A complete NMR spectral assignment of the lipid-free mouse apolipoprotein A-I (apoAI) C-terminal truncation mutant, apoAI(1-216)

ApoAI is the major protein component of the high-density lipoprotein (HDL) that has been a hot subject of interests because of its anti-atherogenic properties. Lipid-free apoAI specifically binds to phospholipids, triggering HDL formation. Here we report a complete backbone assignment and nearly complete sidechain assignment of a C-terminal 24-residue truncation mutant of mouse apoAI, apoAI(1-216), in its lipid-free form.     

Inhibition of free fatty acid mobilization by colchicine

Segments of epididymal adipose tissue from normal male rats were incubated with micromolar concentrations of colchicine for different periods of time up to 4 hr, and the mobilization of free fatty acids (FFA) was measured during a subsequent reincubation. Although pretreatment with colchicine did not alter basal unstimulated FFA release, mobilization of FFA in the presence of epinephrine or theophylline was reduced. However, neither lipolysis, as judged by glycerol production, nor cyclic AMP accumulation was impaired under the same conditions. To assess the possibility that colchicine might limit production of fatty acids by accelerating the entry and metabolism of glucose into adipocytes, the metabolism of glucose by adipose tissue was studied. Pretreatment with colchicine did not affect uptake of glucose nor its oxidation to CO(2), although colchicine-treated tissues did have slightly more [(14)C]glucose incorporated into the glyceride moiety of triglyceride. When adipose tissues pretreated with colchicine were incubated in an albumin-free medium, no reduction in FFA production by colchicine was observed. Because no FFA release occurs in albumin-free media, this experiment suggests that colchicine-induced inhibition of FFA mobilization results from impaired extrusion of FFA from adipose cells.     

Apolipoprotein A-I(Milano): current perspectives

PURPOSE OF REVIEW: Strategies to increase HDL are among the major targets of clinical research in atherosclerosis prevention. The mutant apolipoprotein A-I(Milano) has been associated with a reduced incidence of coronary disease in carriers. Furthermore, recombinant apolipoprotein A-I(Milano) has displayed remarkable atheroprotective activities and the possibility of directly reducing the burden of atherosclerosis in experimental models. This review is aimed at providing an update on the experimental studies in which apolipoprotein A-I(Milano), produced as a recombinant protein, has displayed important effects in the treatment of vascular diseases. RECENT FINDINGS: In the past year, two reports have appeared, indicating that a single-dose administration of recombinant apolipoprotein A-I(Milano) dimers formulated into liposomes can reduce atheromas in models such as the apolipoprotein E-deficient mice and a rabbit model of carotid focal lesion, in which a direct 90 min infusion of the product reduced atheroma up to 30%. This finding was associated with an increase in HDL free cholesterol and the permanence of the recombinant product in the lesion for over 72 h. SUMMARY: Recombinant apolipoprotein A-I(Milano), formulated as synthetic HDL with phospholipids, appears to exert a direct removing effect on arterial cholesterol. This is well evident in experimental animals and, more recently in clinical findings, as indicated by a dramatic increase in HDL free cholesterol after the infusion of different doses of the agent. As the product appears to be well tolerated and non-immunogenic, ongoing phase II studies in patients are being awaited with interest to obtain a 'proof of principle' for 'HDL therapy'.     

GABA(B) receptors function as heterodimers

Our current understanding is that functional GABA(B) receptors exist as heterodimers of two related seven-transmembrane proteins, GABA(B)-R1 and GABA(B)-R2. GABA(B)-R1 requires GABA(B)-R2 to be expressed at the cell surface as a mature glycoprotein. Cloning of the GABA(B) receptor has failed to provide molecular evidence to support the existence of true receptor subtypes. The discovery of the heterodimeric nature of the GABA(B) receptor has already changed the way we think about GPCR function and it is likely that future studies will change our understanding about how receptor subtypes can be formed.     

Thiol-bearing synthetic peptides retain the antioxidant activity of apolipoproteinA-I(Milano)

Apolipoprotein(apo)A-I(Milano) (R173C) and apoA-I(Paris) (R151C) are rare cysteine variants of wild-type (WT) apoA-I that possess novel antioxidant properties on phospholipid surfaces. Yet, the two variants differ in their ability to inhibit lipid peroxidation. In this study, we used synthetic peptides (18mers) to investigate the structural basis for the difference in antioxidant activity between apoA-I(Milano) and apoA-I(Paris). A peptide (aa 167-R173C-184) based on the amphipathic alpha helix harboring the R173C mutation inhibited superoxide anion-mediated oxidation of phospholipid in a dose-dependent manner, but it failed to directly quench superoxide anions in aqueous solution, indicating that the peptide acted at the level of phospholipid to inhibit lipid peroxidation just like the full-length cysteine variant. Peptide 145-R151C-162 based on the helical segment containing R151C exhibited the same capacity as peptide 167-R173C-184 to inhibit lipid peroxidation. Thus, the difference in antioxidant activity between apoA-I(Milano) and apoA-I(Paris) was not governed by the primary amino acid sequence of their individual amphipathic alpha helices, rather contextual constraints within the full-length variants set the difference in antioxidant activity. Cysteine-free peptides were weak inhibitors of lipid peroxidation. These results suggest that thiol-bearing helical peptides based on apoA-I(Milano) may be useful to combat inflammatory related diseases.     

Apolipoprotein A-I(Milano) anion exchange chromatography: Self association and adsorption equilibrium

The self-associative properties of apolipoprotein A-I(Milano) (apoA-I(M)) were investigated in relationship to its anion exchange behavior on Q-Sepharose-HP with and without the addition of urea as a denaturant. Self-association was dependent on protein and urea concentration and both influenced interactions of the protein with the chromatographic surface. In the absence of urea, apoA-I(M) was highly associated and existed primarily as a mixture of homodimer, tetramer and hexamer forms. Under these conditions, since the binding strength was greater for the oligomer forms, broad, asymmetrical peaks were obtained in both isocratic and gradient elution. Adding urea depressed self-association and caused unfolding. This resulted in sharper peaks but also decreased the binding strength. Thus, under these conditions chromatographic elution occurred at lower salt concentrations. The adsorption isotherms obtained at high protein loadings were also influenced by self-association and by the varying binding strength of the differently associated and unfolded forms. The isotherms were thus dependent on protein, urea, and salt concentration. Maximum binding capacity was obtained in the absence of urea, where adsorption of oligomers was shown to be dominant. Adding urea reduced the apparent binding capacity and weakened the apparent binding strength. A steric mass action model accounting for competitive binding of the multiple associated forms was used to successfully describe the equilibrium binding behavior using parameters determined from isocratic elution and isotherm experiments.     

Apolipoprotein A-I(Milano) anion exchange chromatography: mass transfer and adsorption kinetics

The mass transfer and adsorption kinetics of self-associating apolipoprotein A-I(Milano) (apoA-I(M)) was investigated for the two anion exchangers Q-Sepharose-HP and Macro-Prep-HQ. At high salt where no protein binding occurs and without urea, mass transfer was controlled by hindered pore diffusion of multiple associated forms for both materials. Adding urea suppressed self-association, but resulted in higher viscosity and caused unfolding. As a consequence, the effective diffusivity decreased as urea was added and was greater for the larger pore Macro-Prep-HQ resin. At low salt, under strong binding conditions, the adsorption kinetics followed a more complex mechanism. In this case, the kinetics was very slow for both stationary phases up to 2 M urea. However, at higher urea concentrations, the adsorption kinetics for the smaller pore Q-Sepharose-HP matrix became much faster, suggesting a transition from pore- to surface-dominated diffusion. Microscopic observations confirmed that different transport mechanisms were in play below and above 2 M urea, which marked the approximate boundary above which self-association was suppressed and unfolding occurred. The net result was enhanced uptake kinetics at high urea concentrations (e.g., 4 M) where protein unfolding is thought to lead to a more flexible structure that can reptate along the pore surface. Although the observed enhancement was dependent on the pore size and, thus, the surface area of the resin, it was not limited to apoA-I(M). BSA showed a similar trend as a function of urea when its disulfide bonds were reduced.     

Cholesterol: free radical peroxidation and transfer into phospholipid membranes

Cholesterol, when sequestered in saturated liposomes of dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC), undergoes peroxidation thermally initiated either by a lipid-soluble or a water-soluble azo initiator and in both cases the reaction is inhibited effectively by the water-soluble antioxidant, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate (Trolox). Quantitative kinetic methods of autoxidation show that the oxidizability, kp/(2kt)1/2 (where kp and 2kt are the rate constants of radical chain propagation and termination, respectively) of cholesterol in DMPC or DPPC multilamellar liposomes, where kp/(2kt)1/2 is 3.0.10(-3) to 4.3.10(-3) M-1/2 s-1/2 at 37-45 degrees C, is similar to that measured in homogeneous solution in chlorobenzene, where kp/(2kt)1/2 is 3.32.10(-3). However, its oxidizability in smaller unilamellar vesicles of DMPC or DPPC increases by at least 3-times that measured in multilamellar systems. Autoxidation/antioxidant methods show that cholesterol partitions directly from the solid state into DMPC or DPPC liposomes by shaking and this is confirmed by 31P and 2H quadrupole NMR spectra of deuterated cholesterol when membrane bound. Analytical studies indicate that up to 21 mol% cholesterol will partition into the membranes by shaking.     

Apolipoprotein A-I(Milano) unfolds via an intermediate state as studied by differential scanning calorimetry and circular dichroism.

In this study the thermal and denaturant induced denaturation behaviors of apolipoprotein A-I(Milano) (apo A-IM) have been studied by differential scanning calorimetry and circular dichroism spectroscopy, as well as solution properties by analytical ultracentrifugation. Thermal denaturation is dependent on pH, sodium phosphate concentration and NaCl concentration. The protein is highly self-associated at the protein concentrations used in this study. Denaturation of apo A-IM at pH 7.4 and 8.0 occurs in two steps. The midpoint between the transition is at 37 degrees C. The first step at 31 degrees C involves melting of tertiary structure and rearrangement of protein association complexes, i.e. a transition into an intermediate molten globular-like state. Subsequent melting of this intermediate state into an unfolded state occurs at 52 degrees C. At pH 2.8 the protein lacks all tertiary structure and denaturation occurs over a large temperature interval, indicating the induction of a molten globular-like state at low pH.     

Regulation of apoAI processing by procollagen C-proteinase enhancer-2 and bone morphogenetic protein-1

Given the increased prevalence of cardiovascular disease in the world, the search for genetic variations controlling the levels of risk factors associated with the development of the disease continues. Multiple genetic association studies suggest the involvement of procollagen C-proteinase enhancer-2 (PCPE2) in modulating HDL-C levels. Therefore biochemical and mechanistic studies were undertaken to determine whether there might be a basis for a role of PCPE2 in HDL biogenesis. Our studies indicate that PCPE2 accelerates the proteolytic processing of pro-apolipoprotein (apo) AI by enhancing the cleavage of the hexapeptide extension present at the N terminus of apoAI. Surface Plasmon Resonance and immunoprecipitation studies indicate that PCPE2 interacts with BMP-1 and pro-apoAI to form a ternary pro-apoAI/BMP-1/PCPE2 complex. The most favorable interaction among these proteins begins with the association of BMP-1 to pro-apoAI followed by the binding of PCPE2 which further stabilizes the complex. PCPE2 resides, along with apoAI, on the HDL fraction of lipoproteins in human plasma supporting a relationship between HDL and PCPE2. Taken together, the findings from our studies identify a new player in the regulation of apoAI post-translational processing and open a new avenue to the study of mechanisms involved in the regulation of apoAI synthesis, HDL levels, and potentially, cardiovascular disease.     

Biosynthesis and mobilization of poly(3-hydroxybutyrate) [P(3HB)] by Spirulina platensis

Three strains of Spirulina platensis isolated from different locations showed capability of synthesizing poly(3-hydroxybutyrate) [P(3HB)] under nitrogen-starved conditions with a maximum accumulation of up to 10 wt.% of the cell dry weight (CDW) under mixotrophic culture conditions. Intracellular degradation (mobilization) of P(3HB) granules by S. platensis was initiated by the restoration of nitrogen source. This mobilization process was affected by both illumination and culture pH. The mobilization of P(3HB) was better under illumination (80% degradation) than in dark conditions (40% degradation) over a period of 4 days. Alkaline conditions (pH 10-11) were optimal for both biosynthesis and mobilization of P(3HB) at which 90% of the accumulated P(3HB) was mobilized. Transmission electron microscopy (TEM) revealed that the mobilization of P(3HB) involved changes in granule quantity and morphology. The P(3HB) granules became irregular in shape and the boundary region was less defined. In contrast to bacteria, in S. platensis the intracellular mobilization of P(3HB) seems to be faster than the biosynthesis process. This is because in cyanobacteria chlorosis delays the P(3HB) accumulation process.     

Calcium mobilization by nicotinic acid adenine dinucleotide phosphate (NAADP) in rat astrocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to release intracellular Ca(2+) in several types of cells. We have used Ca(2+)-sensitive fluorescent dyes (Fura-2, Fluo-4) to measure intracellular Ca(2+) in astrocytes in culture and in situ. Bath-applied NAADP elicited a reversible and concentration-dependent Ca(2+) rise in up to 90% of astrocytes in culture (EC(50)=7 microM). The NAADP-evoked Ca(2+) rise was maintained in the absence of extracellular Ca(2+), but was suppressed after depleting the Ca(2+) stores of the ER with ATP (20 microM), with cyclopiazonic acid (10 microM) or with ionomycin (5 microM). P(2) receptor antagonist pyridoxalphosphate-6-azophenyl-2'4'-disulfonic acid (PPADS, 100 microM), IP(3) receptor blocker 2-aminoethoxydiphenyl borate (2-APB, 100 microM) and PLC inhibitor U73122 (10 microM) also reduced or suppressed the NAADP-evoked Ca(2+) rise. NAADP still evoked a Ca(2+) response after application of glycyl-l-phenylalanine-beta-naphthylamide (GPN, 200 microM), which permeabilizes lysosomes, or preincubation with H(+)-ATPase inhibitor bafilomycin A1 (4 microM) and of p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP, 2 microM), that impairs mitochondrial Ca(2+) handling. In acute brain slices, NAADP (10 microM) evoked Ca(2+) transients in cerebellar Bergmann glial cells and in hippocampal astrocytes. Our results suggest that NAADP recruits Ca(2+) from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores in mammalian astrocytes, at least partly by activating metabotropic P(2)Y receptors.     

Apolipoprotein A-I(Milano) and apolipoprotein A-I(Paris) exhibit an antioxidant activity distinct from that of wild-type apolipoprotein A-I

Apolipoprotein A-I(Milano) (apoA-I(Milano)) and apoA-I(Paris) are rare cysteine variants of apoA-I that produce a HDL deficiency in the absence of cardiovascular disease in humans. This paradox provides the basis for the hypothesis that the cysteine variants possess a beneficial activity not associated with wild-type apoA-I (apoA-I(WT)). In this study, a unique antioxidant activity of apoA-I(Milano) and apoA-I(Paris) is described. ApoA-I(Milano) was twice as effective as apoA-I(Paris) in preventing lipoxygenase-mediated oxidation of phospholipids, whereas apoA-I(WT) was poorly active. Antioxidant activity was observed using the monomeric form of the variants and was equally effective before and after initiation of oxidative events. ApoA-I(Milano) protected phospholipid from reactive oxygen species (ROS) generated via xanthine/xanthine oxidase (X/Xo) but failed to inhibit X/Xo-induced reduction of cytochrome c. These results indicate that apoA-I(Milano) was unable to directly quench ROS in the aqueous phase. There were no differences between lipid-free apoA-I(Milano,) apoA-I(Paris), and apoA-I(WT) in mediating the efflux of cholesterol from macrophages, indicating that the cysteine variants interacted normally with the ABCA1 efflux pathway. The results indicate that incorporation of a free thiol within an amphipathic alpha helix of apoA-I confers an antioxidant activity distinct from that of apoA-I(WT). These studies are the first to relate gain of function to rare cysteine mutations in the apoA-I primary sequence.     

Increased AT(1) receptor heterodimers in preeclampsia mediate enhanced angiotensin II responsiveness

Several examples of functional G-protein-coupled receptor heterodimers have been identified. However, it is not known whether receptor heterodimerization is involved in the pathogenesis of human disorders. Here we show that in preeclamptic hypertensive women, a significant increase in heterodimerization occurs between the AT(1)-receptor for the vasopressor angiotensin II and the B(2)-receptor for the vasodepressor bradykinin. AT(1)-B(2)-receptor heterodimerization in preeclampsia correlated with a 4-5-fold increase in B(2)-receptor protein levels. Expression of the AT(1)-B(2) heterodimer increased the responsiveness to angiotensin II and conferred resistance in AT(1)-receptors to inactivation by reactive oxygen species raised in normotensive and preeclamptic pregnancies. We suggest that AT(1)-B(2) heterodimers contribute to angiotensin II hypersensitivity in preeclampsia. Moreover, we identify preeclampsia as the first disorder associated with altered G-protein-coupled receptor heterodimerization.