Apolipoprotein A-I mimetic peptide 4F blocks sphingomyelinase-induced LDL aggregation
2015; Elsevier BV; Volume: 56; Issue: 6 Linguagem: Inglês
10.1194/jlr.m059485
ISSN1539-7262
AutoresSu Duy Nguyen, Matti Javanainen, Sami Rissanen, Hongxia Zhao, Jenni Huusko, Annukka M. Kivelä, Seppo Ylä‐Herttuala, Mohamad Navab, Alan M. Fogelman, Ilpo Vattulainen, Petri T. Kovanen, Katariina Öörni,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoLipolytic modification of LDL particles by SMase generates LDL aggregates with a strong affinity for human arterial proteoglycans and may so enhance LDL retention in the arterial wall. Here, we evaluated the effects of apoA-I mimetic peptide 4F on structural and functional properties of the SMase-modified LDL particles. LDL particles with and without 4F were incubated with SMase, after which their aggregation, structure, and proteoglycan binding were analyzed. At a molar ratio of L-4F to apoB-100 of 2.5 to 20:1, 4F dose-dependently inhibited SMase-induced LDL aggregation. At a molar ratio of 20:1, SMase-induced aggregation was fully blocked. Binding of 4F to LDL particles inhibited SMase-induced hydrolysis of LDL by 10% and prevented SMase-induced LDL aggregation. In addition, the binding of the SMase-modified LDL particles to human aortic proteoglycans was dose-dependently inhibited by pretreating LDL with 4F. The 4F stabilized apoB-100 conformation and inhibited SMase-induced conformational changes of apoB-100. Molecular dynamic simulations showed that upon binding to protein-free LDL surface, 4F locally alters membrane order and fluidity and induces structural changes to the lipid layer. Collectively, 4F stabilizes LDL particles by preventing the SMase-induced conformational changes in apoB-100 and so blocks SMase-induced LDL aggregation and the resulting increase in LDL retention. Lipolytic modification of LDL particles by SMase generates LDL aggregates with a strong affinity for human arterial proteoglycans and may so enhance LDL retention in the arterial wall. Here, we evaluated the effects of apoA-I mimetic peptide 4F on structural and functional properties of the SMase-modified LDL particles. LDL particles with and without 4F were incubated with SMase, after which their aggregation, structure, and proteoglycan binding were analyzed. At a molar ratio of L-4F to apoB-100 of 2.5 to 20:1, 4F dose-dependently inhibited SMase-induced LDL aggregation. At a molar ratio of 20:1, SMase-induced aggregation was fully blocked. Binding of 4F to LDL particles inhibited SMase-induced hydrolysis of LDL by 10% and prevented SMase-induced LDL aggregation. In addition, the binding of the SMase-modified LDL particles to human aortic proteoglycans was dose-dependently inhibited by pretreating LDL with 4F. The 4F stabilized apoB-100 conformation and inhibited SMase-induced conformational changes of apoB-100. Molecular dynamic simulations showed that upon binding to protein-free LDL surface, 4F locally alters membrane order and fluidity and induces structural changes to the lipid layer. Collectively, 4F stabilizes LDL particles by preventing the SMase-induced conformational changes in apoB-100 and so blocks SMase-induced LDL aggregation and the resulting increase in LDL retention. Subendothelial retention of LDL by the proteoglycans of the extracellular matrix is the key initiating event in the development of atherosclerosis (1.Skålén K. Gustafsson M. Rydberg E.K. Hulten L.M. Wiklund O. Innerarity T.L. Boren J. Subendothelial retention of atherogenic lipoproteins in early atherosclerosis.Nature. 2002; 417: 750-754Crossref PubMed Scopus (728) Google Scholar, 2.Tabas I. Williams K.J. Boren J. Subendothelial lipoprotein retention as the initiating process in atherosclerosis: update and therapeutic implications.Circulation. 2007; 116: 1832-1844Crossref PubMed Scopus (977) Google Scholar). Modification of the retained LDL by lipolytic and proteolytic enzymes present in the arterial intima induces aggregation and/or fusion of the modified LDL particles (3.Oörni K. Pentikainen M.O. Ala-Korpela M. Kovanen P.T. Aggregation, fusion, and vesicle formation of modified low density lipoprotein particles: molecular mechanisms and effects on matrix interactions.J. Lipid Res. 2000; 41: 1703-1714Abstract Full Text Full Text PDF PubMed Google Scholar). Aggregation of LDL particles increases their binding strength to the proteoglycans, thus enhancing the extracellular retention of LDL in the arterial intima (3.Oörni K. Pentikainen M.O. Ala-Korpela M. Kovanen P.T. Aggregation, fusion, and vesicle formation of modified low density lipoprotein particles: molecular mechanisms and effects on matrix interactions.J. Lipid Res. 2000; 41: 1703-1714Abstract Full Text Full Text PDF PubMed Google Scholar, 4.Oörni K. Posio P. Ala-Korpela M. Jauhiainen M. Kovanen P.T. Sphingomyelinase induces aggregation and fusion of small very low-density lipoprotein and intermediate-density lipoprotein particles and increases their retention to human arterial proteoglycans.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1678-1683Crossref PubMed Scopus (72) Google Scholar). SMase, an enzyme locally secreted by endothelial cells and macrophages in the arteries (5.Marathe S. Schissel S.L. Yellin M.J. Beatini N. Mintzer R. Williams K.J. Tabas I. Human vascular endothelial cells are a rich and regulatable source of secretory sphingomyelinase. Implications for early atherogenesis and ceramide-mediated cell signaling.J. Biol. Chem. 1998; 273: 4081-4088Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 6.Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. Zn2+-stimulated sphingomyelinase is secreted by many cell types and is a product of the acid sphingomyelinase gene.J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar), has been shown to be one of the enzymes responsible for LDL aggregation during atherogenesis (3.Oörni K. Pentikainen M.O. Ala-Korpela M. Kovanen P.T. Aggregation, fusion, and vesicle formation of modified low density lipoprotein particles: molecular mechanisms and effects on matrix interactions.J. Lipid Res. 2000; 41: 1703-1714Abstract Full Text Full Text PDF PubMed Google Scholar). Actually, LDL particles isolated from atherosclerotic arteries resemble LDL aggregates generated by incubation of LDL with SMase in vitro (7.Schissel S.L. Tweedie-Hardman J. Rapp J.H. Graham G. Williams K.J. Tabas I. Rabbit aorta and human atherosclerotic lesions hydrolyze the sphingomyelin of retained low-density lipoprotein. Proposed role for arterial-wall sphingomyelinase in subendothelial retention and aggregation of atherogenic lipoproteins.J. Clin. Invest. 1996; 98: 1455-1464Crossref PubMed Scopus (269) Google Scholar). Importantly, genetic deficiency of secretory SMase associates with a reduction of intimal lipoprotein retention and atherosclerotic lesions in mice, thereby providing evidence for a causal role for secretory SMase in the development of atherosclerotic lesions in vivo (8.Devlin C.M. Leventhal A.R. Kuriakose G. Schuchman E.H. Williams K.J. Tabas I. Acid sphingomyelinase promotes lipoprotein retention within early atheromata and accelerates lesion progression.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 1723-1730Crossref PubMed Scopus (111) Google Scholar). Therapeutic strategies to treat atherosclerosis have been developed using apoA-I mimetic peptides (9.Anantharamaiah G.M. Synthetic peptide analogs of apolipoproteins.Methods Enzymol. 1986; 128: 627-647Crossref PubMed Scopus (81) Google Scholar). These 18 amino acid peptides have no sequence homology to apoA-I, but they possess the same class A amphipathic helical structure as apoA-I (10.Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Yu N. Ansell B.J. Datta G. Garber D.W. et al.Apolipoprotein A-I mimetic peptides.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1325-1331Crossref PubMed Scopus (227) Google Scholar, 11.Navab M. Anantharamaiah G.M. Reddy S.T. Fogelman A.M. Apolipoprotein A-I mimetic peptides and their role in atherosclerosis prevention.Nat. Clin. Pract. Cardiovasc. Med. 2006; 3: 540-547Crossref PubMed Scopus (115) Google Scholar, 12.Getz G.S. Reardon C.A. Apolipoprotein A-I and A-I mimetic peptides: a role in atherosclerosis.J. Inflamm. Res. 2011; 4: 83-92Crossref PubMed Scopus (51) Google Scholar, 13.Anantharamaiah G.M. Mishra V.K. Garber D.W. Datta G. Handattu S.P. Palgunachari M.N. Chaddha M. Navab M. Reddy S.T. Segrest J.P. et al.Structural requirements for antioxidative and anti-inflammatory properties of apolipoprotein A-I mimetic peptides.J. Lipid Res. 2007; 48: 1915-1923Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Among this family of apoA-I mimetics, the most extensively studied peptide 4F is composed of 18 amino acids with the sequence of Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 (10.Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Yu N. Ansell B.J. Datta G. Garber D.W. et al.Apolipoprotein A-I mimetic peptides.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1325-1331Crossref PubMed Scopus (227) Google Scholar, 12.Getz G.S. Reardon C.A. Apolipoprotein A-I and A-I mimetic peptides: a role in atherosclerosis.J. Inflamm. Res. 2011; 4: 83-92Crossref PubMed Scopus (51) Google Scholar). The 4F peptide functionally mimics many of the biological properties of apoA-I, and it is both anti-inflammatory and antiatherogenic (10.Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Yu N. Ansell B.J. Datta G. Garber D.W. et al.Apolipoprotein A-I mimetic peptides.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1325-1331Crossref PubMed Scopus (227) Google Scholar, 11.Navab M. Anantharamaiah G.M. Reddy S.T. Fogelman A.M. Apolipoprotein A-I mimetic peptides and their role in atherosclerosis prevention.Nat. Clin. Pract. Cardiovasc. Med. 2006; 3: 540-547Crossref PubMed Scopus (115) Google Scholar, 12.Getz G.S. Reardon C.A. Apolipoprotein A-I and A-I mimetic peptides: a role in atherosclerosis.J. Inflamm. Res. 2011; 4: 83-92Crossref PubMed Scopus (51) Google Scholar, 13.Anantharamaiah G.M. Mishra V.K. Garber D.W. Datta G. Handattu S.P. Palgunachari M.N. Chaddha M. Navab M. Reddy S.T. Segrest J.P. et al.Structural requirements for antioxidative and anti-inflammatory properties of apolipoprotein A-I mimetic peptides.J. Lipid Res. 2007; 48: 1915-1923Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). In various mouse models of atherosclerosis, 4F significantly reduced the development of atherosclerotic lesions, being most effective during early atherogenesis (14.Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Wagner A.C. Frank J.S. Datta G. Garber D. et al.Oral D-4F causes formation of pre-beta high-density lipoprotein and improves high-density lipoprotein-mediated cholesterol efflux and reverse cholesterol transport from macrophages in apolipoprotein E-null mice.Circulation. 2004; 109: 3215-3220Crossref PubMed Scopus (312) Google Scholar, 15.Li X. Chyu K.Y. Faria Neto J.R. Yano J. Nathwani N. Ferreira C. Dimayuga P.C. Cercek B. Kaul S. Shah P.K. Differential effects of apolipoprotein A-I-mimetic peptide on evolving and established atherosclerosis in apolipoprotein E-null mice.Circulation. 2004; 110: 1701-1705Crossref PubMed Scopus (131) Google Scholar). Several potential mechanisms by which 4F might exert its antiatherogenic properties have been proposed. The 4F peptide induces HDL remodeling, which includes formation of pre-β-HDL with elevated paraoxonase activity, and results in promotion of cholesterol efflux and conversion of proinflammatory HDL into anti-inflammatory HDL (14.Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Wagner A.C. Frank J.S. Datta G. Garber D. et al.Oral D-4F causes formation of pre-beta high-density lipoprotein and improves high-density lipoprotein-mediated cholesterol efflux and reverse cholesterol transport from macrophages in apolipoprotein E-null mice.Circulation. 2004; 109: 3215-3220Crossref PubMed Scopus (312) Google Scholar). The antiatherogenic properties of 4F most likely involve binding and sequestering oxidized lipids, which play a critical role in atherosclerosis (16.Berliner J.A. Leitinger N. Tsimikas S. The role of oxidized phospholipids in atherosclerosis.J. Lipid Res. 2009; 50: S207-S212Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Indeed, 4F avidly binds to oxidized phospholipids and oxidized unsaturated fatty acids with affinity four to six orders of magnitude higher than apoA-I (17.Van Lenten B.J. Wagner A.C. Jung C.L. Ruchala P. Waring A.J. Lehrer R.I. Watson A.D. Hama S. Navab M. Anantharamaiah G.M. et al.Anti-inflammatory apoA-I-mimetic peptides bind oxidized lipids with much higher affinity than human apoA-I.J. Lipid Res. 2008; 49: 2302-2311Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Given the high affinity of 4F for oxidized phospholipids, which might be present in undetectable amounts in freshly isolated lipoproteins (18.Navab M. Hama S.Y. Cooke C.J. Anantharamaiah G.M. Chaddha M. Jin L. Subbanagounder G. Faull K.F. Reddy S.T. Miller N.E. et al.Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: step 1.J. Lipid Res. 2000; 41: 1481-1494Abstract Full Text Full Text PDF PubMed Google Scholar, 19.Navab M. Berliner J.A. Subbanagounder G. Hama S. Lusis A.J. Castellani L.W. Reddy S. Shih D. Shi W. Watson A.D. et al.HDL and the inflammatory response induced by LDL-derived oxidized phospholipids.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 481-488Crossref PubMed Scopus (386) Google Scholar), it is plausible to assume that an apoA-I mimetic peptide would also bind to LDL particles. Indeed, 4F was shown to bind to LDL particles, especially when LDL was supplemented with oxidized lipids (20.Meriwether D. Imaizumi S. Grijalva V. Hough G. Vakili L. Anantharamaiah G.M. Farias-Eisner R. Navab M. Fogelman A.M. Reddy S.T. et al.Enhancement by LDL of transfer of L-4F and oxidized lipids to HDL in C57BL/6J mice and human plasma.J. Lipid Res. 2011; 52: 1795-1809Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Moreover, previous studies have demonstrated that amphipathic α-helix-containing apos block phospholipase C (PLC)-induced LDL aggregation (21.Khoo J.C. Miller E. McLoughlin P. Steinberg D. Prevention of low density lipoprotein aggregation by high density lipoprotein or apolipoprotein A-I.J. Lipid Res. 1990; 31: 645-652Abstract Full Text PDF PubMed Google Scholar, 22.Liu H. Scraba D.G. Ryan R.O. Prevention of phospholipase-C induced aggregation of low density lipoprotein by amphipathic apolipoproteins.FEBS Lett. 1993; 316: 27-33Crossref PubMed Scopus (78) Google Scholar). Based on the previous information, we hypothesized that 4F would affect the susceptibility of LDL to SMase-induced aggregation. We found that 4F interacts with LDL particles and stabilizes them by preventing SMase-induced conformational changes in apoB-100 and can ultimately block SMase-induced LDL aggregation and the resulting increase in LDL retention. The amino acid sequence of L-4F is Ac-DWFKAFYDKVAEKFKEAF-NH2. The inactive control peptide called scrambled L-4F has the same overall amino acid composition as L-4F but in a sequence that does not promote amphipathic α-helix formation (Ac-DWFAKDYFKKAFVEEFAK-NH2). 4F peptides were synthesized by the solid phase peptide synthesis method previously described (23.Datta G. Chaddha M. Hama S. Navab M. Fogelman A.M. Garber D.W. Mishra V.K. Epand R.M. Epand R.F. Lund-Katz S. et al.Effects of increasing hydrophobicity on the physical-chemical and biological properties of a class A amphipathic helical peptide.J. Lipid Res. 2001; 42: 1096-1104Abstract Full Text Full Text PDF PubMed Google Scholar). Human plasma was obtained from healthy volunteers (Finnish Red Cross Blood Service). LDL from human or mouse plasma (d = 1.019–1.050 g/ml) was isolated by sequential ultracentrifugation (24.Havel R.J. Eder H.A. Bragdon J.H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6480) Google Scholar) or by rapid sequential flotation ultracentrifugation using KBr for density adjustment (25.Tong H. Knapp H.R. VanRollins M. A low temperature flotation method to rapidly isolate lipoproteins from plasma.J. Lipid Res. 1998; 39: 1696-1704Abstract Full Text Full Text PDF PubMed Google Scholar). Lipoprotein stock solutions were dialyzed against LDL buffer (150 mM NaCl, 1 mM EDTA, pH 7.4), filtered, stored at 4°C, and used within 2 weeks, during which no changes in protein conformation and particle size were observed. The amounts of lipoproteins are expressed in terms of their total protein concentrations, which were determined by BCA protein assay kit (Pierce, Rockford, IL) using BSA as the standard. To evaluate the effects of the peptides on SMase-induced LDL aggregation, the experiments were performed under several conditions as described subsequently. Human LDL particles (1 mg/ml) were incubated with 200 mU/ml (20.8 nM) of Bacillus cereus SMase (bcSMase) (Sigma-Aldrich) in 20 mM Tris (pH 7.0) buffer containing 150 mM NaCl, 2 mM CaCl2, and MgCl2 at 37°C in the presence or absence of different concentrations of apoA-I mimetic peptide (molar ratio of peptide to apoB-100 ranging from 1:1 to 20:1) for the indicated times. LDL particles (1 mg/ml) were also modified with 50 µg/ml of human recombinant SMase (a kind gift from Genzyme) in 20 mM MES buffer (pH 5.5–6.5) containing 150 mM NaCl and 50 µM ZnCl2 at 37°C in the presence or absence of different concentrations of apoA-I mimetic peptide for the indicated times, after which the lipolysis was stopped by addition of EDTA (final concentration: 10 mM) and samples were placed on ice. The degree of SMase-induced lipolysis was determined by measuring the amounts of phosphorylcholine in the samples using Amplex Red phosphorylcholine kit (Molecular Probes). LDL particles (2 mg/ml) were incubated with L-4F at 10:1 molar ratio of L-4F to apoB-100 in LDL buffer at 37°C for 30 min, followed by extensive dialysis against LDL buffer at 4°C using dialysis membrane with molecular weight cutoff of 12,000–14,000 Da. After removal of the unbound peptides, the L-4F-treated LDL (referred to as 4F-pretreated LDL hereinafter) and control LDL particles (1 mg/ml) that had not been incubated with the 4F peptide were modified with bcSMase as described previously. LDL particles (1.25 mg/ml) were incubated with 200 mU/ml of bcSMase (Sigma-Aldrich) in 20 mM Tris (pH 7.0) containing 150 mM NaCl, 2 mM CaCl2, and MgCl2 at 37°C. After an incubation for 15 min, lipolysis was stopped by the addition of EDTA (final concentration: 10 mM). Native or bcSMase-treated LDL particles (1 mg/ml) were incubated in 20 mM Tris (pH 7.0) containing 150 mM NaCl at 37°C in the presence or absence of L-4F at 10:1 molar ratio of L-4F to apoB-100 for the indicated times. Aggregation of the LDL samples modified as described previously was followed by measuring the absorbance of the LDL samples at 405 nm. The sizes of the aggregated particles were determined by dynamic light scattering (DLS) (ZetasizerNano; Malvern) as described previously (26.Sneck M. Nguyen S.D. Pihlajamaa T. Yohannes G. Riekkola M.L. Milne R. Kovanen P.T. Oorni K. Conformational changes of apoB-100 in SMase-modified LDL mediate formation of large aggregates at acidic pH.J. Lipid Res. 2012; 53: 1832-1839Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The control LDL and 4F-pretreated LDL particles at 10:1 molar ratio of L-4F to apoB-100 (0.1–1.5 mg/ml) were incubated with bcSMase in 20 mM Tris (pH 7.0) containing 150 mM NaCl, 2 mM CaCl2, and MgCl2 at 37°C for 30 min, and the degree of SMase-induced lipolysis was determined by Amplex Red phosphorylcholine kit. Km, Vmax, and Kcat were determined from the Lineweaver-Burk plot. Kcat is the turnover number; the number of substrate molecules each enzyme site converts to product per unit time. The fast-protein liquid chromatography profiles of control LDL and LDL modified under different conditions were analyzed using a high-resolution size-exclusion chromatography (SEC) Superose HR6 column connected to the ÄKTA chromatography system (GE Healthcare). The samples were centrifuged at 10,000 g for 10 min at 4°C, and a 50 µl aliquot of the supernatant was injected into the column and eluted with PBS buffer at a flow rate of 0.5 ml/min. The degree of aggregation is expressed as a percentage of the 280 nm absorbance of peak I of modified LDL to the 280 nm absorbance of peak I of control LDL. Peak areas were calculated by integration of the 280 nm absorbance using the Unicorn 5.2 software. The control and 4F-pretreated LDL particles (1 mg/ml) were modified with bcSMase in 20 mM Tris (pH 7.0) buffer containing 150 mM NaCl, 2 mM MgCl2, and 2 mM CaCl2. Samples of control LDL and 4F-pretreated LDL (50 μg/ml) were analyzed by circular dichroism (CD) at different time points as described previously (26.Sneck M. Nguyen S.D. Pihlajamaa T. Yohannes G. Riekkola M.L. Milne R. Kovanen P.T. Oorni K. Conformational changes of apoB-100 in SMase-modified LDL mediate formation of large aggregates at acidic pH.J. Lipid Res. 2012; 53: 1832-1839Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 27.Nguyen S.D. Oorni K. Lee-Rueckert M. Pihlajamaa T. Metso J. Jauhiainen M. Kovanen P.T. Spontaneous remodeling of HDL particles at acidic pH enhances their capacity to induce cholesterol efflux from human macrophage foam cells.J. Lipid Res. 2012; 53: 2115-2125Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Fluorescence emission spectra were measured for control LDL or 4F-pretreated LDL particles (0.1 mg/ml) in 20 mM Tris (pH 7.0) buffer containing 150 mM NaCl, 2 mM MgCl2, and 2 mM CaCl2 using a PerkinElmer LS 55 Fluorescence Spectrometer. The fluorescence cell holder was thermostatically maintained at 37 ± 0.1°C. The spectra were recorded from 305 to 450 nm with excitation at 295 nm and 5 nm bandwidth for excitation and emission. For each sample, 10 spectra were averaged, and blank measurements were subtracted. Proteoglycans from the intima media of human aortas obtained at autopsy within 24 h of accidental death were prepared essentially by the method of Hurt-Camejo et al. (28.Hurt-Camejo E. Camejo G. Rosengren B. Lopez F. Wiklund O. Bondjers G. Differential uptake of proteoglycan-selected subfractions of low density lipoprotein by human macrophages.J. Lipid Res. 1990; 31: 1387-1398Abstract Full Text PDF PubMed Google Scholar) and characterized as previously (29.Oörni K. Pentikainen M.O. Annila A. Kovanen P.T. Oxidation of low density lipoprotein particles decreases their ability to bind to human aortic proteoglycans. Dependence on oxidative modification of the lysine residues.J. Biol. Chem. 1997; 272: 21303-21311Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). To examine the effect of 4F on the ability of SMase-modified LDL to bind to human aortic proteoglycans, control LDL and 4F-pretreated LDL particles (molar ratio of peptide to apoB-100 ranging from 2.5:1 to 20:1) were modified with bcSMase for 15 min in 20 mM Tris (pH 7.0) buffer containing 150 mM NaCl, 2 mM MgCl2, and 2 mM CaCl2. Aliquots of the control and 4F-pretreated LDL particles were used for proteoglycan binding study. The wells in polystyrene 96-well plates were coated with 100 μl of human aortic proteoglycans (50 μg/ml) or fatty acid-free BSA (5 mg/ml) at 4°C overnight and blocked with 5% fatty acid-free BSA, 1% fat-free milk powder, and 0.05% Tween 20 in PBS for 1 h at 37°C as described previously (4.Oörni K. Posio P. Ala-Korpela M. Jauhiainen M. Kovanen P.T. Sphingomyelinase induces aggregation and fusion of small very low-density lipoprotein and intermediate-density lipoprotein particles and increases their retention to human arterial proteoglycans.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1678-1683Crossref PubMed Scopus (72) Google Scholar). The LDL aliquots were added to the proteoglycan- or BSA-coated wells in 20 mM Tris (pH 7.0) buffer containing 150 mM NaCl, 2 mM MgCl2, and 2 mM CaCl2 and 1% fatty acid-free BSA, and the plate was incubated for 1 h at 37°C. Unbound LDL particles were removed, and the wells were washed with 20 mM Tris (pH 7.0) buffer containing 30 mM NaCl, 2 mM MgCl2, and 2 mM CaCl2, and the bound lipoproteins were detected by a commercial cholesterol kit (Amplex Red; Molecular Probes). Specific binding to proteoglycans was calculated by subtracting the amounts of the lipoproteins bound to the BSA-coated wells from the amounts of lipoproteins bound to the proteoglycan-coated wells. Animal experiments and the protocols were approved by the National Animal Experiment Board. The effect of the peptide on LDL aggregation in vivo was studied in 5-month-old male LDLR−/−ApoB100/100 mice fed with a high-fat, Western-type diet (TD 88173; Harlan Teklad) for 6 weeks. The peptides were dissolved in DMSO, further diluted into NaCl, and 100 µg/mouse of either L-4F (n = 7) or only DMSO (control, n = 6) was injected via tail vein in 150 µl volume. For the injection, the mice were gently sedated with xylazine (0.1 mg/10 g) and ketamin (0.8 mg/10 g). One hour after the injection, the animals were euthanized, and EDTA-Plasma was collected for LDL isolation. LDL particles (0.15 mg/ml) were incubated with 200 mU/ml bcSMase (Sigma-Aldrich) in 20 mM Tris (pH 7.0) buffer containing 150 mM NaCl, 2 mM CaCl2, and MgCl2 at 37°C for indicated times. The sizes of the LDL particles were determined by DLS (ZetasizerNano; Malvern). To study the interactions of 4F with LDL lipids, planar trilayer systems were computationally used as a model for protein-free LDL. The lipids used in the model (see below) were POPC, palmitoyl SM, cholesterol, cholesteryl oleate, and ceramide, whose structures are well known. The model for the structure of the 4F peptide was obtained as a homology model (L3F, L14F) from the 2F peptide (30.Mishra V.K. Anantharamaiah G.M. Segrest J.P. Palgunachari M.N. Chaddha M. Sham S.W. Krishna N.R. Association of a model class A (apolipoprotein) amphipathic alpha helical peptide with lipid: high resolution NMR studies of peptide.lipid discoidal complexes.J. Biol. Chem. 2006; 281: 6511-6519Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) (PDB entry 2F95) by using the Bodil software (31.Lehtonen J.V. Still D.J. Rantanen V.V. Ekholm J. Bjorklund D. Iftikhar Z. Huhtala M. Repo S. Jussila A. Jaakkola J. et al.BODIL: a molecular modeling environment for structure-function analysis and drug design.J. Comput. Aided Mol. Des. 2004; 18: 401-419Crossref PubMed Scopus (189) Google Scholar). The trilayer-4F system was surrounded by water with salt (NaCl; see below). The force fields for POPC, SM, and cholesterol were taken from the Slipids parameter set (32.Jämbeck J.P. Lyubartsev A.P. Derivation and systematic validation of a refined all-atom force field for phosphatidylcholine lipids.J. Phys. Chem. B. 2012; 116: 3164-3179Crossref PubMed Scopus (403) Google Scholar, 33.Jämbeck J.P.M. Lyubartsev A.P. Another piece of the membrane puzzle: extending Slipids further.J. Chem. Theory Comput. 2013; 9: 774-784Crossref PubMed Scopus (201) Google Scholar, 34.Jämbeck J.P.M. Lyubartsev A.P. An extension and further validation of an all-atomistic force field for biological membranes.J. Chem. Theory Comput. 2012; 8: 2938-2948Crossref PubMed Scopus (326) Google Scholar), whereas cholesteryl oleate and ceramide were parameterized to be compatible with it (see supplementary Materials and Methods). The Amber03 force field was used for the peptides, and the TIP3P model was used for water (35.Jorgensen W.L. Chandrasekhar J. Madura J.D. Impey R.W. Klein M.L. Comparison of simple potential functions for simulating liquid water.J. Chem. Phys. 1983; 79: 926-935Crossref Scopus (29895) Google Scholar). All simulations reported in this paper were performed with the GROMACS 4.6.× simulation package (36.Pronk S. Pall S. Schulz R. Larsson P. Bjelkmar P. Apostolov R. Shirts M.R. Smith J.C. Kasson P.M. van der Spoel D. et al.GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit.Bioinformatics. 2013; 29: 845-854Crossref PubMed Scopus (5104) Google Scholar). The peptide structure was equilibrated by simulating it in water and simultaneously gradually removing restraints from its structure (Peptide_water simulations in Table 1). The heavy atoms were first restrained for 100 ps followed by a 20 ns simulation with the backbone atoms restrained. Finally, the unrestrained peptide structure was allowed to relax in water for either 20 ns or 1 µs (see below).TABLE 1The names, descriptions, and durations of the atomistic simulations in this studySimulation SetConfiguration and SimulationsDurationTrilayer_equilBoth trilayer models (with ceramide and with SM) equilibrated in experimental surface tension without the peptide present.Both trilayers: 650 nsPeptide_waterThe 4F peptide simulated in water, while the restraints were gradually discarded.100 ps + 20 ns + 1 µs/20 nsHelix_trilayerTwo helical peptides interacting with the trilayers. Six simulations (one extended) with varying initial configurations for both SM and ceramide.Both trilayers: 5 × (150/200 ns) + 600 nsUnfolded_trilayerOne peptide interacting with the trilayers. The partially unfolded peptide structure was taken from the end of the Peptide_water simulation.Both trilayers: 400 nsFree_energyA total of 21 umbrella windows for both trilayers. One peptide with the trilayer. Initial frames were extracted from the Unfolded_trilayer simulation.Most windows 50 ns, some extended to 200 nsAnnealingThe end structures of the extended Helix_trilayer simulations for the trilayer with SM heated up and subsequently cooled down.3 target temperatures, 100 ns each Open table in a new tab The trilayer systems were constructed by placing a preequilibrated cholesteryl oleate slab of 200 molecules between two lipid monolayers. Each of the monolayers consisted of 100 lipids and their composition followed that of the LDL surface monolayer (37.Hevonoja T
Referência(s)