Dataset on pyrene-labelled apolipoprotein A-I, model development and fitting to monitor oligomeric species of its lipid-free form.

This article contains data for the self-association of pyrene-labelled single Cys-mutants of apolipoprotein A-I (apoA-I). Mathematical models were developed to characterise the self-association events at different cysteine positions on apoA-I obtained as a function of protein concentration based on the multi-parametric spectrum of pyrene, particularly P-value and excimer emissions. The present work complements data related to the article entitled "Analysis of pyrene-labelled apolipoprotein A-I oligomerisation in solution: Spectra deconvolution and changes in P-value and excimer formation" Tárraga et al. [1].

Analysis of pyrene-labelled apolipoprotein A-I oligomerization in solution: Spectra deconvolution and changes in P-value and excimer formation.

ApoA-I is the main protein of HDL which has anti-atherogenic properties attributed to reverse cholesterol transport. It shares with other exchangeable apolipoproteins a high level of structural plasticity. In the lipid-free state, the apolipoprotein amphipathic α-helices interact intra- and inter-molecularly, providing structural stabilization by a complex self-association mechanism. In this study, we employed a multi-parametric fluorescent probe to study the self-association of apoA-I. We constructed six single cysteine mutants spanning positions along three helices: F104C, K107C (H4), K133C, L137C (H5), F225C and K226C (H10); and labelled them with N-Maleimide Pyrene. Taking advantage of its spectral properties, namely formation of an excited dimer (excimer) and polarity-dependent changes in its fluorescence fine structure (P-value), we monitored the apoA-I self-association in its lipid-free form as a function of its concentration. Interactions in helices H5 (K133C) and H10 (F225C and K226C) were highlighted by excimer emission; while polarity changes were reported in helix H4 (K107C), as well as in helices H5 and H10. Mathematical models were developed to enrich data analysis and estimate association constants (KA) and oligomeric species distribution. Furthermore, we briefly discuss the usefulness of the multi-parametric fluorescent probe to monitor different equilibria, even at a single labelling position. Results suggest that apoA-I self-association must be considered to fully understand its physiological roles. Particularly, some contacts that stabilize discoidal HDL particles seem to be already present in the lipid-free apoA-I oligomers.

Understanding the role of apolipoproteinA-I in atherosclerosis. Post-translational modifications synergize dysfunction?

BACKGROUND: The identification of dysfunctional human apolipoprotein A-I (apoA-I) in atherosclerotic plaques suggests that protein structure and function may be hampered under a chronic pro inflammatory scenario. Moreover, the fact that natural mutants of this protein elicit severe cardiovascular diseases (CVD) strongly indicates that the native folding could shift due to the mutation, yielding a structure more prone to misfold or misfunction. To understand the events that determine the failure of apoA-I structural flexibility to fulfill its protective role, we took advantage of the study of a natural variant with a deletion of the residue lysine 107 (K107del) associated with atherosclerosis. METHODS: Biophysical approaches, such as electrophoresis, fluorescence and spectroscopy were used to characterize proteins structure and function, either in native conformation or under oxidation or intramolecular crosslinking. RESULTS: K107del structure was more flexible than the protein with the native sequence (Wt) but interactions with artificial membranes were preserved. Instead, structural restrictions by intramolecular crosslinking impaired the Wt and K107del lipid solubilization function. In addition, controlled oxidation decreased the yield of the native dimer conformation for both variants. CONCLUSIONS: We conclude that even though mutations may alter protein structure and spatial arrangement, the highly flexible conformation compensates the mild shift from the native folding. Instead, post translational apoA-I modifications (probably chronic and progressive) are required to raise a protein conformation with significant loss of function and increased aggregation tendency. GENERAL SIGNIFICANCE: The results learnt from this variant strength a close association between amyloidosis and atherosclerosis.

Dataset of the construction and characterization of stable biological nanoparticles.

This article shows the dataset of clearance assays and the reconstitution of stable biological nano-complexes using both detergent-assisted and spontaneous solubilization of phospholipids by the recombinant purified apolipoprotein A-I (apoA-I). Protein was intra-chain crosslinked in order to introduce steric constrains. Then, native and crosslinked protein function was evaluated by a data collection of dimiristoyl phosphatidyl choline (DMPC) micellization curves. Additionally, resulting particles from spontaneous or detergent-assisted lipid solubilization were characterized by transmission electron microscopy (TEM), size exclusion chromatography (SEC), and native polyacrylamide gel electrophoresis (PAGE). Here we set up an experimental design that may help study protein structure based on its function, since interaction with biological membranes and lipids is an intrinsic activity attributed to many proteins in circulation. In addition, by t-test analysis of collected-data, we examined the formation of lipoprotein particles by native and intra-chain crosslinked proteins under different conditions like temperature and time incubation. Thus, data shown here strengthen the usefulness of an easy, rapid, accessible and inexpensive approach to test protein flexibility related to its function.

Apolipoprotein A-I Helsinki promotes intracellular acyl-CoA cholesterol acyltransferase (ACAT) protein accumulation.

Reverse cholesterol transport is a process of high antiatherogenic relevance in which apolipoprotein AI (apoA-I) plays an important role. The interaction of apoA-I with peripheral cells produces through mechanisms that are still poorly understood the mobilization of intracellular cholesterol depots toward plasma membrane. In macrophages, these mechanisms seem to be related to the modulation of the activity of acyl-CoA cholesterol acyltransferase (ACAT), the enzyme responsible for the intracellular cholesterol ester biosynthesis that is stored in lipid droplets. The activation of ACAT and the accumulation of lipid droplets play a key role in the transformation of macrophages into foam cells, leading to the formation of atheroma or atherosclerotic plaque. ApoA-I Helsinki (or ∆K107) is a natural apoA-I variant with a lysine deletion in the central protein region, carriers of which have increased atherosclerosis risk. We herein show that treatment of cultured RAW macrophages or CHOK1 cells with ∆K107, but not with wild-type apoA-I or a variant containing a similar deletion at the C-terminal region (∆K226), lead to a marked increase (more than 10 times) in the intracellular ACAT1 protein level as detected by western blot analysis. However, we could only detect a slight increase in cholesteryl ester produced by ∆K107 mainly when Chol loading was supplied by low-density lipoprotein (LDL). Although a similar choline-phospholipid efflux is evoked by these apoA-I variants, the change in phosphatidylcholine/sphyngomyelin distribution produced by wild-type apoA-I is not observed with either ∆K107 or ∆K226.

Effect of reconstituted discoidal high-density lipoproteins on lipid mobilization in RAW 264.7 and CHOK1 cells.

Reconstituted discoidal high-density lipoproteins (rHDL) resemble nascent HDL, which are formed at the early reverse cholesterol transport steps, and constitute the initial cholesterol (Chol) acceptors from cell membranes. We have used different sized rHDL containing or not Chol, to test their abilities to promote cholesterol and phospholipid efflux from two different cell lines: Raw 264.7 macrophages and CHOK1 cells. All rHDL and lipid-free apolipoprotein A-I (apoA-I) were found to be bound to CHO and RAW cells. In RAW cells, a positive correlation between cellular binding and Chol removal was found for 78 and 96 Å rHDL. Chol-free rHDL were more effective than Chol-containing ones in binding to RAW cells and promoting Chol removal. These results were more evident in the 96 Å rHDL. On the other hand, rHDL binding to CHO cells was relatively independent of disc size and Chol content. In spite of the fact that apoA-I and rHDL promoted Chol efflux from both cellular lines, only in CHOK1 cells this result was also associated to decrease Chol esterification. Among choline-containing phospholipids, only phosphatidylcholine (PC) (but not sphingomyelin) was detected to be effuxed from both cellular lines. With the only exception of Chol-free 96 Å discs, the other rHDL as well as apoA-I promoted PC efflux from RAW cells. Chol-containing rHDL were more active than Chol-free ones of comparable size to promote PC efflux from RAW macrophages. Regarding CHO cells, only apoA-I and Chol-free 78 Å rHDL were active enough to remove PC.

Membrane insertion topology of the central apolipoprotein A-I region. Fluorescence studies using single tryptophan mutants.

Apolipoprotein A-I (apoAI) contains several amphipathic α-helices. To carry out its function, it exchanges between lipid-free and different lipidated states as bound to membranes or to lipoprotein complexes of different morphology, size, and composition. When bound to membranes or to spherical lipoprotein surfaces, it is thought that most α-helices arrange with their long axis parallel to the membrane surface. However, we previously found that a central region spanning residues 87-112 is exclusively labeled by photoactivable reagents deeply located into the membrane (Córsico et al. (2001) J. Biol. Chem. 276, 16978-16985). A pair of amphipathic α-helical repeats with a particular charge distribution is predicted in this region. In order to study their insertion topology, three single tryptophan mutants, each one containing the tryptophan residue at a selected position in the hydrophobic face of the central Y-helices (W@93, W@104, and W@108), were used. From the accessibility to quenchers located at different membrane depths, distances from the bilayer center of 13.4, 10.5, and 15.7 Å were estimated for positions 93, 104, and 108, respectively. Reported data also indicate that distances between homologous positions (in particular for W@93 and W@104) are very short in dimers in aqueous solution, but they are larger in membrane-bound dimers. Data indicate that an intermolecular central Y-helix bundle would penetrate the membrane perpendicularly to the membrane surface. Intermolecular helix-helix interactions would occur through the hydrophilic helix faces in the membrane-bound bundle but through the hydrophobic faces in the case of dimers in solution.

Membrane organization and regulation of cellular cholesterol homeostasis.

An excess of intracellular free cholesterol (Chol) is cytotoxic, and its homeostasis is crucial for cell viability. Apolipoprotein A-I (apoA-I) is a highly efficient Chol acceptor because it activates complex cellular pathways that tend to mobilize and export Chol from cellular depots. We hypothesize that membrane composition and/or organization is strongly involved in Chol homeostasis. To test this hypothesis, we constructed a cell line overexpressing stearoyl coenzyme A (CoA) desaturase (SCD cells), which modifies plasma membrane (PM) composition by the enrichment of monounsaturated fatty acids, and determined this effect on membrane properties, cell viability, and Chol homeostasis. PM in SCD cells has a higher ratio of phospholipids to sphingomyelin and is slightly enriched in Chol. These cells showed an increase in the ratio of cholesteryl esters to free Chol; they were more resistant to Chol toxicity, and they exported more caveolin than control cells. The data suggest that cell functionality is preserved by regulating membrane fluidity and Chol exportation and storage.

The central type Y amphipathic alpha-helices of apolipoprotein AI are involved in the mobilization of intracellular cholesterol depots.

We studied the role of a central domain of human apolipoprotein AI (apoAI) in cholesterol mobilization and removal from cells. In order to check different protein conformations, we tested different sized and cholesterol-content reconstituted apoAI particles (rHDL). Meanwhile cholesterol-free discs were active to induce mobilization, only small cholesterol-containing rHDL were active. To test the influence of a central domain in such events, we used two apoAI variants: one, with its central Y helix pair replaced by the C-terminal domain, and the other having a lysine deleted in central region. The helix-swapping variant decrease the cholesterol pool available to acyl-CoA cholesterol acyl transferase and increase mobilization of newly synthesized cholesterol. Instead, the deletion mutant had no effect on both events. We conclude that the central domain of apoAI is involved in cholesterol cell traffic and solubilization, and that a Y-type charge distribution in polar face may be required, as well as a correct helices-polar face orientation.

Biophysical characterization of the non-fusogenic interaction between liposomes and the shrimp bifunctional lipoprotein-beta-glucan binding protein HDL/BGBP.

Shrimp High Density Lipoprotein-beta-Glucan Binding Protein (HDL/BGBP) has been studied by its role in nutrition and innate defense. Although the mechanisms of lipid loading are still unknown, HDL-BGBP binds and aggregates phospholipids vesicles in vitro. To gain insights into the HDL-BGBP mechanism of interaction with membranes, we have used fluorescence spectroscopy and electron microscopy. Data show that HDL-BGBP does not induce membrane fusion, leakage nor lipid exchange, although microstructural changes are clearly observed. This work supports a model where protein aggregation leads to liposome clustering. Such interaction may be a critical factor for the activation of the shrimp blood cell in vivo.

Visualization and analysis of apolipoprotein A-I interaction with binary phospholipid bilayers.

Apolipoprotein A-I (apoA-I) interaction with specific cell lipid domains was suggested to trigger cholesterol and phospholipid efflux. We analyzed here apoA-I interaction with dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC) bilayers at a temperature showing phase coexistence. Solid and liquid-crystalline domains were visualized by two-photon fluorescence microscopy on giant unilamellar vesicles (GUVs) labeled with 6-dodecanoyl-2-dimethyl-amino-naphthalene (Laurdan). A decrease of vesicle size was detected as long as they were incubated with lipid-free apoA-I, together with a shape deformation and a relative enrichment in DSPC. Selective lipid removal mediated by apoA-I from different domains was followed in real time by changes in the Laurdan generalized polarization. The data show a selective interaction of apoA-I with liquid-crystalline domains, from which it removes lipids, at a molar ratio similar to the domain compositions. Next, apoA-I was incubated with DMPC/DSPC small unilamellar vesicles, and products were isolated and quantified. Protein solubilized both lipids but formed complexes relatively enriched in the liquid component. We also show changes in the GUV morphology when cooling down. Our results suggest that the most efficient reaction between apoA-I and DMPC/DSPC occurs in particular bilayer conditions, probably when small fluid domains are nucleated within a continuous gel phase and interfacial packing defects are maximal.

The effect of osmotic stress on the biophysical behavior of the Bacillus subtilis membrane studied by dynamic and steady-state fluorescence anisotropy.

The thermotropic behavior of intact bacterial membranes and vesicles prepared from total and polar lipids isolated from Bacillus subtilis cultures grown at 37 degrees C in normal (LB) and hyperosmotic (LBN) conditions was studied using 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), and 2-diethylamino-6-lauroyl-naphthalene (Laurdan) as fluorescent probes. No phase transition of bulk lipids was observed in these preparations at the range of temperature studied. The anisotropy values (r(s)) for DPH and TMA-DPH in purified membranes showed significant differences between the LB and LBN conditions, suggesting that there was an increase in membrane packing during the adaptation to osmotic stress. Furthermore, generalized polarization (GP) parameters for Laurdan indicated small but significant changes in water relaxation at the membrane hydrophobic/hydrophilic interface. Membrane preparations showed r(s) higher values than those of lipid vesicles and a higher temperature dependence of the Laurdan GP parameter. This fact indicates that membrane proteins increase the lipid packing and keep the membrane more sensitive to temperature changes.

Structure of apolipophorin-III in discoidal lipoproteins. Interhelical distances in the lipid-bound state and conformational change upon binding to lipid.

The structure of apolipophorin III in the lipid-bound state and the extent of the conformational change that takes place when the five-helix bundle apolipoprotein binds to a lipoprotein lipid surface were investigated by fluorescence resonance energy transfer in discoidal lipoproteins. Four intramolecular interhelical distances between helix pairs 1-4, 2-4, 3-4, and 5-4 were estimated by fluorescence resonance energy transfer in both the lipid-free and the lipid-bound states. Depending on the helices pairs, the intramolecular interhelical distances increased between 15 and > or = 20 A upon binding of the apolipoprotein to lipid, demonstrating for the first time that binding to lipid is accompanied by a major change in interhelical distances. Using discoidal lipoproteins made with a combination of apolipophorin III molecules containing donor and acceptor groups and apolipophorin III molecules containing neither donor nor acceptor groups, it was possible to obtain information about intermolecular interhelical distances between the helix 4 of one apolipoprotein and the helices 1, 2, 3, and 5 of a second apolipoprotein residing in the same discoidal lipoprotein. Altogether, the estimated intermolecular and intramolecular interhelical distances suggest a model in which the apolipoprotein arranges in pairs of antiparallel and fully extended polypeptide chains surrounding the periphery of the bilayer disc.