On the structure and function of apolipoproteins: more than a family of lipid-binding proteins

Progress in Biophysics & Molecular Biology 83 (2003) 47–68

Review

On the structure and function of apolipoproteins:more than a family of lipid-binding proteins

Victor Martin Bolanos-Garcia*, Ricardo Nunez Miguel

Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK

Abstract

Exchangeable apolipoproteins have been the subject of intense biomedical investigation for decades.However, only in recent years the elucidation of the three-dimensional structure reported for severalmembers of the apolipoprotein family has provided insights into their functions at a molecular level for thefirst time. Moreover, the role of exchangeable apolipoproteins in several cellular events distinct from lipidmetabolism has recently been described. This review summarizes these contributions, which have not onlyallowed the identification of the apolipoprotein domains that determine substrate binding specificity and/oraffinity but also the plausible molecular mechanism(s) involved.r 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Apolipoprotein structure; Lipid-binding proteins; Amyloid formation; Kinetic stability; Amphipathic

helices

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

2. Exchangeable apolipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

2.1. apoA-I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

2.2. apoA-II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

2.3. apoC-I, C-II and C-III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

2.4. apoE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

2.5. Apolipophorin III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

3. Apolipoprotein flexibility is required for lipid binding . . . . . . . . . . . . . . . . . . . . . . 59

*Corresponding author. Tel.: +44-1223-763088; fax: +44-1223-766002.

E-mail address: [email protected] (V.M. Bolanos-Garcia).

0079-6107/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0079-6107(03)00028-2

1. Introduction

Apolipoproteins combine with lipids in order to form different classes of lipoprotein particles.Different combinations of lipid and protein produce lipoprotein particles of different density andsize. Apolipoproteins such as apoA-I, apoA-II, apoA-IV, apoC-I, apoC-II, apoC-III, and apoE3are known as exchangeable apolipoproteins because of their ability to move and exchangebetween lipoprotein particles. These apolipoproteins are found in mammals, including humans,although closely related lipid-binding proteins are also present in insects, for example theapolipophorin III proteins of Locusta migratoria and Manduca sexta.Despite the fact that apolipoprotein exchange among lipoprotein particles has been well

documented during the past 20 years, the specific molecular mechanism(s) that govern this activityremains a subject of intense investigation. Indeed, the function of exchangeable apolipoproteins isnot restricted to lipid-binding activity. During the past 5 years, the elucidation of the role(s) ofthese proteins in a wide range of cellular processes has been described. In this context, the three-dimensional structures recently reported for several apolipoproteins have provided new insightsinto their intrinsic flexibility, stability and lipid affinity and established a framework forunderstanding the mechanism(s) associated with such activity. A previous review describing theseries of structural and physicochemical similarities between human apolipoproteins A-I, A-II, C-I, C-II, C-III and E and some other human lipid-binding proteins such as the cholesteryl estertransfer protein (CETP) and the phospholipid transfer protein (PLTP) was published before thethree-dimensional structures of some of these proteins were solved (Bolanos-Garcia et al., 1997).Here we discuss both the fundamental properties that rule lipid-binding activity of exchangeableapolipoproteins in the light of the structural information now available and their role in othercellular processes.

2. Exchangeable apolipoproteins

2.1. apoA-I

Human apoA-I is significantly associated to high-density lipoprotein (HDL) particles and itseems to regulate HDL particle size distribution (Reschly et al., 2002). This protein also promotescellular cholesterol efflux (Fielding and Fielding, 1995) and interacts with several proteins such asthe scavenger receptor class B type I (SR-BI) (Liu et al., 2002) and the ATP-binding cassettetransporter (ABC) A1 (Arakawa and Yokoyama, 2002). In the latter case, the lipid-free form ofapoA-I stabilizes ABCA1 by protecting it from protease-mediated degradation. ApoA-I also has

4. Apolipoproteins in interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

5. The importance of kinetic stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

6. Apolipoproteins and amyloidogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

7. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

V.M. Bolanos-Garcia, R.N. Miguel / Progress in Biophysics & Molecular Biology 83 (2003) 47–6848

a protective effect on HDL against coronary artery disease, as observed in human apoA-Itransgenic mice (Major et al., 2001). ApoA-I is also a potent activator of lecithin acyl transferase(LCAT), the enzyme that catalyzes cholesterol esterification on HDL (Fielding et al., 1972).LCAT activation is dramatically affected by the specific apoA-I conformational state (Jonas,1998).Human apoA-I consists of 243-aminoacid residues, which can be divided into two domains: an

N-terminal globular domain (encompassing residues 1–43), which is encoded by exon 3 of theapoA-I gene, and a C-terminal domain (residues 44–243), encoded by exon 4 of the same gene. Ina previous review, the C-terminal domain was predicted to contain eight 22-mer and two 11-meramphipathic a-helices, with most of the helices being punctuated by prolines (Bolanos-Garciaet al., 1997). The elucidation of the three-dimensional structure of a truncated human apoA-Iprotein (encompassing residues 44–243) demonstrates that this is the case. The apoA-I (44–243)structure, solved at 3 (A resolution, has provided new insights into the way this protein binds toHDL particles (Borhani et al., 1997, 1999). The structural organization of a human apoA-I(44–243) monomer consists of an array of ten a-helices that form a horseshoe-shape structure, asobserved in Fig. 1A. This figure also shows that a short a-helix at the C-terminal region isseparated from the rest of the molecule by a series of residues that adopt an extended, non-helicalconformation. Helix one encompasses the residues 50–65; helix 2, residues 66–87; helix 3, 88–98;helix 4, 99–120; helix 5, 121–142; helix 6, 143–164; helix 7, 165–186; helix 8, 187–208; helix 9,209–219; and helix 10, 228–243. As mentioned above, the length of most of these helical regions isdetermined by inserted kinks, which are due to regularly spaced proline residues. As shown inFigs. 1B and C, four apoA-I monomers assemble to give place to a tightly associated, twistedelliptical ring. This tetrameric ring can be divided into two pairs of dimers, which relate each otherby a non-crystallographic dyad (Fig. 1C). Each dimer consists of two antiparallel molecules,which form a strip along the length of such a dimer. Two dimers associate to form a tetramer insuch a way that the hydrophobic strip is buried (Fig. 1D).A recently published secondary structure of the apoA-I (1–186) C-terminal truncation mutant

in the presence of SDS micelles indicates a similar location of a-helix segments as those shown in

Fig. 1. (A) Human apoA-I (44–243) adopts a horseshoe-shape structure. (B, C) Two different views of the four apoA-I

monomers that assemble to give place to an elliptical, twisted tetrameric ring. (D) The tetrameric form of apoA-I.

V.M. Bolanos-Garcia, R.N. Miguel / Progress in Biophysics & Molecular Biology 83 (2003) 47–68 49

Fig. 1a (Okon et al., 2001). The extended a-helical array also shows the so-called lipid-boundconformation (Wang, 2002; Li et al., 2002). As shown in Figs. 1A–C, residues 165–208 form acontinuous a-helix with a 12� turn between the putative helices 7 and 8, while a sharp bend occursin the preceding four interhelical regions.It is known that helices 7 and 8 are essential for lipid binding and because they exhibit a large

sequence divergence (50%), they may also contribute to apoA-I association with different HDLsubclasses (Reschly et al., 2002). Additionally, murine and human apoA-I share 65% sequenceidentity and 72% sequence similarity, the largest differences being in the first and last (helix 10)helices (Brouillette et al., 2001). It might be the length of helices 7 and 8 as well as the number ofpossible conformations of the putative interhelical turns between them that makes human apoA-Imore able to accept HDL particles of different radii of curvature, in comparison with murineapoA-I. The latter is structurally more constrained than the human protein due to the presence ofproline residues in this region. Thus, the ability of murine apoA-I to adjust to differences in thesize of the HDL particles is comparatively limited.In addition to the differential lipid-binding activities of apoA-I from different mammals,

there are natural variants of apoA-I that give rise to HDL deficiencies, but with no apparentcardiovascular disease (CVD) in affected subjects. In human beings, apolipoprotein A-I Milanoand apoA-I Paris are the best-known examples (Weisgraber et al., 1980; Bruckert et al., 1997).Approximately 10% of the total plasma pool of apoA-I consists of monomers of apoA-I Paris invivo. Human carriers of both the apoA-I Milano and the apoA-I Paris exhibit mildhypertriglyceridemia in addition to HDL deficiency (Bruckert et al., 1997). Moreover, studiesutilizing mice and rabbits supported clinical studies where the injection of recombinantapoA-I Milano protected them against atherosclerosis (Shah et al., 2001). However, themechanism(s) by which apoA-I Milano exerts its antiatherogenic effects is/are not completelyunderstood.All known human carriers of apoA-I Milano and apoA-I Paris are heterozygous for the

mutations R173C (helix 7) and R151C (helix 6) in the apoA-I protein, respectively (Weisgraberet al., 1980; Bruckert et al., 1997). The free cysteine residue of each mutant is located at thehydrophobic/hydrophilic interface of the respective amphipathic a-helix and its presence disruptsputative salt bridges normally associated with Arg173–Glu169 and Arg151–Glu147, respectively(Franceschini et al., 1985). The introduction of a cysteine residue in a normally cysteine-freeapolipoprotein allows the formation of homodimers and heterodimers with apoA-II (Vadivelooet al., 1993). Dimerization of the cysteine variants results in the inhibition of HDL maturationpartially due to the impaired activation of LCAT. ApoA-I Milano and, to a lesser extent, apoA-IParis are also potent inhibitors of lipid peroxidation, protecting phospholipid surfaces fromwater-soluble and lipophilic free radical initiators (Bielicki and Oda, 2002). In contrast, nativeapoA-I is a comparatively poor inhibitor of oxidative events in vivo. Thus, the apoA-I Milano-and apoA-I Paris-mediated inhibition of lipid peroxidation is an effective mechanism of avoidingthe propagation of phospholipid peroxides.It has been suggested that apoA-I Milano is twice as effective as apoA-I Paris in protecting

phospholipids from oxidation, due to a conformational constraint governed by adjacentamphipathic a-helices located in its C-terminal lipid-binding domain (Bielicki and Oda, 2002).However, the difference in antioxidant activity between these apoA-I mutants might also bedetermined by sequences located outside of these specific helices (Bielicki and Oda, 2002). Thus, a


systematic, structural analysis of these mutants is needed in order to fully understand themolecular determinants that govern their activity as inhibitors of lipid peroxidation.

2.2. apoA-II

Human apoA-II is a major component of HDL particles and it is mainly synthesized in theliver. ApoA-II plays a significant role in HDL metabolism. It displaces apoA-I from the surface ofHDL particles, which could result in the impairment of reverse cholesterol transport induced byapoA-I as well as the modification of HDL particles remodeling. As a consequence of apoA-Idisplacement, the risk of developing atherosclerosis increases (Lagocki and Scanu, 1980). HumanapoA-II consists of 77-aminoacid residues that form a homodimer linked through a disulfidebridge (Vadiveloo et al., 1993). However, the formation of a heterodimer with apoE and apoH hasalso been reported (Sprecher et al., 1984). Here we will not describe apoH although its structurehas been solved (Schwarzenbacher et al., 1999) and it belongs to the exchangeable apolipoproteinfamily. The domain organization of apoH is completely different and it does not show the typicalamphipathic a-helices found in most of the family members.Two high-resolution crystal structures of human apoA-II have recently been published

(Tricerri et al., 2001). One structure corresponds to native, lipid-free human apoA-II, while theother is an apoA-II/b-octyl-glucopiranoside complex. The native, lipid-free apoA-II crystalstructure solved at 2 (A resolution shows that it consists of disulfide-bonded homodimers with ahierarchical organization. Each apoA-II protomer exhibits three long a-helices, which arepunctuated by proline residues in a similar way as observed in apoA-I (44–243) (although lessuniformly). Short 310 helices of 3–4 residues in length are also present between the long a-helices.Each homodimer contains three hydrophobic patches, encompassing residues 6–29, 41–53and 60–70 from each chain. Two homodimers associate to form a tetramer and these tetramersassociate to form a dodecamer (Fig. 2A). The two homodimers in a tetramer interactvery extensively, and such interaction takes place through the hydrophobic patches mentionedbefore.

Fig. 2. (A) The lipid-free apoA-II crystal structure shows that it consists of disulfide-bonded homodimers with a

hierarchical organization. (B) Crystal structure of the apoA-II/b-octyl-glucopiranoside complex, which supports the so-called belt-model. (C) The lipid surrogate molecules in the apoA-II/b-octyl-glucopiranoside complex are shown in aball–stick representation.


In contrast with the lipid-free apoA-II crystal structure, the apoA-II/b-octyl-glucopiranosidecomplex shows very different global conformations (i.e, a variety of curvatures, as shown inFigs. 2B and C). In this case, two copies of 16 homodimers form an irregular double-strandedhelical array. Thus, the structure of the apoA-II/b-octyl-glucopiranoside complex provides thestrongest support, so far, for the so-called ‘‘belt model’’, initially proposed to explain the relativeorientation of lipid acyl chains in apolipoprotein binding to discoidal particles (Segrest et al.,1999; Tricerri et al., 2001). In general terms, the belt model proposed that apolipoproteinmolecules wrap around the circumference of the corresponding discoidal lipoprotein particle, insuch a way that acyl side chains lie parallel to local protein helical axes.The structure of this complex also shows that the orientation of the a-helices places

hydrophobic patches on the same side of the dimer planes, as observed in the lipid-free apoA-IIcrystal structure. However, monomers interact with each other at a lesser extent in comparisonwith the free form. This is in part due to the presence of an average of eight b-octyl-glucopiranoside molecules at each tetramer interface.In brief, the arrangement of helices in both apoA-II structures is very similar to that observed in

the apoA-I structure (Figs. 1A–C), although in that case the helices are longer. However, thedifferences between the lipid-free and apoA-II/b-octyl-glucopiranoside complex include adifferential ability to adopt different curvatures and a different head-to-tail-specific oligomeriza-tion. Moreover, the orientation of the octyl side chains of b-octyl-glucopiranoside molecules tolocal helical axes of the protein might partially reflect the types of interactions that takeplace during apolipoprotein–lipid association. Besides, a comparison between the structureof the lipid-free and the apoA-II/b-octyl-glucopiranoside complex provides new insights into thewell-established fact that a-helix content of exchangeable apolipoproteins increases uponassociation with lipids (Clement-Collin et al., 1999; Brouillette et al., 1984; Kawooya et al.,1986) and may also explain how apoA-II can completely displace apoA-I molecules from HDLparticles.

2.3. apoC-I, C-II and C-III

Apolipoproteins C-I, C-II and C-III have an inhibitory or stimulatory effect on a variety ofreceptors and enzymes involved in lipoprotein metabolism. These distinct effects on the majorlipoprotein metabolic pathways imply that changes in human apolipoprotein C gene expressionmight play an important role in the etiology of human hyperlipidemias. For example, patientswith apoC-I and apoC-II deficiency show markedly decreased levels of cholesterol ester,suggesting that apoC-I deficiency in HDL may modulate LCAT activity (Dumon and Clerc,1986). On the other hand, the role of apoC-II as an activator of lipoprotein lipase (LPL) has beendemonstrated in patients with genetic defects in either the structure or the production of apoC-II(Kinnunen et al., 1977). However, it is equally important to determine which of these effectsobserved in vitro can be extended to the in vivo situation, because several in vitro effects of apoCson receptors and enzymes appear non-specific or secondary such as the displacement of otheractivating or inhibiting components of the lipoprotein particle.The analysis of human apoC locus showed that it is located on chromosome 19 downstream

from the apoE gene and in the same transcriptional orientation (Davison et al., 1986). The apoC-I

gene is primarily expressed in the liver, but lower amounts are also expressed in the lung, skin,


testes, and spleen (Lauer et al., 1988). ApoC-II spans a region of 3.4 kb and is primarily expressedin the liver and intestine (Myklebost et al., 1984). An additional gene within the apoE/C-I/C-IIgene cluster, designated as the apoC-II-linked gene, was first discovered in mice and more recentlyfound in humans (Allan et al., 1995). This human gene was designated apoC-IV because of itsproperties and location (i.e, 555 bp upstream from apoC-II). The human apoC-III gene is locatedin a cluster that also contains the apoA-1 and apoA-IV genes on the long arm of chromosome 11and is mainly expressed in the liver and intestine (Jong et al., 1999).ApoC-I is synthesized with a 26-residue signal peptide that is cleaved cotranslationally in the

rough endoplasmic reticulum (Shulmann et al., 1975). The remaining single-chain polypeptide of57-aminoacid residues has a molecular mass of 6.6 kDa. ApoC-I has a high content of lysine(16%mol) and contains no histidine, tyrosine, cysteine, or carbohydrate modification (Rozeket al., 1995). Due to its composition, plasma apoC-I exhibits the highest isoelectric point (pI)among the HDL-associated apolipoproteins. ApoC-I inhibits both phospholipase A2 and hepaticlipase (Conde-Knape et al., 2002; Kinnunen and Ehnholm, 1976) and activates lecithin:cholesterolacyltransferase (Soutar et al., 1975), even though to a much less extent compared with apoA-I.ApoC-I is the most potent inhibitor of the apoE-mediated binding of b-VLDL to the LDLreceptor and the LDL receptor-related protein (Kowal et al., 1990). Since it has also been reportedthat apoC-I deficiency can lead to a significant increase in levels of atherogenic VLDL- and LDL-like particles (Jong et al., 1996), the function in vivo of apoC-I might not be restricted to theregulation of the uptake of potentially atherogenic lipoprotein particles. Besides, the N-terminalfragment (residues 1–38) of baboon and human apoC-I has been reported to act as inhibitors ofCETP in vitro (Buchko et al., 2000; Gautier et al., 2000).The bulk of apoC-I (i.e., approx. 80%) is associated with HDL in vivo. In contrast, no

detectable levels of apoC-I are detected in LDL. This asymmetrical distribution of apoC-I mayexplain the different abilities of plasma LDL and HDL to inhibit CETP. ApoC-I over-expressionin transgenic mice has also been associated with protection from obesity and insulin resistance(Jong et al., 2001) and one of its alleles, the H2, is associated with Alzheimer’s disease(Petit-Turcotte et al., 2001).The structures of two apoC-I fragments in the presence of SDS to mimic the lipoprotein

environment have been solved by NMR techniques (Rozek et al., 1995). These fragmentscorrespond to residues 7–24 and 35–53. In the absence of SDS, both peptides did not show well-defined structure but in the presence of this detergent at submicellar concentrations, a dramaticincrease in the a-helix content was observed in both peptides (i.e, from zero to 69% and from 4%to 99%, respectively) (Rozek et al., 1995). The three-dimensional structures of these apoC-Ipeptides (shown in Fig. 3A) were obtained using distance geometry calculations and simulatedannealing. Both structures correspond to amphipathic a-helices class A2 that are not significantlystabilized by the formation of salt bridges (Bolanos-Garcia et al., 1997). Instead, the mainstabilizing force is due to hydrophobic interactions (Rozek et al., 1995). More recently, the samegroup reported the structure of an apoC-I fragment (residues 1–38) in the presence of saturatingamounts of SDS (Rozek et al., 1997). The backbone conformation of residues 14–21 superimposeswith the corresponding residues reported for the peptide 7–24. In contrast with this peptide, theapoC-I (1–38) structure showed a bend around residues K12 and E13. Taken together, thestructure of all these apoC-I fragments allows us to propose the structure of the intact humanapoC-I protein.


Concerning apoC-II, the sequence analysis of this gene in families with familial hyperchylo-micronemia has revealed a variety of molecular defects. For example, a single base changeresulted in the introduction of a premature stop that led to the synthesis of truncated forms ofapoC-II that were either not secreted or rapidly cleared from the circulation (Xiong et al., 1991).Moreover, a donor splice-site mutation in the first base of the second intron of the apoC-II genecaused abnormal splicing of apoC-II mRNA and was associated with low levels of apoC-II inplasma (Okubo et al., 1997). However, there are at least two apoC-II genetic variants (apoC-II SanFrancisco and the apoC-II K193T mutation), where a direct relationship between these mutantforms of apoC-II and lipoprotein abnormalities could not be established (Jong et al., 1999). Theexpressed product, apoC-II, is synthesized with a 22-residue signal peptide that is cleavedcotranslationally in the rough endoplasmic reticulum. The remaining single polypeptide chain of79-aminoacid residues has a calculated molecular mass of 8.8 kDa. The structure of mature, full-length human apoC-II in complex with SDS micelles, solved by NMR spectroscopy (MacRaildet al., 2001) (Fig. 4), shows that apoC-II contains three regions with a-helical conformationspanning residues 16–36, 50–56, and 63–77. The lipase-activating region of apoC-II is localized tothe C-terminal one-third of the sequence, from about residue 56, whereas the N-terminal two-thirds of the sequence is involved in lipid binding (MacPhee et al., 1999). Seven residues presenton this region are fully conserved in different species, while deletion of the COOH-terminalresidues 76–79 impairs the ability of the protein to activate LPL (MacPhee et al., 2000).Replacement of Y63, I66, D69 or Q70 by alanine lowered the affinity for LPL and the catalytic

activity of the LPL–apoC-II complex, while substitution of A59 by glycine, or T62 and G65 byalanine, did not change the activation, indicating that these residues are outside the LPL-bindingsite. Interestingly, these mutants retained some activating ability, but replacement of Y63 byphenylalanine or tryptophan and Q70 by glutamate caused almost complete loss of activity. Thus,the main LPL-binding site in apoC-II consists of 4 residues located on one face of the third a-helixclosest to the C-terminus. Since inactive mutants did not compete with wild-type apoC-II, it is

Fig. 3. Proposed structure of apoC-I based on the NMR structures of the fragments 7–24 and 35–53 in the presence of

SDS. Hydrophobic residues are shown in a space–fill representation.


likely that the productive apoC-II/LPL interaction may be dependent on substrate molecules.Moreover, it has also been proposed that binding of apoC-II to LPL induces a conformationalchange in the lipase or modifies its orientation at the lipid–water interface. Consequently, LPLmight be more efficient in binding substrate molecules or releasing products of lipolysis (fattyacids) (Bengtsson and Olivecrona, 1982).ApoC-III is the most abundant C apolipoprotein in human plasma (i.e., at a concentration of

12mg/dl) and it corresponds up to 60% of total protein mass of HDL particles. It is mainlysynthesized in the liver and in minor extent by the intestine as a 99-aminoacid peptide. Aftercleavage of the 20-aminoacid-residue signal peptide, the mature apoC-III protein consists of 79residues with a molecular mass of 8.8 kDa. Three isoforms have been identified by isoelectricfocusing, which differ in their degree of O-linked sialylation at the threonine residue in position74: apoC-III-0 (no sialic acid), apoC-III-1 (1mol sialic acid), and apoC-III-2 (2mol sialic acid)(Ito et al., 1989). ApoC-III inhibits the lipolysis of triglyceride-rich lipoproteins, hampering theinteraction of these lipoproteins with a heparan sulfate proteoglycan–LPL complex (Schaeferet al., 1985). Indeed, such inhibition of lipolysis of triglyceride-rich lipoproteins possiblycontributes to the development of hypertriglyceridemia. Interestingly, a complete apoC-IIIdeficiency has been reported in families with an increased prevalence of premature coronary heartdisease (Jong and Havekes, 2000).ApoC-III has also been postulated to act as a modulator of the receptor-mediated clearance of

lipoproteins (Aalto-Setala et al., 1996). As previously shown for apoC-II, apoC-III also inhibitsLCAT activity, probably by displacing the activating apolipoproteins from the lipoprotein surface(Subbaiah et al., 1991) and it is also able to stimulate CETP activity on recombinant HDLparticles (Sparks and Pritchard, 1989).Thrombin cleavage of mature apoC-III results in a N-terminal domain, residues 1–40, and a C-

terminal domain, residues 41–79, corresponding to the products of exons 3 and 4, respectively(Sparrow et al., 1977). A series of functional analyses have demonstrated that the binding of

Fig. 4. NMR-solved structure of apoC-II in complex with SDS micelles. Residues corresponding to the lipase-

activating region are shown in a space–fill representation.


apoC-III to surface phospholipids of lipoprotein particles is mediated by an amphipathic a-helixat residues 50–69, which reside in the C-terminal domain (Trieu and McConathy, 1995).Since no three-dimensional structure of apoC-III has been reported, the sequence of human

apoC-III was compared with all protein sequences deposited in SWISS-PROT by using the Blastv2.0 program. A PSI-BLAST search was used to produce an alignment between apoC-III andother apolipoproteins, which highlights conserved residues in this family. Homologous proteinswith known structure were identified using the FUGUE (Shi et al., 2001) homology recognitionserver. FUGUE searches for homologues in the structural profile library derived from thestructure-based alignments in the HOMSTRAD database (de Bakker et al., 2001) and using theenvironment-specific substitution tables automatically generates the best alignments for the tophits. The alignment produced by FUGUE for the highest scoring hit was formatted with JOY(Mizuguchi et al., 1998) and analyzed visually to highlight the conservation of structurally andfunctionally important residues (Fig. 5A). Furthermore, a model of human apoC-III wasconstructed with MODELLER (Sali and Blundell, 1993) and validated with PROCHECK(Laskowski et al., 1993), VERIFY3D (Bowie et al., 1991) and JOY (Mizuguchi et al., 1998).Visual inspection was carried out using three-dimensional graphics software. All these programsrevealed that no further modifications were needed for the model. Overall, the method followedhere was the same as the one recently described (Miguel et al., 2002).The apoC-III modeled structure, which is shown in Fig. 5B, exhibits a fold similar to the one

observed in other apoC proteins, such as apoC-I and apoC-II. The apoC-III model also shows thecluster of residues in its C-terminal end that are essential for lipoprotein binding (Fig. 5C).

Fig. 5. (A) Sequence alignment of human apoC-III with human apoA-II, the top FUGUE hit. (B) Structure of apoC-

III generated by comparative modeling using the structure of apoA-II as a template. (C) The residues highlighted (balls

and sticks) are those determining binding of apoC-III to phospholipids located in the surface of lipoproteins.


A recently identified human apoC-IV gene has been over-expressed in transgenic mice (Allanand Taylor, 1996). Under normal conditions, the apoC-IV gene is poorly expressed in humanliver, most likely as a consequence of a TATA-less promoter. Human apoC-IV transgenic micewere hypertriglyceridemic compared with their non-transgenic littermates, owing to anaccumulation of TG-rich VLDL particles. Because there was little change in serum cholesterollevels in these transgenic mice, it is thought that apoC-IV may interfere with the clearance ofVLDL triglycerides via an inhibitory effect on lipolysis, a mechanism previously observed inapoC-II and apoC-III (Allan and Taylor, 1996). Since investigations on this apolipoprotein havejust begun, no structure of this protein is yet available.

2.4. apoE

In addition to its role in lipoprotein metabolism, apoE has been associated with other functionssuch as an antioxidant (Miyata and Smith, 1996), platelet aggregation inhibitor (Riddell et al.,1997), antiproliferative (Ishigami et al., 1998), neuroprotective (Kitagawa et al., 2002) andimmunomodulator (Zhou et al., 1998). Moreover, apoE has been recently identified in cell signaltransduction as an inducer of Akt/protein kinase B phosphorylation (Laffont et al., 2002).Possible apoE-mediated events on human longevity and the onset of Alzheimer’s disease have alsobeen reported (Schachter et al., 1994; Katzman, 1994). Whereas the apoE3 isoform is consideredthe ‘physiological’ apolipoprotein, the apoE2 and E4 isoforms alter lipid metabolism. These threeisoforms differ at aminoacid positions 112 and 158. The isoform apoE3 (which contains theresidues cysteine and arginine at positions 112 and 158, respectively) mediates the interaction ofapoE3–containing lipoproteins with the low-density lipoprotein (LDL) receptor and thechylomicron remnant apoE3-receptor. In consequence, apoE3 plays a critical role in determiningthe metabolic fate of several classes of lipoproteins and plays a central role in cholesterolmetabolism (Miyata and Smith, 1996). In contrast, the isoform apoE2 (Cys112 and Cys158) hasbeen associated with type III hyperlipidemia, while subjects with apoE4 (Arg112 and Arg158)are more likely to develop neurodegenerative disorders such as Alzheimer’s disease (Poeggeleret al., 2001).Human apoE consists of 299-aminoacid residues organized in two independently folded

domains: a N-terminal domain (residues 1–191), responsible for the binding of apoE to the LDLreceptor and a C-terminal domain, residues 216–299, which mediates the binding of apoE to thesurface of apolipoprotein particles. The N-terminal domain (1–191) is rich in basic aminoacids,can exist as a monomer in solution and its free energy of stabilization (DGunfold) is approximately10 kcal/mol, which is typical of other globular proteins. This N-terminal domain shows four a-helices, encompassing residues 24–42, 54–81, 87–122 and 130–164, respectively, which areorganized in an antiparallel helix bundle connected by loop regions (Wilson et al., 1991). It alsocontains the residues 136–150, which correspond to the LDL-receptor-binding region. The crystalstructure of the N-terminal domain of apoE, solved at 2.5 (A resolution, was the first humanapolipoprotein structure reported and is shown in Fig. 6.More recently, the structure of a mutant (K146E) of the N-terminal domain of human apoE3,

solved at a higher resolution (i.e, 1.9 (A), has been reported (PDB entry1EA8). Both crystalstructures showed the topology of the four helix bundles to be very similar to that found inhuman apoA-I.


As mentioned above, the residues R136, H140, R142, K143, R145, K146, R147 and R150correspond to the LDL-receptor-binding region and are located in the helix 4 of the N-terminaldomain. There are natural variants for positions 136, 142, 145 and 146. The substitution of thesebasic aminoacids by neutral or acidic residues results in a defective binding to the LDL receptor.Since no single substitutions completely abolish binding activity, multiple interactions must takeplace during apoE–LDL receptor interaction. Moreover, the basic residues of helix 4 mentionedabove may have a cooperative effect during this interaction. The crystal structure of the N-terminal domain of apoE3 may also help to explain the effect of residue substitutions (i.e, inisoforms E2 and E4) indicating that they are due to the disruption of stabilizing interactions andconformational changes, respectively.The C-terminal domain (216–299) of apoE contains the major lipid-binding elements and

exhibits properties typical of other soluble apolipoproteins. Moreover, it displays a relatively lowenergy of stabilization (DGunfold is approximately 3–4 kcal/mol), it is responsible for the self-association of apoE in the absence of lipids, and it forms tetramers in solution. Three a-helices arepredicted in this C-terminal region of apoE3, encompassing residues 204–221, 223–263, and266–287. The three-dimensional structure of this C-terminal domain remains unknown.A discussion of the implications of both the topology and intrinsic flexibility of the helix bundle

of the N-terminal domain of apoE for lipid binding is presented in the next section of this work.

2.5. Apolipophorin III

Apolipophorin III (apoLp-III), an exchangeable apolipoprotein found in many insect species,functions in transport of diacylglycerol from the fat body lipid storage depot to flight muscles inthe adult life stage.The structural and biophysical information available show that the lipid-free apolipophorin III

shares the elongated up-and-down amphipathic a-helix-bundle fold present in exchangeable

Fig. 6. (A) The crystal structure of the N-terminal domain of apoE, residues 1–191, shows this domain consists of a

typical four a-helix bundles. (B) Spatial disposition of the hydrophobic residues in the core of the structure. (C) Topview of the a-helix-bundle fold.


apolipoproteins. As in the case of human exchangeable apolipoproteins, proline residues serve todelineate the N-termini of several helices (specifically, helices 5 and 7). As shown in Fig. 7, the a-helices present in apolipophorin III of M. sexta encompass the following aminoacids residues:a-helix 1, residues 10–30; a-helix 2, residues 40–66; a-helix 3, residues 70–93; a-helix 4, residues104–128; and a-helix 5, residues 140–164. Based on this structure (Breiter et al., 1991), it has beenproposed that a dramatic conformational change is induced in the helix bundle when contactingwith a lipid surface, allowing the opening of the protein about ‘‘hinged’’ loops that connect helicalsegments (Wang et al., 2002). Such a conformational change involves the movement of helices 1,2, and 5 in one direction and helices 3 and 4 in the other around ‘‘hinges’’ located in the loopsbetween helices 2 and 3 (residues 67–69) and between helices 4 and 5 (residues 122–128). Thus, theopen, lipid-associated conformation corresponds to an extended a-helical structure with acontinuous hydrophobic surface that resembles the structure of human helix bundles ofapolipoproteins A-I and E, as observed by Wang and collaborators. The implications of thisdomain organization, in the context of lipid-binding activity, are presented and discussed in thenext section.

3. Apolipoprotein flexibility is required for lipid binding

Exchangeable apolipoproteins are the only known examples where this class of conformationaladaptation upon ligand binding takes place. This raises the question of why other helix-bundleproteins, such as chemokines or cytochrome c; are not able to bind to lipid surfaces. It has beenproposed that other a-helix bundles not associated with lipid binding are unable to open becauseof their intrinsic bundle topology (Wang et al., 2002) For example, cytochrome c is an up-and-down bundle with long loops that form short b-strands (Kamtekar and Hecht, 1995), whilechemokines are up-up-down-down helix bundles (Rozwarski et al., 1994). The structuralconstraints of these two different a-helix-bundle topologies do not permit a simple concertedopening because the long loops form knots, the short b-sheet connecting the long loops will beotherwise disrupted, or the helix–helix interactions are particularly favorable. Besides, the shorthelix 310 of apolipophorin III could also play an important role in lipid surface recognition. Thishelix 310 connects a-helix 3 and a-helix 4 in the bundle and is located at one end of the molecule,exposed to solvent. It adopts an orientation approximately perpendicular to the long axis of the

Fig. 7. (A) The NMR structure of the lipid-free apolipophorin III protein exhibits the up-and-down amphipathic a-helix-bundle fold present in other exchangeable apolipoproteins. (B) Top view of the a-helix bundle.


bundle and its conservation in four distinct lepidopeteran species suggests that it has an importantfunctional role. Interestingly, a short, similar helix is also found in the N-terminal domain ofhuman apoE and L. migratoria apolipophorin-III, but not in other helix bundles. The short helixof the N-terminal domain of apolipoprotein E comprises the most conserved sequence in thisprotein, although no specific biological function for this region has been reported so far.The free energy of unfolding of most lipid-free apolipoproteins (typically, DG ¼ 4:5 kcal/mol)

supports the idea that buried polar residues in the lipid-free helix bundle modulate apolipoproteinconversion between lipid-free and lipid-associated states (Gursky and Atkinson, 1996). Incontrast, other helix bundles, such as the ROP dimer, adopt a more stable up-and-down four a-helix bundles with no short helix (DG ¼ 17:1 kcal/mol for the dimeric form) (Vlassi et al., 1999).Therefore, the high stability of this a-helix bundle would preclude opening in the presence oflipids. The driving force for recovery of the a-helix-bundle conformation upon release from alipoprotein surface is the reestablishment of non-specific interhelical hydrophobic interactions.This structural reorganization occurs because an exposed hydrophobic surface in the openconformation is thermodynamically unfavorable. Thus, the reestablishment of helix–helixhydrophobic contacts to restore the helix-bundle fold revert this situation.A characteristic property of exchangeable apolipoproteins is their ability to form discoidal

lipoprotein particles. On the basis of the size and stoichiometry of discoidal lipoproteins, and thesurface properties of the apolipoproteins, it has been generally accepted that the apolipoproteinsspread along the periphery of the lipid disks, interacting with the phospholipid acyl chains.This model was first described for apoA-I and later extended to apoE (Nichols et al., 1984;De Pauw et al., 1995). More recently, Soulages and Arrese (2001) proposed a model in which thea-helices of apolipophorin-III are arranged perpendicular to the phospholipid acyl chainssurrounding the periphery of the discoidal bilayer. Since five a-helices of this insect apolipoproteinare perpendicular to the acyl chains, they cannot be accommodated in the thickness of abilayer. Thus, it was deduced that this apolipoprotein molecule probably adopts a highlyextended conformation. Nowadays, there is experimental evidence that supports this model, suchas the crystal structure of apoA-II in complex with a lipid surrogate (Kumar et al., 2002)and molecular dynamics simulations on discoidal HDL particles containing apoA-I (Klonet al., 2002).

4. Apolipoproteins in interfaces

The analysis of several exchangeable apolipoproteins in interfaces, using a system that mimicsthe one found in lipoprotein particles, has recently been described (Bolanos-Garcia et al., 1999,2001). These analyses combined the use of Langmuir–Blodgett monolayers with Brewster anglemicroscopy (BAM) to observe the series of changes that take place during lateral compression ofthe protein monolayer. Such studies have revealed that most of the molecular rearrangements aredue to phase changes, where each phase presents a distinctive molecular organization. Forexample, in experiments where human apoC-I was deposited onto a hydrophilic subphase, severalphase transitions were observed during monolayer compression. As a result of such analysis,a model to explain the conformational change that might take place during the interaction ofapoC-I with lipids was proposed (Bolanos-Garcia et al., 1999). Grazing incidence X-ray


diffraction experiments of apoC-I monolayers have recently confirmed that both a-helices remainstable at high lateral pressures (Bolanos-Garcia, unpublished results).A more recent analysis of other apolipoproteins in monolayers, including apoA-I, apoA-II,

apoC-III and apoE, allowed the observation of similar phase transitions. Some of thesetransitions were due to the desorption of a-helical segments out of the surface of the monolayer(Bolanos-Garcia et al., 2001). It has been proposed that the desorbed a-helical regions are those oflower amphiphilicity and/or length. Besides, the conformational changes of human apolipopro-teins observed in Langmuir–Blodgett monolayers are consistent with experimental data obtainedwith other techniques, including fluorescence spectroscopy (Ryan et al., 1993), near UV circulardichroism spectroscopy (Weers et al., 1994), surface plasmon resonance spectroscopy (Soulageset al., 1995), Fourier-transformed IR spectroscopy (Raussens et al., 1996) and disulfide-bondengineering (Narayanaswami et al., 1996). All these studies support a mechanism based on theconformational opening of the a-helix bundle upon lipid binding. This conformationaladaptability, that raises the question of which structural features in both, the lipid-free andlipid-bound helix-bundle conformations regulate this process, remains as a subject of intensestudies.

5. The importance of kinetic stability

The behavior of exchangeable apolipoproteins in monolayers is consistent with their relativelow thermodynamic stability in the lipid-free state. In a series of unfolding studies using circulardichroism, it was found that apolipoproteins A-II, C-III, E-3, and A-I show an isochromatic pointat 204–205 nm, suggesting a two-state transition character (Bolanos-Garcia et al., 2001). Theseresults also showed that preservation of the a-helix structure is temperature dependent and itsstabilization is enthalpy-driven. Besides, it is known that exchangeable apolipoproteins exhibitproperties similar to those associated with the molten globule state: a compact shape in solutionwith well-defined secondary structure content, the lack of specific tertiary interactions, a broadthermal unfolding profile and a relaxed affinity for various ligands. Thus, it has been proposedthat some of these properties might mediate protein–lipid interactions (Soulages and Bendavid,1998). Moreover, Epand and colleagues reported that the calorimetric transition of apoA-1/DMPC complexes is strongly dependent on the heating rate used. This observation as well askinetic data obtained by spectroscopic techniques indicated a slow non-equilibrium unfolding(Epand, 1982). Therefore, the unfolding of other DMPC-containing reconstituted lipoproteinsmay also be determined by kinetic factors (Epand, 1982). As a consequence of such behavior,molecular models of discoidal lipoproteins (such as nascent HDL) and of the structure of thetransition state will have to be described in detail in order to elucidate the molecular interactionsthat govern lipoprotein stability. A comparison of the free energy of activation, determined forthe unfolding of apoA-1 and apoC-1 on DMPC disks with the free energy of stability of typicalglobular proteins, shows that even in lipoproteins lacking the high packing specificity that mightcontribute to protein stability, their kinetic stability may be comparable to the thermodynamicstability of globular proteins (Privalov, 1989). These observations have led to the recognition ofkinetic stability as an important mechanism to control folding and function in exchangeableapolipoproteins, in a similar way to that reported for other globular proteins (Baker and Agard,


1994). This kinetic control may help to maintain lipoprotein stability in the absence of highpacking specificity and might also contribute to regulate the rates of apolipoprotein exchangeamong lipoprotein particles.

6. Apolipoproteins and amyloidogenesis

One of the most striking properties of exchangeable apolipoproteins recently described is that inthe lipid-free form, they self-associate to form twisted ribbon-like fibrils with all of the hallmarksof amyloid fibrils, including binding to Congo Red with red-green birefringence under cross-polarized light, binding to thioflavin T, and increased content of b-structure. In vitro amyloidformation can be compared with the in vivo deposition of amyloid involving apolipoproteinssuch as apoA-I, apoA-II, apoA-IV, apoC-I, apoC-II and apoE (Petit-Turcotte et al., 2001;Cedazo-Minguez and Cowburn, 2001; Bergstrom et al., 2001; Hatters and Howlett, 2002), as wellas two apolipoprotein-like proteins, a-synuclein and serum amyloid A (Kessler et al., 2003; Kindyet al., 1999). In fact, the well-characterized ability of apoC-II to form amyloid fibrils has beenused as a model to examine the factors that could control the growth of amyloid fibrils in vivo(Pham et al., 2002; Nettleton et al., 1998). In order to explain the prevalence of apolipoproteins inamyloid formation, it has been suggested to be due to their limited conformational stability or thespecific secondary structure content in their lipid-free form, as observed in other proteins thatpromote amyloid formation, or being the result of macromolecular crowding (Hatters et al.,2002a). Besides, apolipoprotein derivatives that form amyloid fibrils are frequently mutantisoforms or truncated products, as noticed in apoA-I (Andreola et al., 2003; Obici et al., 1999),apoA-II (Yazaki et al., 2001), and apoE (Carter et al., 2001; Corder et al., 1993). Thesemodifications may destabilize the lipid-binding conformation(s) leading to amyloid formation invivo. Interestingly, it has also recently been reported that the protein clusterin, an extracellularchaperone, is a potent inhibitor of amyloid formation by apoC-II. The same group suggested thatsuch inhibition involves the interaction of clusterin with transient amyloid nuclei, which results inthe dissociation of the monomeric subunits (Hatters et al., 2002b).

7. Conclusions and future perspectives

The determination of the three-dimensional structures of several human exchangeableapolipoproteins provides the basis for the understanding of the molecular mechanisms involvedin lipid binding. Since all these structures were solved in microenvironments that are differentfrom the one found in ‘‘natural’’ lipoprotein particles, the question of how these proteins act invivo remains open. However, it is reasonable to expect that in the near future the elucidation of anentire lipoprotein particle at a high resolution will be achieved.On the other hand, the elucidation of the role of this family of proteins in diverse cellular

processes will continue and it is likely that their association with other functions will be described.With the first draft of human and mice genomes available, it is also possible that new genes andisoforms will be identified in the apo gene cluster. Undoubtedly, these achievements will boost ourunderstanding of lipid metabolism and other apolipoprotein-mediated events under normal and


pathological conditions. They will open new frontiers for biochemical, pharmacological andmedical research.

Acknowledgements

Dr. V. Bolanos-Garcia acknowledges The Wellcome Trust for their support (InternationalFellows Program, 60125). We are grateful to Prof. Tom L. Blundell for multiple discussions andsuggestions as well as to Dr. J.L. Soulages, Dr. J. Venkatesh Pratap and Mr. Matthew Daniels fortheir critical review of the manuscript.

References

Aalto-Setala, K., Weinstock, P.H., Bisgaier, C.L., Wu, L., Smith, J.D., Breslow, J.L., 1996. Further characterization of

the metabolic properties of triglyceride-rich lipoproteins from human and mouse apoC-III transgenic mice. J. Lipid

Res. 37, 1802–1811.

Allan, C.M., Taylor, J.M., 1996. Expression of a novel human apolipoprotein (apoC-IV) causes hypertriglyceridemia in

transgenic mice. J. Lipid Res. 37, 1510–1518.

Allan, C.M., Walker, D., Segrest, J.P., Taylor, J.M., 1995. Identification and characterization of a new human gene

(APOC4) in the apolipoprotein E, C-I, and C-II gene locus. Genomics 28, 291–300.

Andreola, A., Bellotti, V., Giorgetti, S., Mangione, P., Obici, L., Stoppini, M., Torres, J., Monzani, E., Merlini, G.,

Sunde, M., 2003. Conformational switching and fibrillogenesis in the amyloidogenic fragment of apolipoprotein A-I.

J. Biol. Chem 278, 2444–2451.

Arakawa, R., Yokoyama, S., 2002. Helical apolipoproteins stabilize ATP-binding cassette transporter A1 by protecting

it from thiol protease-mediated degradation. J. Biol. Chem. 277, 22426–22429.

Baker, D., Agard, D.A., 1994. Kinetics versus thermodynamics in protein folding. Biochemistry 33, 7505–7509.

Bengtsson, G., Olivecrona, T., 1982. Activation of lipoprotein lipase by apolipoprotein CII. Demonstration of an effect

of the activator on the binding of the enzyme to milk-fat globules. FEBS Lett. 147, 183–187.

Bergstrom, J., Murphy, C., Eulitz, M., Weiss, D.T., Westermark, G.T., Solomon, A., Westermark, P., 2001.

Codeposition of apolipoprotein A-IV and transthyretin in senile systemic (ATTR) amyloidosis. Biochem. Biophys.

Res. Commun. 285, 903–908.

Bielicki, J.K., Oda, M.N., 2002. Apolipoprotein A-I(Milano) and apolipoprotein A-I(Paris) exhibit an antioxidant

activity distinct from that of wild-type apolipoprotein A-I. Biochemistry 41, 2089–2096.

Bolanos-Garcia, V., Soriano-Garcia, M., Mas-Oliva, J., 1997. CETP and exchangeable apoproteins: common features

in lipid binding activity. Mol. Cellular Biochem. 175, 1–10.

Bolanos-Garcia, V.M., Mas-Oliva, J., Ramos, S., Castillo, R., 1999. Phase transitions in monolayers of human

Apolipoprotein C-I. J. Phys. Chem. B. 103, 6236–6242.

Bolanos-Garcia, V.M., Ramos, S., Castillo, R., Xicohtencatl-Cortes, J., Mas-Oliva, J., 2001. Monolayers of

apolipoproteins at the air/water interface. J. Phys. Chem. B 105, 5757–5765.

Borhani, D.W., Rogers, D.P., Engler, J.A., Brouillette, C.G., 1997. Crystal structure of truncated human

apolipoprotein A-I suggests a lipid-bound conformation. Proc. Natl. Acad. Sci. USA 94, 12291–12296.

Borhani, D.W., Engler, J.A., Brouillette, C.G., 1999. Crystallization of truncated human apolipoprotein A-I in a novel

conformation. Acta Crystallogr. D 55, 1578–1583.

Bowie, J.U., Luthy, R., Eisenberg, D., 1991. A method to identify protein sequences that fold into a known three-

dimensional structure. Science 253, 164–170.

Breiter, D.R., Kanost, M.R., Benning, M.M., Wesenberg, G., Law, J.H., Wells, M.A., Rayment, I.,

Holden, H.M., 1991. Molecular structure of an apolipoprotein determined at 2.5- (A resolution. Biochemistry. 30,

603–608.


Brouillette, C.G., Jones, J.L., Ng, T.C., Kercret, H., Chung, B.H., Segrest, J.P., 1984. Structural studies of

apolipoprotein A-I/phosphatidylcholine recombinants by high-field proton NMR, nondenaturing gradient gel

electrophoresis, and electron microscopy. Biochemistry 23, 359–367.

Brouillette, C.G., Anantharamaiah, G.M., Engler, J.A., Borhani, D.W., 2001. Structural models of human

apolipoprotein A-I: a critical analysis and review. Biochim. Biophys. Acta 1531, 4–46.

Bruckert, E., von Eckardstein, A., Funke, H., Beucler, I., Wiebusch, H., Turpin, G., Assmann, G., 1997. The

replacement of arginine by cysteine at residue 151 in apolipoprotein A-I produces a phenotype similar to that of

apolipoprotein A-I Milano. Atherosclerosis 128, 121–128.

Buchko, G.W., Rozek, A., Kanda, P., Kennedy, M.A., Cushley, R.J., 2000. Structural studies of a baboon (Papio sp)

plasma protein inhibitor of cholesteryl ester transferase. Prot. Sci. 8, 1548–1558.

Carter, D.B., Dunn, E., McKinley, D.D., Stratman, N.C., Boyle, T.P., Kuiper, S.L., Oostveen, J.A., Weaver, R.J.,

Boller, J.A., Gurney, M.E., 2001. Human apolipoprotein E4 accelerates b-amyloid deposition in APPsw transgenicmouse brain. Ann. Neurol. 50, 468–475.

Cedazo-Minguez, A., Cowburn, R.F., 2001. Apolipoprotein E: a major piece in the Alzheimer’s disease puzzle. J. Cell.

Mol. Med. 5, 254–266.

Clement-Collin, V., Leroy, A., Monteilhet, C., Aggerbeck, L.P., 1999. Mimicking lipid-binding-induced conforma-

tional changes in the human apolipoprotein E N-terminal receptor binding domain effects of low pH and propanol.

Eur. J. Biochem. 264, 358–368.

Conde-Knape, K., Bensadoun, A., Sobel, J.H., Cohn, J.S., Shachter, N.S., 2002. Overexpression of apoC-I in apoE-null

mice: severe hypertriglyceridemia due to inhibition of hepatic lipase. J. Lipid Res. 43, 2136–2145.

Corder, E.H., Saunders, A.M., Strittmatter, W.J., Schmechel, D.E., Gaskell, P.C., Small, G.W., Roses, A.D., Haines,

J.L., Pericak-Vance, M.A., 1993. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in

late onset families. Science 261, 921–923.

Davison, P.J., Norton, P., Wallis, S.C., Gill, L., Cook, M., Williamson, R., Humphries, S.R., 1986. There are two gene

sequences for human apolipoprotein C1 (APO C1) on chromosome 19, one of which is 4 kb from the gene for apoE.

Biochem. Biophys. Res. Commun. 136, 876–884.

de Bakker, P.I., Bateman, A., Burke, D.F., Miguel, R.N., Mizuguchi, K., Shi, J., Shirai, H., Blundell, T.L., 2001.

HOMSTRAD: adding sequence information to structure-based alignments of homologous protein families.

Bioinformatics 17, 748–749.

De Pauw, M., Vanloo, B., Weisgraber, K., Rosseneu, M., 1995. Comparison of lipid-binding and lecithin: Cholesterol

acyltransferase activation of the amino- and carboxyl-terminal domains of human apolipoprotein E3. Biochemistry

34, 10953–10960.

Dumon, M.F., Clerc, M., 1986. Preliminary report on a case of apolipoprotein CI and CII deficiency. Clin. Chim. Acta

157, 239–248.

Epand, R.M., 1982. The apparent preferential interactions of human plasma high-density apolipoprotein A-1 with

gel-state phospholipids. Biophys. Biochem. Acta 712, 146–151.

Fielding, C.J., Fielding, P.E., 1995. Molecular physiology of reverse cholesterol transport. J. Lipid Res. 36, 211–228.

Fielding, C.J., Shore, V.G., Fielding, P.E., 1972. A protein cofactor of lecithin: cholesterol acyltransferase. Biochem.

Biophys. Res. Commun. 46, 1493–1498.

Franceschini, G., Vecchio, G., Gianfranceschi, G., Magani, D., Sirtori, CR., 1985. Apolipoprotein AI Milano.

Accelerated binding and dissociation from lipids of a human apolipoprotein variant. J. Biol. Chem. 260,

16321–16325.

Gautier, T., Masson, D., Pais de Barros, J-P., Athias, A., Gambert, P., Aunis, D., Metz-Boutigue, M-H., Lagrost, L.,

2000. Human apolipoprotein C-I accounts for the ability of plasma high density lipoproteins to inhibit the

cholesteryl ester transfer protein activity. J. Biol. Chem. 275, 37504–37509.

Gursky, O., Atkinson, D., 1996. Thermal unfolding of human high-density apolipoprotein A-1: implications for a

lipid-free molten globular state. Proc. Natl. Acad. Sci. USA 93, 2991–2995.

Hatters, D.M., Howlett, G.J., 2002. The structural basis for amyloid formation by plasma apolipoproteins: a review.

Eur. Biophys. J. 31, 2–8.

Hatters, D.M., Minton, A.P., Howlett, G.J., 2002a. Macromolecular crowding accelerates amyloid formation by

human apolipoprotein C-II. Biochemistry 277, 7824–7830.


Hatters, D.M., Wilson, M.R., Easterbrook-Smith, S.B., Howlett, G.J., 2002b. Suppression of apolipoprotein C-II

amyloid formation by the extracellular chaperone, clusterin. Eur. J. Biochem. 269, 2789–2794.

Ishigami, M., Swerfeger, D.K., Granholm, N.A., Hui, D.Y., 1998. Apolipoprotein E inhibits platelet-derived growth

factor-induced vascular smooth muscle cell migration and proliferation by suppressing signal transduction and

preventing cell entry to G1 phase. J. Biol. Chem. 273, 20156–20161.

Ito, Y., Breslow, J.L., Chait, B.T., 1989. Apolipoprotein C-III-0 lacks carbohydrate residues: use of mass spectrometry

to study apolipoprotein structure. J. Lipid Res. 30, 1781–1787.

Jonas, A., 1998. Regulation of lecithin cholesterol acyltransferase activity. Prog. Lipid Res. 37, 209–234.

Jong, M.C., Havekes, L.M., 2000. Insights into apolipoprotein C metabolism from transgenic and gene-targeted mice.

Int. J. Tissue React. 22, 59–66.

Jong, M.C., Dahlmans, V.E.H., van Gorp, P.J.J., Willems van Dijk, K., Koopmans, S-J., Chan, L., Hofker, M.H.,

Havekes, L.M., 1996. In the absence of the low density lipoprotein receptor, human apolipoprotein

C1 overexpression in transgenic mice inhibits the hepatic uptake of very low density lipoproteins via a receptor-

associated protein-sensitive pathway. J. Clin. Invest. 98, 2259–2267.

Jong, M.C., Hofker, M.H., Havekes, L.M., 1999. Role of ApoCs in lipoprotein metabolism. Functional differences

between ApoC1, ApoC2, and ApoC3. Arterioscler. Thromb. Vasc. Biol. 19, 472–484.

Jong, M.C., Voshol, P.J., Muurling, M., Dahlmans, V.E., Romijn, J.A., Pijl, H., Havekes, L.M., 2001. Protection from

obesity and insulin resistance in mice overexpressing human apolipoprotein C1. Diabetes 50, 2779–2785.

Kamtekar, S., Hecht, M.H., 1995. Protein motifs 7. The four-helix bundle: what determines a fold? FASEB J. 9,

1013–1022.

Katzman, R., 1994. Apolipoprotein E and Alzheimer’s disease. Curr. Opin. Neurobiol. 4, 703–707.

Kawooya, J.K., Meredith, S.C., Wells, M.A., Kezdy, F.J., Law, J.H., 1986. Physical and surface properties of insect

apolipophorin III. J. Biol. Chem. 261, 13588–13591.

Kessler, J.C., Rochet, J.C., Lansbury Jr, P.T., 2003. The N-terminal repeat domain of a-synuclein inhibits b-sheet andamyloid fibril formation. Biochemistry 42, 672–678.

Kindy, M.S., Yu, J., Guo, J.T., Zhu, H., 1999. Apolipoprotein serum amyloid A in Alzheimer’s disease. J. Alzheimer

Dis. 1, 155–167.

Kinnunen, P.K.J., Ehnholm, C., 1976. Effect of serum and C apoproteins from very low density lipoproteins on human

postheparin plasma hepatic lipase. FEBS Lett. 65, 354–357.

Kinnunen, P.K., Jackson, R.L., Smith, L.C., Gotto, A.M., Sparrow, J.T., 1977. Activation of lipoprotein lipase

by native and synthetic fragments of human plasma apolipoprotein C-II. Proc. Natl. Acad. Sci. USA 74,

4848–4851.

Kitagawa, K., Matsumoto, M., Hori, M., Yanagihara, T., 2002. Neuroprotective effect of apolipoprotein E against

ischemia. Ann. N.Y. Acad. Sci. 977, 468–475.

Klon, A.E., Segrest, J.P., Harvey, S.C., 2002. Molecular dynamics simulations on discoidal HDL particles suggest a

mechanism for rotation in the apo A-I belt model. J. Mol. Biol. 324, 703–721.

Kowal, R.C., Herz, J., Weisgraber, K.H., Mahley, R.W., Brown, M.S., Goldstein, J.L., 1990. Opposing effects of

apolipoprotein E and C on lipoprotein binding to the low density lipoprotein receptor-related protein. J. Biol. Chem.

265, 10771–10779.

Kumar, M.S., Carson, M., Hussain, M.M., Murthy, H.K.M., 2002. Structures of apolipoprotein A-II and a

lipid-surrogate complex provide insights into apolipoprotein–lipid interactions. Biochemistry 41, 11681–11691.

Laffont, I., Takahashi, M., Shibukawa, Y., Honke, K., Shuvaev, V.V., Siest, G., Visvikis, S., Taniguchi, N., 2002.

Apolipoprotein E activates Akt pathway in neuro-2a in an isoform-specific manner. Biochem. Biophys. Res.

Commun. 292, 83–87.

Lagocki, P.A., Scanu, A.M., 1980. In vitro modulation of the apolipoprotein composition of high density lipoprotein

displacement of apolipoprotein A-I from high density lipoprotein by apolipoprotein A-II. J. Biol. Chem. 255,

3701–3706.

Laskowski, R.A., MacArthur, M.W., Moss, D.S., Thornton, J.M., 1993. PROCHECK: a program to check the

stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291.

Lauer, S., Walker, D., Elshourbagy, N.A., Reardon, C.A., Levy-Wilson, B., Taylor, J.M., 1988. Two copies of the

human apolipoprotein C-I gene are linked closely to the apolipoprotein E gene. J. Biol. Chem. 263, 7277–7286.


Li, H.H., Lyles, D.S., Pan, W., Alexander, E., Thomas, M.J., Sorci-Thomas, M.G., 2002. ApoA-I structure on discs

and spheres variable helix registry and conformational states. J. Biol. Chem. 277, 39093–39101.

Liu, T., Krieger, M., Kan, H.Y., Zannis, V.I., 2002. The effects of mutations in helices 4 and 6 of ApoA-I on scavenger

receptor class B type I (SR-BI)-mediated cholesterol efflux suggest that formation of a productive complex

between reconstituted high density lipoprotein and SR-BI is required for efficient lipid transport. J. Biol. Chem. 277,

21576–21584.

MacPhee, C.E., Howlett, G.J., Sawyer, W.H., 1999. Mass spectrometry to characterize the binding of a peptide to a

lipid surface. Anal. Biochem. 275, 22–29.

MacPhee, C.E., Hatters, D.M., Sawyer, W.H., Howlett, G.J., 2000. Apolipoprotein C-II 39-62 activates lipoprotein

lipase by direct lipid-independent binding. Biochemistry 39, 3433–3440.

MacRaild, C.A., Hatters, D.M., Howlett, G.J., Gooley, P.R., 2001. NMR structure of human apolipoprotein C-II in

the presence of sodium dodecyl sulfate. Biochemistry 40, 5414–5421.

Major, A.S., Dove, D.E., Ishiguro, H., Su, Y.R., Brown, A.M., Liu, L., Carter, K.J., Linton, M.F., Fazio, S., 2001.

Increased cholesterol efflux in apolipoprotein AI (ApoAI)-producing macrophages as a mechanism for reduced

atherosclerosis in ApoAI((�/�)) mice. Arterioscler. Thromb. Vasc. Biol. 21, 1790–1795.Miguel, R.N., Shi, J., Mizuguchi, K., 2002. Protein fold recognition and comparative modeling using HOMSTRAD,

JOY and FUGUE. In: Tsigelny, I. (Ed.), Protein Structure Prediction: Bioinformatic Approach. International

University Line Publishers, La Jolla, CA.

Miyata, M., Smith, J.D., 1996. Apolipoprotein E allele-specific antioxidant activity and effects on cytotoxicity by

oxidative insults and b-amyloid peptides. Nat. Genet. 14, 55–61.Mizuguchi, K., Deane, C.M., Blundell, T.L., Johnson, M.S., Overington, J.P., 1998. JOY: protein sequence-structure

representation and analysis. Bioinformatics 14, 617–623.

Myklebost, O., Williamson, B., Markham, A.F., Myklebost, S.R., Rogers, J., Woods, D.E., Humphries, S.E.,

1984. The isolation and characterization of cDNA clones for human apolipoprotein CII. J. Biol. Chem. 259,

4401–4404.

Narayanaswami, V., Wang, J., Kay, C.M., Scraba, D.G., Ryan, R.O., 1996. Disulfide bond engineering to monitor

conformational opening of apolipophorin III during lipid binding. J. Biol. Chem. 271, 26855–26862.

Nettleton, E.J., Sunde, M., Lai, Z., Kelly, J.W., Dobson, C.M., Robinson, C.V., 1998. Protein subunit interactions and

structural integrity of amyloidogenic transthyretins: evidence from electrospray mass spectrometry. J. Mol. Biol.

281, 553–564.

Nichols, A.V., Gong, E.L., Blanche, P.J., Forte, T.M., Shore, V.G., 1984. Interaction of model discoidal complexes of

phosphatidylcholine and apolipoprotein A-I with plasma components physical and chemical properties of the

transformed complexes. Biochim. Biophys. Acta 793, 325–337.

Obici, L., Bellotti, V., Mangione, P., Stoppini, M., Arbistini, E., Vega, L., Zorzoli, I., Anesi, E., Zanotti, G., Campana,

C., Vigano, M., Merlini, G., 1999. The new apolipoprotein A-I variant Leu(174)-Ser causes hereditary cardiac

amyloidosis, and the amyloid fibrils are constituted by the 93-residue N-terminal polypeptide. Am. J. Pathol. 155,

695–702.

Okon, M., Frank, P.G., Marcel, Y.L., Cushley, R.J., 2001. Secondary structure of human apolipoprotein A-I(1–186) in

lipid-mimetic solution. FEBS Lett. 487, 390–396.

Okubo, M., Hasegawa, Y., Aoyama, Y., Murase, T., 1997. A G11 to C mutation in a donor splice site of intron 2 in the

apolipoprotein (apo) C-II gene in a patient with apo C-II deficiency: a possible interaction between apo C-II

deficiency and apo E4 in a severely hypertriglyceridemic patient. Atherosclerosis 130, 153–160.

Petit-Turcotte, C., Stohl, S.M., Beffert, U., Cohn, J.S., Aumont, N., Tremblay, M., Dea, D., Yang, L., Porier, J.,

Shachter, N.S., 2001. Apolipoprotein C-I expression in the brain in Alzheimer’s disease. Neurobiol. Dis. 8, 953–963.

Pham, C.L., Hatters, D.M., Lawrence, L.J., Howlett, G.J., 2002. Cross-linking and amyloid formation by N- and

C-terminal cysteine derivatives of human apolipoprotein C-II. Biochemistry 41, 14313–14322.

Poeggeler, B., Miravalle, L., Zagorski, M.G., Wisniewski, T., Chyan, Y.J., Zhang, Y., Shao, H., Bryant-Thomas, T.,

Vidal, R., Frangione, B., Ghiso, J., Pappolla, M.A., 2001. Melatonin reverses the profibrillogenic activity of

apolipoprotein E4 on the Alzheimer amyloid Ab peptide. Biochemistry 40, 14995–15001.Privalov, PL., 1989. Thermodynamic problems of protein structure. Annu. Rev. Biophys. Biophys. Chem. 18,

47–69.


Raussens, V., Narayanaswami, V., Goormaghtigh, E., Ryan, R.O., Ruysschaert, J-M., 1996. Hydrogen/deuterium

exchange kinetics of apolipophorin-III in lipid-free and phospholipid-bound states an analysis by Fourier transform

infrared spectroscopy. J. Biol. Chem. 271, 23089–23093.

Reschly, E.J., Sorci-Thomas, M.G., Davidson, W.S., Meredith, S.C., Reardon, C.A., Getz, G.S., 2002. Apolipoprotein

A-I a-helices 7 and 8 modulate high density lipoprotein subclass distribution. J. Biol. Chem. 277, 9645–9654.Riddell, D.R., Graham, A., Owen, J.S., 1997. Apolipoprotein E inhibits platelet aggregation through the

l-arginine:nitric oxide pathway: implications for vascular disease. J. Biol. Chem. 272, 89–95.

Rozek, A., Buchko, G.W., Cushley, R.J., 1995. Conformation of two peptides corresponding to human apolipoprotein

C-I residues 7–23 and 35–53 in the presence of sodium dodecyl sulfate by CD and NMR spectroscopy. Biochemistry

324, 7401–7408.

Rozek, A., Buchko, G.W., Kanda, A., Cushley, R.J., 1997. Conformational studies of the N-terminal lipid-associating

domain of human apolipoprotein C-I by CD and 1H NMR spectroscopy. Protein Sci. 6, 1858–1868.

Rozwarski, D.A., Gronenborn, A.M., Clore, G.M., Bazan, J.F., Bohm, A., Wlodawer, A., Hatada, M., Karplus, P.A.,

1994. Structural comparisons among the short-chain helical cytokines. Structure 2, 159–173.

Ryan, R.O., Oikawa, K., Kay, C.M., 1993. Conformational, thermodynamic, and stability properties ofManduca sexta

apolipophorin III. J. Biol. Chem. 268, 1525–1530.

Sali, A., Blundell, T.L., 1993. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234,

779–815.

Schachter, F., Faure-Delanef, L., Guenot, F., Rouger, H., Froguel, P., Lesueur-Ginot, L., Cohen, D., 1994. Genetic

associations with human longevity at the APOE and ACE loci. Nat. Genet. 6, 29–32.

Schaefer, E.J., Ordovas, J., Law, S., Ghiselli, G., Kashyap, L., Srivastava, L., Heaton, W.H., Albers, J., Connor, W.,

Lindgren, F., 1985. Familial apolipoprotein A-I and C-III deficiency: variant II. J. Lipid Res. 26, 1089–1101.

Schwarzenbacher, R., Zeth, K., Diederichs, K., Gries, A., Kostner, G.M., Laggner, P., Prassl, R., 1999. Crystal

structure of human b2-glycoprotein I: implications for phospholipid binding and the antiphospholipid syndrome.EMBO J. 18, 6228–6239.

Segrest, J.P., Jones, M.K., Klon, A.E., Sheldahl, C.J., Hellinger, M., De Loof, H., Harvey, S.C., 1999. A detailed

molecular belt model for apolipoprotein A-I in discoidal high density lipoprotein. J. Biol. Chem. 274, 31755–31758.

Shah, P.K., Yano, J., Reyes, O., Chyu, K.Y., Kaul, S., Bisgaier, C.L., Drake, S., Cercek, B., 2001. High-dose

recombinant apolipoprotein A-I(Milano) mobilizes tissue cholesterol and rapidly reduces plaque lipid and

macrophage content in apolipoprotein E-deficient mice potential implications for acute plaque stabilization.

Circulation 103, 3047–3050.

Shi, J., Blundell, T.L., Mizuguchi, K., 2001. FUGUE: sequence-structure homology recognition using environment-

specific substitution tables and structure-dependent gap penalties. J. Mol. Biol. 310, 243–257.

Shulmann, R.S., Herbert, P.N., Wehrly, K., Frederickson, D.S., 1975. The complete amino acid sequence of C-I

(apoLP–Ser), an apolipoprotein from human very low density lipoproteins. J. Biol. Chem. 250, 182–190.

Soulages, J.L., Arrese, E.L., 2001. Interaction of the a-helices of apolipophorin III with the phospholipid acyl chains indiscoidal lipoprotein particles: a fluorescence quenching study. Biochemistry 40, 14279–14290.

Soulages, J.L., Bendavid, O.J., 1998. The lipid binding activity of the exchangeable apolipoprotein apolipophorin-III

correlates with the formation of a partially folded conformation. Biochemistry 37, 10203–10210.

Soulages, J.L., Salamon, Z., Wells, M.A., Tollin, G., 1995. Low concentrations of diacylglycerol promote the binding of

apolipophorin III to a phospholipid bilayer: a surface plasmon resonance spectroscopy study. Proc. Natl. Acad. Sci.

USA 92, 5650–5654.

Soutar, A.K., Garner, C.W., Baker, H.N., Sparrow, J.T., Jackson, R.L., Gotto Jr, A.M., Smith, L.C., 1975. Effect of

the human plasma apolipoproteins and phosphatidylcholine acyl donor on the activity of lecithin: cholesterol

acyl-transferase. Biochemistry 14, 3057–3064.

Sparks, D.L., Pritchard, P.H., 1989. Transfer of cholesteryl ester into high density lipoprotein by cholesteryl ester

transfer protein: effect of HDL lipid and apolipoprotein content. J. Lipid Res. 30, 1491–1498.

Sparrow, J.T., Pownall, H.J., Hsu, F-J., Blumenthal, L.D., Culwell, A.R., Gotto, A.M., 1977. Lipid binding by

fragments of apolipoprotein CIII-1 obtained by thrombin cleavage. Biochemistry 16, 5427–5431.

Sprecher, D.L., Taam, L., Brewer Jr, H.B., 1984. Two-dimensional electrophoresis of human plasma apolipoproteins.

Clin. Chem. 30, 2084–2092.


Subbaiah, P.V., Norum, R.A., Bagdade, J.D., 1991. Effect of apolipoprotein activators on the specificity of

lecithin:cholesterol acyltransferase: determination of cholesteryl esters formed in A-I/C-III deficiency. J. Lipid Res.

32, 1601–1609.

Tricerri, M.A., Behling Agree, A.K., Sanchez, S.A., Bronski, J., Jonas, A., 2001. Arrangement of apolipoprotein A-I in

reconstituted high-density lipoprotein disks: an alternative model based on fluorescence resonance energy transfer

experiments. Biochemistry 40, 5065–5074.

Trieu, V.N., McConathy, W.J., 1995. APOC-III-b-galactosidase hybrid distinguishes between VLDL and LDL

phospholipids. Biochem. Biophys. Res. Commun. 211, 754–760.

Vadiveloo, P.K., Allan, C.M., Murray, B.J., Fidge, N.H., 1993. Interaction of apolipoprotein AII with the putative

high-density lipoprotein receptor. Biochemistry 32, 9480–9485.

Vlassi, M., Cesareni, G., Kokkinidis, M., 1999. A correlation between the loss of hydrophobic core packing interactions

and protein stability. J. Mol. Biol. 285, 817–827.

Wang, G., 2002. How the lipid-free structure of the N-terminal truncated human apoA-I converts to the lipid-bound

form: new insights from NMR and X-ray structural comparison. FEBS Lett. 529, 157–161.

Wang, J., Sykes, B.D., Ryan, R.O., 2002. Structural basis for the conformational adaptability of apolipophorin III, a

helix-bundle exchangeable apolipoprotein. Proc. Natl. Acad. Sci. USA 99, 1188–1193.

Weers, P.M.M., Kay, C.M., Oikawa, K., Wientzek, M., Van der Horst, D.J., Ryan, R.O., 1994. Factors affecting the

stability and conformation of Locusta migratoria apolipophorin III. Biochemistry 33, 3617–3624.

Weisgraber, K.H., Bersot, T.P., Mahley, R.W., Franceschini, G., Sirtori, C.R., 1980. A-I milano apoprotein isolation

and characterization of a cysteine-containing variant of the A-I apoprotein from human high density lipoproteins.

J. Clin. Invest. 66, 901–907.

Wilson, C., Wardell, M.R., Weisgraber, K.H., Mahley, R.W., Agard, D.A., 1991. Three-dimensional structure of the

LDL receptor-binding domain of human apolipoprotein E. Science 252, 1817–1822.

Xiong, W., Li, W-H., Posner, I., Yamamura, T., Yamamoto, A., Gotto Jr, A.M., Chan, L., 1991. No severe bottleneck

during human evolution: evidence from two apolipoprotein C-II deficiency alleles. Am. J. Hum. Genet. 48, 383–389.

Yazaki, M., Liepnieks, J.J., Yamashita, T., Guenther, B., Skinner, M., Benson, M.D., 2001. Renal amyloidosis caused

by a novel stop-codon mutation in the apolipoprotein A-II gene. Kidney Int. 60, 1658–1665.

Zhou, X.H., Paulsson, G., Stemme, S., Hansson, G.K., 1998. Hypercholesterolemia is associated with a T helper (Th) 1

Th2 switch of autoimmune response in atherosclerotic apoE knockout mice. J. Clin. Invest. 101, 1717–1725.


Documents

On the structure and function of apolipoproteins: more than a family of lipid-binding proteins