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Apolipoprotein (apo) A-I is the major protein component of high-density lipoprotein (HDL) and plays key roles in the Reverse Cholesterol Transport pathway. In the past decade, reconstituted HDL (rHDL) has been employed as a therapeutic agent for treatment of atherosclerosis. The ability of rHDL to promote cholesterol efflux from peripheral cells has been documented to reduce the size of atherosclerotic plaque lesions. However, development of apoA-I rHDL-based therapeutics for human use requires a cost effective process to generate an apoA-I product that meets "Good Manufacturing Practice" standards. Methods available for production and isolation of unmodified recombinant human apoA-I at scale are cumbersome, laborious and complex. To overcome this obstacle, a streamlined two-step procedure has been devised for isolation of recombinant untagged human apoA-I from E. coli that takes advantage of its ability to re-fold to a native conformation following denaturation. Heat treatment of a sonicated E. coli supernatant fraction induced precipitation of a large proportion of host cell proteins (HCP), yielding apoA-I as the major soluble protein. Reversed-phase HPLC of this material permitted recovery of apoA-I largely free of HCP and endotoxin. Purified apoA-I possessed α-helix secondary structure, formed rHDL upon incubation with phospholipid and efficiently promoted cholesterol efflux from cholesterol loaded J774 macrophages.

ObjectiveApolipoprotein A-V (apoA-V) is a low-abundance plasma protein that modulates triacylglycerol homeostasis. Gene transfer studies were undertaken in apoa5 (-/-) mice to define the mechanism underlying the correlation between the single-nucleotide polymorphism c.553G>T in APOA5 and hypertriglyceridemia.Approach and resultsAdeno-associated virus (AAV) 2/8-mediated gene transfer of wild-type apoA-V induced a dramatic lowering of plasma triacylglycerol in apoa5 (-/-) mice, whereas AAV2/8-Gly162Cys apoA-V (corresponding to the c.553G>T single-nucleotide polymorphism: rs2075291; p.Gly185Cys when numbering includes signal sequence) had a modest effect. Characterization studies revealed that plasma levels of wild-type and G162C apoA-V in transduced mice were similar and within the physiological range. Fractionation of plasma from mice transduced with AAV2/8-G162C apoA-V indicated that, unlike wild-type apoA-V, >50% of G162C apoA-V was recovered in the lipoprotein-free fraction. Nonreducing SDS-PAGE immunoblot analysis provided evidence that G162C apoA-V present in the lipoprotein-free fraction, but not that portion associated with lipoproteins, displayed altered electrophoretic mobility consistent with disulfide-linked heterodimer formation. Immunoprecipitation followed by liquid chromatography/mass spectrometry of human plasma from subjects homozygous for wild-type APOA5 and c.553G>T APOA5 revealed that G162C apoA-V forms adducts with extraneous plasma proteins including fibronectin, kininogen-1, and others.ConclusionsSubstitution of Cys for Gly at position 162 of mature apoA-V introduces a free cysteine that forms disulfide bonds with plasma proteins such that its lipoprotein-binding and triacylglycerol-modulation functions are compromised.

A fusion protein comprising an α-CD20 single chain variable fragment (scFv) antibody, a spacer peptide, and human apolipoprotein (apo) A-I was constructed and expressed in Escherichia coli. The lipid interaction [...]

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Introduction Apolipoprotein (apo) A-I, the predominant apolipoprotein found on high-density lipoprotein (HDL), plays a central role in reverse cholesterol transport (RCT), the pathway by which cholesterol is removed from peripheral [...], Mechanisms to increase plasma high-density lipoprotein (HDL) or to promote egress of cholesterol from cholesterol-loaded cells (e.g., foam cells from atherosclerotic lesions) remain an important target to regress heart disease. Reconstituted HDL (rHDL) serves as a valuable vehicle to promote cellular cholesterol efflux in vitro and in vivo. rHDL were prepared with wild type apolipoprotein (apo) A-I and the rare variant, apoA-I Milano (M), and each apolipoprotein was reconstituted with phosphatidylcholine (PC) or sphingomyelin (SM). The four distinct rHDL generated were incubated with CHO cells, J774 macrophages, and BHK cells in cellular cholesterol efflux assays. In each cell type, apoA-I(M) SM-rHDL promoted the greatest cholesterol efflux. In BHK cells, the cholesterol efflux capacities of all four distinct rHDL were greatly enhanced by increased expression of ABCG1. Efflux to PC-containing rHDL was stimulated by transfection of a nonfunctional ABCA1 mutant (W590S), suggesting that binding to ABCA1 represents a competing interaction. This interpretation was confirmed by binding experiments. The data show that cholesterol efflux activity is dependent upon the apoA-I protein employed, as well as the phospholipids constituent of the rHDL. Future studies designed to optimize the efflux capacity of therapeutic rHDL may improve the value of this emerging intervention strategy. Key words: apolipoprotein, ABCA1, ABCG1, reverse cholesterol transport, macrophage. Les mecanismes permettant d'augmenter la quantite de lipoproteines de haute densite (HDL) du plasma ou de favoriser la sortie du cholesterol des cellules qui en sont chargees (cellules spumeuses des lesions atherosclereuses) demeurent des cibles importantes permettant de faire regresser les maladies cardiaques. La HDL reconstituee (rHDL) sert de vehicule valable pour promouvoir l'efflux cellulaire de cholesterol in vitro et in vivo. Des rHDL ont ete preparees a partir de l'apolipoproteine (apo) A-I sauvage et de son variant rare, l'apoA-I Milano (M), et chaque apolipoproteine a ete reconstituee avec de la phosphatidylcholine (PC) ou de la sphingomyeline (SM). Les quatre rHDL distinctes generees ont ete incubees avec des cellules CHO, des macrophages J774 et des cellules BHK lors de dosages d'efflux de cholesterol cellulaire. Dans chaque type de cellules, la SM-rHDL de l'apoA-I(M) favorisait l'efflux de cholesterol le plus important. Chez les cellules BHK, les capacites d'efflux de cholesterol des quatre rHDL distinctes etaient grandement accrues par l'augmentation de l'expression d'ABCG1. L'efflux de rHDL contenant de la PC etait stimule par la transfection d'un mutant d'ABCA1 non fonctionnel (W590S), suggerant que la liaison a ABCA1 represente une interaction competitive. Cette interpretation a ete confirmee par des experiences de liaison. Les donnees montrent que l' activite d' efflux de cholesterol est dependante de l'apoA-I utilisee ainsi que du phospholipide de la rHDL. Des etudes futures concues pour optimiser la capacite d'efflux de rHDL therapeutiques peuvent ameliorer la valeur de cette strategie d' intervention en emergence. Mots-cles : apolipoproteine, ABCA1, ABCG1, transport inverse de cholesterol, macrophage. [Traduit par la Redaction]

The ability of apolipoprotein A-V to interact with two members of the low density lipoprotein receptor family, the low density lipoprotein receptor-related protein and the mosaic type-1 receptor, SorLA is explored to investigate the molecular basis. It is demonstrated that the apolipoprotein A-V influences the plasma lipid homeostasis by enhancing receptor-mediated endocytosis of triacylglycerol-rich lipoproteins.

A combined N- and C-terminal truncation variant of human apolipoprotein A-I (apoA-I) is designed, expressed in Escherichia coli, isolated and characterized. The results suggest that, in the absence of the C-terminal residues 187-243, removal of residues 1-43 does not induce a major structural reorganization while retaining a protein fold that is consistent with a globular amphipathic alpha-helix bundle.

A computer-assisted sequence analysis was used along with limited proteolysis studies to identify N-terminal domain in full-length human apolipoprotein A-V (apoA-V). Lipid-free apoA-V was found to adopt a relatively stable, monomeric, asymmetric tertiary fold that is consistent with an amphipathic [alpha]-helix bundle.

BACKGROUND: The development of water-soluble nanodevices extends the potential use of compounds developed for other purposes (e.g. antifungal drugs or antibiotics) for applications in agriculture. For example, the broad-spectrum, water-insoluble, macrolide polyene antibiotic amphotericin B (AMB) could be used to inhibit phytopathogenic fungi. A new formulation embedding AMB in nanodisks (NDs) enhances antibiotic solubility and confers protection against environmental damage. In the present study, AMB-NDs were tested for efficacy against several phytopathogenic fungi in vitro and on infected living plants (chickpea and wheat). RESULTS: Compared with AMB in dimethylsulfoxide (DMSO), AMB-NDs increased the sensitivity of several fungal species to this antimycotic in vitro. Sensitivity varied with fungal species as well as with the forma specialis. Phytophthora cinnamomi, previously reported as insensitive to other polyene antimycotics, remained unaffected at the doses examined. Some effect against disease symptoms were obtained with AMB-NDs against fusarium wilt in chickpea, whereas the results were highly variable in wheat, depending on both the species and treatment regimen. CONCLUSION: The results confirm that formulation of AMB into ND increases its effectiveness against phytopathogenic fungi in vitro, opening the possibility for its use on infected plants in the field. Copyright © 2011 Society of Chemical Industry [ABSTRACT FROM AUTHOR]