggest that the presence of a core triglyceride modifies the highly curved SEM surface packing and thereby the thermodynamics of apoA-I binding in a manner that compensates for the exothermic heat generated by alpha-helix formation.

Apolipoprotein A-I↗

PubMed2005

Class B scavenger receptor-mediated intestinal absorption of dietary beta-carotene and cholesterol.

There is now a general consensus that the intestinal absorption of water-insoluble, dietary lipids is protein-mediated, but the assignment of protein(s) to this function is still a matter of debate. To address this issue, we measured beta-carotene and cholesterol absorption in wild-type and SR-BI knockout mice and the uptake of these lipids in vitro using brush border membrane (BBM) vesicles. From the comparison of the in vivo and in vitro results we conclude that both BBM-resident class B scavenger receptors, SR-BI and CD36, can facilitate the absorption of beta-carotene and cholesterol. SR-BI is essential for beta-carotene absorption, at least in mice on a high fat diet. This is due to the fact that the absorption of beta-carotene is restricted to the duodenum and SR-BI is the predominant receptor in the mouse duodenum. In contrast, SR-BI may be involved but is not essential for cholesterol absorption in the small intestine. The question of whether SR-BI contributes to cholesterol absorption in vivo is still unresolved. Transfection of COS-7 cells with SR-BI or CD36 confers on these cells lipid uptake properties closely resembling those of enterocytes and BBM vesicles. Both scavenger receptors facilitate the uptake of dietary lipids such as beta-carotene, free and esterified cholesterol, phospholipids, and fatty acids into COS-7 cells. This lipid uptake is effected from three different lipid donor particles: mixed bile salt micelles, phospholipid small unilamellar vesicles, and trioleoylglycerol emulsions which are all likely to be present in the small intestine. Ezetimibe, a representative of a new class of drugs that inhibit intestinal cholesterol absorption, blocks SR-BI- and CD36-facilitated uptake of cholesterol into COS-7 cells.

Animal Feed↗

PubMed2005

New HIV Drugs in Development, 2005.

Since the introduction of zidovudine 18 years ago, the treatment of HIV has been rapidly evolving. Current therapies target the HIV retrovirus successfully but contain their own perils. Active therapies with reduced adverse effects and long-term activity in the presence of, or reduced susceptibility to, antiviral resistance continue to be needed. Furthermore, more convenient agents that may facilitate adherence to therapy are a goal of new drug development. This review addresses these clinical needs, with a discussion of several new drugs currently in the clinical trial pipeline.

Journal Article↗

PubMed2005

Mechanisms of cholesterol-lowering effects of dietary insoluble fibres: relationships with intestinal and hepatic cholesterol parameters.

Fibres with a range of abilities to perturb cholesterol homeostasis were used to investigate how the serum cholesterol-lowering effects of insoluble dietary fibres are related to parameters of intestinal cholesterol absorption and hepatic cholesterol homeostasis in mice. Cholestyramine, chitosan and cellulose were used as examples of fibres with high, intermediate and low bile acid-binding capacities, respectively. The serum cholesterol levels in a control group of mice fed a high fat/high cholesterol (HFHC) diet for 3 weeks increased about 2-fold to 4.3 mm and inclusion of any of these fibres at 7.5 % of the diet prevented this increase from occurring. In addition, the amount of cholesterol accumulated in hepatic stores due to the HFHC diet was reduced by treatment with these fibres. The three kinds of fibres showed similar hypocholesterolaemic activity; however, cholesterol depletion of liver tissue was greatest with cholestyramine. The mechanisms underlying the cholesterol-lowering effect of cholestyramine were (1) decreased cholesterol (food) intake, (2) decreased cholesterol absorption efficiency, and (3) increased faecal bile acid and cholesterol excretion. The latter effects can be attributed to the high bile acid-binding capacity of cholestyramine. In contrast, incorporation of chitosan or cellulose in the diet reduced cholesterol (food) intake, but did not affect either intestinal cholesterol absorption or faecal sterol output. The present study provides strong evidence that above all satiation and satiety effects underlie the cholesterol-lowering properties of insoluble dietary fibres with moderate or low bile acid-binding capabilities.

Animals↗

PubMed2004

Two-step mechanism of binding of apolipoprotein E to heparin: implications for the kinetics of apolipoprotein E-heparan sulfate proteoglycan complex formation on cell surfaces.

The interaction of apolipoprotein E (apoE) with cell-surface heparan sulfate proteoglycans is an important step in the uptake of lipoprotein remnants by the liver. ApoE interacts predominantly with heparin through the N-terminal binding site spanning the residues around 136-150. In this work, surface plasmon resonance analysis was employed to investigate how amphipathic alpha-helix properties and basic residue organization in this region modulate binding of apoE to heparin. The apoE/heparin interaction involves a two-step process; apoE initially binds to heparin with fast association and dissociation rates, followed by a step exhibiting much slower kinetics. Circular dichroism and surface plasmon resonance experiments using a disulfide-linked mutant, in which opening of the N-terminal helix bundle was prevented, demonstrated that there is no major secondary or tertiary structural change in apoE upon heparin binding. Mutations of Lys-146, a key residue for the heparin interaction, greatly reduced the favorable free energy of binding of the first step without affecting the second step, suggesting that electrostatic interaction is involved in the first binding step. Although lipid-free apoE2 tended to bind less than apoE3 and apoE4, there were no significant differences in rate and equilibrium constants of binding among the apoE isoforms in the lipidated state. Discoidal apoE3-phospholipid complexes using a substitution mutant (K143R/K146R) showed similar binding affinity to wild type apoE3, indicating that basic residue specificity is not required for the effective binding of apoE to heparin, unlike its binding to the low density lipoprotein receptor. In addition, disruption of the alpha-helix structure in the apoE heparin binding region led to an increased favorable free energy of binding in the second step, suggesting that hydrophobic interactions contribute to the second binding step. Based on these results, it seems that cell-surface heparan sulfate proteoglycan localizes apoE-enriched remnant lipoproteins to the vicinity of receptors by fast association and dissociation.

Apolipoprotein E3↗

PubMed2004

Influence of ApoA-I structure on the ABCA1-mediated efflux of cellular lipids.

The influence of apolipoprotein (apo) A-I structure on ABCA1-mediated efflux of cellular unesterified (free) cholesterol (FC) and phospholipid (PL) is not well understood. To address this issue, we used a series of apoA-I mutants to examine the contributions of various domains in the molecule to ABCA1-mediated FC and PL efflux from mouse J774 macrophages and human skin fibroblasts. Irrespective of the cell type, deletion or disruption of the C-terminal lipid-binding domain of apoA-I drastically reduced the FC and PL efflux ( approximately 90%), indicating that the C-terminal amphipathic alpha-helix is required for high affinity microsolubilization of FC and PL. Deletion in the N-terminal region of apoA-I also reduced the lipid efflux ( approximately 30%) and increased the K(m) about 2-fold compared with wild type apoA-I, whereas deletion of the central domain (Delta123-166) had no effect on either K(m) or V(max). These results indicate that ABCA1-mediated lipid efflux is relatively insensitive to the organization of the apoA-I N-terminal helix-bundle domain. Alterations in apoA-I structure caused parallel changes in its ability to bind to a PL bilayer and to induce efflux of FC and PL. Overall, these results are consistent with a two-step model for ABCA1-mediated lipid efflux. In the first step, apoA-I binds to ABCA1 and hydrophobic alpha-helices in the C-terminal domain of apoA-I insert into the region of the perturbed PL bilayer created by the PL transport activity of ABCA1, thereby allowing the second step of lipidation of apoA-I and formation of nascent high density lipoprotein particles to occur.

ATP Binding Cassette Transporter 1↗

PubMed2004

Structure of human apolipoprotein A-IV: a distinct domain architecture among exchangeable apolipoproteins with potential functional implications.

Apolipoprotein A-IV (apoA-IV) is an exchangeable apolipoprotein that shares many functional similarities with related apolipoproteins such as apoE and apoA-I but has also been implicated as a circulating satiety factor. However, despite the fact that it contains many predicted amphipathic alpha-helical domains, relatively little is known about its tertiary structure. We hypothesized that apoA-IV exhibits a characteristic functional domain organization that has been proposed to define apoE and apoA-I. To test this, we created truncation mutants in a bacterial system that deleted amino acids from either the N- or C-terminal ends of human apoA-IV. We found that apoA-IV was less stable than apoA-I but was more highly organized in terms of its cooperativity of unfolding. Deletion of the extreme N and C termini of apoA-IV did not significantly affect the cooperativity of unfolding, but deletions past amino acid 333 on the C terminus or amino acid 61 on the N terminus had major destabilizing effects. Functionally, apoA-IV was less efficient than apoA-I at clearing multilamellar phospholipid liposomes and promoting ATP-binding cassette transporter A1-mediated cholesterol efflux. However, deletion of a C-terminal region of apoA-IV, which is devoid of predicted amphipathic alpha helices (amino acids 333-376) stimulated both of these activities dramatically. We conclude that the amphipathic alpha helices in apoA-IV form a single, large domain that may be similar to the N-terminal helical bundle domains of apoA-I and apoE but that apoA-IV lacks the C-terminal lipid-binding and cholesterol efflux-promoting domain present in these apolipoproteins. In fact, the C terminus of apoA-IV appears to reduce the ability of apoA-IV to interact with lipids and promote cholesterol efflux. This indicates that, although apoA-IV may have evolved from gene duplication events of ancestral apolipoproteins and shares the basic amphipathic helical building blocks, the overall localization of functional domains within the sequence is quite different from apoA-I and apoE.

ATP-Binding Cassette Transporters↗

PubMed2004

Aromatic residue position on the nonpolar face of class a amphipathic helical peptides determines biological activity.

The apolipoprotein A-I mimetic peptide 4F (Ac-DWFKAFYDKVAEKFKEAF-NH(2)), with four Phe residues on the nonpolar face of the amphipathic alpha-helix, is strongly anti-inflammatory, whereas two 3F analogs (3F(3) and 3F(14)) are not. To understand how changes in helix nonpolar face structure affect function, two additional 3F analogs, Ac-DKLKAFYDKVFEWAKEAF-NH(2) (3F-1) and Ac-DKWKAVYDKFAEAFKEFL-NH(2) (3F-2), were designed using the same amino acid composition as 3F(3) and 3F(14). The aromatic residues in 3F-1 and 3F-2 are near the polar-nonpolar interface and at the center of the nonpolar face of the helix, respectively. Like 4F, but in contrast to 3F(3) and 3F(14), these peptides effectively inhibited lytic peptide-induced hemolysis, oxidized phospholipid-induced monocyte chemotaxis, and scavenged lipid hydroperoxides from low density lipoprotein. High pressure liquid chromatography retention times and monolayer exclusion pressures indicated that there is no direct correlation of peptide function with lipid affinity. Fluorescence studies suggested that, although the peptides bind phospholipids similarly, the Trp residue in 4F, 3F-1, and 3F-2 is less motionally restricted than in 3F(3) and 3F(14). Based on these results and molecular modeling studies, we propose that the arrangement of aromatic residues in class A amphipathic helical molecules regulates entry of reactive oxygen species into peptide-phospholipid complexes, thereby reducing the extent of monocyte chemotaxis, an important step in atherosclerosis.

Amino Acid Sequence↗

PubMed2004

Apolipoprotein A-I-stimulated apolipoprotein E secretion from human macrophages is independent of cholesterol efflux.

Apolipoprotein A-I (apoA-I)-mediated cholesterol efflux involves the binding of apoA-I to the plasma membrane via its C terminus and requires cellular ATP-binding cassette transporter (ABCA1) activity. ApoA-I also stimulates secretion of apolipoprotein E (apoE) from macrophage foam cells, although the mechanism of this process is not understood. In this study, we demonstrate that apoA-I stimulates secretion of apoE independently of both ABCA1-mediated cholesterol efflux and of lipid binding by its C terminus. Pulse-chase experiments using (35)S-labeled cellular apoE demonstrate that macrophage apoE exists in both relatively mobile (E(m)) and stable (E(s)) pools, that apoA-I diverts apoE from degradation to secretion, and that only a small proportion of apoA-I-mobilized apoE is derived from the cell surface. The structural requirements for induction of apoE secretion and cholesterol efflux are clearly dissociated, as C-terminal deletions in recombinant apoA-I reduce cholesterol efflux but increase apoE secretion, and deletion of central helices 5 and 6 decreases apoE secretion without perturbing cholesterol efflux. Moreover, a range of 11- and 22-mer alpha-helical peptides representing amphipathic alpha-helical segments of apoA-I stimulate apoE secretion whereas only the C-terminal alpha-helix (domains 220-241) stimulates cholesterol efflux. Other alpha-helix-containing apolipoproteins (apoA-II, apoA-IV, apoE2, apoE3, apoE4) also stimulate apoE secretion, implying a positive feedback autocrine loop for apoE secretion, although apoE4 is less effective. Finally, apoA-I stimulates apoE secretion normally from macrophages of two unrelated subjects with genetically confirmed Tangier Disease (mutations C733R and c.5220-5222delTCT; and mutations A1046D and c.4629-4630insA), despite severely inhibited cholesterol efflux. We conclude that apoA-I stimulates secretion of apoE independently of cholesterol efflux, and that this represents a novel, ABCA-1-independent, positive feedback pathway for stimulation of potentially anti-atherogenic apoE secretion by alpha-helix-containing molecules including apoA-I and apoE.

ATP Binding Cassette Transporter 1↗

PubMed2004

Identification of an apolipoprotein A-I structural element that mediates cellular cholesterol efflux and stabilizes ATP binding cassette transporter A1.

Synthetic peptides were used in this study to identify a structural element of apolipoprotein (apo) A-I that stimulates cellular cholesterol efflux and stabilizes the ATP binding cassette transporter A1 (ABCA1). Peptides (22-mers) based on helices 1 (amino acids 44-65) and 10 (amino acids 220-241) of apoA-I had high lipid binding affinity but failed to mediate ABCA1-dependent cholesterol efflux, and they lacked the ability to stabilize ABCA1. The addition of helix 9 (amino acids 209-219) to either helix 1 (creates a 1/9 chimera) or 10 (9/10 peptide) endowed cholesterol efflux capability and ABCA1 stabilization activity similar to full-length apoA-I. Adding helix 9 to helix 1 or 10 had only a small effect on lipid binding affinity compared with the 22-mer peptides, indicating that helix length and/or determinants on the polar surface of the amphipathic alpha-helices is important for cholesterol efflux. Cholesterol efflux was specific for the structure created by the 1/9 and 9/10 helical combinations, as 33-mers composed of helices 1 and 3 (1/3), 2/9, and 4/9 failed to mediate cholesterol efflux in an ABCA1-dependent manner. Transposing helices 9 and 10 (10/9 peptide) did not change the class Y structure, hydrophobicity, or amphiphilicity of the helical combination, but the topography of negatively charged amino acids on the polar surface was altered, and the 10/9 peptide neither mediated ABCA1-dependent cholesterol efflux nor stabilized ABCA1 protein. These results suggest that a specific structural element possessing a linear array of acidic residues spanning two apoA-I amphipathic alpha-helices is required to mediate cholesterol efflux and stabilize ABCA1.

ATP Binding Cassette Transporter 1↗

PubMed2004

Alpha-helix formation is required for high affinity binding of human apolipoprotein A-I to lipids.

Apolipoprotein (apo) A-I is thought to undergo a conformational change during lipid association that results in the transition of random coil to alpha-helix. Using a series of deletion mutants lacking different regions along the molecule, we examined the contribution of alpha-helix formation in apoA-I to the binding to egg phosphatidylcholine (PC) small unilamellar vesicles (SUV). Binding isotherms determined by gel filtration showed that apoA-I binds to SUV with high affinity and deletions in the C-terminal region markedly decrease the affinity. Circular dichroism measurements demonstrated that binding to SUV led to an increase in alpha-helix content, but the helix content was somewhat less than in reconstituted discoidal PC.apoA-I complexes for all apoA-I variants, suggesting that the helical structure of apoA-I on SUV is different from that in discs. Isothermal titration calorimetry showed that the binding of apoA-I to SUV is accompanied by a large exothermic heat and deletions in the C-terminal regions greatly decrease the heat. Analysis of the rate of release of heat on binding, as well as the kinetics of quenching of tryptophan fluorescence by brominated PC, indicated that the opening of the N-terminal helix bundle is a rate-limiting step in apoA-I binding to the SUV surface. Significantly, the correlation of thermodynamic parameters of binding with the increase in the number of helical residues revealed that the contribution of alpha-helix formation upon lipid binding to the enthalpy and the free energy of the binding of apoA-I is -1.1 and -0.04 kcal/mol per residue, respectively. These results indicate that alpha-helix formation, especially in the C-terminal regions, provides the energetic source for high affinity binding of apoA-I to lipids.

Apolipoprotein A-I↗

PubMed2004

Helix orientation of the functional domains in apolipoprotein e in discoidal high density lipoprotein particles.

Human apolipoprotein E (apoE) mediates high affinity binding to the low density lipoprotein receptor when present on a lipidated complex. In the absence of lipid, however, apoE does not bind the receptor. Whereas the x-ray structure of lipid-free apoE3 N-terminal (NT) domain is known, the structural organization of its lipid-associated, receptor-active conformation is poorly understood. To study the organization of apoE amphipathic alpha-helices in a lipid-associated state, single tryptophan-containing apoE3 variants were employed in fluorescence quenching studies. The relative positions of the Trp residues with respect to the phospholipid component of apoE/lipid particles were established from the degree of quenching by phospholipids bearing nitroxide groups at various positions along their fatty acyl chains. Four apoE3-NT variants bearing Trp reporter groups at positions 141, 148, 155, or 162 within helix 4 and two apoE3 variants containing single Trp at positions 257 or 264 in the C-terminal (CT) domain, were reconstituted into phospholipid-containing discoidal complexes. Parallax analysis revealed that each engineered Trp residue in helix 4 of apoE3-NT, as well as those in the CT domain of apoE, localized approximately 5 A from the center of the bilayer. Circular dichroism studies revealed that lipid association induces additional helix formation in apoE. Protease protection assays suggest the flexible loop segment between the NT and CT domains may transition from unstructured to helix upon lipid association. Taken together, these data support a model wherein the alpha-helices in the receptor-binding region and the CT domain of apoE align perpendicular to the fatty acyl chains of the phospholipid bilayer. In this alignment, the residues of helix 4 are arrayed in a positively charged, curved helical segment for optimal receptor interaction.

Apolipoproteins E↗

PubMed2004

Scavenger receptor class B type I-mediated cholesteryl ester-selective uptake and efflux of unesterified cholesterol. Influence of high density lipoprotein size and structure.

Scavenger receptor (SR)-BI catalyzes the selective uptake of cholesteryl ester (CE) from high density lipoprotein (HDL) by a two-step process that involves the following: 1) binding of HDL to the receptor and 2) diffusion of the CE molecules into the cell plasma membrane. We examined the effects of the size of discoidal HDL particles containing wild-type (WT) apoA-I on selective uptake of CE and efflux of cellular free (unesterified) cholesterol (FC) from COS-7 cells expressing SR-BI to determine the following: 1) the influence of apoA-I conformation on the lipid transfer process, and 2) the contribution of receptor binding-dependent processes to the overall efflux of cellular FC. Large (10 nm diameter) reconstituted HDL bound to SR-BI better (B(max) approximately 420 versus 220 ng of apoA-I/mg cell protein), delivered more CE, and promoted more FC efflux than small ( approximately 8 nm) particles. When normalized to the number of reconstituted HDL particles bound to the receptor, the efficiencies of either CE uptake or FC efflux with these particles were the same indicating that altering the conformation of WT apoA-I modulates binding to the receptor (step 1) but does not change the efficiency of the subsequent lipid transfer (step 2); this implies that binding induces an optimal alignment of the WT apoA-I.SR-BI complex so that the efficiency of lipid transfer is always the same. FC efflux to HDL is affected both by binding of HDL to SR-BI and by the ability of the receptor to perturb the packing of FC molecules in the cell plasma membrane.

Animals↗

PubMed2004

Contributions of domain structure and lipid interaction to the functionality of exchangeable human apolipoproteins.

Exchangeable apolipoproteins function in lipid transport as structural components of lipoprotein particles, cofactors for enzymes and ligands for cell-surface receptors. Recent findings with apoA-I and apoE suggest that the tertiary structures of these two members of the human exchangeable apolipoprotein gene family are related. Characteristically, these proteins contain a series of proline-punctuated, 11- or 22-amino acid, amphipathic alpha-helical repeats that can adopt a helix bundle conformation in the lipid-free state. The amino- and carboxyl-terminal regions form separate domains with the latter being primarily responsible for lipid binding. Interaction with lipid induces changes in the conformation of the amino-terminal domain leading to alterations in function; for example, opening of the amino-terminal four-helix bundle in apolipoprotein E upon lipid binding is associated with enhanced receptor-binding activity. The concept of a two-domain structure for the larger exchangeable apolipoproteins is providing new molecular insights into how these apolipoproteins interact with lipids and other proteins, such as receptors. The ways in which structural changes induced by lipid interaction modulate the functionality of these apolipoproteins are reviewed.

Amino Acid Sequence↗