**2. Biogenesis of HDL**

HDL is synthesized through a complex pathway (Zannis et al., 2004a). The first step involves an ABCA1 mediated transfer of cellular phospholipids and cholesterol to lipid poor apoA-I extracellularly. The lipidated apoA-I is gradually converted to discoidal particles that are remodelled in the plasma compartment by the esterification of cholesterol by the enzyme LCAT (Zannis et al., 2006a) and are converted to spherical HDL particles. The cholesteryl esters formed are transferred to very low-density lipoproteins/intermediatedensity lipoproteins/low density lipoproteins (VLDL/IDL/LDL) by the cholesteryl ester transfer protein (CETP) (Barter et al., 2003). Additional remodelling of HDL involves transfer of phospholipids from VLDL/LDL to HDL by the phospholipid transfer protein (PLTP) (Lusa et al., 1996), cholesterol efflux from cells or delivery of cholesteryl esters to cells mediated by the SR-BI (Krieger, 2001) as well as cholesterol efflux mediated by the cell surface transporter ATP binding cassette transporter G1 (ABCG1) (Wang et al., 2004). Finally hydrolysis of lipids of HDL is mediated by various lipases [lipoprotein lipase (LpL), hepatic lipase (HL), endothelial lipase (EL)] (Breckenridge et al., 1982; Brunzell and Deeb, 2001; Ishida et al., 2003; Krauss et al., 1974). Mutations in any of these proteins may affect the biogenesis, maturation and the functions of HDL (Fig. 1).

Fig. 1. Schematic representation of the pathway of the biogenesis and catabolism of HDL Numbers 1-11 indicate key cell membrane or plasma proteins shown to influence HDL levels or composition as follows: 1) apoA-I; 2) ABCA1; 3) LCAT; 4) CETP; 5) HL; 6) EL; 7) PLTP; 8) lipoproteins lipase; 9) SR-BI; 10) LDL receptor; 11) ABCG1. The figure is modified from ref. (Zannis et al., 2004b; Zannis et al., 2006b).

#### **3. Proteins involved in the biogenesis, remodeling and signaling of HDL**

**3.1 Apolipoprotein A-I and its structure in solution and in discoidal and spherical HDL** ApoA-I is synthesized by the liver and the intestine in humans (Williamson et al., 1992) and it is the major protein component of the HDL. ApoA-I along with several other proteins participate in the biogenesis and remodeling of HDL as well as in signaling pathways induced by apoA-I and HDL (Yuhanna et al., 2001; Zannis et al., 2004a). ApoA-I contains 22 or 11

HDL is synthesized through a complex pathway (Zannis et al., 2004a). The first step involves an ABCA1 mediated transfer of cellular phospholipids and cholesterol to lipid poor apoA-I extracellularly. The lipidated apoA-I is gradually converted to discoidal particles that are remodelled in the plasma compartment by the esterification of cholesterol by the enzyme LCAT (Zannis et al., 2006a) and are converted to spherical HDL particles. The cholesteryl esters formed are transferred to very low-density lipoproteins/intermediatedensity lipoproteins/low density lipoproteins (VLDL/IDL/LDL) by the cholesteryl ester transfer protein (CETP) (Barter et al., 2003). Additional remodelling of HDL involves transfer of phospholipids from VLDL/LDL to HDL by the phospholipid transfer protein (PLTP) (Lusa et al., 1996), cholesterol efflux from cells or delivery of cholesteryl esters to cells mediated by the SR-BI (Krieger, 2001) as well as cholesterol efflux mediated by the cell surface transporter ATP binding cassette transporter G1 (ABCG1) (Wang et al., 2004). Finally hydrolysis of lipids of HDL is mediated by various lipases [lipoprotein lipase (LpL), hepatic lipase (HL), endothelial lipase (EL)] (Breckenridge et al., 1982; Brunzell and Deeb, 2001; Ishida et al., 2003; Krauss et al., 1974). Mutations in any of these proteins may affect

Fig. 1. Schematic representation of the pathway of the biogenesis and catabolism of HDL Numbers 1-11 indicate key cell membrane or plasma proteins shown to influence HDL levels or composition as follows: 1) apoA-I; 2) ABCA1; 3) LCAT; 4) CETP; 5) HL; 6) EL; 7) PLTP; 8) lipoproteins lipase; 9) SR-BI; 10) LDL receptor; 11) ABCG1. The figure is modified

**3. Proteins involved in the biogenesis, remodeling and signaling of HDL** 

**3.1 Apolipoprotein A-I and its structure in solution and in discoidal and spherical HDL** ApoA-I is synthesized by the liver and the intestine in humans (Williamson et al., 1992) and it is the major protein component of the HDL. ApoA-I along with several other proteins participate in the biogenesis and remodeling of HDL as well as in signaling pathways induced by apoA-I and HDL (Yuhanna et al., 2001; Zannis et al., 2004a). ApoA-I contains 22 or 11

the biogenesis, maturation and the functions of HDL (Fig. 1).

from ref. (Zannis et al., 2004b; Zannis et al., 2006b).

**2. Biogenesis of HDL** 

amino acid repeats which are organized in amphipathic a-helices (Nolte and Atkinson, 1992). Based on the crystal structure of apoA-I in solution (Borhani et al., 1997) a belt model was proposed to explain the structure of apoA-I on discoidal HDL particles. In this model, two antiparallel molecules of apoA-I are wrapped like a belt around a discoidal bilayer containing 160 phospholipid molecules and shields the hydrophobic fatty acid chains of the phospholipids. Analysis of the 93 Å spherical HDL in solution by small angle neutron scattering (SANS) showed that three molecules of apoA-I fold around a central lipid core that has 88.4 Å x 62.8 Å dimensions to form a spheroidal HDL (sHDL) particle (Wu et al., 2011).

### **3.2 Interactions of ApoA-I with ABCA1 are the first step in the biogenesis of HDL**

ABCA1 is a ubiquitous protein that belongs to the ABC family of transporters and is expressed abundantly in the liver, macrophages, brain and various other tissues (Kielar et al., 2001). ABCA1 was shown to promote the efflux of cellular phospholipids and cholesterol to lipid free or minimally lipidated apoA-I and other apolipoproteins and amphipathic peptides, but it does not promote efflux to spherical HDL particles (Remaley et al., 2001; Wang et al., 2000). The functional interactions between apoA-I and ABCA-1 are important for the biogenesis of HDL. In the abscence of either apoA-I (Matsunaga et al., 1991) or ABCA-1 (Brunham et al., 2006) HDL is not formed. Adenovirus mediated gene transfer of apoA-I mutants in apoA-I-/- mice showed that deletion of the C-terminal region of apoA-I prevented the formation of HDL (Chroni et al., 2007). The ability of ABCA1 to promote cholesterol efflux from macrophages is very important for the prevention of formation of foam cells in the atherosclerotic lesions (Van Eck et al., 2002). Mutations resulting in inactivation of ABCA1 are present in patients with Tangier disease (Brunham et al., 2006). The deficiency is associated with very low levels of total plasma and HDL cholesterol and abnormal lipid deposition in various tissues (Christiansen-Weber et al., 2000; McNeish et al., 2000). The ABCA1 deficiency in humans or experimental animals may contribute to accelerated atherosclerosis (Joyce et al., 2002; Singaraja et al., 2003). Inactivation of the ABCA1 gene in macrophages increases the susceptibility to atherosclerosis (Van Eck et al., 2002; Van Eck et al., 2006). Specific amino acid substitutions found in the Danish general population were predictors of ischemic heart disease and reduced life expectancy (Frikke-Schmidt et al., 2008).

#### **3.3 Interactions of lipid-bound ApoA-I with LCAT stabilize the nascent HDL**

Plasma LCAT is a 416 amino acid long enzyme that is synthesized and secreted by the liver and esterifies the free cholesterol of HDL and LDL. ApoA-I is a potent activator of LCAT (Fielding et al., 1972). Following esterification, the cholesteryl esters formed become part of the lipid core and the discoidal HDL is converted to mature spherical HDL (Chroni et al., 2005a).

Mutations in LCAT are associated with two phenotypes in humans. The familiar LCAT deficiency (FLD) is characterized by the inability of the mutant LCAT to esterify cholesterol on HDL and LDL and causes accumulation of discoidal HDL in the plasma. The fish eye disease (FED) is characterized by the inability of mutant LCAT to esterify cholesterol on HDL only. Both diseases are characterized by low HDL levels due to the inability of LCAT to convert the nascent immature pre-β and discoidal particles to mature spherical HDL (Santamarina-Fojo et al., 2001).

Pleiotropic Functions of HDL Lead to Protection from Atherosclerosis and Other Diseases 177

**4. Phenotypes of humans and experimental animals having apoA-I mutations** 

Several apoA-I mutations have been described in the general population that are associated with low plasma HDL levels. Most of the mutations affect the interaction of apoA-I with LCAT. Eight mutations between residues 26 and 107 and one on residue 173, have been associated with amyloidosis and low HDL levels (Sorci-Thomas and Thomas, 2002; Zannis et al., 1993) and one mutation on residue 164 is associated with increased risk for ischemic heart disease and reduced life expectancy (Haase et al., 2011). The in vivo interactions of representative naturally occurring apoA-I mutants with LCAT were studied by adenovirusmediated gene transfer in apoA-I deficient mice. The mutants apoA-I(Leu141Arg)Pisa and apoA-I(Leu159Arg)FIN produced only small amounts of HDL that formed mostly preβ1 and small size α4 HDL particles. The apoA-I(Arg151Cys)Paris and apoA-I(arg160Leu)Oslo formed discoidal HDL particles. These studies indicated that apoA-I(Leu141Arg)Pisa and apoA-I(Leu159Arg)FIN mutation may inhibit an early step in the biogenesis of HDL due to insufficient esterification of the cholesterol of the preβ1-HDL particles by the endogenous LCAT. The LCAT insufficiency appears to result from depletion of the plasma LCAT mass (Koukos et al., 2007). A remarkable finding of these studies was that all the aberrant phenotypes were corrected by treatment with exogenous LCAT. This indicates that LCAT administration could be a potential therapeutic intervention to correct low-HDL conditions in humans that are caused by these and other unidentified mutations (Amar et al., 2009).

**4.2 Specific bioengineered mutations in ApoA-I may cause dyslipidemia** 

Four bioengineered mutations in apoA-I have been studied by adenovirus-mediated gene transfer in apoA-I deficient mice. Mutants apoA-I[Δ(62-78)], apoA-I [Glu110Ala/Glu111Ala] and apoA-I[Asp89Ala/Glu90Ala/Glu92Ala], caused combined hyperlipidemia, characterized by elevated plasma cholesterol and severe hypertriglyceridemia (Chroni et al., 2004; Chroni et al., 2005b; Kateifides et al., 2011). An apoA-I[Δ89-99] mutant induced high plasma cholesterol, but did not affect plasma triglyceride levels (Chroni et al., 2005b).

Fig. 2. The pathway of HDL biogenesis and functions of HDL. a-e represent sites of possible disruption of the HDL biogenesis pathway. Different subpopulations of HDL that have been

generated due to these defects may have different functions.

**4.1 Natural apoA-I mutations** 

#### **3.4 Interactions of lipid-bound ApoA-I with SR-BI**

SR-BI is an 82 kDa membrane glycoprotein primarily expressed in the liver, steroidogenic tissues and endothelial cells but is also found in other tissues. The most important property of SR-BI is considered to be its ability to act as the HDL receptor (Acton et al., 1996). SR-BI mediates both selective uptake of cholesteryl esters and other lipids from HDL to cells (Acton et al., 1996; Stangl et al., 1999; Thuahnai et al., 2001), as well as efflux of unesterified cholesterol (Gu et al., 2000). Transgenic mice expressing SR-BI in the liver had greatly decreased apoA-I and HDL levels as well as increased clearance of VLDL and LDL (Ueda et al., 1999) and were protected from atherosclerosis (Arai et al., 1999). SR-BI deficient mice had decreased HDL cholesterol clearance (Out et al., 2004), two fold increased plasma cholesterol and presence of large size abnormal apolipoprotein E (apoE) enriched particles that were distributed in the HDL/IDL/LDL region (Rigotti et al., 1997). The SR-BI deficiency in the background of LDL receptor (LDLr) deficient or apoE deficient mice accelerated dramatically the development of atherosclerosis (Huszar et al., 2000; Trigatti et al., 1999). The double deficient mice for apoE and SR-BI developed occlusive coronary atherosclerosis, cardiac hypertrophy, myocardial infarctions, cardiac dysfunction and died prematurely (mean age of death ~6 weeks) (Braun et al., 2002; Trigatti et al., 1999). The SR-BI deficiency reduced greatly cholesteryl ester levels in the steroidogenic tissues that utilize HDL cholesterol for synthesis of steroid hormones (Ji et al., 1999). It also decreased secretion of biliary cholesterol by approximately 50% (Mardones et al., 2001; Rigotti et al., 1997). The SR-BI deficiency also caused defective maturation of oocytes and red blood cells due to accumulation of cholesterol in the plasma membrane of progenitor cells (Holm et al., 2002; Trigatti et al., 1999) and caused infertility in the female but not the male mice (Trigatti et al., 1999; Yesilaltay et al., 2006). Interactions of HDL with SR-BI in endothelial cells triggers signaling mechanisms discussed below that involve activation of endothelial nitric oxide synthase (eNOS) and release of NO that causes vasodilation (Mineo et al., 2003; Yuhanna et al., 2001). Human subjects have been identified with a P297S substitution in SR-BI. Heterozygote carriers for this mutation had increased HDL levels, decreased adrenal stereoidogenesis and dysfunctional platelets but did not develop atherosclerosis (Vergeer et al., 2011).

A [Gly2Ser]SR-BI substitution in humans is associated with decreased follicular progesterone levels in Caucasian women and non viable fetuses 42 days post embryo transfer. Another single nucleotide polymorphism (SNP) (rs10846744) was associated with gestational sacs and fetal heart beats and with poor fetal viability in African-American women (Yates et al., 2011).

#### **3.5 Interactions of HDL with ABCG1**

ABCG1 is a 67 kDa protein which is a member of ABC family of half transporters. ABCG1 is expressed in the spleen, thymus, lung, brain, endothelial cells and other tissues (Savary et al., 1996) and promotes cholesterol efflux from cells to HDL but not to lipid free apoA-I (Nakamura et al., 2004; Vaughan and Oram, 2005). The absence of ABCG1 in mice causes cholesterol accumulation in various tissues (Kennedy et al., 2005) and selective deletion of both ABCA1 and ABCG1 genes in macrophages further increases cholesterol accumulation and results in severe atherosclerosis (Out et al., 2008; Yvan-Charvet et al., 2007).
