Targeted intracellular delivery of resveratrol to glioblastoma cells using apolipoprotein E-containing reconstituted HDL as a nanovehicle.

The objective of this study is to transport and deliver resveratrol to intracellular sites using apolipoprotein E3 (apoE3). Reconstituted high-density lipoprotein (rHDL) bearing resveratrol (rHDL/res) was prepared using phospholipids and the low-density lipoprotein receptor (LDLr)-binding domain of apoE3. Biophysical characterization revealed that resveratrol was partitioned into the phospholipid bilayer of discoidal rHDL/res particles (~19 nm diameter). Co-immunoprecipitation studies indicated that the LDLr-binding ability of apoE3 was retained. Cellular uptake of resveratrol to intracellular sites was evaluated in glioblastoma A-172 cells by direct fluorescence using chemically synthesized NBD-labeled resveratrol (res/NBD) embedded in rHDL/res. Competition and inhibition studies indicate that the uptake is by receptor mediated endocytosis via the LDLr, with co-localization of apoE3 and res/NBD in late endosomes/lysosomes. We propose that rHDL provides an ideal hydrophobic milieu to sequester resveratrol and that rHDL containing apoE3 serves as an effective "nanovehicle" to transport and deliver resveratrol to targeted intracellular sites.

A pyrene based fluorescence approach to study conformation of apolipoprotein E3 in macrophage-generated nascent high density lipoprotein.

Apolipoprotein E3 (apoE3) is an anti-atherogenic apolipoprotein with the ability to exist in lipid-free and lipoprotein-associated states. During atherosclerosis, its function in promoting cholesterol efflux from macrophages via the ATP-binding cassette transporter A1 (ABCA1) takes a prominent role, leading to generation of nascent high density lipoprotein (nHDL) particles. The objective of this study is to understand the conformation adopted by apoE3 in macrophage-generated nHDL using a fluorescence spectroscopic approach involving pyrene. Pyrene-labeled recombinant human apoE3 displayed a robust ability to stimulate ABCA1-mediated cholesterol efflux from cholesterol-loaded J774 macrophages (which do not express apoE), comparable to that elicited by unlabeled apoE3. The nHDL recovered from the conditioned medium revealed the presence of apoE3 by immunoblot analysis. A heterogeneous population of nHDL bearing exogenously added apoE3 was generated with particle size varying from ∼12 to ∼19 nm in diameter, corresponding to molecular mass of ∼450 to ∼700 kDa. The lipid: apoE3 ratio varied from ∼60:1 to 10:1. A significant extent of pyrene excimer emission was noted in nHDL, indicative of spatial proximity between Cys112 on neighboring apoE3 molecules similar to that noted in reconstituted HDL. Cross-linking analysis using Cys-specific cross-linkers revealed the predominant presence of dimers. Taken together the data indicate a double belt arrangement of apoE molecules on nHDL. A similar organization of the C-terminal tail of apoE on nHDL was noted when pyrene-apoEA277C(201-299) was used as the cholesterol acceptor. These studies open up the possibility of using exogenously labeled apoE3 to generate nHDL for structural and conformational analysis.

Biochemical and biophysical characterization of recombinant rat apolipoprotein E: similarities to human apolipoprotein E3.

Apolipoprotein E (apoE) is an anti-atherogenic protein that plays a critical role in maintaining plasma cholesterol and triglyceride homeostasis by virtue of its ability to act as a ligand for the low-density lipoprotein receptor (LDLr) family of proteins. In this study, we characterized the biochemical and biophysical features of recombinant rat apoE, in comparison with those of human apoE3. Rat apoE was overexpressed in Escherichia coli using a codon optimized system and purified by affinity chromatography. SDS-PAGE and RP-HPLC of rat apoE confirmed the purity, while immunoblot verified the identity and cross-reactivity with the LDLr-binding region of apoE3. The α-helical content was calculated to be ~45% by circular dichroism spectroscopy. The protein exists in a predominantly tetrameric form in lipid-free state. Chemical denaturation studies reveal that the unfolding pattern is biphasic with mid points of denaturation corresponding to 0.8 and 2.2 M guanidine hydrochloride, suggesting the presence of two domains. Rat apoE converts DMPC vesicles to smaller DMPC/apoE complexes with a first order rate constant of 0.12 min(-1). It has the ability to bind the LDLr and to heparin. Our studies indicate that although its sequence resembles apoE4, an isoform of apoE3, rat apoE displays the biophysical behavior of apoE3.

The extent of pyrene excimer fluorescence emission is a reflector of distance and flexibility: analysis of the segment linking the LDL receptor-binding and tetramerization domains of apolipoprotein E3.

Pyrene is a spatially sensitive probe that displays an ensemble of monomeric fluorescence emission peaks (375-405 nm) and an additional band (called excimer) at ~460 nm when two fluorophores are spatially proximal. We examined if there is a correlation between distance between two pyrenes on an α-helical structure and excimer/monomer (e/m) ratio. Using structure-guided design, pyrene maleimide was attached to pairs of Cys residues separated by ~5 Å increments on helix 2 of the N-terminal domain of apolipoprotein E3 (apoE3). Fluorescence spectral analysis revealed an intense excimer band when the probes were ~5 Å from each other with an e/m ratio of ~3.0, which decreased to ~1.0 at 20 Å. An inverse correlation between e/m ratio and the distance between pyrenes was observed, with the probe and helix flexibility also contributing to the extent of excimer formation. We verified this approach by estimating the distance between T57C and C112 (located on helices 2 and 3, respectively) to be 5.2 Å (4.9 Å from NMR and 5.7 Å from the X-ray structure). Excimer formation was also noted to a significant extent with probes located in the linker segment, suggesting spatial proximity (10-15 Å) to corresponding sites on neighboring molecules in the tetrameric configuration of apoE. We infer that oligomerization via the C-terminal domain juxtaposes the linker segments from neighboring apoE molecules. This study offers new insights into the conformation of tetrameric apoE and presents the use of pyrene as a powerful probe for studying protein spatial organization.

The positional specificity of EXXK motifs within an amphipathic α-helix dictates preferential lysine modification by acrolein: implications for the design of high-density lipoprotein mimetic peptides.

Despite the ability of acrolein to damage proteins, factors governing its reactivity with the ε-amino group of lysine are poorly understood. We used a small 26-mer α-helical peptide (ATI-5261) to evaluate the influence of acidic glutamate (E) residues on site-specific lysine modification by acrolein and if this targeting played a major role in inhibiting the cholesterol efflux activity of the peptide. Exposure of ATI-5261 to acrolein resulted in N-(3-formyl-3,4-dehydropiperidino) (FDP)-lysine adducts at positions 5 and 25 and led to a concentration-dependent reduction in cholesterol efflux activity (55 ± 7 and 83 ± 3% decrease with 5:1 and 20:1 acrolein:peptide molar ratios, respectively). Amino acid substitution (K → R) experiments and mass spectrometry revealed neither K5 nor K25 was preferentially modified by acrolein, despite the location of K5 within a putative EXXK motif. Moreover, both lysine residues remained equally reactive when the lipidated peptide was exposed to acrolein. In contrast, placement of EXXK in the center of ATI-5261 resulted in site-specific modification of lysine. The latter was dependent on glutamate, thus establishing that acidic residues facilitate lysine modification and form the molecular basis of the EXXK motif. Preferential targeting of lysine, however, failed to augment the inhibitory effect of the aldehyde. Overall, the inhibitory effects of acrolein on cholesterol efflux activity were largely dependent on the number of lysine residue modifications and cross-linking of α-helical strands that restricted dissociation of the peptide to active forms.