Abstract:Nanometer scale apolipoprotein A-I stabilized phospholipid disk complexes (nanodisks; ND) have been formulated with the polyene antibiotic amphotericin B (AMB). The present studies were designed to evaluate if a peptide can substitute for the function of the apolipoprotein component of ND with respect to particle formation and stability. An 18-residue synthetic amphipathic α-helical, solubilized vesicles comprised of egg phosphatidylcholine (egg PC), dipentadecanoyl PC or dimyristoylphosphatidylcholine (DMPC) … Show more
“…Immunoblot analysis of a portion of selected column fractions revealed that Wnt3a and apoA-I co-elute from the column with an apparent molecular weight in the range of 100 kDa. This value is consistent with previous studies of ND [17, 23–25] wherein evidence of discoidal morphology was obtained by electron microscopy or atomic force microscopy. By contrast, when a control sample containing apoA-I and Wnt3a, but no phospholipid, was chromatographed, apoA-I was recovered in the column eluate but Wnt3a was lost (data not shown).Fig.…”
BackgroundWnt proteins modulate development, stem cell fate and cancer through interactions with cell surface receptors. Wnts are cysteine-rich, glycosylated, lipid modified, two domain proteins that are prone to aggregation. The culprit responsible for this behavior is a covalently bound palmitoleoyl moiety in the N-terminal domain.ResultsBy combining murine Wnt3a with phospholipid and apolipoprotein A-I, ternary complexes termed nanodisks (ND) were generated. ND-associated Wnt3a is soluble in the absence of detergent micelles and gel filtration chromatography revealed that Wnt3a co-elutes with ND. In signaling assays, Wnt3a ND induced β-catenin stabilization in mouse fibroblasts as well as hematopoietic stem and progenitor cells (HSPC). Prolonged exposure of HSPC to Wnt3a ND stimulated proliferation and expansion of Lin− Sca-1+ c-Kit+ cells. Surprisingly, ND lacking Wnt3a contributed to Lin− Sca-1+ c-Kit+ cell expansion, an effect that was not mediated through β-catenin.ConclusionsThe data indicate Wnt3a ND constitute a water-soluble transport vehicle capable of promoting ex vivo expansion of HSPC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0218-5) contains supplementary material, which is available to authorized users.
“…Immunoblot analysis of a portion of selected column fractions revealed that Wnt3a and apoA-I co-elute from the column with an apparent molecular weight in the range of 100 kDa. This value is consistent with previous studies of ND [17, 23–25] wherein evidence of discoidal morphology was obtained by electron microscopy or atomic force microscopy. By contrast, when a control sample containing apoA-I and Wnt3a, but no phospholipid, was chromatographed, apoA-I was recovered in the column eluate but Wnt3a was lost (data not shown).Fig.…”
BackgroundWnt proteins modulate development, stem cell fate and cancer through interactions with cell surface receptors. Wnts are cysteine-rich, glycosylated, lipid modified, two domain proteins that are prone to aggregation. The culprit responsible for this behavior is a covalently bound palmitoleoyl moiety in the N-terminal domain.ResultsBy combining murine Wnt3a with phospholipid and apolipoprotein A-I, ternary complexes termed nanodisks (ND) were generated. ND-associated Wnt3a is soluble in the absence of detergent micelles and gel filtration chromatography revealed that Wnt3a co-elutes with ND. In signaling assays, Wnt3a ND induced β-catenin stabilization in mouse fibroblasts as well as hematopoietic stem and progenitor cells (HSPC). Prolonged exposure of HSPC to Wnt3a ND stimulated proliferation and expansion of Lin− Sca-1+ c-Kit+ cells. Surprisingly, ND lacking Wnt3a contributed to Lin− Sca-1+ c-Kit+ cell expansion, an effect that was not mediated through β-catenin.ConclusionsThe data indicate Wnt3a ND constitute a water-soluble transport vehicle capable of promoting ex vivo expansion of HSPC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0218-5) contains supplementary material, which is available to authorized users.
“…18 Previous studies have shown that a truncated fragment of apolipoprotein and their oligomers can form a nanodisc by the interaction with lipids. 19–23 Additionally, some amphipathic α -helical peptides have been found to form nanodiscs. 24,25 These facts suggested that an amphipathic structure of these proteins and peptides is essential to form lipid nanodiscs.…”
There is a growing interest in the use of lipid bilayer nanodiscs for various biochemical and biomedical applications. Among the different types of nanodiscs, the unique features of synthetic polymer-based nanodiscs have attracted additional interest. A styrene–maleic acid (SMA) copolymer demonstrated to form lipid nanodiscs has been used for structural biology related studies on membrane proteins. However, the application of SMA polymer based lipid nanodiscs is limited because of the strong absorption of the aromatic group interfering with various experimental measurements. Thus, there is considerable interest in the development of other molecular frameworks for the formation of polymer-based lipid nanodiscs. In this study, we report the first synthesis and characterization of a library of polymethacrylate random copolymers as alternatives to SMA polymer. In addition, we experimentally demonstrate the ability of these polymers to form lipid bilayer nanodiscs through the fragmentation of lipid vesicles by means of light scattering, electron microscopy, differential scanning calorimetry, and solution and solid-state NMR experiments. We further demonstrate a unique application of the newly developed polymer for kinetics and structural characterization of the aggregation of human islet amyloid polypeptide (also known as amylin) within the lipid bilayer of the polymer nanodiscs using thioflavin-T-based fluorescence and circular dichroism experiments. Our results demonstrate that the reported new styrene-free polymers can be used in high-throughput biophysical experiments. Therefore, we expect that the new polymer nanodiscs will be valuable in the structural studies of amyloid proteins and membrane proteins by various biophysical techniques.
“…Further studies will be required to evaluate ATRA-ND as an in vivo delivery vehicle for this potent inducer of cell differentiation and apoptosis. Since ND particles containing amphotericn B have been generated using apoliprotein A-I (Oda et al, 2006) or a synthetic amphipathic peptide (Tufteland et al, 2007), it is anticipated that ATRA-ND can be generated that contain one of several potential alternate scaffold proteins / peptides.…”
Nanodisks are nanoscale, disk-shaped phospholipid bilayers whose edge is stabilized by association of apolipoprotein molecules. Self assembled ND particles enriched with all-trans-retinoic acid (ATRA) [phospholipid:ATRA molar ratio = 5.5:1] were generated wherein all reaction components were solubilized. ATRA-ND migrated as a single band (Stokes' diameter ∼20 nm) on native gradient polyacrylamide gel electrophoresis. ATRA, phospholipid and apolipoprotein co-eluted from a Sepharose 6B gel filtration column, consistent with stable integration of ATRA into the ND particle milieu. Spectroscopic analysis of ATRA-ND in buffer yielded an absorbance spectrum characteristic of ATRA. ATRA-ND mediated time-dependent inhibition of cultured HepG2 cell growth more effectively than free ATRA. The nanoscale size of the formulation particles and the stable integration of biologically active ATRA suggest ND represent a potentially useful vehicle for solubilization and in vivo delivery of ATRA.
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