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40 Sereyrath Ngeth and Gordon A. Francis

The active interaction of HDL proteins, particularly apolipoprotein A-I (apoA-I), with cells to remove cellular lipids is the critical step in the initial production of HDL, without which no HDL is available for further passive removal of cell cholesterol or subsequent steps of the RCT pathway.

Studies of a rare genetic cause of extremely low HDL levels, Tangier disease, indicated phospholipid [5] and cholesterol [5, 6] efflux to apoA-I and other HDL apoproteins [7] was impaired from Tangier disease cells. The defect in Tangier disease was subsequently identified as mutations in the ATPbinding cassette transporter AI (ABCA1) [8–12]. ABCA1 is a member of the large superfamily of ABC membrane transporters [13], and is present on the surface of most cells to stimulate the delivery of cell phospholipids and cholesterol to apoA-I and other HDL apolipoproteins including apoA-II and apoE [14]. The critical importance of functional ABCA1 in determining plasma HDL cholesterol (HDL-C) levels is suggested by the near-absence of HDL particles in individuals with mutations in both ABCA1 alleles (Tangier disease), and approximately 1/2 normal plasma HDL-C levels in individuals with one functional ABCA1 allele [15, 16]. It therefore appears that subsequent steps in the RCT pathway cannot compensate for an initial decrease in ABCA1 activity to normalize HDL-C levels. Tangier disease homozygotes and heterozygotes are consequently at increased risk of developing atherosclerosis [17, 18].

ABCA1 is a full ABC transporter containing two transmembrane domains and two ATP-binding domains [13]. ABCA1 expression is regulated at multiple levels (reviewed in Ref. [14]). Cellular cholesterol accumulation leads to increased ABCA1 expression [11, 19] through oxysterol-dependent activation of liver X receptor (LXR), a nuclear receptor that binds to the promoter region of the ABCA1 gene to activate its expression [20–22]. ABCA1 expression is therefore an additional way by which cells attempt to maintain cholesterol balance. Increased ABCA1 expression correlates with increased cholesterol efflux to HDL apoproteins [23, 24], as well as cell surface binding of apoA-I [25, 26].

ApoA-I is an amphipathic protein containing ten α-helical domains [27]. Lipid-free/lipid-poor apoA-I as well as other amphipathic HDL apolipoproteins including apoA-II, apoA-IV, apoC, and apoE have been shown to simulate ABCA1-mediated lipid efflux to generate nascent HDL particles [24]. However, the nature of this critical interaction between HDL apoproteins and ABCA1, and whether this definitely requires a direct apoA-I–ABCA1 interaction, remains unclear. Here we summarize recent literature regarding the nature of the apoA-I–ABCA1 interaction, and the regulation of ABCA1 activity by apoA-I-dependent and -independent phosphorylation mechanisms. Although apoA-I is the most abundant HDL apoprotein, and the protein used in most in vitro studies of apoprotein–ABCA1 interactions, the results of these studies in many cases have also been found to apply to the interactions of ABCA1 with other lipid-poor HDL apoproteins.

42 Sereyrath Ngeth and Gordon A. Francis

properties suggestive of an interaction with membrane lipids, rather than a membrane protein receptor [35].

Studies with ABCA1 mutants have also shone light on the nature of the molecular interaction between apoA-I and ABCA1. Chambenoit et al. [35] have shown that increased ABCA1 expression does not linearly correlate to increased apoA-I binding and that apoA-I cell surface binding is dependent on a fully functional ABCA1 protein because cells expressing an ATPasedefective ABCA1 fail to bind apoA-I. The conclusion was that cells lacking functional ABCA1 would not be able to flip PS to the outer leaflet of the PM, thereby inhibiting the generation of a suitable lipid environment required for apoA-I docking. In support of this is the observation that the expression of ABCA1 correlates with the amount of exofacial PS exposed on the cell surface [35] and the requirement for PS flipping to release cellular phospholipids to apoA-I [36]. Another study, however, showed that apoptosisinduced exofacial PS flipping was not sufficient to promote lipid efflux to apoA-I, and that annexin V, a PS-binding protein, did not compete for apoA-I cell surface binding [37]. A more recent study showed treatment of ABCA1expressing human fibroblasts with phosphatidylcholine-specific phospholipase C or sphingomyelinase had no effect on the ability of apoA-I to associate with ABCA1, suggesting alterations in PM phospholipid content by ABCA1 do not predict apoA-I binding to cells [38].

Direct Association/Receptor–Ligand Model

Studies using chemical cross-linkers have suggested that apoA-I binds directly to cell surface ABCA1 to form a high affinity complex that is required for ABCA1-mediated lipid efflux [25, 31, 39] (Fig. 3.1B). ApoA-I has been shown to cross-link to ABCA1 in an aqueous environment [40] and at a distance as little as 3 Å away, indicating a very close association between the two proteins [41]. One ABCA1 transporter seems to bind to one molecule of apoA-I; however, ABCA1 has been proposed to oligomerize into a homotetrameric complex binding four molecules of apoA-I [42]. In support of the direct association model, inhibition of ABCA1 by glibenclamide has been shown to inhibit phospholipid and cholesterol efflux as well as crosslinking of apoA-I to ABCA1, suggesting a close relationship between apoA-I cross-linking and lipid efflux [26].

Studies with ABCA1 mutants have also provided mounting evidence for the direct binding of apoA-I to ABCA1. Fitzgerald et al. [40] have shown that four naturally occurring ABCA1 mutants harboring missense mutations in the two extracellular loops of ABCA1 fail to promote cholesterol efflux and had diminished ability to cross-link to apoA-I, even though the ABCA1 mutant proteins were properly targeted to the PM [40]. This would imply that a direct interaction between apoA-I and ABCA1 is required for efflux. Interestingly, one other ABCA1 mutant (W590S) that failed to promote lipid efflux retained full cross-linking ability with apoA-I, suggesting that apoA-I

binding is necessary but not sufficient to stimulate cholesterol efflux [40]. Further studies with the W590S mutant revealed that apoA-I binds and dissociates from the defective ABCA1 transporter at a similar rate to wild-type ABCA1, but the dissociated apoA-I was without lipids, implying that dissociation is not dependent on lipid transfer [39]. An explanation for the inability of the W590S mutant to efflux lipids may be its’ inability to drive the flipping of PS to the exofacial side of the PM, proposed to be a requirement for lipid efflux in the indirect association model [36]. Hence, the apoA-I– ABCA1 interaction is complex and may involve the association of apoA-I with lipid domains and ABCA1.

Panagotopulos and colleagues observed that helix 10 of apoA-I is crucial for apolipoprotein-mediated cholesterol efflux, and that two apoA-I mutants containing intact helix 10 exhibited markedly reduced lipid-binding affinity, but promoted cholesterol efflux much like wild-type apoA-I [43]. To explain this observation, the authors propose a hybrid model integrating the pro- tein–protein and lipid domain interaction hypotheses. They speculate that helix 10 functions to tether apoA-I to the ABCA1-generated lipid domain such that apoA-I can diffuse laterally until it comes into contact with ABCA1 to form a productive complex, leading to the lipidation of apoA-I [43]. This hybrid model (Fig. 3.1C) would explain why apoA-I does not readily crosslink to ABCA1 at lower temperatures, as the lack of membrane fluidity would prevent the initial insertion and lateral diffusion of apoA-I [40].

The actual mechanism of ABCA1 in flipping lipids to the cell surface remains unknown. It may act primarily as a phospholipid flippase between the inner and outer PM leaflets, with cholesterol moving secondarily to maintain a constant phospholipid:cholesterol ratio in the outer leaflet, or ABCA1 might facilitate the simultaneous movement of both phospholipids and cholesterol. Further studies are required to resolve this question.

ApoA-I–ABCA1 Interaction Domains

ABCA1 is primarily found on the cell surface, consistent with its function as a cell surface lipid transporter, but it has also been found to localize to the Golgi and endolysosomal compartments [31, 44, 45]. Due to its presence in intracellular compartments as well as at the cell surface, it has been proposed that ABCA1-mediated lipid efflux may include ABCA1 internalization, possibly along with apoA-I, to intracellular sites and back to the PM for release of nascent HDL. Consistent with this idea was the report that ABCA1 preferentially mediates the removal of cholesterol from late endosomes–lyso- somes [46] that would otherwise be esterified by acyl CoA:cholesterol acyltransferase [47]. As well, Golgi to PM vesicular transport is increased during apoA-I-mediated cholesterol efflux [48] and cellular lipid export from the Golgi to the PM is defective in ABCA1-deficient cells [45]. Alternatively, intracellular ABCA1 recycling may be mainly a mechanism to regulate

44 Sereyrath Ngeth and Gordon A. Francis

ABCA1 degradation [14, 44]. ApoA-I has been shown to be internalized and resecreted by phagocytic cells [49], consistent with a possible role in late endocytic vesicular trafficking and mobilization of intracellular lipid pools [50].

Regardless of the mechanism of ABCA1–apoA-I-dependent lipid efflux, the initial interaction of apoA-I with ABCA1 likely occurs at the PM. Since ABCA1 is localized mainly to the PM, it is thought that it functions primarily at the cell surface to efflux lipids, consistent with a direct role in the binding of apoA-I to ABCA1 at the cell surface. For this interaction to occur, ABCA1 must be properly trafficked to the PM in order to bind and facilitate lipid efflux to apoA-I. ABCA1 mutants (R587W, Q597R, L1379F, V1704D) that are retained in the endoplasmic reticulum and fail to localize to the PM do not efflux lipids or bind apoA-I [36, 51, 52]. Studies with ABCA1 mutants have revealed that amino acid residues in the two large extracellular loops of ABCA1 may form critical apoA-I-binding domains. Indeed, many of the Tangier disease-causing ABCA1 missense mutations have been mapped to these two domains [8–10, 18, 53]. Fitzgerald et al. [40] observed that four of five mutants carrying a mutation in either of the extracellular loops were properly translocated to the PM, but lacked the ability to cross-link with apoA-I, implying that these extracellular loops play an important role in mediating apoA-I binding. Another ABCA1-dependent requirement for apoA-I binding is functional ATPase domains. Engineered ATPase-deficient mutants that are correctly folded and targeted to the PM do not elicit apoA-I cell surface binding as assessed by fluorescence photobleaching [35]. This lack of apoA-I binding may be due to the inability of the ABCA1 mutant to create a PM lipid domain required for apoA-I docking [35]. Alternatively, ATP binding/hydrolysis may be required to induce a conformational change of ABCA1 that enables the direct binding of apoA-I to ABCA1, as suggested by lack of cross-linking of apoA-I to an ATPase-deficient ABCA1 mutant [26]. In agreement with this idea is the observation that the W590S ABCA1 mutant that fails to promote lipid efflux but is properly targeted to the PM and had a functional ATPase domain exhibited enhanced cross-linking activity to apoA-I [40, 51].

ABCA1 has been shown to promote lipid efflux to other exchangeable apolipoproteins associated with HDL particles, namely apoA-II, apoA-IV, apoC-I, apoC-II, apoC-III, and apoE [24]. This is seemingly implausible if apolipoproteins interact with ABCA1 in a classical receptor–ligand type complex, considering that the apolipoproteins do not share a similar primary amino acid sequence. Several of these proteins, however, have been shown to compete effectively with apoA-I for cross-linking and are themselves crosslinked to ABCA1 [39, 54]. The common feature between these apolipoproteins is that they contain multiple amphipathic helical domains, suggesting that the secondary structure rather than the primary amino acid sequence is the structural element that allows an apolipoprotein to interact with ABCA1. In support of this theory, Fitzgerald and colleagues showed that synthetic amphipathic apolipoproteins were able to block the cross-linking of apoA-I

to ABCA1, whereas nonamphipathic lipid binding proteins did not [39]. Similar to this theory, the scavenger receptor class B type I has been shown to bind various apolipoproteins and the major motif found to mediate the interaction was amphipathic helices [55]. Hence, it is proposed that apolipoproteins may interact with ABCA1 by way of a hydrophobic groove within the ABCA1 transporter, an interaction that is seen between protein kinase A (PKA) and its amphipathic helical anchoring proteins [39]. ApoA-I likely interacts with a hydrophobic domain of ABCA1; however, some of the contact points between apoA-I and ABCA1 may occur in an aqueous environment because cross-linking was achievable with a water-soluble agent [40].

Interestingly, ABCA1 contains a cytoplasmic domain that is critical for promoting cholesterol efflux and apoA-I binding. Alterations in the highly conserved C-terminal VFVNFA motif resulted in defective lipid efflux as well as impaired apoA-I cross-linking [56]. Since the VFVNFA domain faces the cytosol, the authors speculate that this domain may be involved in protein–protein interactions that are responsible for recruiting additional factors to form an efflux complex [56]. ABCA1-expressing cells treated with a peptide mimetic of the VFVNFA motif inhibited ABCA1-mediated cholesterol efflux [56]. Clearly, less is known about the apoA-I–ABCA1 interaction domain than the molecular association of apoA-I to ABCA1. Additional studies designed to map and identify the critical amino acid residues on apolipoproteins and ABCA1 that interact will be necessary to fully understand the role of apolipoprotein binding to the ABCA1 transporter in lipid efflux. Perhaps obtaining the crystal structure of ABCA1 could best answer the question of what are the interaction domains between apoA-I and ABCA1.

Phosphorylation Events Modifying ABCA1 Activity and the Role of HDL Apolipoproteins

Constitutive and ApoA-I-Dependent Phosphorylation

of ABCA1

In addition to the indirect or direct interactions between ABCA1 and apoA-I mediating cellular lipid efflux at the PM, numerous phosphorylation and dephosphorylation events affect ABCA1 activity, several of which are dependent on apoA-I binding to cells. See et al. [57] reported that PKA-dependent phosphorylation of serines 1042 and 2054 in the nucleotide binding domains of ABCA1 occurs constitutively (i.e., independent of apoA-I binding), and that decreased phosphorylation of ABCA1 through mutation of serine 2054 reduces apoA-I-mediated phospholipid efflux but not apoA-I binding or ABCA1 protein stability. Lawn et al. [11] showed that cyclic AMP (cAMP) could increase ABCA1 expression and cell surface levels of ABCA1 protein. Haidar et al. [58] subsequently showed that treatment of human fibroblasts with 8-bromo cAMP increased ABCA1 phosphorylation, and concluded that

46 Sereyrath Ngeth and Gordon A. Francis

abnormal folding of ABCA1 caused by mutations might prevent phosphorylation of critical serine–threonine residues [58]. Further studies from this group suggested apoA-I also activates ABCA1 phosphorylation through a cAMP/PKA-dependent pathway under conditions where ABCA1 is present in high levels (following treatment with an LXR agonist), an effect that was reduced in Tangier disease fibroblasts [59]. These results suggested binding of apoA-I to ABCA1 may also activate cAMP signaling through a G proteincoupled ABCA1 transporter [59].

Earlier results showed that apoA-I-dependent lipid release is dependent on activation of protein kinase C (PKC) [60–62]. Yamauchi et al. [63] have recently provided evidence that PKC inhibitors suppress the stabilization of ABCA1 and cellular lipid release mediated by apoA-I. They showed that digestion of sphingomyelin increased ABCA1 protein levels, and that inhibition of phosphatidylcholine phospholipase C (PC-PLC) inhibits this effect. They also proposed PKC-dependent phosphorylation of ABCA1 by apoA-I, as suggested by inhibition of this effect by PKC-specific inhibitors [63]. They conclude that apoA-I activates PKC following depletion of the PM of sphingomyelin, thereby activating PC-PLC and generating diacylglycerol that activates PKC, leading to the phosphorylation and stabilization of ABCA1.

Yet another phosphorylation mechanism activated by the apoA-I interaction with cells is an increase in the tyrosine kinase Janus kinase 2 (JAK2) activity [64]. JAK2 activity was required for apoA-I binding and apoA-I- dependent efflux of cellular lipids, but did not directly affect ABCA1 phosphorylation or cholesterol translocase activity of ABCA1 [64]. These results suggest JAK2 is activated by apoA-I, and that JAK2 promotes the productive interaction between apoA-I and ABCA1 to mobilize cellular lipids, possibly by enhancing the activity of another partner protein that facilitates the binding of apoA-I to ABCA1.

Lastly, phosphorylation sites in conserved regions of a cytoplasmic domain of ABCA1 downstream of the first nucleotide domain have been identified that are phosphorylated by protein kinase 2, leading to decreased apoA-I and apoA-II binding, ABCA1 flippase activity, and phospholipid plus cholesterol efflux [65]. These results demonstrate an inhibitory mechanism of ABCA1 phosphorylation independent of, but subsequently inhibiting, apoA-I binding.

Protection by apoA-I against Calpain-Dependent Proteolysis of ABCA1

Another remarkable facet of the apoA-I–ABCA1 interaction is the demonstration that HDL apoproteins serve to protect ABCA1 against proteolysis, thereby increasing ABCA1 activity and further facilitating apoproteinmediated lipid efflux [66–68]. Arakawa and Yokoyama [66] demonstrated that incubation of apoA-I with cultured THP-1 cells increased ABCA1 protein levels without increasing ABCA1 message, and that free apoA-II did the same thing, however intact HDL particles did not [66]. Thiol protease inhibitors

could also prevent ABCA1 degradation and enhanced cholesterol efflux, suggesting a possible mechanism by which the apoproteins could be stabilizing ABCA1. Wang and colleagues subsequently reported that calpain-dependent proteolysis is involved in the degradation of ABCA1 through a proline, glutamate, serine, and threonine (PEST)-rich cytoplasmic sequence of the transporter, and that deletion of this PEST sequence or treatment of cells with apoA-I could inhibit this proteolysis and increase cell surface ABCA1 [67]. They also demonstrated that infusion of apoA-I increased ABCA1 protein levels in the liver and peritoneal macrophages of C57BL/6J mice [67]. This group subsequently reported that phosphorylation of threonine residues within this PEST sequence is required for calpain-dependent proteolysis of ABCA1, and that phosphorylation of this sequence is reduced by apoA-I. In this way, apoA-I modulates yet another mechanism of ABCA1 phosphorylation, by somehow decreasing phosphorylation of the PEST sequence, to increase ABCA1 activity. Further studies suggest the PEST sequence of ABCA1 is required for the internalization of the transporter, to mediate efflux of the portion of total cholesterol efflux derived from intracellular pools [69].

Conclusions

The interactions of apoA-I with ABCA1 that mediate nascent HDL particle formation are the rate-limiting step in the RCT pathway. Evidence is presented for both an indirect and direct interaction of apoA-I with the cell surface, mediated by ABCA1, which facilitates the delivery of cellular phospholipids and cholesterol to apoA-I to generate HDL particles. The complexity of this partnership is demonstrated by the numerous phosphorylation events mediated in part by apoA-I interactions with cells that enhance or reduce ABCA1 activity. Further research into this critical interaction will enhance our understanding of HDL particle formation and our ability to utilize this pathway for the treatment and prevention of atherosclerosis.

Acknowledgments: This work is supported by CIHR operating grant MOP12660. Gordon. A. Francis is a Senior Scholar of the Alberta Heritage Foundation for Medical Research.

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