Solubilization Of Lipids Research Articles

Steroidal scaffolds as FXR and GPBAR1 ligands: from chemistry to therapeutical application.

Bile acids (BAs) are experiencing a new life. Next to their ancestral roles in lipid digestion and solubilization, BAs are today recognized signaling molecules involved in many physiological functions. These signaling pathways involve the activation of metabolic nuclear receptors, mainly the BA sensor FXR, and the dedicated membrane G protein-coupled receptor, GPBAR1 (TGR5). As a consequence, the discovery of GPBAR1/FXR selective or dual modulators represents an important answer to the urgent demand of new pharmacological opportunity for several human diseases including dyslipidemia, cholestasis, nonalcoholic steatohepatitis, Type 2 diabetes and inflammation. Targeted oriented discovery of natural compounds and medicinal chemistry manipulation have allowed the development of promising drug candidates.

Solubilization of G protein-coupled receptors: a convenient strategy to explore lipid-receptor interaction.

G protein-coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across cell membranes and are major drug targets. Since GPCRs are integral membrane proteins, their structure and function are modulated by membrane lipids. In particular, membrane cholesterol is an important lipid in the context of GPCR function. Solubilization of integral membrane proteins is a process in which the proteins and lipids in native membranes are dissociated in the presence of a suitable amphiphilic detergent. Interestingly, solubilization offers a convenient approach to monitor lipid-receptor interaction as it results in differential extents of lipid solubilization, thereby allowing to assess the role of specific lipids on receptor function. In this review, we highlight how this solubilization strategy is utilized to decipher novel information about the structural stringency of cholesterol necessary for supporting the function of the serotonin(1A) receptor. We envision that insight in GPCR-lipid interaction would result in better understanding of GPCR function in health and disease.

Digestion of phospholipids after secretion of bile into the duodenum changes the phase behavior of bile components.

Bile components play a significant role in the absorption of dietary fat, by solubilizing the products of fat digestion. The absorption of poorly water-soluble drugs from the gastrointestinal tract is often enhanced by interaction with the pathways of fat digestion and absorption. These processes can enhance drug absorption. Thus, the phase behavior of bile components and digested lipids is of great interest to pharmaceutical scientists who seek to optimize drug solubilization in the gut lumen. This can be achieved by dosing drugs after food or preferably by formulating the drug in a lipid-based delivery system. Phase diagrams of bile salts, lecithin, and water have been available for many years, but here we investigate the association structures that occur in dilute aqueous solution, in concentrations that are present in the gut lumen. More importantly, we have compared these structures with those that would be expected to be present in the intestine soon after secretion of bile. Phosphatidylcholines are rapidly hydrolyzed by pancreatic enzymes to yield equimolar mixtures of their monoacyl equivalents and fatty acids. We constructed phase diagrams that model the association structures formed by the products of digestion of biliary phospholipids. The micelle-vesicle phase boundary was clearly identifiable by dynamic light scattering and nephelometry. These data indicate that a significantly higher molar ratio of lipid to bile salt is required to cause a transition to lamellar phase (i.e., liposomes in dilute solution). Mixed micelles of digested bile have a higher capacity for solubilization of lipids and fat digestion products and can be expected to have a different capacity to solubilize lipophilic drugs. We suggest that mixtures of lysolecithin, fatty acid, and bile salts are a better model of molecular associations in the gut lumen, and such mixtures could be used to better understand the interaction of drugs with the fat digestion and absorption pathway.

Advances in Bile Acids-Mediated Liver Injury and Liver Regeneration

Bile acids are endogenous molecules that originate from the liver and transport via bile to the intestines. They normally regulate cholesterol homeostasis, stimulate lipid solubilization and mediate metabolic signaling. Early studies implicated that disorders of bile acids compositions and concentrations can cause liver injury. Several hydrophobic bile acids are toxic and ample increases of them in liver may induce cell inflammation, apoptosis and necrosis. While the hydrophilic bile acid, such as ursodeoxycholic acid, has a therapeutic effect on cholestatic liver diseases. Further more, recent researches demonstrate that bile acids have also been implicated in stimulation of liver regeneration. The antagonistic regulation of liver injury and liver regeneration by bile acids may correlate with its composition and concentration. This review will focus on both how different bile acids and different bile acid concentrations play a critical role in liver injury and regeneration.

Bile Acids Modulate Signaling by Functional Perturbation of Plasma Membrane Domains

Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.

Soaping Up Type 2 Diabetes With Bile Acids?

Bile acids (BA) are amphipathic molecules whose physicochemical properties facilitate the solubilization of biliary cholesterol and intestinal lipids. As detergents, excessive concentrations of BA, such as in cholestatic diseases, are toxic, imposing a tight control of BA pool size and in particular its hydrophobicity level. Hence, BA synthesis is under negative control by BA themselves, which act as signaling molecules via specific pathways, including the G protein–coupled BA receptor (TGR5) and the nuclear receptors farnesoid X receptor (FXR), vitamin D receptor, and pregnane X receptor as well as different kinase signaling pathways. Recently, BA were identified as regulators of glucose, lipid, and energy metabolism with unexpected roles for TGR5 and FXR therein (1). BA are synthesized from cholesterol by the hepatic parenchymal cells through the classical and alternative pathways with cholesterol 7 α-hydroxylase (Cyp7a1) and cholesterol 27 α-hydroxylase (Cyp27a1) as first enzymes, respectively. In humans, the ratio of the primary BA cholic acid (CA)/chenodeoxycholic acid (CDCA) determines the hydrophobicity of the BA pool, a step regulated by the activity of cholesterol 12α-hydroxylase (Cyp8b1), which adds an additional OH group at C12 (Fig. 1). CDCA and CA are conjugated to glycine or taurine and secreted via the bile canaliculus into the intestine, where they are transformed through dehydroxylation by the intestinal flora into the more hydrophobic secondary BA lithocholic acid (LCA) and deoxycholic acid (DCA), respectively, before returning to the liver via the enterohepatic cycle. The expression and activity of the hepatic BA synthesis enzymes is negatively controlled by BA acting via FXR in both intestine (via FGF19 …

Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane

The zwitterionic detergent CHAPS, a derivative of the bile salts, is widely used in membrane protein solubilization. It is a “facial” detergent, having a hydrophilic side and a hydrophobic back. The objective of this work is to characterize the interaction of CHAPS with a cell membrane. To this aim, erythrocytes were incubated with a wide range of detergent concentrations in order to determine CHAPS partition behavior, and its effects on membrane lipid order, hemolytic effects, and the solubilization of membrane phospholipids and cholesterol. The results were compared with those obtained with the nonionic detergent Triton X-100. It was found that CHAPS has a low affinity for the erythrocyte membrane (partition coefficient K=0.06mM−1), and at sub-hemolytic concentrations it causes little effect on membrane lipid order. CHAPS hemolysis and phospholipid solubilization are closely correlated. On the other side, binding of Triton X-100 disorders the membrane at all levels, and has independent mechanisms for hemolysis and solubilization. Differential behavior was observed in the solubilization of phospholipids and cholesterol. Thus, the detergent resistant membranes (DRM) obtained with the two detergents will have different composition. The behaviors of the two detergents are related to the differences in their molecular structures, suggesting that CHAPS does not penetrate the lipid bilayer but binds in a flat position on the erythrocyte surface, both in intact and cholesterol depleted erythrocytes. A relevant result for Triton X-100 is that hemolysis is not directly correlated with the solubilization of membrane lipids, as it is usually assumed.

Helical domains that mediate lipid solubilization and ABCA1-specific cholesterol efflux in apolipoproteins C-I and A-II

Many of the apolipoproteins in HDL can elicit cholesterol efflux via ABCA1, a critical initial step in HDL formation. Recent work has indicated that omnipresent amphipathic helices play a critical role, and these have been studied intensively in the most common HDL protein, apolipoprotein (apo)A-I. However, little information exists about helical domain arrangement in other apolipoproteins. We studied two of the smallest apolipoproteins known to interact with ABCA1, human apoA-II and apoC-I, in terms of ability to reorganize phospholipid (PL) bilayers and to promote ABCA1-mediated cholesterol. We found that both proteins contained helical domains that were fast and slow with respect to solubilizing PL. ABCA1-medated efflux required a minimum of a bihelical polypeptide comprised of at least one each of a slow and fast lipid reorganizing domain. In both proteins, the fast helix was located at the C terminus preceded by a slow helix. Helical placement in apoC-I was not critical for ABCA1 activity, but helix swaps in apoA-II dramatically disrupted cholesterol efflux, indicating that the tertiary structure of the longer apolipoprotein is important for the pathway. This work has implications for a more complete molecular understanding of apolipoprotein-mediated cholesterol efflux.

Microstructural Investigation Of Lipid Solubilized Microemulsions Using Laser Light Scattering

Solubilization of solid lipids in oil-in-water microemulsion is an important step in the preparation of lipid nanoparticles. Oil in water microemulsion has been prepared using Tween-80 (T-80) as a surfactant and isopropyl myristate (IPM) as an oil phase with a view to utilize them for the preparation of Solid Lipid Nanoparticles (SLN). The microstructure of the microemulsions were evaluated using laser light scattering studies. From light scattering studies, it was observed that the intensity of scattered light increases on increasing the concentration of IPM at a fixed concentration of T-80 (15%), reflecting an increase in the size of the micelles. Dynamic light scattering studies show that the hydrodynamic diameter of the micelles increases on increasing the concentration of IPM. Phospholipon® 90 G (lipid) solubilized microemulsions were prepared using 1:1 w/w mixture of lipid and IPM as the oil phase. DLS studies suggest that addition of lipid did not alter the size of microemulsion droplets significantly.

Effect of Aeration Rate on the Hydrolysis and Acidification of Waste Activated Sludge in Thermophilic Aerobic Conditions

This study investigated the effect of aeration rates on the hydrolysis process of Waste Activated Sludge (WAS) with thermophilic aerobic microbes and explained by the change of solubilization of lipids, carbohydrates and proteins in sludge under different aeration rates (0.03 vvm, 0.05 vvm, 0.07 vvm, 0.09 vvm, 0.11 vvm). The results revealed that with the increase of aeration rate, the accumulation of volatile fatty acids (VFAs) in the treated sludge was decreased. Only 2 142 mg COD/L was accumulated at the ventilation rate of 0.11 vvm, while the highest accumulation which was 4 088 mg/L at the ventilation rate of 0.05 vvm. Further investigation showed that under optimal aeration rate which was 0.05 vvm, theromophilic aerobic microbes facilitated the organism hydrolysis and increased the biodegradability of WAS significantly. The concentration of carbohydrates was improved remarkably from 70 mg COD/L to 560 mg COD/L compared with the control (the process without aeration) at 65°C. Meanwhile, the concentration of protein was increased stably due to the high activity of protease, and reached the peak of 1 320 mg COD/L after 72h, then decline at the later period. The maximal soluble chemical oxygen demand (SCOD) was 5 600 mg/L and VFAs was 4 088 mg COD/L, which would be beneficial to the followed digestion process. Therefore, appropriate aeration is efficient to improve the accumulation of soluble organic matters and VFAs in WAS.

Challenges in the Development of Functional Assays of Membrane Proteins

Lipid bilayers are natural barriers of biological cells and cellular compartments. Membrane proteins integrated in biological membranes enable vital cell functions such as signal transduction and the transport of ions or small molecules. In order to determine the activity of a protein of interest at defined conditions, the membrane protein has to be integrated into artificial lipid bilayers immobilized on a surface. For the fabrication of such biosensors expertise is required in material science, surface and analytical chemistry, molecular biology and biotechnology. Specifically, techniques are needed for structuring surfaces in the micro- and nanometer scale, chemical modification and analysis, lipid bilayer formation, protein expression, purification and solubilization, and most importantly, protein integration into engineered lipid bilayers. Electrochemical and optical methods are suitable to detect membrane activity-related signals. The importance of structural knowledge to understand membrane protein function is obvious. Presently only a few structures of membrane proteins are solved at atomic resolution. Functional assays together with known structures of individual membrane proteins will contribute to a better understanding of vital biological processes occurring at biological membranes. Such assays will be utilized in the discovery of drugs, since membrane proteins are major drug targets.

Improved detection of advanced oxidation protein products in plasma

BackgroundOxidative stress has been associated with many diseases and can among others be assessed as increased levels of advanced oxidation protein products (AOPP). Current AOPP methods suffer from poor reproducibility and accuracy due to precipitation of lipids in plasma samples. We therefore aimed to develop a robust method in which plasma lipids are solubilized. MethodsPlasma was diluted with citric acid, and AOPP measured as absorbance at 340nm. The method was optimized and validated, and then used to analyze AOPP levels in plasma from healthy control subjects (HC), and in three patients groups; chronic kidney disease (CKD), primary Sjögren's Syndrome (pSS) and systemic lupus erythematosus (SLE). ResultsAOPP was detected with improved precision compared to established methods where lipids precipitate. Within- and between days variations were less than 1.4% and 2.2%, respectively. A control chart was established and the long-term reproducibility followed over six months. ConclusionsThis improved method detects plasma AOPP with significantly better reproducibility and accuracy compared to previously reported methods. Solubilization of plasma lipids before spectrophotometric measure of AOPP levels is novel. It prevents both loss of lipoproteins due to precipitation and overestimation as a result of light scattering.

Impact of Self-association on Function of Apolipoprotein A-I

Self-association is an inherent property of the lipid-free forms of several exchangeable apolipoproteins, including apolipoprotein A-I (apoA-I), the main protein component of high density lipoproteins (HDL) and an established antiatherogenic factor. Monomeric lipid-free apoA-I is believed to be the biologically active species, but abnormal conditions, such as specific natural mutations or oxidation, produce an altered state of self-association that may contribute to apoA-I dysfunction. Replacement of the tryptophans of apoA-I with phenylalanines (ΔW-apoA-I) leads to unusually large and stable self-associated species. We took advantage of this unique solution property of ΔW-apoA-I to analyze the role of self-association in determining the structure and lipid-binding properties of apoA-I as well as ATP-binding cassette A1 (ABCA1)-mediated cellular lipid release, a relevant pathway in atherosclerosis. Monomeric ΔW-apoA-I and wild-type apoA-I activated ABCA1-mediated cellular lipid release with similar efficiencies, whereas the efficiency of high order self-associated species was reduced to less than 50%. Analysis of specific self-associated subclasses revealed that different factors influence the rate of HDL formation in vitro and ABCA1-mediated lipid release efficiency. The α-helix-forming ability of apoA-I is the main determinant of in vitro lipid solubilization rates, whereas loss of cellular lipid release efficiency is mainly caused by reduced structural flexibility by formation of stable quaternary interactions. Thus, stabilization of self-associated species impairs apoA-I biological activity through an ABCA1-mediated mechanism. These results afford mechanistic insights into the ABCA1 reaction and suggest self-association as a functional feature of apoA-I. Physiologic mechanisms may alter the native self-association state and contribute to apoA-I dysfunction.

Bile acids as colon carcinogens and coffee ingredients as antagonists

High dietary fat is associated with increased colon cancer risk. A possible mechanism is that dietary fat stimulates hepatocytes to secrete bile acids into bile canaliculi which are necessary for solubilization and absorption of lipids in the gut (Hewitt et al. 2007; Hoehme et al. 2010; Hofmann 1999a, b; Modica et al. 2008). Bile acids have been reported to act as tumor promoters in the colon (Degirolamo et al. 2011; Gadaleta et al. 2010; Modica et al. 2008), most probably via generation of reactive oxygen species, a mechanism of central interest for our journal (Bhusari et al. 2010; Bolt and Hengstler 2010; Degirolamo et al. 2011; Kawai et al. 2010; Kell 2010; Kumar and Gill 2009; Michalowicz 2010). The editors are happy that Carol Bernstein and colleagues from the University of Arizona contributed a study that gives additional insight into the role of bile acids as colon carcinogens (Bernstein et al. 2011; this issue). They demonstrated that deoxycholic acid at high physiological levels induced colon carcinomas in mice. Addition of the antioxidative compound, chlorogenic acid to the diet clearly reduced tumor induction by deoxycholic acid, which is present at relatively high concentrations in coffee. Importantly, Bernstein et al. controlled the concentration of deoxycholic acid in feces of their mice by mass spectrometry and compared it to the human situation. Concentrations in the exposed mice were 4.6 mg deoxycholic acid/g dry feces compared to only 0.3 mg/g in control mice. In humans, deoxycholic acid concentrations range between 2.3 and 4.1 mg/g dry feces for non-controlled diets and may increase to 6.4 mg/g for high fat diets. Therefore, the data of Bernstein and colleagues demonstrate that deoxycholic acid may contribute to human colon carcinogenesis.

Fluid and Condensed ApoA-I/Phospholipid Monolayers Provide Insights into ApoA-I Membrane Insertion

Apolipoprotein A-I (ApoA-I) is a protein implicated in the solubilization of lipids and cholesterol from cellular membranes. The study of ApoA-I in phospholipid (PL) monolayers brings relevant information about ApoA-I/PL interactions. We investigated the influence of PL charge and acyl chain organization on the interaction with ApoA-I using dipalmitoyl-phosphatidylcholine, dioleoyl-phosphatidylcholine and dipalmitoyl-phosphatidylglycerol monolayers coupled to ellipsometric, surface pressure, atomic force microscopy and infrared (polarization modulation infrared reflection–absorption spectroscopy) measurements. We show that monolayer compressibility is the major factor controlling protein insertion into PL monolayers and show evidence of the requirement of a minimal distance between lipid headgroups for insertion to occur, Moreover, we demonstrate that ApoA-I inserts deepest at the highest compressibility of the protein monolayer and that the presence of an anionic headgroup increases the amount of protein inserted in the PL monolayer and prevents the steric constrains imposed by the spacing of the headgroup. We also defined the geometry of protein clusters into the lipid monolayer by atomic force microscopy and show evidence of the geometry dependence upon the lipid charge and the distance between headgroups. Finally, we show that ApoA-I helices have a specific orientation when associated to form clusters and that this is influenced by the character of PL charges. Taken together, our results suggest that the interaction of ApoA-I with the cellular membrane may be driven by a mechanism that resembles that of antimicrobial peptide/lipid interaction.

Fluorescence Turn On by Cholate Aggregates

Bile salts, including sodium cholate (NaCh), are amphiphilic molecules with a concave hydrophilic side and a convex hydrophobic side. By forming aggregates in aqueous solution, these natural surfactants fulfill vital biological roles in the solubilization of cholesterol, lipids, and fat-soluble vitamins and thus are involved in the transport and absorption of important biological molecules. Following our success with the encapsulation of fluorescent protein chromophore (FP) analogs by synthetic hydrophobic and hydrophilic hosts, based upon substitution patterns, we now report the binding and turn on of other analogs by bile salt aggregates, observations which may lead to new tools for studying trafficking in these important systems.

Effect of Antibiotic Growth Promoters on Intestinal Microbiota in Food Animals: A Novel Model for Studying the Relationship between Gut Microbiota and Human Obesity?

As more children and adults become obese in the U.S. and other countries, obesity-associated diseases are becoming more prevalent worldwide (Poirier et al., 2006). Major chronic diseases linked to obesity include heart disease, stroke, and type 2 diabetes (Kahn et al., 2006; Poirier et al., 2006; Van Gaal et al., 2006). Thus, development of effective preventive and therapeutic strategies against obesity will ultimately reduce the burden of cardiovascular diseases and diabetes. Factors contributing to obesity development are complex. Although it is obvious that human genetics plays an important role in determining body weight, it is widely accepted that the increase in the prevalence of obesity over the past 30 years cannot be attributed to changes in human genome so other factors are responsible for obesity. Recent human and mice studies (Reviewed in DiBaise et al., 2008; Tilg et al., 2009) strongly support a concept that the gut microbiota together with host genotype and lifestyle contribute to the development of obesity. These studies suggest manipulating the microbial populations in the gut may be one means to control body weight. To develop such microbiota-manipulating strategies to aid in weight loss, it is critical to identify potential keystone microorganisms from more than 1,000 different species in the gut. However, there are three major limitations in these pioneering studies which ultimately slow progress in this field. First, previous studies on the relationship between microbiota and obesity (Ley et al., 2005, 2006; Turnbaugh et al., 2006) only analyzed fecal samples which represent the microbiota from the large intestine. However, the small intestine is the principal site for digestion, nutrient assimilation and energy harvest, which is directly relevant to body weight gain. In addition, the microbiota have been observed to vary significantly between small intestine and large intestine (Hayashi et al., 2005; Dumonceaux et al., 2006). Thus, use of fecal sample from small intestine is critical to reveal the direct relationship between gut microbiota and obesity development. Second, the potential role of microbiota in obesity development has focused on the utilization of indigestible polysaccharides in colon. However, many metabolic functions of microbiota, such as fat digestion, are not captured by only considering polysaccharide utilization. For example, the deconjugation of bile salt complexes by bile salt hydrolases, which are produced by many commensal bacteria (e.g., Lactobacillus), could reduce lipid solubilization and absorption and even lower cholesterol levels in humans (Begley et al., 2006; Ridlon et al., 2006). Lastly, due to technical difficulties, these studies used fecal biota as a surrogate for the entire gut microflora. However, fecal biota may not contain the mucosa-associated microbial populations that are in close contact with the underlying gut epithelium and play a different but important role in nutrient assimilation (Zoetendal et al., 2002; Eckburg et al., 2005). Thus, to identify specific obesity-associated microorganisms, it is essential to develop appropriate animal model and organ systems to overcome the above limitations in studying the relationship between gut microbiota and human obesity.

Lymphatic absorption of α-linolenic acid in rats fed flaxseed oil-based emulsion

The bioavailability of α-linolenic acid (ALA) from flaxseed oil in an emulsified form v. a non-emulsified form was investigated by using two complementary approaches: the first one dealt with the characterisation of the flaxseed oil emulsion in in vitro gastrointestinal-like conditions; the second one compared the intestinal absorption of ALA in rats fed the two forms of the oil. The in vitro study on emulsified flaxseed oil showed that decreasing the pH from 7·3 to 1·5 at the physiological temperature (37°C) induced instantaneous oil globule coalescence. Some phase separation was observed under acidic conditions that vanished after further neutralisation. The lecithin used to stabilise the emulsions inhibited TAG hydrolysis by pancreatic lipase. In contrast, lipid solubilisation by bile salts (after lipase and phospholipase hydrolysis) was favoured by preliminary oil emulsification. The in vivo absorption of ALA in thoracic lymph duct-cannulated rats fed flaxseed oil, emulsified or non-emulsified, was quantified. Oil emulsification significantly favoured the rate and extent of ALA recovery as measured by the maximum ALA concentration in the lymph (Cmax=14mg/ml at 3h in the emulsion group v. 9mg/ml at 5h in the oil group; P<0·05). Likewise, the area under the curve of the kinetics was significantly higher in the emulsion group (48mg×h/ml for rats fed emulsion v. 26mg×h/ml for rats fed oil; P<0·05). On the whole, ALA bioavailability was improved with flaxseed oil ingested in an emulsified state. Data obtained from the in vitro studies helped to partly interpret the physiological results.

Influence of N-terminal helix bundle stability on the lipid-binding properties of human apolipoprotein A-I

As the principal component of high-density lipoprotein (HDL), apolipoprotein (apo) A-I plays essential roles in lipid transport and metabolism. Because of its intrinsic conformational plasticity and flexibility, the molecular details of the tertiary structure of lipid-free apoA-I have not been fully elucidated. Previously, we demonstrated that the stability of the N-terminal helix bundle structure is modulated by proline substitution at the most hydrophobic region (residues around Y18) in the N-terminal domain. Here we examine the effect of proline substitution at S55 located in another relatively hydrophobic region compared to most of the helix bundle domain to elucidate the influences on the helix bundle structure and lipid interaction. Fluorescence measurements revealed that the S55P mutation had a modest effect on the stability of the bundle structure, indicating that residues around S55 are not pivotally involved in the helix bundle formation, in contrast to the insertion of proline at position 18. Although truncation of the C-terminal domain (Δ190–243) diminishes the lipid binding of apoA-I molecule, the mutation S55P in addition to the C-terminal truncation (S55P/Δ190–243) restored the lipid binding, suggesting that the S55P mutation causes a partial unfolding of the helix bundle to facilitate lipid binding. Furthermore, additional proline substitution at Y18 (Y18P/S55P/Δ190–243), which leads to a drastic unfolding of the helix bundle structure, yielded a greater lipid binding ability. Thus, proline substitutions in the N-terminal domain of apoA-I that destabilized the helix bundle promoted lipid solubilization. These results suggest that not only the hydrophobic C-terminal helical domain but also the stability of the N-terminal helix bundle in apoA-I are important modulators of the spontaneous solubilization of membrane lipids by apoA-I, a process that leads to the generation of nascent HDL particles.

Reaction of a Phospholipid Monolayer with Gas-Phase Ozone at the Air−Water Interface: Measurement of Surface Excess and Surface Pressure in Real Time

The reaction between gas-phase ozone and monolayers of the unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC, on aqueous solutions has been studied in real time using neutron reflection and surface pressure measurements. The reaction between ozone and lung surfactant, which contains POPC, leads to decreased pulmonary function, but little is known about the changes that occur to the interfacial material as a result of oxidation. The results reveal that the initial reaction of ozone with POPC leads to a rapid increase in surface pressure followed by a slow decrease to very low values. The neutron reflection measurements, performed on an isotopologue of POPC with a selectively deuterated palmitoyl strand, reveal that the reaction leads to loss of this strand from the air-water interface, suggesting either solubilization of the product lipid or degradation of the palmitoyl strand by a reactive species. Reactions of (1)H-POPC on D(2)O reveal that the headgroup region of the lipids in aqueous solution is not dramatically perturbed by the reaction of POPC monolayers with ozone supporting degradation of the palmitoyl strand rather than solubilization. The results are consistent with the reaction of ozone with the oleoyl strand of POPC at the air-water interface leading to the formation of OH radicals. The highly reactive OH radicals produced can then go on to react with the saturated palmitoyl strands leading to the formation of oxidized lipids with shorter alkyl tails.