Biomedical subjects

Jere P Segrest

Publications and source records attributed to Jere P Segrest.

15 recordsLinked to original sources

PubMed2006

Novel changes in discoidal high density lipoprotein morphology: a molecular dynamics study.

ApoA-I is a uniquely flexible lipid-scavenging protein capable of incorporating phospholipids into stable particles. Here we report molecular dynamics simulations on a series of progressively smaller discoidal high density lipoprotein particles produced by incremental removal of palmitoyloleoylphosphatidylcholine via four different pathways. The starting model contained 160 palmitoyloleoylphosphatidylcholines and a belt of two antiparallel amphipathic helical lipid-associating domains of apolipoprotein (apo) A-I. The results are particularly compelling. After a few nanoseconds of molecular dynamics simulation, independent of the starting particle and method of size reduction, all simulated double belts of the four lipidated apoA-I particles have helical domains that impressively approximate the x-ray crystal structure of lipid-free apoA-I, particularly between residues 88 and 186. These results provide atomic resolution models for two of the particles produced by in vitro reconstitution of nascent high density lipoprotein particles. These particles, measuring 95 angstroms and 78 angstroms by nondenaturing gradient gel electrophoresis, correspond in composition and in size/shape (by negative stain electron microscopy) to the simulated particles with molar ratios of 100:2 and 50:2, respectively. The lipids of the 100:2 particle family form minimal surfaces at their monolayer-monolayer interface, whereas the 50:2 particle family displays a lipid pocket capable of binding a dynamic range of phospholipid molecules.

Apolipoprotein A-I↗

PubMed2006

Association of a model class A (apolipoprotein) amphipathic alpha helical peptide with lipid: high resolution NMR studies of peptide.lipid discoidal complexes.

Class A amphipathic helical peptides have been shown to mimic apolipoprotein A-I, the major protein component of high density lipoproteins and have been shown to inhibit atherosclerosis in several dyslipidemic mouse models. Previously we reported the NMR structure of Ac-18A-NH2, the base-line model class A amphipathic helical peptide in a 50% (v/v) trifluoroethanol-d3/water mixture, a membrane-mimic environment (Mishra, V. K., Palgunachari, M. N., Anantharamaiah, G. M., Jones, M. K., Segrest, J. P., and Krishna, N. R. (2001) Peptides 22, 567-573). The peptide Ac-18A-NH2 forms discoidal nascent high density lipoprotein-like particles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Because subtle structural changes in the peptide.lipid complexes have been shown to be responsible for their antiatherogenic properties, we undertook high resolution NMR studies to deduce detailed structure of recombinant peptide.1,2-dimyristoyl-sn-glycero-3-phosphocholine complexes. The peptide adopts a well defined amphipathic alpha helical structure in association with the lipid at a 1:1 peptide:lipid weight ratio. Nuclear Overhauser effect spectroscopy revealed a number of intermolecular close contacts between the aromatic residues in the hydrophobic face of the helix and the lipid acyl chain protons. The pattern of observed peptide-lipid nuclear Overhauser effects is consistent with a parallel orientation of the amphipathic alpha helix, with respect to the plane of the lipid bilayer, on the edge of the disc (the belt model). Based on the results of chemical cross-linking and molecular modeling, we propose that peptide helices are arranged in a head to tail fashion to cover the edge of the disc. This arrangement of peptides is also consistent with the pKa values of the Lys residues determined previously. Taken together, these results provide for the first time a high resolution structural view of the peptide.lipid discoidal complexes formed by a class A amphipathic alpha helical peptide.

Amino Acid Sequence↗

PubMed2006

Synthetic peptides: managing lipid disorders.

PURPOSE OF REVIEW: Recent publications related to the potential use of synthetic peptides for the management of lipid disorders and their vascular complications are reviewed. RECENT FINDINGS: The potential use of synthetic peptides for the management of lipid disorders and their vascular complications has emerged in recent years. These peptides are models of apolipoproteins, but are much smaller in size than the apolipoproteins. Oral peptides that improve the antiinflammatory properties of HDLs have been shown to potently inhibit atherosclerosis in mouse models. Injection of a peptide with a class A amphipathic helix in a rat model of diabetes dramatically reduced endothelial sloughing and improved vasoreactivity. Injected synthetic peptides have also been described that dramatically lower plasma cholesterol and restore endothelial function in a rabbit model of familial hypercholesterolemia. These studies suggest the therapeutic potential for synthetic peptides in the management of lipid disorders and their vascular complications. SUMMARY: Synthetic peptides much smaller than exchangeable human plasma apolipoproteins but with physical and chemical characteristics similar to the plasma apolipoproteins have shown promise in the management of lipid disorders and their vascular complications in animal models. The initial success of these animal studies suggests that synthetic peptides have the potential to emerge as a new therapeutic class of agents in the management of patients with lipid disorders.

Animals↗

PubMed2005

A mass spectrometric determination of the conformation of dimeric apolipoprotein A-I in discoidal high density lipoproteins.

Discoidal forms of high density lipoproteins (HDL) are critical intermediates between lipid-poor apolipoprotein A-I (apo A-I), the major protein constituent of HDL, and the mature spherical forms that comprise the bulk of circulating particles. Thus, many studies have focused on understanding apoA-I structure in discs reconstituted in vitro. Recent theoretical and experimental work supports a "belt" model for apoA-I in which repeating amphipathic helical domains run parallel to the plane of the lipid disc. However, disc-associated apoA-I can adopt several tertiary arrangements that are consistent with a belt orientation. To distinguish among these, we cross-linked near-neighbor Lys groups in homogeneous 96 A discs containing exactly two molecules of apoA-I. After delipidation and tryptic digestion, mass spectrometry was used to identify 9 intermolecular and 11 intramolecular cross-links. The cross-linking pattern strongly suggests a "double-belt" molecular arrangement for apoA-I in which two apoA-I molecules wrap around the lipid bilayer disc forming two stacked rings in an antiparallel orientation with helix 5 of each apoA-I in juxtaposition (LL5/5 orientation). The data also suggests the presence of an additional double-belt orientation with a shifted helical registry (LL5/2 orientation). Furthermore, a 78 A particle with two molecules of apoA-I fit a similar double-belt motif with evidence for conformational changes in the N-terminus and the region near helix 5. A comparison of this work to a previous study is suggestive that a third molecule of apoA-I can form a hairpin in larger particles containing three molecules of apoA-I.

Amino Acid Sequence↗

PubMed2005

Assembly of lipoprotein particles containing apolipoprotein-B: structural model for the nascent lipoprotein particle.

Apolipoprotein B (apoB) is the major protein component of large lipoprotein particles that transport lipids and cholesterol. We have developed a detailed model of the first 1000 residues of apoB using standard sequence alignment programs (ClustalW and MACAW) and the MODELLER6 package for three-dimensional homology modeling. The validity of the apoB model was supported by conservation of disulfide bonds, location of all proline residues in turns and loops, and conservation of the hydrophobic faces of the two C-terminal amphipathic beta-sheets, betaA (residues 600-763) and betaB (residues 780-1000). This model suggests a lipid-pocket mechanism for initiation of lipoprotein particle assembly. In a previous model we suggested that microsomal triglyceride transfer protein might play a structural role in completion of the lipid pocket. We no longer think this likely, but instead propose a hairpin-bridge mechanism for lipid pocket completion. Salt-bridges between four tandem charged residues (717-720) in the turn of the hairpin-bridge and four tandem complementary residues (997-1000) at the C-terminus of the model lock the bridge in the closed position, enabling the deposition of an asymmetric bilayer within the lipid pocket.

Amino Acid Sequence↗

PubMed2004

Double belt structure of discoidal high density lipoproteins: molecular basis for size heterogeneity.

We recently proposed an all-atom model for apolipoprotein (apo) A-I in discoidal high-density lipoprotein in which two monomers form stacked antiparallel helical rings rotationally aligned by interhelical salt-bridges. The model can be derived a priori from the geometry of a planar bilayer disc that constrains the hydrophobic face of a continuous amphipathic alpha helix in lipid-associated apoA-I to a plane inside of an alpha-helical torus. This constrains each apoA-I monomer to a novel conformation, that of a slightly unwound, curved, planar amphipathic alpha 11/3 helix (three turns per 11 residues). Using non-denaturing gradient gel electrophoresis, we show that dimyristoylphosphocholine discs containing two apoA-I form five distinct particles with maximal Stokes diameters of 98 A (R2-1), 106 A (R2-2), 110 A (R2-3), 114 A (R2-4) and 120 A (R2-5). Further, we show that the Stokes diameters of R2-1 and R2-2 are independent of the N-terminal 43 residues (the flexible domain) of apoA-I, while the flexible domain is necessary and sufficient for the formation of the three larger complexes. On the basis of these results, the conformation of apoA-I on the R2-2 disc can be modeled accurately as an amphipathic helical double belt extending the full length of the lipid-associating domain with N and C-terminal ends in direct contact. The smallest of the discs, R2-1, models as the R2-2 conformation with an antiparallel 15-18 residue pairwise segment of helixes hinged off the disc edge. The conformations of full-length apoA-I on the flexible domain-dependent discs (R2-3, R2-4 and R2-5) model as the R2-2 conformation extended on the disc edge by one, two or three of the 11-residue tandem amphipathic helical repeats (termed G1, G2 and G3), respectively, contained within the flexible domain. Although we consider these results to favor the double belt model, the topographically very similar hairpin-belt model cannot be ruled out entirely.

Apolipoprotein A-I↗

PubMed2004

Apolipoprotein B-containing lipoprotein particle assembly: lipid capacity of the nascent lipoprotein particle.

We previously proposed that the N-terminal 1000-residue betaalpha(1) domain of apolipoprotein B (apoB) forms a bulk lipid pocket homologous to that of lamprey lipovitellin. In support of this "lipid pocket" hypothesis, we demonstrated that apoB:1000 (residues 1-1000) is secreted by a stable transformant of McA-RH7777 cells as a monodisperse particle with high density lipoprotein 3 (HDL(3)) density. In contrast, apoB:931 (residues 1-931), missing only 69 residues of the sequence homologous to lipovitellin, was secreted as a particle considerably more dense than HDL(3). In the present study we have determined the stoichiometry of the lipid component of the apoB:931 and apoB:1000 particles. The secreted [(3)H]glycerol-labeled apoB:1000 particles, isolated by nondenaturing gradient gel electrophoresis, contained 50 phospholipid (PL) and 11 triacylglycerol (TAG) molecules/particle. In contrast, apoB:931 particles contained only a few molecules of PL and were devoid of TAG. The unlabeled apoB:1000 particles, isolated by immunoaffinity chromatography, contained 56 PL, 8 TAG, and 7 cholesteryl ester molecules/particle. The surface to core lipid ratio of apoB:1000-containing particles was approximately 4:1 and was not affected by oleate supplementation. Although very small amounts of microsomal triglyceride transfer protein (MTP) were associated with apoB:1000 particles, it never approached a 1:1 molar ratio of MTP to apoB. These results support a model in which (i) the first 1000 amino acid residues of apoB are competent to complete the lipid pocket without a structural requirement for MTP; (ii) a portion, or perhaps all, of the amino acid residues between 931 and 1000 of apoB-100 are critical for the formation of a stable, bulk lipid-containing nascent lipoprotein particle, and (iii) the lipid pocket created by the first 1000 residues of apoB-100 is PL-rich, suggesting a small bilayer type organization and has a maximum capacity on the order of 50 molecules of phospholipid.

Animals↗

PubMed2004

An apolipoprotein AI mimetic peptide: membrane interactions and the role of cholesterol.

The 18-amino acid amphipathic helical peptide Ac-DWFKAFYDKVAEKFKEAF-NH(2) promotes the separation of cholesterol from the phospholipid, resulting in the formation of cholesterol crystallites, even at mole fractions of cholesterol as low as 0.3. The peptide exerts a greater degree of penetration into membranes of pure phosphatidylcholine in the absence of cholesterol than into bilayers of phosphatidylcholine and cholesterol. The circular dichroism spectrum of the peptide in buffer indicates that it self-associates, leading to the formation of structures with higher helical content. However, in the presence of lipid, the peptide remains helical over a larger concentration range. The peptide undergoes a thermal transition on heating. Cholesterol has little effect on the secondary structure of the peptide; however, increased Trp emission intensity in the absence of cholesterol indicates a deeper penetration of the helix upon removal of cholesterol from the membrane. The results with these model systems demonstrate changes in peptide-lipid interactions that may be related to the observed biological properties of this peptide.

Amino Acid Sequence↗

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↗

PubMed2003

Human apolipoprotein AI mimetic peptides for the treatment of atherosclerosis.

The effects of apolipoprotein (Apo) AI mimetic peptide synthesized from D- and L-amino acids on atherosclerotic lesion formation were investigated in low-density lipoprotein (LDL) receptor-deficient mice on a Western diet and in apoE null mice. In addition, their effects on the inflammatory changes induced in LDL-receptor mice fed a Western diet following influenza A infection were studied. When apolipoprotein AI mimetic peptides synthesized from either D- or L-amino acids were administered to LDL-receptor null mice, only peptides synthesized from D-amino acids were stable in the circulation and enhanced the ability of high-density lipoprotein (HDL) to protect LDL against oxidation. Administration of the peptide D-4F to LDL-receptor null mice and apoE null mice decreased lesion size. Additionally, in LDL receptor null mice after influenza infection, D-4F treatment increased plasma HDL levels and paraoxonase activity, and inhibited increased in LDL-cholesterol and peak levels of interleukin-6 post-infection. Injection of female mice with male macrophages, and subsequent measurement of the male 'sry' gene, revealed a marked increase in macrophage traffic into the aortic arch after infection that was prevented by administration of D-4F. This indicates that: (i) oral D-4F has powerful anti-atherosclerotic properties, and (ii) the loss of the anti-inflammatory properties of HDL after influenza infection in mice is associated with increased arterial macrophage traffic that can be prevented by administration of D-4F.

Animals↗

PubMed2002

Molecular dynamics simulations on discoidal HDL particles suggest a mechanism for rotation in the apo A-I belt model.

Apolipoprotein A-I (apo A-I) is the major protein component of high-density lipoprotein (HDL) particles. Elevated levels of HDL in the bloodstream have been shown to correlate strongly with a reduced risk factor for atherosclerosis. Molecular dynamics simulations have been carried out on three separate model discoidal high-density lipoprotein particles (HDL) containing two monomers of apo A-I and 160 molecules of palmitoyloleoylphosphatidylcholine (POPC), to a time-scale of 1ns. The starting structures were on the basis of previously published molecular belt models of HDL consisting of the lipid-binding C-terminal domain (residues 44-243) wrapped around the circumference of a discoidal HDL particle. Subtle changes between two of the starting structures resulted in significantly different behavior during the course of the simulation. The results provide support for the hypothesis of Segrest et al. that helical registration in the molecular belt model of apo A-I is modulated by intermolecular salt bridges. In addition, we propose an explanation for the presence of proline punctuation in the molecular belt model, and for the presence of two 11-mer helical repeats interrupting the otherwise regular pattern of 22-mer helical repeats in the lipid-binding domain of apo A-I.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2002

Comparative models for human apolipoprotein A-I bound to lipid in discoidal high-density lipoprotein particles.

We have constructed a series of models for apolipoprotein A-I (apo A-I) bound to discoidal high-density lipoprotein (HDL) particles, based upon the molecular belt model [Segrest, J. P., et al. (1999) J. Biol. Chem. 274, 31755-31758] and helical hairpin models [Rogers, D. P., et al. (1998) Biochemistry 37, 11714-11725], and compared these with picket fence models [Phillips, J. C., et al. (1997) Biophys. J. 73, 2337-2346]. Molecular belt models for discoidal HDL particles with differing diameters are presented, illustrating that the belt model can explain the discrete changes in HDL particle size observed experimentally. Hairpin models are discussed for the binding of apo A-I to discoidal HDL particles with diameters identical to those for the molecular belt model. Two models are presented for the binding of three monomers of apo A-I to a 150 A diameter discoidal HDL particle. In one model, two monomers of apo A-I bind to the exterior of the HDL particle in an antiparallel belt, with a third monomer of apo A-I bound to the disk in a hairpin conformation. In the second model, all three monomers of apo A-I are bound to the discoidal HDL particle in a hairpin conformation. Previously published experimental data for each model are reviewed, with FRET favoring either the belt or hairpin models over the picket fence models for HDL particles with diameters of 105 A. Naturally occurring mutations appear to favor the belt model for the 105 A particles, while the 150 A HDL particles favor the presence of at least one hairpin.

Apolipoprotein A-I↗

PubMed2002

Identification of the proteoglycan binding site in apolipoprotein B48.

An initial event in atherosclerosis is the retention of lipoproteins within the intima of the vessel wall. Previously we identified Site B (residues 3359-3369) in apolipoprotein (apo) B100 as the proteoglycan binding sequence in low density lipoproteins (LDLs) and showed that the atherogenicity of apoB-containing lipoproteins is linked to their affinity for artery wall proteoglycans. However, both apoB100- and apoB48-containing lipoproteins are equally atherogenic even though Site B lies in the carboxyl-terminal half of apoB100 and is absent in apoB48. If binding to proteoglycans is a key step in atherogenesis, apoB48-containing lipoproteins must bind to proteoglycans via other proteoglycan binding sites in the amino-terminal 48% of apoB. In vitro studies have identified five clusters of basic amino acids in delipidated apoB48 that bind negatively charged glycosaminoglycans. To determine which of these sites is functional on LDL particles, we analyzed the proteoglycan binding activity of recombinant human LDLs from transgenic mice or rat hepatoma cells. Substitution of neutral amino acids for the basic amino acids in Site B-Ib (residues 84-94) abolished the proteoglycan binding activity of recombinant apoB53. Carboxyl-truncated apoB80 bound biglycan with higher affinity than apoB100 and apoB48. ApoB80 in which Site B was mutated had the same affinity for proteoglycans as apoB48. These data support the hypothesis that the carboxyl terminus of apoB100 "masks" Site B-Ib, the amino-terminal proteoglycan binding site, and that this site is exposed in carboxyl-truncated forms of apoB. The presence of a proteoglycan binding site in the amino-terminal region of apoB may explain why apoB48- and apoB100-containing lipoproteins are equally atherogenic.

Amino Acid Sequence↗

PubMed2002

The N-terminal 1000 residues of apolipoprotein B associate with microsomal triglyceride transfer protein to create a lipid transfer pocket required for lipoprotein assembly.

Apolipoprotein (apo) B, the major protein component of the atherogenic low-density lipoprotein (LDL), has a pentapartite structure, NH2-betaalpha1-beta1-alpha2-beta2-alpha3-COOH, the beta domains containing multiple amphipathic beta strands and the alpha domains containing multiple amphipathic alpha helixes. We recently reported that the first 1000 residues of human apoB-100 have sequence and amphipathic motif homologies to the lipid-pocket of lamprey lipovitellin (LV) [Segrest, J. P., Jones, M. K., and Dashti, N. (1999) J. Lipid Res. 40, 1401-1416]. The lipid-pocket of LV is a small triangular space lined by three antiparallel amphipathic beta sheets, betaA, betaB, and betaD. The betaA and betaB sheets are joined together by an antiparallel alpha helical bundle, alpha domain. We proposed [Segrest, J. P., Jones, M. K., and Dashti, N. (1999) J. Lipid Res. 40, 1401-1416] that formation of a LV-like lipid-pocket is necessary for lipid-transfer to apoB-containing lipoprotein particles and that this pocket is formed by association of the region of the betaalpha1 domain homologous to the betaA and betaB sheets of LV with a betaD-like amphipathic beta sheet from microsomal triglyceride transfer protein (MTP). To test this hypothesis, we generated four truncated cDNA constructs terminating at or near the juncture of the betaalpha1 and beta1 domains: Residues 1-800 (apoB:800), 1-931 (apoB:931), 1-1000 (apoB:1000), and 1-1200 (apoB:1200). Characterization of particles secreted by stable transformants of the McA-RH7777 cell line demonstrated that (i) ApoB:800, missing the betaB domain, was secreted as a lipid-poor aggregate. (ii) ApoB:931, containing most, but not all, of the betaB domain, was secreted as lipid-poor particles unassociated with MTP. (iii) ApoB:1000, containing the entire betaB domain, was secreted as a relatively lipid-rich particle associated hydrophobically with MTP. (iv) ApoB:1200, containing the betaalpha1 domain plus 200 residues of the beta1 domain, was secreted predominantly as a lipid-poor particle but also as a minor relatively lipid-rich, MTP-associated particle. We thus have captured an intermediate in apoB-containing particle assembly, a lipid transfer competent pocket formed by association of the complete betaalpha1 domain of apoB with MTP.

Animals↗

PubMed2002

The role of non-LDL:non-HDL particles in atherosclerosis.

Elevated concentrations of circulating apolipoprotein B (apoB)-containing lipoproteins, other than low-density lipoprotein (LDL), have been implicated as causative agents for the development of atherosclerosis. A form of dyslipidemia, the atherogenic lipoprotein profile, that consists of elevated intermediate-density lipoprotein (IDL), triglycerides (TGs), dense LDL and dense very low density lipoprotein (VLDL), and low high density lipoprotein-2, occurs in 40% to 50% of patients with coronary artery disease (CAD). The recently released Adult Treatment Panel III guidelines suggest that because elevated TGs are an independent CAD risk factor, some TG-rich lipoproteins, commonly called remnant lipoproteins, must be atherogenic. Relevant to this series on diabetes, a number of studies have shown that in type 2 diabetes, the severity of CAD is positively related to the numbers of TG-rich particles in the plasma. Although less clear, other studies in type 2 diabetes suggest that elevated levels of lipoprotein (a) [Lp(a)] may also be independently associated with CAD. In this article, we summarize evidence for the role of apoB-containing lipoprotein particles other than LDL in the development of atherosclerosis and discuss methods of quantification and possible pharmacologic interventions for lowering their plasma concentrations. The particles reviewed include the TG-rich lipoproteins: VLDL and its remnants, chylomicron remnants and IDL, and the C-rich lipoprotein: Lp(a).

Apolipoproteins B↗