Recent studies of Tangier disease have shown that the ATP-binding cassette transporter A1 (ABCA1)/apolipoprotein A-I (apoA-I) interaction is critical for high density lipoprotein particle formation, apoA-I integrity, and proper reverse cholesterol transport. However, the specifics of this interaction are unknown. It has been suggested that amphipathic helices of apoA-I bind to a lipid domain created by the ABCA1 transporter. Alternatively, apoA-I may bind directly to ABCA1 itself. To better understand this interaction, we created several truncation mutants of apoA-I and then followed up with more specific point mutants and helix translocation mutants to identify and characterize the locations of apoA-I required for ABCA1-mediated cholesterol efflux. We found that deletion of residues 221-243 (helix 10) abolished ABCA1-mediated cholesterol efflux from cultured RAW mouse macrophages treated with 8-bromo-cAMP. Point mutations in helix 10 that affected the helical charge distribution reduced ABCA1-mediated cholesterol efflux versus the wild type. We noted a strong positive correlation between cholesterol efflux and the lipid binding characteristics of apoA-I when mutations were made in helix 10. However, there was no such correlation for helix translocations in other areas of the protein as long as helix 10 remained intact at the C terminus. From these observations, we propose an alternative model for apolipoprotein-mediated efflux.

Specialized chromatin structures such as nucleosomes with specific histone modifications decorate exons in eukaryotic genomes, suggesting a functional connection between chromatin organization and the regulation of pre-mRNA splicing. Through profiling the functional location of Poly (ADP) ribose polymerase, we observed that it is associated with the nucleosomes at exon/intron boundaries of specific genes, suggestive of a role for this enzyme in alternative splicing. Poly (ADP) ribose polymerase has previously been implicated in the PARylation of splicing factors as well as regulation of the histone modification H3K4me3, a mark critical for co-transcriptional splicing. In light of these studies, we hypothesized that interaction of the chromatin-modifying factor, Poly (ADP) ribose polymerase with nucleosomal structures at exon–intron boundaries, might regulate pre-mRNA splicing. Using genome-wide approaches validated by gene-specific assays, we show that depletion of PARP1 or inhibition of its PARylation activity results in changes in alternative splicing of a specific subset of genes. Furthermore, we observed that PARP1 bound to RNA, splicing factors and chromatin, suggesting that Poly (ADP) ribose polymerase serves as a gene regulatory hub to facilitate co-transcriptional splicing. These studies add another function to the multi-functional protein, Poly (ADP) ribose polymerase, and provide a platform for further investigation of this protein’s function in organizing chromatin during gene regulatory processes.

Human apolipoprotein E (apoE) mediates high affinity binding to the low density lipoprotein receptor when present on a lipidated complex. In the absence of lipid, however, apoE does not bind the receptor. Whereas the x-ray structure of lipid-free apoE3 N-terminal (NT) domain is known, the structural organization of its lipidassociated, receptor-active conformation is poorly understood. To study the organization of apoE amphipathic ␣-helices in a lipid-associated state, single tryptophan-containing apoE3 variants were employed in fluorescence quenching studies. Apolipoprotein E (ApoE) is a key regulator of plasma cholesterol homeostasis. Its interactions with the low density lipoprotein receptor (LDLR) 1 family and cell surface heparan sulfate proteoglycans (1-3) are a critical step for the cellular uptake of apoE-containing lipoproteins. Transgenic mice overexpressing apoE manifest decreased plasma cholesterol levels on chow diet and a marked resistance to hypercholesterolemia on a high cholesterol/fat diet (4). On the other hand, apoE-deficient subjects display features of type III hyperlipoproteinemia (5) and apoE null mice exhibit massive accumulation of remnant lipoproteins (6), documenting the direct relevance of apoE in lipoprotein metabolism. However, structural features of lipidassociated apoE responsible for its receptor interaction properties are not understood at the molecular level. ApoE comprises two independently folded structural and functional domains that are linked by a protease-sensitive loop segment. The globular 22 kDa N-terminal (NT) domain houses the LDLR recognition site, whereas the 10 kDa Cterminal (CT) domain bears high affinity lipoprotein-binding and self-association sites (7,8). The NT domain is composed of four elongated amphipathic ␣-helices organized as an upand-down helix bundle in the absence of lipid (9, 10). Helix 4 of the NT domain (residues 131-164) harbors key residues required for interaction with lipoprotein receptors (1). However, lipid association is a requirement for apoE (full-length or NT domain) to display receptor recognition capability, with apoE bound to model lipid particles displaying receptor binding ability comparable with that of native apoE-containing lipoproteins (11). Upon interaction with lipid it has been proposed that the NT domain helix bundle "opens" to expose the hydrophobic faces of its amphipathic helices to potential lipid surface binding sites, thereby achieving a receptor-active conformation (1, 12). Monolayer surface balance studies at the air/water interface provide evidence that the NT domain occupies a larger surface area than can be accounted for by its globular 4-helix bundle conformation (13), consistent with adoption of an "open" conformation. Subsequent fluorescence energy transfer analysis revealed that helical segments in the NT domain realign as a function of the transition from lipid-free helix bundle to lipid-bound state (12,14). Attenuated total reflectance Fourier-transformed infrared spectroscopic studies (15) indicate that ap...

Apolipoprotein (apo) A-I is the major protein constituent of human high-density lipoprotein (HDL) and is likely responsible for many of its anti-atherogenic properties. Since distinct HDL size subspecies may play different roles in interactions critical for these properties, a key question concerns how apoA-I can adjust its conformation in response to changes in HDL particle size. A prominent hypothesis states that apoA-I contains a flexible "hinge domain" that can associate/dissociate from the lipoprotein as its diameter fluctuates. Although flexible domains clearly exist within HDL-bound apoA-I, this hypothesis has not been directly tested by assessing the ability of such domains to modulate their contacts with the lipid surface. In this work, discoidal HDL particles of different size were reconstituted with a series of human apoA-I mutants containing a single reporter tryptophan residue within each of its 22 amino acid amphipathic helical repeats. The particles also contained nitroxide spin labels, potent quenchers of tryptophan fluorescence, attached to the phospholipid acyl chains. We then measured the relative exposure of each tryptophan probe with increasing quencher concentrations. We found that, although there were modest structural changes across much of apoA-I, only helices 5, 6, and 7 exhibited significant differences in terms of exposure to lipid between large (96 A) and small (78 A) HDL particles. From these results, we present a model for a putative hinge domain in the context of recent "belt" and "hairpin" models of apoA-I structure in discoidal HDL particles.

Apolipoprotein A-I (apoA-I) is the major protein associated with high density lipoprotein (HDL), and its plasma levels have been correlated with protection against atherosclerosis. Unfortunately, the structural basis of this phenomenon is not fully understood. Over 25 years of study have produced two general models of apoA-I structure in discoidal HDL complexes. The "belt" model states that the amphipathic helices of apoA-I are aligned perpendicular to the acyl chains of the lipid bilayer, whereas the "picket fence" model argues that the helices are aligned parallel with the acyl chains. To distinguish between the two models, various single tryptophan mutants of apoA-I were analyzed in reconstituted, discoidal HDL particles composed of phospholipids containing nitroxide spin labels at various positions along the acyl chain. We have previously used this technique to show that the orientation of helix 4 of apoA-I is most consistent with the belt model. In this study, we performed additional control experiments on helix 4, and we extended the results by performing the same analysis on the remaining 22-mer helices (helices 1, 2, 5, 6, 7, 8, and 10) of human apoA-I. For each helix, two different mutants were produced that each contained a probe Trp occurring two helical turns apart. In the belt model, the two Trp residues in each helix should exhibit maximal quenching at the same nitroxide group position on the lipid acyl chains. For the picket fence model, maximal quenching should occur at two different levels in the bilayer. The results show that the majority of the helices are in an orientation that is consistent with a belt model, because most Trp residues localized to a position about 5 Å from the center of the bilayer. This study corroborates a belt hypothesis for the majority of the helices of apoA-I in phospholipid discs.Because of sedentary lifestyles and high fat diets, atherosclerosis remains one of the leading causes of death in the Western world. It has been demonstrated repeatedly that levels of high density lipoprotein (HDL) 1 and its major protein constituent apolipoprotein A-I (apoA-I) are inversely correlated with the incidence of heart disease (1). Apo-AI, a 28-kDa protein made up of 11-and 22-mer amphipathic helices (Ref. 2; reviewed in Ref. 3), is critical to the formation and stability of the HDL particle in circulation. The process of reverse cholesterol transport is thought to remove excess cholesterol from extra-hepatic tissues such as the arterial wall and return it to the liver for processing (4, 5). ApoA-I performs many critical functions in this pathway. In the lipid poor form, it can interact with the cell surface under the control of the ATP-binding cassette A1 transporter (6). Once apoA-I accumulates phospholipids and cholesterol from the cell surface, it likely forms discoidal complexes that interact with enzymes such as lecithin:cholesterol acetyl transferase and cholesterol ester transfer protein to form the spherical HDL particles commonly found in plasma. These are the particles ...