Apolipoprotein AI (apoA-I) is the principal acceptor of lipids from ATP-binding cassette transporter A1, a process that yields nascent high density lipoproteins. Analysis of lipidated apoA-I conformation yields a belt or twisted belt in which two strands of apoA-I lie antiparallel to one another. In contrast, biophysical studies have suggested that a part of lipid-free apoA-I was arranged in a 4-helix bundle. To understand how lipid-free apoA-I opens from a bundle to a belt while accepting lipid it was necessary to have a more refined model for the conformation of lipid-free apoA-I. This study reports the conformation of lipid-free human apoA-I using lysine-to-lysine chemical cross-linking in conjunction with disulfide cross-linking achieved using selective cysteine mutations. After proteolysis cross-linked peptides were verified by sequencing using tandem mass spectrometry. The resulting structure is compact with roughly 4 helical regions, amino acids 44 through 186, bundled together. C- and N-terminal ends, amino acids 1-43 and 187-243, respectively, are folded such that they lie close to one another. An unusual feature of the molecule is the high degree of connectivity of lysine40 with 6 other lysines, lysines that are close, e.g., lysine59, to distant lysines, e.g., lysine239, that are at the opposite end of the primary sequence. These results are compared and contrasted with other reported conformations for lipid-free human apoA-I and an NMR study of mouse apoA-I.

Background: Extracellular matrix protein PCPE2 is linked to alterations in HDL size and concentration. Results: PCPE2 protects against diet-induced atherosclerosis by promoting HDL catabolism, reverse cholesterol transport, and SR-BI-mediated uptake of HDL-cholesteryl ester. Conclusion: PCPE2 mediates HDL function by reducing lipid and immune cell accumulation in the artery. Significance: These findings establish a role for the extracellular matrix glycoprotein PCPE2 in SR-BI-mediated HDL function and the prevention of atherosclerosis.

Purpose of reviewThe purpose of this study is to provide an update on the role HDL apolipoprotein A-I plays in reducing the risk of cardiovascular disease (CVD) and how it relates to reverse cholesterol transport (RCT).Recent findingsDespite numerous studies showing that plasma HDL cholesterol concentrations are correlated with a reduced risk of CVD, pharmacologic elevation of HDL has not shown any beneficial effects to date. In contrast, studies correlating the measure of an individual's plasma cholesterol efflux capacity show greater promise as a tool for assessing CVD risk. Although ATP-binding cassette transporter 1-mediated lipidation of apoA-I is considered the principal source of plasma HDL, it represents only one side of the RCT pathway. Equally important is the second half of the RCT pathway in which the liver scavenger receptor class B1 selectively removes HDL cholesteryl esters for excretion. The combined action of the two enzyme systems is reflected in the overall steady-state concentration of plasma HDL cholesterol. For example, reduced ATP-binding cassette transporter 1-mediated production of nascent HDL lowers plasma HDL concentration, just as an increase in cholesteryl ester uptake by scavenger receptor class B1 reduces HDL levels. Thus, the complexity of intravascular HDL metabolism suggests that steady-state plasma HDL concentrations do not provide adequate information regarding an individual's HDL quality or function. Herein, we describe a new player, procollagen C-endopeptidase enhancer 2, which shows atheroprotective function and influences both sides of RCT by enhancing production and catabolism of HDL cholesteryl esters.SummaryThe discovery of a new molecule, procollagen C-endopeptidase enhancer 2, implicated in the regulation of HDL cholesteryl ester concentrations suggests that the extracellular matrix and the proteins that regulate its function represent a new and as yet unexplored realm of HDL cholesterol metabolism.

The first step in removing cholesterol from a cell is the ATP-binding cassette transporter 1 (ABCA1)-driven transfer of cholesterol to lipid-free or lipid-poor apolipoprotein A-I (apoA-I), which yields cholesterol-rich nascent high-density lipoprotein (nHDL) that then matures in plasma to spherical, cholesteryl ester-rich HDL. However, lipid-free apoA-I has a three-dimensional (3D) conformation that is significantly different from that of lipidated apoA-I on nHDL. By comparing the lipid-free apoA-I 3D conformation of apoA-I to that of 9–14 nm diameter nHDL, we formulated the hypothetical helical domain transitions that might drive particle formation. To test the hypothesis, ten apoA-I mutants were prepared that contained two strategically placed cysteines several of which could form intramolecular disulfide bonds and others that could not form these bonds. Mass spectrometry was used to identify amino acid sequence and intramolecular disulfide bond formation. Recombinant HDL (rHDL) formation was assessed with this group of apoA-I mutants. ABCA1-driven nHDL formation was measured in four mutants and wild-type apoA-I. The mutants contained cysteine substitutions in one of three regions: the N-terminus, amino acids 34 and 55 (E34C to S55C), central domain amino acids 104 and 162 (F104C to H162C), and the C-terminus, amino acids 200 and 233 (L200C to L233C). Mutants were studied in the locked form, with an intramolecular disulfide bond present, or unlocked form, with the cysteine thiol blocked by alkylation. Only small amounts of rHDL or nHDL were formed upon locking the central domain. We conclude that both the N- and C-terminal ends assist in the initial steps in lipid acquisition, but that opening of the central domain was essential for particle formation.

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