RESUMEN
Apolipoprotein A1 (APOA1), the major protein of high-density lipoprotein (HDL), contains 10 helical repeats that play key roles in protein-protein and protein-lipid interactions. The current structural model for HDL proposes that APOA1 forms an antiparallel dimer in which helix 5 in monomer 1 associates with helix 5 in monomer 2 along a left-left (LL5/5) interface, forming a protein complex with a 2-fold axis of symmetry centered on helix 5. However, computational studies suggest that other orientations are possible. To test this idea, we used a zero-length chemical cross-linking reagent that forms covalent bonds between closely apposed basic and acidic residues. Using proteolytic digestion and tandem mass spectrometry, we identified amino acids in the central region of the antiparallel APOA1 dimer of HDL that were in close contact. As predicted by the current model, we found six intermolecular cross-links that were consistent with the antiparallel LL5/5 registry. However, we also identified three intermolecular cross-links that were consistent with the antiparallel LL5/4 registry. The LL5/5 is the major structural conformation of the two complexes in both reconstituted discoidal HDL particles and in spherical HDL from human plasma. Molecular dynamic simulations suggest that that LL5/5 and LL5/4 APOA1 dimers possess similar free energies of dimerization, with LL5/5 having the lowest free energy. Our observations indicate that phospholipidated APOA1 in HDL forms different antiparallel dimers that could play distinct roles in enzyme regulation, assembly of specific protein complexes, and the functional properties of HDL in humans.
Asunto(s)
Apolipoproteína A-I/metabolismo , Lipoproteínas HDL/metabolismo , Multimerización de Proteína , Secuencia de Aminoácidos , Apolipoproteína A-I/química , Reactivos de Enlaces Cruzados/química , Humanos , Modelos Moleculares , Isótopos de Nitrógeno , Péptidos/químicaRESUMEN
Excessive inflammation after myocardial infarction (MI) can promote infarct expansion and adverse left ventricular (LV) remodeling. L-4F, a mimetic peptide of apolipoprotein A-I (apoA-I), exhibits anti-inflammatory and anti-atherogenic properties; however, whether L-4F imparts beneficial effects after myocardial infarction (MI) is unknown. Here we demonstrate that L-4F suppresses the expansion of blood, splenic, and myocardial pro-inflammatory monocytes and macrophages in a mouse model of reperfused MI. Changes in immune cell profiles were accompanied by alleviation of post-MI LV remodeling and dysfunction. In vitro, L-4F also inhibited pro-inflammatory and glycolytic gene expression in macrophages. In summary, L-4F treatment prevents prolonged and excessive inflammation after MI, in part through modulation of pro-inflammatory monocytes and macrophages, and improves post-MI LV remodeling. These data suggest that L-4F could be a used as a therapeutic adjunct in humans with MI to limit inflammation and alleviate the progression to heart failure.
Asunto(s)
Apolipoproteína A-I/metabolismo , Monocitos/patología , Infarto del Miocardio/fisiopatología , Péptidos/farmacología , Remodelación Ventricular/efectos de los fármacos , Animales , Plasticidad de la Célula/efectos de los fármacos , Inflamación/patología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Células RAW 264.7 , Sístole/efectos de los fármacos , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
High-density lipoprotein (HDL) and its main protein, apolipoprotein AI (apoAI), have established benefits in various cells, but whether these cytoprotective effects of HDL pertain to renal cells is unclear. We investigated the in vitro consequences of exposing damaged podocytes to normal apoAI, HDL, and apoAI mimetic (L-4F), and the in vivo effects of L-4F on kidney and atherosclerotic injury in a podocyte-specific injury model of proteinuria. In vitro, primary mouse podocytes were injured by puromycin aminonucleoside (PAN). Cellular viability, migration, production of reactive oxygen species (ROS), apoptosis, and the underlying signaling pathway were assessed. In vivo, we used a proteinuric model, Nphs1-hCD25 transgenic (NEP25+) mice, which express human CD25 on podocytes. Podocyte injury was induced by using immunotoxin (LMB2) and generated a proteinuric atherosclerosis model, NEP25+:apoE-/- mice, was generated by mating apoE-deficient (apoE-/-) mice with NEP25+ mice. Animals received L-4F or control vehicle. Renal function, podocyte injury, and atherosclerosis were assessed. PAN reduced podocyte viability, migration, and increased ROS production, all significantly lessened by apoAI, HDL, and L-4F. L-4F attenuated podocyte apoptosis and diminished PAN-induced inactivation of Janus family protein kinase-2/signal transducers and activators of transcription 3. In NEP25+ mice, L-4F significantly lessened overall proteinuria, and preserved podocyte expression of synaptopodin and cell density. Proteinuric NEP25+:apoE-/- mice had more atherosclerosis than non-proteinuric apoE-/- mice, and these lesions were significantly decreased by L-4F. Normal human apoAI, HDL, and apoAI mimetic protect against podocyte damage. ApoAI mimetic provides in vivo beneficial effects on podocytes that culminate in reduced albuminuria and atherosclerosis. The results suggest supplemental apoAI/apoAI mimetic may be a novel candidate to lessen podocyte damage and its complications.
Asunto(s)
Apolipoproteína A-I/farmacología , Enfermedades Renales/metabolismo , Podocitos , Sustancias Protectoras/farmacología , Proteinuria/metabolismo , Animales , Células Cultivadas , Humanos , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Enfermedades Renales/patología , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Lipoproteínas HDL/farmacología , Ratones , Ratones Transgénicos , Podocitos/efectos de los fármacos , Podocitos/metabolismo , Puromicina Aminonucleósido/efectos adversosRESUMEN
Apolipoprotein A-I is the major protein component of high-density lipoproteins and fulfils important functions in lipid metabolism. Its structure consists of a chain of tandem domains of amphipathic helices. Using this protein as a template membrane scaffolding protein, class A amphipathic helical peptides were designed to support the amphipathic helix theory and later as therapeutic tools in biomedicine. Here, we investigated the lipid interactions of two apolipoprotein-A-I-derived class A amphipathic peptides, 14A (Ac-DYLKA FYDKL KEAF-NH2) and 18A (Ac-DWLKA FYDKV AEKLK EAF- NH2), including the disc-like supramolecular structures they form with phospholipids. Thus, the topologies of 14A and 18A in phospholipid bilayers have been determined by oriented solid-state NMR spectroscopy. Whereas at a peptide-to-lipid ratio of 2 mol% the peptides align parallel to the bilayer surface, at 7.5 mol% disc-like structures are formed that spontaneously orient in the magnetic field of the NMR spectrometer. From a comprehensive data set of four 15N- or 2H-labeled positions of 14A, a tilt angle, which deviates from perfectly in-planar by 14°, and a model for the peptidic rim structure have been obtained. The tilt and helical pitch angles are well suited to cover the hydrophobic chain region of the bilayer when two peptide helices form a head-to-tail dimer. Thus, the detailed topology found in this work agrees with the peptides forming the rim of nanodiscs in a double belt arrangement.
Asunto(s)
Apolipoproteína A-I/química , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación ProteicaRESUMEN
Ac-hE18A-NH2 is a dual-domain apoE mimetic peptide that possesses the putative receptor binding domain from apoE (LRKLRKRLLR, denoted hE; residues 141-150) covalently attached to lipid-associating peptide 18A. Like apoE, Ac-hE18A-NH2 reduces plasma cholesterol in animal models and exhibits anti-inflammatory properties independent of its cholesterol-reducing effect. Ac-hE18A-NH2 has already undergone phase I clinical trials as a lipid-lowering agent. To explore the therapeutic potential more, we designed and synthesized new analogues by linking É-aminohexanoic acid, octanoic acid, or myristic acid to LRRLRRRLLR-18A-NH2 ([R]hE18A-NH2) and examined the cholesterol-lowering potency in animals. The modified peptides effectively reduced plasma cholesterol in apoE-null mice fed standard chow or a Western diet; the myristyl analogue was the most effective. A single administration of the myristyl analogue reduced plasma total and LDL cholesterol in a dose-dependent manner in hypercholesterolemic cynomolgus macaques for up to 1 week despite the continuation of a cholesterol-supplemented diet. The myristyl peptide (7.4 mg/kg) reduced total and LDL cholesterol at 24 h by 64% and 74%, respectively; plasma HDL levels were modestly reduced and returned to baseline by day 7. These new analogues should exhibit enhanced potency at lower doses than Ac-hE18A-NH2, which may make them attractive therapeutic candidates for clinical trials.
Asunto(s)
Apolipoproteínas E/química , Colesterol/sangre , Péptidos/química , Péptidos/farmacología , Animales , LDL-Colesterol/sangre , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Agar , Femenino , Haplorrinos , Humanos , Péptidos y Proteínas de Señalización Intercelular , Metabolismo de los Lípidos/efectos de los fármacos , Macaca , Masculino , Ratones , Ratones Noqueados , Péptidos/sangreRESUMEN
Structural interactions that enable electron transfer to cytochrome-P450 (CYP450) from its redox partner CYP450-reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane-bound functional complex to reveal interactions between the full-length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome-b5 (cyt-b5 ), Arg 125 on the C-helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein-protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Mononucleótido de Flavina/metabolismo , Nanoestructuras/química , Péptidos/química , Sistema Enzimático del Citocromo P-450/química , Mononucleótido de Flavina/química , Modelos Moleculares , Oxidación-ReducciónRESUMEN
Although membrane environment is known to boost drug metabolism by mammalian cytochromeâ P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide-based lipid nanodiscs to "trap" the lipid boundaries of microsomal cytochromeâ P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The protein-induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/química , Esfingomielinas/química , Animales , Humanos , Cinética , Lípidos de la Membrana/química , Proteínas de la Membrana/metabolismo , Simulación de Dinámica Molecular , Nanopartículas/química , Unión ProteicaRESUMEN
PURPOSE OF REVIEW: Unregulated uptake of oxidized LDL by macrophages to form foam cells is the hallmark for atherosclerosis. The paraoxonase (PON) family of enzymes plays a critical role in attenuating atherosclerotic lesion formation by hydrolyzing lipid peroxides (LOOHs) and preventing the oxidation of LDL particles and by enhancing HDL-mediated cholesterol efflux. Findings in recent years suggest novel mechanisms by which PON isoforms interact with macrophages to regulate cholesterol metabolism and cellular function. RECENT FINDINGS: The association of PON with HDL particles facilitates binding of the particle to macrophages and ABCA1-dependent cholesterol efflux. The hydrolysis of membrane phospholipids by PON generates lysophosphatidylcholine which is shown to regulate expression of cholesterol transport proteins. The PON family also regulates multiple aspects of macrophage function. PON attenuates inflammation and prevents induction of apoptosis via activation of a scavenger receptor class B type-1-dependent signaling mechanism. PON limits macrophage-dependent oxidant formation by preventing the activation of the membrane-associated NADPH oxidase and by stabilizing mitochondria. PON also promotes the differentiation of macrophages to an anti-inflammatory phenotype. This function appears to be independent of PON enzymatic activity and, rather, is dependent on the ability of endogenous sulfhydryls to neutralize pro-inflammatory peroxides. SUMMARY: In recent years, the therapeutic efficacy of HDL-based therapies has been subject to dispute. Pharmacological approaches that target an increase in the expression and/or activity of PON may facilitate macrophage cholesterol metabolism and attenuate inflammatory injury.
Asunto(s)
Arildialquilfosfatasa/metabolismo , Colesterol/metabolismo , Macrófagos/inmunología , Animales , Humanos , Inflamación/enzimología , Inflamación/inmunología , Inflamación/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Mitocondrias/metabolismo , Estrés OxidativoRESUMEN
The site and mechanism of action of the apoA-I mimetic peptide 4F are incompletely understood. Transintestinal cholesterol efflux (TICE) is a process involved in the clearance of excess cholesterol from the body. While TICE is responsible for at least 30% of the clearance of neutral sterols from the circulation into the intestinal lumen, few pharmacological agents have been identified that modulate this pathway. We show first that circulating 4F selectively targets the small intestine (SI) and that it is predominantly transported into the intestinal lumen. This transport of 4F into the SI lumen is transintestinal in nature, and it is modulated by TICE. We also show that circulating 4F increases reverse cholesterol transport from macrophages and cholesterol efflux from lipoproteins via the TICE pathway. We identify the cause of this modulation of TICE either as 4F being a cholesterol acceptor with respect to enterocytes, from which 4F enhances cholesterol efflux, or as 4F being an intestinal chaperone with respect to TICE. Our results assign a novel role for 4F as a modulator of the TICE pathway and suggest that the anti-inflammatory functions of 4F may be a partial consequence of the codependent intestinal transport of both 4F and cholesterol.
Asunto(s)
Apolipoproteína A-I/genética , Aterosclerosis/metabolismo , Colesterol/metabolismo , Péptidos/metabolismo , Animales , Apolipoproteína A-I/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Transporte Biológico , Colesterol/sangre , Humanos , Inflamación/metabolismo , Inflamación/patología , Intestino Delgado/metabolismo , Lipoproteínas/metabolismo , Macrófagos/metabolismoRESUMEN
The apoA-I (apolipoprotein A-I) mimetic peptide 4F favours the differentiation of human monocytes to an alternatively activated M2 phenotype. The goal of the present study was to test whether the 4F-mediated differentiation of MDMs (monocyte-derived macrophages) requires the induction of an oxidative metabolic programme. 4F treatment induced several genes in MDMs that play an important role in lipid metabolism, including PPARγ (peroxisome-proliferator-activated receptor γ) and CD36. Addition of 4F was associated with a significant increase in FA (fatty acid) uptake and oxidation compared with vehicle treatment. Mitochondrial respiration was assessed by measurement of the OCR (oxygen-consumption rate). 4F increased basal and ATP-linked OCR as well as maximal uncoupled mitochondrial respiration. These changes were associated with a significant increase in ΔΨm (mitochondrial membrane potential). The increase in metabolic activity in 4F-treated MDMs was attenuated by etomoxir, an inhibitor of mitochondrial FA uptake. Finally, addition of the PPARγ antagonist T0070907 to 4F-treated MDMs reduced the expression of CD163 and CD36, cell-surface markers for M2 macrophages, and reduced basal and ATP-linked OCR. These results support our hypothesis that the 4F-mediated differentiation of MDMs to an anti-inflammatory phenotype is due, in part, to an increase in FA uptake and mitochondrial oxidative metabolism.
Asunto(s)
Apolipoproteína A-I/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Péptidos/farmacología , Antiinflamatorios/farmacología , Benzamidas/farmacología , Materiales Biomiméticos/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Metabolismo Energético , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Macrófagos/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Consumo de Oxígeno , PPAR gamma/antagonistas & inhibidores , Piridinas/farmacologíaRESUMEN
PURPOSE OF REVIEW: Randomized clinical trials have assessed the effects of several classes of drugs on plasma cholesterol levels in patients with coronary artery disease. Agents including niacin, fibrates and statins significantly lower LDL-cholesterol, but tolerance issues and undesirable side-effects are common. Residual risk may also be present in patients with persistently low HDL-cholesterol despite a reduction in LDL-cholesterol. Recent trials of drugs that increase circulating HDL-cholesterol have also been disappointing. RECENT FINDINGS: Ongoing efforts target the development of new pharmacotherapies to reduce circulating levels of atherogenic lipoproteins. The goal of this review is to discuss recent advances in the treatment of coronary artery disease and other vascular diseases characterized by an increase in circulating atherogenic lipoproteins. These include the development of inhibitors of ATP citrate lyase and proprotein convertase subtilisin/kexin type 9. We also discuss recent developments in HDL therapy, including the clinical assessment of cholesteryl ester transfer protein inhibitors and apolipoprotein E mimetic peptides. SUMMARY: Several new classes of drug are undergoing clinical evaluation that show promise for atherogenic lipoprotein reduction in patients who are statin intolerant.
Asunto(s)
Aterosclerosis/tratamiento farmacológico , Hipolipemiantes/farmacología , Lipoproteínas/metabolismo , Animales , Aterosclerosis/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Hipolipemiantes/uso terapéutico , Metabolismo de los Lípidos/efectos de los fármacos , Terapia Molecular DirigidaRESUMEN
We previously reported that i) a Western diet increased levels of unsaturated lysophosphatidic acid (LPA) in small intestine and plasma of LDL receptor null (LDLR(-/-)) mice, and ii) supplementing standard mouse chow with unsaturated (but not saturated) LPA produced dyslipidemia and inflammation. Here we report that supplementing chow with unsaturated (but not saturated) LPA resulted in aortic atherosclerosis, which was ameliorated by adding transgenic 6F tomatoes. Supplementing chow with lysophosphatidylcholine (LysoPC) 18:1 (but not LysoPC 18:0) resulted in dyslipidemia similar to that seen on adding LPA 18:1 to chow. PF8380 (a specific inhibitor of autotaxin) significantly ameliorated the LysoPC 18:1-induced dyslipidemia. Supplementing chow with LysoPC 18:1 dramatically increased the levels of unsaturated LPA species in small intestine, liver, and plasma, and the increase was significantly ameliorated by PF8380 indicating that the conversion of LysoPC 18:1 to LPA 18:1 was autotaxin dependent. Adding LysoPC 18:0 to chow increased levels of LPA 18:0 in small intestine, liver, and plasma but was not altered by PF8380 indicating that conversion of LysoPC 18:0 to LPA 18:0 was autotaxin independent. We conclude that i) intestinally derived unsaturated (but not saturated) LPA can cause atherosclerosis in LDLR(-/-) mice, and ii) autotaxin mediates the conversion of unsaturated (but not saturated) LysoPC to LPA.
Asunto(s)
Aterosclerosis/metabolismo , Dislipidemias/metabolismo , Mucosa Intestinal/metabolismo , Lisofosfolípidos/metabolismo , Animales , Aorta/efectos de los fármacos , Aterosclerosis/sangre , Aterosclerosis/inducido químicamente , Benzoxazoles/farmacología , Grasas de la Dieta/efectos adversos , Dislipidemias/sangre , Dislipidemias/inducido químicamente , Femenino , Fosfolipasas A2 Grupo IB/metabolismo , Absorción Intestinal/efectos de los fármacos , Intestinos/efectos de los fármacos , Yeyuno/efectos de los fármacos , Yeyuno/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Lisofosfatidilcolinas/farmacología , Lisofosfolípidos/química , Lisofosfolípidos/farmacología , Masculino , Ratones , Hidrolasas Diéster Fosfóricas/metabolismo , Piperazinas/farmacología , Receptores de LDL/deficienciaRESUMEN
Despite strong evidence that HDL-cholesterol levels predict atherosclerotic events in a population, attempts at using and HDL-based treatment strategy have not yet been successful. Most of the efforts to date have focused on raising plasma HDL-cholesterol levels. This brief review focuses on a different strategy, which is based on the use of 18-amino acid apoA-I-mimetic peptides. The story of these peptides spans decades and illustrates the remarkable complexity of HDL-based treatment strategies, but suggests that such a strategy may still be successful.
Asunto(s)
HDL-Colesterol/sangre , Péptidos/uso terapéutico , Peptidomiméticos/uso terapéutico , Animales , HumanosRESUMEN
Reduced levels of HDL cholesterol (HDL-C) are a strong independent predictor of coronary artery disease (CAD) risk. The major anti-atherogenic function of HDL is to mediate reverse cholesterol transport. This response is highly dependent on apoA-I and apoE, protein components of HDL. Randomized clinical trials have assessed effects of several classes of drugs on plasma cholesterol levels in CAD patients. Agents including cholestyramine, fibrates, niacin, and statins significantly lower LDL cholesterol (LDL-C) and induce modest increases in HDL-C, but tolerance issues and undesirable side effects are common. Additionally, residual risk may be present in patients with persistently low HDL-C and other complications despite a reduction in LDL-C. These observations have fueled interest in the development of new pharmacotherapies that positively impact circulating lipoproteins. The goal of this review is to discuss the therapeutic potential of synthetic apolipoprotein mimetic peptides. These include apoA-I mimetic peptides that have undergone initial clinical assessment. We also discuss newer apoE mimetics that mediate the clearance of atherogenic lipids from the circulation and possess anti-inflammatory properties. One of these (AEM-28) has recently been given orphan drug status and is undergoing clinical trials.
Asunto(s)
Apolipoproteína A-I , Apolipoproteínas E , Materiales Biomiméticos/uso terapéutico , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Hipolipemiantes/uso terapéutico , Animales , HDL-Colesterol/sangre , Enfermedad de la Arteria Coronaria/sangre , HumanosRESUMEN
BACKGROUND: It is critical to develop new metrics to determine whether HDL is cardioprotective in humans. One promising approach is HDL particle concentration (HDL-P), the size and concentration of HDL in plasma. However, the 2 methods currently used to determine HDL-P yield concentrations that differ >5-fold. We therefore developed and validated an improved approach to quantify HDL-P, termed calibrated ion mobility analysis (calibrated IMA). METHODS: HDL was isolated from plasma by ultracentrifugation, introduced into the gas phase with electrospray ionization, separated by size, and quantified by particle counting. We used a calibration curve constructed with purified proteins to correct for the ionization efficiency of HDL particles. RESULTS: The concentrations of gold nanoparticles and reconstituted HDLs measured by calibrated IMA were indistinguishable from concentrations determined by orthogonal methods. In plasma of control (n = 40) and cerebrovascular disease (n = 40) participants, 3 subspecies of HDL were reproducibility measured, with an estimated total HDL-P of 13.4 (2.4) µmol/L. HDL-C accounted for 48% of the variance in HDL-P. HDL-P was significantly lower in participants with cerebrovascular disease (P = 0.002), and this difference remained significant after adjustment for HDL cholesterol concentrations (P = 0.02). CONCLUSIONS: Calibrated IMA accurately determined the concentration of gold nanoparticles and synthetic HDL, strongly suggesting that the method could accurately quantify HDL particle concentration. The estimated stoichiometry of apolipoprotein A-I determined by calibrated IMA was 3-4 per HDL particle, in agreement with current structural models. Furthermore, HDL-P was associated with cardiovascular disease status in a clinical population independently of HDL cholesterol.
Asunto(s)
Apolipoproteína A-I/sangre , HDL-Colesterol/sangre , Lipoproteínas HDL/sangre , Factores de Edad , Apolipoproteína A-I/aislamiento & purificación , Trastornos Cerebrovasculares/sangre , HDL-Colesterol/aislamiento & purificación , Femenino , Oro/química , Humanos , Iones/química , Lipoproteínas HDL/aislamiento & purificación , Masculino , Nanopartículas del Metal/química , Tamaño de la Partícula , Reproducibilidad de los Resultados , Factores Sexuales , UltracentrifugaciónRESUMEN
Lipid-bilayer nanodiscs provide a stable, native-like membrane environment for the functional and structural studies of membrane proteins and other membrane-binding molecules. Peptide-based nanodiscs having unique properties are developed for membrane protein studies and other biological applications. While the self-assembly process rendering the formation of peptide-nanodiscs is attractive, it is important to understand the stability and suitability of these nanodisc systems for membrane protein studies. In this study, we investigated the nanodiscs formation by the anti-inflammatory and tumor-suppressing peptide AEM28. AEM28 is a chimeric peptide containing a cationic-rich heparan sulfate proteoglycan- (HSPG)-binding domain from human apolipoprotein E (hapoE) (141-150) followed by the 18A peptide's amino acid sequence. AEM28-based nanodiscs made with different types of lipids were characterized using various biophysical techniques and compared with the nanodiscs formed using 2F or 4F peptides. Variable temperature dynamic light-scattering and 31P NMR experiments indicated the fusion and size heterogeneity of nanodiscs at high temperatures. The suitability of AEM28 and Ac-18A-NH2- (2F-) based nanodiscs for studying membrane proteins is demonstrated by reconstituting and characterizing a drug-metabolizing enzyme, cytochrome-P450 (CYP450), or the redox complex CYP450-CYP450 reductase. AEM28 and 2F were also tested for their efficacies in solubilizing E. coli membranes to understand the possibility of using them for detergent-free membrane protein isolation. Our experimental results suggest that AEM28 nanodiscs are suitable for studying membrane proteins with a net positive charge, whereas 2F-based nanodiscs are compatible with any membrane proteins and their complexes irrespective of their charge. Furthermore, both peptides solubilized E. coli cell membranes, indicating their use in membrane protein isolation and other applications related to membrane solubilization.
Asunto(s)
Proteínas de la Membrana , Nanoestructuras , Humanos , Proteínas de la Membrana/química , Nanoestructuras/química , Escherichia coli/metabolismo , Péptidos/química , Membrana Dobles de Lípidos/químicaRESUMEN
We recently reported that levels of unsaturated lysophosphatidic acid (LPA) in the small intestine significantly correlated with the extent of aortic atherosclerosis in LDL receptor-null (LDLRâ»/â») mice fed a Western diet (WD). Here we demonstrate that WD increases unsaturated (but not saturated) LPA levels in the small intestine of LDLRâ»/â» mice and causes changes in small intestine gene expression. Confirmation of microarray analysis by quantitative RT-PCR showed that adding transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to WD prevented many WD-mediated small intestine changes in gene expression. If instead of feeding WD, unsaturated LPA was added to chow and fed to the mice: i) levels of LPA in the small intestine were similar to those induced by feeding WD; ii) gene expression changes in the small intestine mimicked WD-mediated changes; and iii) changes in plasma serum amyloid A, total cholesterol, triglycerides, HDL-cholesterol levels, and the fast-performance liquid chromatography lipoprotein profile mimicked WD-mediated changes. Adding Tg6F (but not control tomatoes) to LPA-supplemented chow prevented the LPA-induced changes. We conclude that: i) WD-mediated systemic inflammation and dyslipidemia may be in part due to WD-induced increases in small intestine LPA levels; and ii) Tg6F reduces WD-mediated systemic inflammation and dyslipidemia by preventing WD-induced increases in LPA levels in the small intestine.
Asunto(s)
Dieta/efectos adversos , Dislipidemias/etiología , Dislipidemias/prevención & control , Intestino Delgado/metabolismo , Lisofosfolípidos/metabolismo , Peptidomiméticos/metabolismo , Solanum lycopersicum/genética , Animales , Apolipoproteína A-I/metabolismo , Dislipidemias/sangre , Dislipidemias/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/sangre , Inflamación/etiología , Inflamación/metabolismo , Inflamación/prevención & control , Intestino Delgado/efectos de los fármacos , Lisofosfolípidos/administración & dosificación , Lisofosfolípidos/sangre , Lisofosfolípidos/farmacología , Ratones , Plantas Modificadas Genéticamente , Receptores de LDL/deficiencia , Mundo OccidentalRESUMEN
Transgenic tomato plants were constructed with an empty vector (EV) or a vector expressing an apoA-I mimetic peptide, 6F. EV or 6F tomatoes were harvested, lyophilized, ground into powder, added to Western diet (WD) at 2.2% by weight, and fed to LDL receptor-null (LDLR(-/-)) mice at 45 mg/kg/day 6F. After 13 weeks, the percent of the aorta with lesions was 4.1 ± 4%, 3.3 ± 2.4%, and 1.9 ± 1.4% for WD, WD + EV, and WD + 6F, respectively (WD + 6F vs. WD, P = 0.0134; WD + 6F vs. WD + EV, P = 0.0386; WD + EV vs. WD, not significant). While body weight did not differ, plasma serum amyloid A (SAA), total cholesterol, triglycerides, and lysophosphatidic acid (LPA) levels were less in WD + 6F mice; P < 0.0295. HDL cholesterol and paroxonase-1 activity (PON) were higher in WD + 6F mice (P = 0.0055 and P = 0.0254, respectively), but not in WD + EV mice. Plasma SAA, total cholesterol, triglycerides, LPA, and 15-hydroxyeicosatetraenoic acid (HETE) levels positively correlated with lesions (P < 0.0001); HDL cholesterol and PON were inversely correlated (P < 0.0001). After feeding WD + 6F: i) intact 6F was detected in small intestine (but not in plasma); ii) small intestine LPA was decreased compared with WD + EV (P < 0.0469); and iii) small intestine LPA 18:2 positively correlated with the percent of the aorta with lesions (P < 0.0179). These data suggest that 6F acts in the small intestine and provides a novel approach to oral apoA-I mimetic therapy.
Asunto(s)
Apolipoproteína A-I/química , Péptidos/química , Péptidos/uso terapéutico , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/tratamiento farmacológico , Colesterol/sangre , Femenino , Ácidos Hidroxieicosatetraenoicos/sangre , Intestino Delgado/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Lisofosfolípidos/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Péptidos/genética , Péptidos/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Triglicéridos/sangreRESUMEN
The cationic single domain peptide mR18L has demonstrated lipid-lowering and anti-atherogenic properties in different dyslipidemic mouse models. Lipopolysaccharide (LPS)-mediated inflammation is considered as one of the potential triggers for atherosclerosis. Here, we evaluated anti-inflammatory effects of mR18L peptide against LPS-mediated inflammation. First, we tested the efficacy and tolerance of 1, 2.5 and 5mg/kg mR18L in normolipidemic rats stimulated with 5mg/kg LPS. LPS and then mR18L were injected in different intraperitoneal regions. By 2h post LPS, mR18L inhibited LPS-mediated plasma TNF-α elevation at all doses, with the effect being stronger for 2.5mg/kg (P<0.05 vs. 1mg/kg, non-significant vs. 5mg/kg). In a similar model, 2.5mg/kg mR18L reduced LPS-mediated inflammation in the liver, as assessed by microscopic examination of liver sections and measurements of iNOS expression in the liver tissue. In plasma, 2.5mg/kg mR18L decreased levels of TNF-α and IL-6, decreased endotoxin activity and enhanced HDL binding to LPS. In another similar experiment, mR18L administered 1h post LPS, prevented elevation of plasma triglycerides by 6h post LPS and increased plasma activity of anti-oxidant enzyme paraoxonase 1, along with noted trends in reducing plasma levels of endotoxin and IL-6. Surface plasmon resonance study revealed that mR18L readily binds LPS. We conclude that mR18L exerts anti-endotoxin activity at least in part due to direct LPS-binding and LPS-neutralizing effects. We suggest that anti-endotoxin activity of mR18L is an important anti-inflammatory property, which may increase anti-atherogenic potential of this promising orally active lipid-lowering peptide.
Asunto(s)
Hipolipemiantes/farmacología , Inflamación/prevención & control , Lípidos/sangre , Lipopolisacáridos/toxicidad , Hígado/efectos de los fármacos , Péptidos/farmacología , Animales , Cationes , Inflamación/inducido químicamente , Hígado/patología , Ratas , Resonancia por Plasmón de SuperficieRESUMEN
OBJECTIVE: The apolipoprotein A-I (apoA-I) mimetic peptide 4F favors the differentiation of human monocytes to an anti-inflammatory phenotype and attenuates lipopolysaccharide (LPS)-induced inflammatory responses. We investigated the effects of LPS on the Toll-like receptor (TLR) signaling pathway in 4F-differentiated monocyte-derived macrophages. METHODS AND RESULTS: Monocyte-derived macrophages were pretreated with 4F or vehicle for 7 days. 4F downregulated cell-surface TLRs (4, 5, and 6) as determined by flow cytometry. 4F attenuated the LPS-dependent upregulation of genes encoding TLR1, 2, and 6 and genes of the MyD88-dependent (CD14, MyD88, TRAF6, interleukin-1 receptor-associated kinase 4, and inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta) and MyD88-independent (interferon regulatory factor 3, TANK-binding kinase 1, and Toll-interleukin 1 receptor domain-containing adaptor-inducing interferon-ß) pathways as determined by microarray analysis and quantitative reverse transcriptase polymerase chain reaction. Functional analyses of monocyte-derived macrophages showed that 4F reduced LPS-dependent TLR4 recycling, phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, activation and translocation of nuclear factor-κB and inhibited the secretion of tumor necrosis factor-α and interleukin-6 induced by LPS or lipoteichoic acid. These changes were associated with depletion of cellular cholesterol and caveolin, components of membrane lipid rafts. CONCLUSIONS: These data suggest that disruption of rafts by 4F alters the assembly of TLR-ligand complexes in cell membranes and inhibits proinflammatory gene expression in monocyte-derived macrophages, thus attenuating the responsiveness of macrophages to LPS.