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Objective- We have shown that ABCA1 (ATP-binding cassette protein A1) mediates unfolding of the apoA1 (apolipoprotein A1) N-terminal helical hairpin during apoA1 lipidation. Others have shown that an acidic pH exposes the hydrophobic surface of apoA1. We postulated that the V-ATPase (vacuolar ATPase) proton pump facilitates apoA1 unfolding and promotes ABCA1-mediated cholesterol efflux. Approach and Results- We found that V-ATPase inhibitors dose-dependently decreased ABCA1-mediated cholesterol efflux to apoA1 in baby hamster kidney cells and RAW264.7 cells; and similarly, siRNA knockdown of ATP6V 0 C inhibited ABCA1-mediated cholesterol efflux to apoA1 in RAW264.7 cells. Although ABCA1 expression did not alter total cellular levels of V-ATPase, ABCA1 increased the cell surface levels of the V 0 A1 and V 1 E1 subunits of V-ATPase. We generated a fluorescein isothiocyanate/Alexa647 double-labeled fluorescent ratiometric apoA1 pH indicator whose fluorescein isothiocyanate/Alexa647 emission ratio decreased as the pH drops. We found that ABCA1 induction in baby hamster kidney cells led to acidification of the cell-associated apoA1 pH indicator, compared with control cells without ABCA1 expression. The V-ATPase inhibitor bafilomycin A1 dose-dependently inhibited the apoA1 pH shift in ABCA1-expressing cells, without affecting the levels of cell-associated apoA1. However, we were not able to detect ABCA1-mediated extracellular proton release. We showed that acidic pH facilitated apoA1 unfolding, apoA1 solubilization of phosphatidycholine:phosphatidyserine liposomes, and increased lipid fluidity of these liposomes. Conclusions- Our results support a model that ABCA1 recruits V-ATPase to the plasma membrane where V-ATPase mediates apoA1 acidification and membrane remodeling that promote apoA1 unfolding and ABCA1-mediated HDL (high-density lipoprotein) biogenesis and lipid efflux.

Objective: Cholesterol metabolism is a dynamic process involving intracellular trafficking, cholesterol esterification, and cholesterol ester hydrolysis. Our objective was to identify genes that regulate macrophage cholesterol metabolism., Approaches and Results: We performed quantitative trait loci mapping of free and esterified cholesterol levels and the ratio of esterified to free cholesterol in acetylated low-density lipoprotein-loaded bone marrow-derived macrophages from an AKR×DBA/2 strain intercross. Ten distinct cholesterol modifier loci were identified, and bioinformatics was used to prioritize candidate genes. The strongest locus was located on distal chromosome 1, which we named Mcmm1 (macrophage cholesterol metabolism modifier 1). This locus harbors the Soat1 (sterol O-acyltransferase 1) gene, encoding Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), which esterifies free cholesterol. The parental AKR strain has an exon 2 deletion in Soat1, which leads to a 33 amino acid N-terminal truncation in ACAT1. CRISPR/Cas9 editing of DBA/2 embryonic stem cells was performed to replicate the AKR strain Soat1 exon 2 deletion, while leaving the remainder of the genome unaltered. DBA/2 stem cells and stem cells heterozygous and homozygous for the Soat1 exon 2 deletion were differentiated into macrophages and loaded with acetylated low-density lipoprotein. DBA/2 stem cell-derived macrophages accumulated less free cholesterol and more esterified cholesterol relative to cells heterozygous and homozygous for the Soat1 exon 2 deletion., Conclusions: A Soat1 deletion present in AKR mice, and resultant N-terminal ACAT1 truncation, was confirmed to be a significant modifier of macrophage cholesterol metabolism. Other Mcmm loci candidate genes were prioritized via bioinformatics., (© 2017 American Heart Association, Inc.)

Objective: To gain insight into the mechanism by which ABCA1 generates nascent high-density lipoprotein., Approach and Results: HEK293 cells were stably transfected with ABCA1 vectors, encoding wild type, and the W590S and C1477R Tangier disease mutation isoforms, along with the K939M ATP-binding domain mutant. Apolipoprotein AI (ApoAI) binding, plasma membrane remodeling, cholesterol efflux, apoAI cell surface unfolding, and apoAI cell surface lipidation were determined, the latter 2 measured using novel fluorescent apoAI indicators. The W590S isoform had decreased plasma membrane remodeling and lipid efflux activities, and the C1477R isoform had decreased apoAI binding, and lipid efflux activities, whereas the K939M isoform did not bind apoAI, remodel the membrane, or efflux cholesterol. However, all ABCA1 isoforms led to apoAI unfolding at the cell surface, which was higher for the isoforms that increased apoAI binding. ApoAI lipidation was not detected on ABCA1-expressing cells, only in the conditioned medium, consistent with rapid release of nascent high-density lipoprotein from ABCA1-expressing cells., Conclusions: We identified a third activity of ABCA1, the ability to unfold the N terminus of apoAI on the cell surface. Our results support a model in which unfolded apoAI on the cell surface is an intermediate in its lipidation and that, once apoAI is lipidated, it forms an unstable structure that is rapidly released from the cells to generate high-density lipoprotein.

Objective: Nascent high-density lipoprotein (HDL) particles form from cellular lipids and extracellular lipid-free apolipoprotein AI (apoAI) in a process mediated by ATP-binding cassette transporter A1 (ABCA1). We have sought out compounds that inhibit nascent HDL biogenesis without affecting ABCA1 activity., Methods and Results: Reconstituted HDL (rHDL) formation and cellular cholesterol efflux assays were used to show that 2 compounds that bond via hydrogen with phospholipids inhibit rHDL and nascent HDL production. In rHDL formation assays, the inhibitory effect of compound 1 (methyl 3α-acetoxy-7α,12α-di[(phenylaminocarbonyl)amino]-5β-cholan-24-oate), the more active of the 2, depended on its ability to associate with phospholipids. In cell assays, compound 1 suppressed ABCA1-mediated cholesterol efflux to apoAI, the 18A peptide, and taurocholate with high specificity, without affecting ABCA1-independent cellular cholesterol efflux to HDL and endocytosis of acetylated low-density lipoprotein and transferrin. Furthermore, compound 1 did not affect ABCA1 activity adversely, as ABCA1-mediated shedding of microparticles proceeded unabated and apoAI binding to ABCA1-expressing cells increased in its presence., Conclusion: The inhibitory effects of compound 1 support a 3-step model of nascent HDL biogenesis: plasma membrane remodeling by ABCA1, apoAI binding to ABCA1, and lipoprotein particle assembly. The compound inhibits the final step, causing accumulation of apoAI in ABCA1-expressing cells.

Objective: Apolipoprotein A-I (apoAI) acts as an ABCA1-dependent acceptor of cellular phospholipids and cholesterol during the biogenesis of HDL, but this activity is susceptible to oxidative inactivation by myeloperoxidase. We tried to determine which residues mediated this inactivation and create an oxidant-resistant apoAI variant., Methods and Results: Mass spectrometry detected the presence of tryptophan, methionine, tyrosine, and lysine oxidation in apoAI recovered from human atheroma. We investigated the role of these residues in the myeloperoxidase-mediated loss of apoAI activity. Site-directed mutagenesis and chemical modification were used to create variants of apoAI which were tested for ABCA1-dependent cholesterol acceptor activity and oxidative inactivation. We previously reported that tyrosine modification is not required for myeloperoxidase-induced loss of apoAI function. Lysine methylation did not alter the sensitivity of apoAI to myeloperoxidase, whereas site-specific substitution of apoAI methionine to valine increased the sensitivity of apoAI to myeloperoxidase. ApoAI tryptophan residues were identified as essential in apoAI function and oxidant sensitivity as substitution of all four apoAI tryptophan residues to leucine led to loss of function, but the conservative substitution to phenylalanine retained full function and was resistant to oxidative inactivation., Conclusions: Tryptophan modification of apoAI is primarily responsible for the myeloperoxidase-mediated loss of the cholesterol acceptor activity of apoAI.

Objective: To determine the mechanism by which expression of the murine ABCA1 gene is highly induced by cAMP analogues., Methods and Results: ABCA1 mRNA turnover cannot account for its induction by cAMP. Thus cAMP induction of ABCA1 mRNA occurs at a transcriptional level. Shotgun cloning DNA fragments from the murine ABCA1 locus identified a strong cAMP responsive enhancer located in the first intron, which led to 25- to 100-fold cAMP-mediated induction of reporter gene activity. Deletions and mutations of this enhancer led to the identification a cAMP-responsive element (CRE) that was essential for the cAMP induction. Furthermore, the capacity of this CRE site to mediate the cAMP induction required the presence of a STAT3/4 element located 81 bp away. A dominant-negative CREB expression vector inhibited the cAMP induction of ABCA1, demonstrating that CREB was required for cAMP induction of ABCA1 expression in RAW264.7 cells., Conclusions: Phospho-CREB1 controls the cAMP-mediated induction of murine ABCA1 gene expression through a CRE site acting in cooperation with a nearby STAT element. This CRE site is not conserved in the human ABCA1 gene, explaining why human ABCA1 is not strongly stimulated by cAMP analogs.

Objective: ABCA1 mediates cellular cholesterol and phospholipid efflux to apolipoprotein A-I and other apolipoprotein acceptors. In this study, we analyzed the effect of the immunosuppressant cyclosporin A on the ABCA1-mediated lipid effluxes reactions., Methods and Results: Cyclosporin A acted as a potent inhibitor of ABCA1 activity in several cell lines. Using the RAW264.7 mouse macrophage cell line, in which ABCA1 and its associated cholesterol efflux activity are inducible by cAMP analogues, cyclosporin A inhibition of cholesterol efflux to apolipoprotein A-I was rapidly reversible after its removal from the culture media, implying that ABCA1 levels were not drastically reduced by cyclosporin A. In fact, cyclosporin A treatment decreased ABCA1 turnover and yielded a 2-fold increase in cell-surface ABCA1. Despite the increase in cell-surface ABCA1, cyclosporin A decreased apolipoprotein A-I uptake, resecretion, and degradation in RAW cells. Finally, consistent with the inhibition of ABCA1 in vitro, cyclosporin A treatment induced a 33% reduction of high-density lipoprotein (HDL) levels in mice., Conclusions: ABCA1 inhibition by cyclosporin A supports a role for ABCA1 endocytic trafficking in ABCA1-mediated lipid efflux and could explain in part the low HDL levels observed in some patients with transplants.