Increasing STEM Skills, Knowledge and Interest Among Diverse Students: Results from an Intensive Summer Research Program at the University of California, San Francisco.

This study evaluates the effectiveness of the UCSF Summer Student Research Program (SSRP) in enhancing research-related skills, academic outcomes, and post-baccalaureate aspirations of underrepresented minority (URM) and non-URM undergraduate students in biomedical sciences and STEM fields. The SSRP, spanning 9 weeks, provides immersive research experiences, structured mentorship, trainings, seminars, and STEM education. Pre- and post-program survey data from eight cohorts (N = 315) were analyzed using paired-sample t-tests, MANOVA, and content analysis. Results demonstrate significant gains in critical thinking skills, research abilities, science identity, applied science skills, and readiness for a research career. Notably, participants exhibited improvements in understanding the research process, scientific thinking, science writing, and problem-solving. URM and non-URM students experienced similar gains, highlighting the program's inclusivity. The SSRP also positively influenced students' postgraduate aspirations. Some participants expressed heightened interest in pursuing Master of Arts, Ph.D., and M.D. degrees, indicating increased clarity and motivation towards advanced education and research careers. Furthermore, 87% of participants expressed a high likelihood of engaging in future research endeavors, underscoring the program's sustained impact on research interest. This study underscores the transformative potential of a well-structured, intensive summer research program in significantly enhancing academic outcomes for URM and non-URM students alike. These findings align with the persistence framework, emphasizing the importance of early research experiences, active learning, and learning communities in fostering student success. The SSRP's effectiveness in improving research skills and post-baccalaureate aspirations suggests its potential in diversifying the STEM fields, biomedical sciences and health-related professions.

Insights into the C-terminal domain of apolipoprotein E from chimera studies with apolipophorin III.

Apolipoprotein E3 (apoE) is a critical cholesterol transport protein in humans and is composed of two domains: a well characterized N-terminal (NT) domain that harbors the low-density lipoprotein LDL receptor, and a less understood C-terminal (CT) domain that is the site of protein oligomerization and initiation of lipid binding. To better understand the domain structure of apoE, the CT domain was fused to apolipophorin III (apoLp-III), a single-domain, monomeric apolipoprotein of insect origin, to yield a chimeric protein, apoLp-III/CT-apoE. Recombinant apoLp-III/CT-apoE maintained an overall helical content similar to that of the parent proteins, while chemical induced unfolding studies indicated that its structural integrity was not compromised. Analysis using 1-anilinonaphthalene-8-sulfonic acid (ANS), a sensitive fluorescent indicator of exposed hydrophobic sites and protein folding, demonstrated that whereas apoLp-III provided few ANS binding sites, apoLp-III/CT-apoE harbored an abundance of ANS binding sites. Thus, this indicated tertiary structure formation in CT-apoE when part of the chimera. Size-exclusion chromatography and chemical crosslinking analysis demonstrated that while apoLp-III is monomeric, the chimeric protein formed large oligomeric complexes, similar to native apoE3. Compared to apoLp-III, the chimera showed a two-fold enhancement in phospholipid vesicle solubilization rates and a significantly improved ability to bind to lipolyzed low-density lipoprotein, preventing the onset of lipoprotein aggregation at concentrations comparable to that of parent CT-apoE. These results confirm that high lipid binding and self-association sites are located in the CT domain of apoE, and that these properties can be transferred to an unrelated apolipoprotein, demonstrating that these properties operate independently from the NT domain.

Cellular Uptake and Clearance of Oxidatively-modified Apolipoprotein E3 by Cerebral Cortex Endothelial Cells.

Apolipoprotein E3 (apoE3) plays a critical role in the metabolism of lipoproteins and lowers plasma lipid levels by serving as a ligand for the low-density lipoprotein receptor (LDLr) family of proteins and by promoting macrophage cholesterol efflux. The current study examines the effect of acrolein (an endogenously generated metabolite and an environmental pollutant) modification on the structure and function of apoE3. Acrolein modification was confirmed in Western blots by reactivity with acrolein-lysine-specific antibody and by the presence of oligomeric species due to cross-linking. LC-MS/MS analysis revealed modification of 10 out of 12 lysines in apoE3, with Nε-(3-methylpyridinium)-lysine being the predominant form of modification, and Lys75 being a 'hot spot' in terms of susceptibility to oxidation. Circular dichroism spectroscopy showed no major change in overall secondary structure compared to unmodified apoE3. Reconstituted high density lipoprotein (HDL) bearing acrolein modified apoE3 showed loss of binding to soluble LDLr; however, incubation with mouse endothelioma bEnd.3 cells showed that it was internalized. Incubation with excess LDL did not abolish cellular uptake of acrolein modified apoE3, suggesting alternative mechanism(s) not involving LDLr. Incubation with anti-CD36 antibody did not show a decrease in internalization while incubation with anti- lectin-like oxidized LDL receptor 1 (LOX1) showed partial internalization. However, incubation with anti-scavenger receptor class B type I (SRB1) antibody abolished internalization of acrolein modified apoE3. Taken together, our studies suggest that acrolein modification of apoE3 at lysine residues leads to increase in net negative charge, and as a consequence, results in clearance by LOX1 and SRB1 on endothelial cells. Overall, oxidative modification of apoE3 likely impairs its role in regulating plasma cholesterol homeostasis, eventually leading to lipid disorders.

Transfer of C-terminal residues of human apolipoprotein A-I to insect apolipophorin III creates a two-domain chimeric protein with enhanced lipid binding activity.

Apolipophorin III (apoLp-III) is an insect apolipoprotein (18kDa) that comprises a single five-helix bundle domain. In contrast, human apolipoprotein A-I (apoA-I) is a 28kDa two-domain protein: an α-helical N-terminal domain (residues 1-189) and a less structured C-terminal domain (residues 190-243). To better understand the apolipoprotein domain organization, a novel chimeric protein was engineered by attaching residues 179 to 243 of apoA-I to the C-terminal end of apoLp-III. The apoLp-III/apoA-I chimera was successfully expressed and purified in E. coli. Western blot analysis and mass spectrometry confirmed the presence of the C-terminal domain of apoA-I within the chimera. While parent apoLp-III did not self-associate, the chimera formed oligomers similar to apoA-I. The chimera displayed a lower α-helical content, but the stability remained similar compared to apoLp-III, consistent with the addition of a less structured domain. The chimera was able to solubilize phospholipid vesicles at a significantly higher rate compared to apoLp-III, approaching that of apoA-I. The chimera was more effective in protecting phospholipase C-treated low density lipoprotein from aggregation compared to apoLp-III. In addition, binding interaction of the chimera with phosphatidylglycerol vesicles and lipopolysaccharides was considerably improved compared to apoLp-III. Thus, addition of the C-terminal domain of apoA-I to apoLp-III created a two-domain protein, with self-association, lipid and lipopolysaccharide binding properties similar to apoA-I. The apoA-I like behavior of the chimera indicate that these properties are independent from residues residing in the N-terminal domain of apoA-I, and that they can be transferred from apoA-I to apoLp-III.

Low-density lipoprotein receptor-related protein 1 is a novel modulator of radial glia stem cell proliferation, survival, and differentiation.

The LDL family of receptors and its member low-density lipoprotein receptor-related protein 1 (LRP1) have classically been associated with a modulation of lipoprotein metabolism. Current studies, however, indicate diverse functions for this receptor in various aspects of cellular activities, including cell proliferation, migration, differentiation, and survival. LRP1 is essential for normal neuronal function in the adult CNS, whereas the role of LRP1 in development remained unclear. Previously, we have observed an upregulation of LewisX (LeX) glycosylated LRP1 in the stem cells of the developing cortex and demonstrated its importance for oligodendrocyte differentiation. In the current study, we show that LeX-glycosylated LRP1 is also expressed in the stem cell compartment of the developing spinal cord and has broader functions in the developing CNS. We have investigated the basic properties of LRP1 conditional knockout on the neural stem/progenitor cells (NSPCs) from the cortex and the spinal cord, created by means of Cre-loxp-mediated recombination in vitro. The functional status of LRP1-deficient cells has been studied using proliferation, differentiation, and apoptosis assays. LRP1 deficient NSPCs from both CNS regions demonstrated altered differentiation profiles. Their differentiation capacity toward oligodendrocyte progenitor cells (OPCs), mature oligodendrocytes and neurons was reduced. In contrast, astrocyte differentiation was promoted. Moreover, LRP1 deletion had a negative effect on NSPCs proliferation and survival. Our observations suggest that LRP1 facilitates NSPCs differentiation via interaction with apolipoprotein E (ApoE). Upon ApoE4 stimulation wild type NSPCs generated more oligodendrocytes, but LRP1 knockout cells showed no response. The effect of ApoE seems to be independent of cholesterol uptake, but is rather mediated by downstream MAPK and Akt activation. GLIA 2016 GLIA 2016;64:1363-1380.

Acrolein modification impairs key functional features of rat apolipoprotein E: identification of modified sites by mass spectrometry.

Apolipoprotein E (apoE), an antiatherogenic apolipoprotein, plays a significant role in the metabolism of lipoproteins. It lowers plasma lipid levels by acting as a ligand for the low-density lipoprotein receptor (LDLr) family of proteins, in addition to playing a role in promoting macrophage cholesterol efflux in atherosclerotic lesions. The objective of this study is to examine the effect of acrolein modification on the structure and function of rat apoE and to determine the sites and nature of modification by mass spectrometry. Acrolein is a highly reactive aldehyde, which is generated endogenously as one of the products of lipid peroxidation and is present in the environment in pollutants such as tobacco smoke and heated oils. In initial studies, acrolein-modified apoE was identified by immunoprecipitation using an acrolein-lysine specific antibody in the plasma of 10-week old male rats that were exposed to filtered air (FA) or low doses of environmental tobacco smoke (ETS). While both groups displayed acrolein-modified apoE in the lipoprotein fraction, the ETS group had higher levels in the lipid-free fraction compared with the FA group. This observation provided the rationale to further investigate the effect of acrolein modification on rat apoE at a molecular level. Treatment of recombinant rat apoE with a 10-fold molar excess of acrolein resulted in (i) a significant decrease in lipid-binding and cholesterol efflux abilities, (ii) impairment in the LDLr- and heparin-binding capabilities, and (iii) significant alterations in the overall stability of the protein. The disruption in the functional abilities is attributed directly or indirectly to acrolein modification yielding an aldimine adduct at K149 and K155 (+38); a propanal adduct at K135 and K138 (+56); an N(ε)-(3-methylpyridinium)lysine (MP-lysine) at K64, K67, and K254 (+76), and an N(ε)-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) derivative at position K68 (+94), as determined by matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). The loss of function may also be attributed to alterations in the overall fold of the protein as noted by changes in the guanidine HCl-induced unfolding pattern and to protein cross-linking. Overall, disruption of the structural and functional integrity of apoE by oxidative modification of essential lysine residues by acrolein is expected to affect its role in maintaining plasma cholesterol homeostasis and lead to dysregulation in lipid metabolism.

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.

Apolipoprotein E3 Containing Nanodiscs as Vehicles for Transport and Targeted Delivery of Flavonoid Luteolin.

Luteolin is a flavonoid that possesses multiple beneficial biological properties, such as anticancer, antioxidant, and anti-inflammatory effects. The objective of this study is to test the hypothesis that luteolin can be transported across a cell via a nanodisc delivery system and delivered to intracellular sites. Luteolin was incorporated into reconstituted high-density lipoprotein complexes made up of apolipoprotein E3 (apoE3) N-terminal domain (apoE3NT) and 1,2-dimystrioyl-sn-glycero-3-phosphocholine. ApoE3NT confers the ability on nanodiscs to traverse the plasma membrane via low-density lipoprotein receptor or scavenger receptor-B1. Physicochemical characterization revealed that the nanodiscs were 17-22 nm in diameter as demonstrated by native polyacrylamide gel electrophoresis and dynamic lightering analysis and ∼660 kDa in size, with a luteolin content of ∼4 luteolin molecules/nanodisc. Luteolin appeared to be embedded in the nonpolar core of nanodiscs, as revealed by fluorescence quenching and polarization analysis and spectroscopic characterization. The presence of luteolin did not affect the ability of apoE3NT to mediate binding and cellular uptake of luteolin containing nanodiscs in macrophages, as inferred from immunofluorescence analysis that revealed apoE- and lipid-related fluorescence as punctate perinuclear vesicles and from flow cytometry studies. Lastly, luteolin appeared to be localized in the nucleus, having escaped the lysosomes following disassembly of the nanodiscs as suggested by fluorescence spectroscopy and microscopy analyses. Taken together, nanodiscs offer the potential to effectively transport luteolin and potentially therapeutic drugs into perinuclear sites in cells, where they can be available to enter the nucleus.

Molecular dynamics simulation of apolipoprotein E3 lipid nanodiscs.

Nanodiscs are binary discoidal complexes of a phospholipid bilayer circumscribed by belt-like helical scaffold proteins. Using coarse-grained and all-atom molecular dynamics simulations, we explore the stability, size, and structure of nanodiscs formed between the N-terminal domain of apolipoprotein E3 (apoE3-NT) and variable number of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) molecules. We study both parallel and antiparallel double-belt configurations, consisting of four proteins per nanodisc. Our simulations predict nanodiscs containing between 240 and 420 DMPC molecules to be stable. The antiparallel configurations exhibit an average of 1.6 times more amino acid interactions between protein chains and 2 times more ionic contacts, compared to the parallel configuration. With one exception, DMPC order parameters are consistently larger in the antiparallel configuration than in the parallel one. In most cases, the root mean square deviation of the positions of the protein backbone atoms is smaller in the antiparallel configuration. We further report nanodisc size, thickness, radius of gyration, and solvent accessible surface area. Combining all investigated parameters, we hypothesize the antiparallel protein configuration leading to more stable and more rigid nanodiscs than the parallel one.

Retention of α-helical structure by HDL mimetic peptide ATI-5261 upon extensive dilution represents an important determinant for stimulating ABCA1 cholesterol efflux with high efficiency.

ATI-5261 is a novel, single-helix peptide that stimulates cellular cholesterol efflux with high potency similar to native apolipoproteins on a molar basis. Presently we investigated structural features of the peptide that conferred cholesterol efflux activity. Analogs of ATI-5261 with amino acids arranged in reverse order or with individual arginine (R) to glutamine (Q) substitutions (i.e. R3Q, R14Q, or R23Q) stimulated ABCA1 dependent cholesterol efflux similar to ATI-5261. Consequently, neither the presence of specific positively charged residues nor their specific arrangement along the length of the peptide was necessary for mediating cholesterol efflux. Similarly, peptides composed of all d-amino acids stimulated cholesterol efflux efficiently, indicating a stereospecific component was not required for promotion of cholesterol efflux from macrophages. Removal of two or more positively charged residues (R3, 14→Q and R3, 14, 23→Q) however, greatly reduced the ability of ATI-5261 to mediate cellular cholesterol efflux. This was accompanied by a loss of α-helical structure upon dilution, indicating the secondary structure of individual peptide strands was important for stimulating cholesterol efflux. Surprisingly, peptides with removal of two or more positively charged residues retained the ability to bind phospholipid and adopt an α-helical structure. These data indicate that the propensity of a hydrophobic peptide to form an amphipathic α-helix is not sufficient to mediate cellular cholesterol efflux. Efficient stimulation of cholesterol efflux requires that ATI-5261 retain α-helical structure upon dilution.

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.

Isoform-specific modification of apolipoprotein E by 4-hydroxynonenal: protective role of apolipoprotein E3 against oxidative species.

High levels of 4-hydroxynonenal (HNE), arising from lipid peroxidation, and HNE-modified proteins have been identified in postmortem brains of ageing and Alzheimer's disease (AD) patients. The goal of this study is to understand the effect of HNE modification on the structure and function of recombinant apolipoprotein E3 (apoE3) and apolipoprotein E4 (apoE4), which play a critical role in brain cholesterol homeostasis. The two isoforms differ in a single amino acid at position 112: Cys in apoE3 and Arg in apoE4. Immunoblot with HNE-specific antibody indicates HNE modification of apoE3 and apoE4 with a major band at ~ 36 kDa, while LC-MS/MS revealed Michael addition at His140 (60-70% abundance) and His299 (3-5% abundance) in apoE3 and apoE4, and Cys112 adduct in apoE3 (75% abundance). Circular dichroism spectroscopy revealed no major differences in the overall secondary structure or helical content between unmodified and HNE-modified apoE. HNE modification did not affect their ability to promote cholesterol efflux from J774.1 macrophages. However, it led to a 3-fold decrease in their ability to bind lipids and 25-50% decrease in the ability of cerebral cortex endothelial cells to uptake lipoproteins bearing HNE-modified HNE-apoE3 or HNE-apoE4 as noted by fluorescence microscopy and flow cytometry. Taken together, the data indicate that HNE modification impairs lipid binding and cellular uptake of both isoforms, and that apoE3, bearing a Cys, offers a protective role by sequestering lipid peroxidation products that would otherwise cause indiscriminate damage to biomolecules. ApoE4, lacking Cys, is unable to protect against oxidative damage that is commensurate with ageing.

Molecular Dynamics Simulation of Apolipoprotein E3 Lipid Nanodiscs.

Nanodiscs are binary discoidal complexes of a phospholipid bilayer circumscribed by belt-like helical scaffold proteins. Using coarse-grained and all-atom molecular dynamics simulations, we explore the stability, size, and structure of nanodiscs formed between the N-terminal domain of apolipoprotein E3 (apoE3-NT) and variable number of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) molecules. We study both parallel and antiparallel double-belt configurations, consisting of four proteins per nanodisc. Our simulations predict nanodiscs containing between 240 and 420 DMPC molecules to be stable. The antiparallel configurations exhibit an average of 1.6 times more amino acid interactions between protein chains and 2 times more ionic contacts, compared to the parallel configuration. With one exception, DMPC order parameters are consistently larger in the antiparallel configuration than in the parallel one. In most cases, the root mean square deviation of the positions of the protein backbone atoms is smaller in the antiparallel configuration. We further report nanodisc size, thickness, radius of gyration, and solvent accessible surface area. Combining all investigated parameters, we hypothesize the antiparallel protein configuration leading to more stable and more rigid nanodiscs than the parallel one.

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.

Apolipoprotein E LDL receptor-binding domain-containing high-density lipoprotein: a nanovehicle to transport curcumin, an antioxidant and anti-amyloid bioflavonoid.

Curcumin is an antioxidant and anti-inflammatory bioflavonoid that has been recently identified as an anti-amyloid agent as well. To make it more available in its potent form as a potential amyloid disaggregation agent, we employed high-density lipoproteins (HDL), which are lipid-protein complexes that transport plasma cholesterol, to transport curcumin. The objective of this study was to employ reconstituted HDL containing human apoE3 N-terminal (NT) domain, as a vehicle to transport curcumin. The NT domain serves as a ligand to mediate binding and uptake of lipoprotein complexes via the low-density lipoprotein receptor (LDLr) family of proteins located at the cell surface. Reconstituted HDL was prepared with phospholipids and recombinant apoE3-NT domain in the absence or presence of curcumin. Non-denaturing polyacrylamide gel electrophoresis indicated that the molecular mass and Stokes' diameter of HDL bearing curcumin were ~670kDa and ~17nm, respectively, while electron microscopy revealed the presence of discoidal particles. Fluorescence emission spectra of HDL bearing (the intrinsically fluorescent) curcumin indicated that the wavelength of maximal fluorescence emission (λ(max)) of curcumin was ~495nm, which is highly blue-shifted compared to λ(max) of curcumin in solvents of varying polarity (λ(max) ranging from 515-575nm) or in aqueous buffers. In addition, an enormous enhancement in fluorescence emission intensity was noted in curcumin-containing HDL compared to curcumin in aqueous buffers. Curcumin fluorescence emission was quenched to a significant extent by lipid-based quenchers but not by aqueous quenchers. These observations indicate that curcumin has partitioned efficiently into the hydrophobic milieu of the phospholipid bilayer of HDL. Functional assays indicated that the LDLr-binding ability of curcumin-containing HDL with apoE3-NT is similar to that of HDL without curcumin. Taken together, we report that apoE-containing HDL has a tremendous potential as a 'nanovehicle' with a homing device to transport curcumin to target sites.

HDL mimetic peptide ATI-5261 forms an oligomeric assembly in solution that dissociates to monomers upon dilution.

ATI-5261 is a 26-mer peptide that stimulates cellular cholesterol efflux with high potency. This peptide displays high aqueous solubility, despite having amphipathic α-helix structure and a broad nonpolar surface. These features suggested to us that ATI-5261 may adopt a specific form in solution, having favorable structural characteristics and dynamics. To test this, we subjected ATI-5261 to a series of biophysical studies and correlated self-association with secondary structure and activity. Gel-filtration chromatography and native gel electrophoresis indicated ATI-5261 adopted a discrete self-associated form of low molecular weight at concentrations >1 mg/mL. Formation of a discrete molecular species was verified by small-angle X-ray scattering (SAXS), which further revealed the peptide formed a tetrameric assembly having an elongated shape and hollow central core. This assembly dissociated to individual peptide strands upon dilution to concentrations required for promoting high-affinity cholesterol efflux from cells. Moreover, the α-helical content of ATI-5261 was exceptionally high (74.1 ± 6.8%) regardless of physical form and concentration. Collectively, these results indicate ATI-5261 displays oligomeric behavior generally similar to native apolipoproteins and dissociates to monomers of high α-helical content upon dilution. Optimizing self-association behavior and secondary structure may prove useful for improving the translatability and efficacy of apolipoprotein mimetic peptides.

Pyrene: a probe to study protein conformation and conformational changes.

The review focuses on the unique spectral features of pyrene that can be utilized to investigate protein structure and conformation. Pyrene is a fluorescent probe that can be attached covalently to protein side chains, such as sulfhydryl groups. The spectral features of pyrene are exquisitely sensitive to the microenvironment of the probe: it exhibits an ensemble of monomer fluorescence emission peaks that report on the polarity of the probe microenvironment, and an additional band at longer wavelengths, the appearance of which reflects the presence of another pyrene molecule in spatial proximity (~10 Å). Its high extinction coefficient allows us to study labeled proteins in solution at physiologically relevant concentrations. The environmentally- and spatially-sensitive features of pyrene allow monitoring protein conformation, conformational changes, protein folding and unfolding, protein-protein, protein-lipid and protein-membrane interactions.

Pyrene fluorescence analysis offers new insights into the conformation of the lipoprotein-binding domain of human apolipoprotein E.

The C-terminal domain (CT) of apolipoprotein E (apoE), a critical protein involved in cholesterol transport in the plasma and brain, plays an important role in high-affinity lipoprotein binding. Although high-resolution structural information is available for the N-terminal domain of apoE, the structural organization of the CT (residues 201-299) is largely unknown. In this study, we employ site-specific fluorescence labeling with pyrene maleimide to gain insight into the structure and conformation of apoE CT in its naturally self-associated state in buffer at physiologically relevant concentrations (5-50 microg/mL). Pyrene is a highly sensitive fluorophore that reports on spatial proximity between desired sites by displaying unique spectral features. Pyrene was covalently attached to single cysteine-containing recombinant human apoE CT at position 223 or 255 to probe the first predicted helical segment and at position 277 to monitor the terminal predicted helical segment. Regardless of the location of the probe, all three pyrene-labeled apoE CT variants display an intense and dramatic fluorescence excimer band at 460 nm, a signature feature of pyrene, which indicates that two pyrene moieties are within 10 A of each other. In addition, an intense peak at 387 nm (indicative of a highly hydrophobic environment) was noted in all cases. Fluorescence emission quenching by potassium iodide indicates that the accessibility to the probes was restricted at these locations. The possibility that the hydrophobicity of the pyrene moiety was the driving force for helix-helix interaction was excluded because pyrene located at position 209, which is predicted to be located in a nonhelical segment, did not display the above intense unique features. Lastly, denaturation studies suggest that the terminal helix unfolds prior to the first predicted helix in apoE CT. Our studies indicate that there are extensive intermolecular helix-helix contacts throughout the entire CT in the lipid-free state with two apoE CT molecules oriented parallel to each other to form a dimer, which dimerizes further to yield a tetramer. Such an organization allows helix-helix interactions to be replaced by helix-lipid interactions upon encountering a lipoprotein surface, with the terminal helix likely initiating the binding interaction. This study presents the possibility of employing pyrene fluorophores as powerful new alternatives to obtain conformational information of proteins at physiologically relevant concentrations.

A new HDL mimetic peptide that stimulates cellular cholesterol efflux with high efficiency greatly reduces atherosclerosis in mice.

Here, we report the creation of a single-helix peptide (ATI-5261) that stimulates cellular cholesterol efflux with K(m) molar efficiency approximating native apolipoproteins. Anti-atherosclerosis activity of ATI-5261 was evaluated in LDLR(-/-) and apolipoprotein (apo)E(-/-) mice approximately 5-7 months of age, following 13-18 weeks on a high-fat Western diet (HFWD). Treatment of fat-fed LDLR(-/-) mice with daily intraperitoneal injections of ATI-5261 (30 mg/kg) for 6 weeks reduced atherosclerosis by 30%, as judged by lesion area covering the aorta (7.9 +/- 2 vs.11.3 +/- 2.5% control, P = 0.011) and lipid-content of aortic sinus plaque (25 +/- 5.8 vs. 33 +/- 4.9% control, P = 0.014). In apoE(-/-) mice, the peptide administered 30 mg/kg ip on alternate days for 6 weeks reduced atherosclerosis by approximately 45% (lesion area = 15 +/- 7 vs. 25 +/- 8% control, P = 0.00016; plaque lipid-content = 20 +/- 6 vs. 32 +/- 8% control, P < 0.0001). Similar reductions in atherosclerosis were achieved using ATI-5261:POPC complexes. Single intraperitoneal injection of ATI-5261 increased reverse cholesterol transport from macrophage foam-cells to feces over 24-48 h. In summary, relatively short-term treatment of mice with the potent cholesterol efflux peptide ATI-5261 reduced substantial atherosclerosis. This was achieved using an L-amino acid peptide, in the presence of severe hypercholesterolemia/HFWD, and did not require daily injections or formulation with phospholipids when administered via intraperitoneal injection.