Vinod K. Mishra

Only the Two End Helixes of Eight Tandem Amphipathic Helical Domains of Human Apo A-I Have Significant Lipid Affinity Implications for HDL Assembly

Human apolipoprotein A-I (apo A-I) possesses multiple tandem repeating 22-mer amphipathic alpha-helixes. Computer analysis and studies of model synthetic peptides and recombinant protein-lipid complexes of phospholipids have suggested that apo A-I interacts with HDL surface lipids through cooperation among its individual amphipathic helical domains. To delineate the overall lipid-associating properties of apo A-I, the first step is to understand the lipid-associating properties of individual amphipathic helical domains. To this end, we synthesized and studied each of the eight tandem repeating 22-mer domains of apo A-I: residues 44-65, 66-87, 99-120, 121-142, 143-164, 165-186, 187-208, and 220-241. Among the 22-mers, only the N- and C-terminal peptides (44-65 and 220-241) were effective in clarifying multilamellar vesicles (MLVs) of dimyristoylphosphatidylcholine (DMPC). These two peptides also exhibited the highest partition coefficient into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine liposomes, the highest exclusion pressure for penetration into an egg yolk phosphatidylcholine monolayer, and the greatest reduction in the enthalpy of the gel-to-liquid crystalline phase transition of DMPC MLVs. These results suggest that the strong, lipid-associating properties of apo A-I are localized to the N- and C-terminal amphipathic domains. Although each of the eight peptides studied has an amphipathic structure, models based on changes in residual effective amino acid hydrophobicity resulting from differing depths of helix penetration into the lipid are best able to explain the high lipid affinity possessed by the two terminal domains. Differential scanning calorimetry (DSC) studies showed that on a molar basis, apo A-I is about 10 times more effective than the most effective peptide analyzed in reducing the enthalpy of the gel-to-liquid crystalline phase transition of DMPC MLVs. Because previous proteolysis experiments coupled with the present DSC results suggest that the lipid-associating domains of apo A-I are distributed throughout the length of the 243 amino acid residues, we propose that the terminal amphipathic helical domains are involved in the initial binding of apo A-I to the lipid surface to form HDL particles, followed by cooperative binding of the middle six amphipathic helical domains, perhaps aided by salt-bridge formation between adjacent helixes arranged in an antiparallel orientation.

apoB-100 has a pentapartite structure composed of three amphipathic alpha-helical domains alternating with two amphipathic beta-strand domains. Detection by the computer program LOCATE.

Due to the great length of apolipoprotein (apo) B-100, the localization of lipid-associating domains in this protein has been difficult. To address this question, we developed a computer program called Locate that searches amino acid sequences to identify potential amphipathic alpha-helixes and beta-strands by using sets of rules for helix and strand termination. A series of model chimeric protein test datasets were created by tandem linking of amino acid sequences of multiple proteins containing four different secondary structural motifs: motif A (exchangeable plasma apolipoproteins); motif G (globular alpha-helical proteins); motif C (coiled-coil alpha-helical proteins); and motif B (beta pleated-sheet proteins). These four test datasets, as well as randomly scrambled sequences of each dataset, were analyzed by Locate using increasingly stringent parameters. Using intermediately stringent parameters under which significant numbers of amphipathic helixes were found only in the unscrambled motif A, two dense clusters of putative lipid-associating amphipathic helixes were located precisely in the middle and at the C-terminal end of apoB-100 (a sparse cluster of class G* helixes is located at the N-terminus). The dense clusters are located between residues 2103 through 2560 and 4061 through 4338 and have densities of 2.4 and 2.2 amphipathic helixes per 100 residues, respectively; under these conditions, motif A has a density of 1.4 amphipathic helixes per 100 residues. These two domains correspond closely to the two major apoB-100 lipid-associated domains at residues 2100 through 2700 and 4100 through 4500 using the principle of releasability of tryptic peptides from trypsin-treated intact low-density lipoprotein. The classes of amphipathic helixes identified within these two putative lipid-associating domains are considerably more diverse than those found in the exchangeable plasma apolipoproteins. Interestingly, apoB-48 terminates at the N-terminal edge of the middle cluster. By using a similar strategy for analysis of amphipathic beta-strands, we discovered that the two gap regions between the three amphipathic helix clusters are highly enriched in putative amphipathic beta-strands, while the three amphipathic helical domains are essentially devoid of this putative lipid-associating motif. We propose, therefore, that apoB-100 has a pentapartite structure, NH2-alpha 1-beta 1-alpha 2-beta 2-alpha 3-COOH, with alpha 1 representing a globular domain.

Structural requirements for antioxidative and anti-inflammatory properties of apolipoprotein A-I mimetic peptides

Recently, attention has been focused on pharmacological treatments that increase HDL cholesterol to prevent coronary artery disease. Despite three decades of extensive research of human apolipoprotein A-I (apoA-I), the major protein component of HDL, the molecular basis for its antiatherogenic and anti-inflammatory functions remain elusive. Another protein component of HDL, apoA-II, has structural features similar to those of apoA-I but does not possess atheroprotective properties. To understand the molecular basis for the effectiveness of apoA-I, we used model synthetic peptides. We designed analogs of the class A amphipathic helical motif in apoA-I that is responsible for solubilizing phospholipids. None of these analogs has sequence homology to apoA-I, but all are similar in their lipid-associating structural motifs. Although all of these peptide analogs interact with phospholipids to form peptide:lipid complexes, the biological properties of these analogs are different. Physical-chemical and NMR studies of these peptides have enabled the delineation of structural requirements for atheroprotective and anti-inflammatory properties in these peptides. It has been shown that peptides that interact strongly with lipid acyl chains do not have antiatherogenic and anti-inflammatory properties. In contrast, peptides that associate close to the lipid head group (and hence do not interact strongly with the lipid acyl chain) are antiatherogenic and anti-inflammatory. Understanding the structure and function of apoA-I and HDL through studies of the amphipathic helix motif may lead to peptide-based therapies for inhibiting atherosclerosis and other related inflammatory lipid disorders.

An Oral ApoJ Peptide Renders HDL Antiinflammatory in Mice and Monkeys and Dramatically Reduces Atherosclerosis in Apolipoprotein E–Null Mice

Objective—To determine the properties of a peptide synthesized from D-amino acids corresponding to residues 113 to 122 in apolipoprotein (apo) J. Methods and Results—In contrast to D-4F, D- [113–122]apoJ showed minimal self-association and helicity in the absence of lipids. D-4F increased the concentration of apoA-I with pre-&bgr; mobility in apoE-null mice whereas D- [113–122]apoJ did not. After an oral dose D- [113–122]apoJ more slowly associated with lipoproteins and was cleared from plasma much more slowly than D-4F. D- [113–122]apoJ significantly improved the ability of plasma to promote cholesterol efflux and improved high-density lipoprotein (HDL) inflammatory properties for up to 48 hours after a single oral dose in apoE-null mice, whereas scrambled D- [113–122]apoJ did not. Oral administration of 125 &mgr;g/mouse/d of D- [113–122]apoJ reduced atherosclerosis in apoE-null mice (70.2% reduction in aortic root sinus lesion area, P=4.3×10−13; 70.5% reduction by en face analysis, P=1.5×10−6). In monkeys, oral D- [113–122]apoJ rapidly reduced lipoprotein lipid hydroperoxides (LOOH) and improved HDL inflammatory properties. Adding 250 ng/mL of D-[113–122]apoJ (but not scrambled D- [113–122]apoJ) to plasma in vitro reduced LOOH and increased paraoxonase activity. Conclusions—Oral D- [113–122]apoJ significantly improves HDL inflammatory properties in mice and monkeys and inhibits lesion formation in apoE-null mice.

Inhibition of Lipopolysaccharide-Induced Inflammatory Responses by an Apolipoprotein AI Mimetic Peptide

Previous studies suggest that high-density lipoprotein and apoAI inhibit lipopolysaccharide (LPS)-induced inflammatory responses. The goal of the current study was to test the hypothesis that the apoAI mimetic peptide L-4F exerts antiinflammatory effects similar to apoAI. Pretreatment of human umbilical vein endothelial cells (HUVECs) with LPS induced the adhesion of THP-1 monocytes. Incubation of cells with LPS and L-4F (1 to 50 &mgr;g/mL) reduced THP-1 adhesion in a concentration-dependent manner. This response was associated with a significant reduction in the synthesis of cytokines, chemokines, and adhesion molecules. L-4F reduced vascular cell adhesion molecule-1 expression induced by LPS or lipid A, whereas a control peptide (Sc-4F) showed no effect. In contrast to LPS treatment, L-4F did not inhibit IL-1&bgr;- or tumor necrosis factor-&agr;–induced vascular cell adhesion molecule-1 expression. The inhibitory effect of L-4F on LPS induction of inflammatory markers was associated with reduced binding of LPS to its plasma carrier molecule, lipopolysaccharide binding protein, and decreased binding of LPS to HUVEC monolayers. LPS and L-4F in HUVEC culture medium were fractionated by fast protein liquid chromatography and were localized to the same fractions, suggesting a physical interaction between these molecules. Proinflammatory responses to LPS are associated with the binding of lipid A to cell surface receptors. The current studies demonstrate that L-4F reduces the expression of inflammatory markers induced by LPS and lipid A and suggest that apoAI peptide mimetics may be useful in the treatment of inflammation associated with endotoxemia.

Oral Small Peptides Render HDL Antiinflammatory in Mice and Monkeys and Reduce Atherosclerosis in ApoE Null Mice

A peptide containing only 4 amino acid residues (KRES) that is too small to form an amphipathic helix, reduced lipoprotein lipid hydroperoxides (LOOH), increased paraoxonase activity, increased plasma HDL-cholesterol levels, rendered HDL antiinflammatory, and reduced atherosclerosis in apoE null mice. KRES was orally effective when synthesized from either L or D-amino acids suggesting that peptide-protein interactions were not required. Remarkably, changing the order of 2 amino acids (from KRES to KERS) resulted in the loss of all biologic activity. Solubility in ethyl acetate and interaction with lipids, as determined by differential scanning calorimetry, indicated significant differences between KRES and KERS. Negative stain electron microscopy showed that KRES formed organized peptide-lipid structures whereas KERS did not. Another tetrapeptide FREL shared many of the physical-chemical properties of KRES and was biologically active in mice and monkeys when synthesized from either L- or D-amino acids. After oral administration KRES and FREL were found associated with HDL whereas KERS was not. We conclude that the ability of peptides to interact with lipids, remove LOOH and activate antioxidant enzymes associated with HDL determines their antiinflammatory and antiatherogenic properties regardless of their ability to form amphipathic helixes.

Estimates of HIV incidence from household-based prevalence surveys

Objective:To estimate HIV incidence in the general population in countries where there have been two recent household-based HIV prevalence surveys (the Dominican Republic, Mali, Niger, Tanzania, and Zambia). Methods:We applied a validated method to estimate HIV incidence using HIV prevalence measurement in two surveys. Results:We estimate incidence among men and women aged 15–44 years to be: 0.5/1000 person-years at risk in the Dominican Republic 2002–2007, 1.1/1000 in Mali 2001–2006, 0.6/1000 in Niger 2002–2006, 3.4/1000 in Tanzania 2004–2008, and 11.2/1000 in Zambia 2002–2007. The groups most at risk in these epidemics are typically 15–24-year-old women and 25–39-year-old men. Incidence appears to have declined in recent years in all countries, but only significantly among men in the Dominican Republic and Tanzania and women in Zambia. Conclusion:Using prevalence measurements to estimate incidence reveals the current level and age distribution of new infections and the trajectory of the HIV epidemic. This information is more useful than prevalence data alone and should be used to help determine priorities for interventions.

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 α 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 α 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 α helical peptide.

ApoA-I mimetic peptides with differing ability to inhibit atherosclerosis also exhibit differences in their interactions with membrane bilayers.

Two homologous apoA-I mimetic peptides, 3F-2 and 3F14, differ in their in vitro antiatherogenic properties (Epand, R. M., Epand, R. F., Sayer, B. G., Datta, G., Chaddha, M., and Anantharamaiah, G. M. (2004) J. Biol. Chem. 279, 51404-51414). In the present work, we demonstrate that the peptide 3F-2, which has more potent anti-inflammatory activity in vitro when administered intraperitoneally to female apoE null mice (20 μg/mouse/day) for 6 weeks, inhibits atherosclerosis (lesion area 15,800 ± 1000 μm2, n = 29), whereas 3F14 does not (lesion area 20,400 ± 1000 μm2, n = 26) compared with control saline administered (19,900 ± 1400 μm2, n = 22). Plasma distribution of the peptides differs in that 3F-2 preferentially associates with high density lipoprotein, whereas 3F14 preferentially associates with apoB-containing particles. After intraperitoneal injection of 14C-labeled peptides, 3F14 reaches a higher maximal concentration and has a longer half-time of elimination than 3F-2. A study of the effect of these peptides on the motional and organizational properties of phospholipid bilayers, using several NMR methods, demonstrates that the two peptides insert to different extents into membranes. 3F-2 with aromatic residues at the center of the nonpolar face partitions closer to the phospholipid head group compared with 3F14. In contrast, only 3F14 affects the terminal methyl group of the acyl chain, decreasing the 2H order parameter and at the same time also decreasing the molecular motion of this methyl group. This dual effect of 3F14 can be explained in terms of the cross-sectional shape of the amphipathic helix. These results support the proposal that the molecular basis for the difference in the biological activities of the two peptides lies with their different interactions with membranes.

Apolipoprotein E Mimetics and Cholesterol-Lowering Properties

Apolipoprotein E (apoE) is a ligand for clearance of lipoprotein remnants such as chylomicrons and very low-density lipoproteins. It has anti-atherogenic and anti-inflammatory properties. Therefore, there is extensive ongoing research to create peptides that can mimic properties of apoE. A number of synthetic peptides that encompass different regions of apoE have been studied for inhibiting inflammatory states, including Alzheimer disease. However, peptides that clear atherogenic lipoproteins, analogous to apoE, via enhanced hepatic uptake have not been previously reviewed. Toward this end, we describe the design and studies of a dual-domain apoE mimetic peptide, Ac-hE18A-NH2. This peptide consists of residues 141–150, the putative receptor-binding region of human apoE, covalently linked to a well characterized class A amphipathic helix, 18A, which has no sequence homology to any other exchangeable apolipoprotein sequences. It demonstrates dramatic effects in reducing plasma cholesterol levels in dyslipidemic mouse and rabbit models. We discuss the scientific rationale and review the literature for the design and efficacy of the peptide. Analogous to apoE, this peptide bypasses the low-density lipoprotein receptor for the hepatic uptake of atherogenic lipoproteins via heparan sulfate proteoglycan (HSPG). ApoE mimetics such as Ac-hE18A-NH2 may therefore restore or replace ligands in genetically induced hyperlipidemias to enable reduction in atherogenic lipoproteins via HSPG even in the absence of functional low-density lipoprotein receptors. Therefore, this and similar peptides may be useful in the treatment of dyslipidemic disorders such as familial hyperlipidemia and atherosclerosis.