© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd doi:10.1111/tra.12177


Binding Domain-Driven Intracellular Trafficking of Sterols for Synthesis of Steroid Hormones, Bile Acids and Oxysterols Andrew Midzak1∗ and Vassilios Papadopoulos1,2,3,4∗ 1 Research

Institute of the McGill University Health Centre, McGill University, Montreal, Quebec, Canada of Medicine, McGill University, Montreal, Quebec, Canada 3 Department of Biochemistry, McGill University, Montreal, Quebec, Canada 4 Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada 2 Department

Corresponding authors: Andrew Midzak, [email protected] and Vassilios Papadopoulos, [email protected]

Abstract Steroid hormones, bioactive oxysterols and bile acids are all derived from

cholesterol metabolism to steroid hormones and bile acid, relating it to

the biological metabolism of lipid cholesterol. The enzymatic pathways

both lipid- and protein-based mechanisms facilitating intracellular and

generating these compounds have been an area of intense research for

intraorganellar cholesterol movement and delivery to these pathways. In

almost a century, as cholesterol and its metabolites have substantial

particular, we examine evidence for the involvement of specific protein

impacts on human health. Owing to its high degree of hydrophobicity

domains involved in cholesterol binding, which impact cholesterol move-

and the chemical properties that it confers to biological membranes, the

ment and metabolism in steroidogenesis and bile acid synthesis. A better

distribution of cholesterol in cells is tightly controlled, with subcellular

understanding of the physical mechanisms by which these protein- and

organelles exhibiting highly divergent levels of cholesterol. The manners

lipid-based systems function is of fundamental importance to under-

in which cells maintain such sterol distributions are of great interest in

standing physiological homeostasis and its perturbation.

the study of steroid and bile acid synthesis, as limiting cholesterol sub-

Keywords cholesterol, mitochondria, protein motifs, steroidogenesis,

strate to the enzymatic pathways is the principal mechanism by which

steroids, sterol trafficking

production of steroids and bile acids is regulated. The mechanisms by

Received 25 April 2014, revised and accepted for publication 28 May

which cholesterol moves within cells, however, remain poorly under-

2014, uncorrected manuscript published online 30 May 2014, pub-

stood. In this review, we examine the subcellular machinery involved in

lished online 7 July 2014

The lipid cholesterol has generated some of the greatest research interest regarding the chemicals that make up life. Providing profoundly critical physical properties as a component of the membranes that separate the biotic from the abiotic, cholesterol exerts significant physiological effects in its own right. Moreover, cholesterol is enzymatically metabolized in cells to structurally diverse families of steroids, oxysterols and bile acids, each of which have specific and highly potent signaling capabilities. Our

understanding of the ways through which cells handle cholesterol and respond to cholesterol metabolite signaling has expanded enormously over the past half century. However, the manner in which cells synthesize steroids and other cholesterol metabolites, despite the progress made, has yet to fully integrate the advances of cholesterol biophysics and cellular biology into its theoretical framework. These issues have captured the interest of researchers in the field (1), and in this review, we look to extend on this www.traffic.dk 895

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Figure 1: Cholesterol and its oxidized metabolites. Cholesterol is a cycloperhydropentanophenanthrene-ringed structure that can be chemically modified to yield molecules possessing potent biological activity. Shown in the center is cholesterol, with the carbon atoms indicated by numbers and the rings designated by letters. In vertebrates, cholesterol can be oxidized to form oxysterols by enzymatic reactions or by auto-oxidation in all cells (e.g. 27-hydroxycholesterol and 7α-hydroxycholesterol), or they can be oxidized to bile acids in hepatocytes (e.g. cholic acid and chenodeoxycholate, right ), or oxidized to steroid hormones in steroidogenic cells (e.g. pregnenolone and cortisol, left ). In arthropods, differential enzymatic systems lead to the metabolism of cholesterol to ecdysteroids (e.g. 20-hydroxyecdysone, top). Note that only steroids lack the aliphatic tail of cholesterol; in other metabolites, increased aqueous solubility is achieved by oxidation of the aliphatic tail. Not shown for clarity are additional chemical modifications of cholesterol with biological relevance, such as lipidation and sulfation.

previous work and offer our own critical assessment of the relationship between sterol movement and sterol metabolism. We shall begin with a description of steroidogenesis and the role of mitochondrial cholesterol transport in this process. We then step back and describe the current understanding of general cellular cholesterol transport, placing emphasis on the biophysical behavior of cholesterol in lipid and aqueous media. Finally, we describe the proposed role of lipid-binding domains in steroidogenesis and cellular cholesterol traffic, highlighting both the strengths and weaknesses of our current knowledge of these processes. 896

Steroidogenesis, Oxysterol and Bile Acid Synthesis, and Mitochondrial Cholesterol Metabolism All vertebrate steroids are metabolic products of cholesterol. The structural characteristics of cholesterol and its oxygenated products confer varying chemical properties to the molecules (Figure 1). Cholesterol is characterized by (i) a fused cyclopentanophenanthrene four-ring backbone with a single double bond between carbons 5′ and 6′ ; (ii) a single stereospecific hydroxylation at the 3′ carbon (3β-OH); and (iii) a saturated carbon tail attached to Traffic 2014; 15: 895–914

Protein Motifs in Sterol Traffic and Metabolism

carbon 17′ of the D ring. In contrast, vertebrate steroid hormones (2,3) and bile acids (4) are oxygenated at multiple positions on the cyclopentanophenanthrene backbone. Steroids differ from bile acids by retaining a double bond at either the 4′ or 5′ carbons and, most importantly, steroid hormones are oxygenated at the 17′ carbon rather than possessing an extended aliphatic tail. This last characteristic greatly increases steroid hydrophilicity and makes this class of lipids structurally distinct from the oxysterols and bile acids, which retain the aliphatic tail of cholesterol (though it itself is oxygenated). This class of lipids is also distinct from the compounds that have been referred to as steroids in the lower metazoans [e.g. ecdysteroids in insects (5)], but that more closely resemble oxysterols and bile as they retain the sterol carbon tail. While oxysterols and bile acids are still functionally grouped by their chemical structure, the contemporary definition of each class of steroids is based on the nuclear receptor(s) to which it binds rather than on its chemical structure (6,7). The structures of steroids were biochemically determined in the early 20th century (8) and with extensive work conducted to establish precursor/product relationships in mammalian tissues. This work led to the general mapping of the chemical metabolism of sterols and steroids, and it provided the framework for establishing the pathways of steroidogenesis; however, it was not until the 1980s, with the isolation and cloning of steroidogenic enzymes, that the steroidogenic pathways were defined. This work has been recently, and authoritatively, reviewed by Miller and Auchus (3). Briefly, steroidogenic enzymes fall into two broad families: the cytochrome P450 (CYP) and hydroxysteroid dehydrogenase/ketosteroid reductase (HSD/KSR) enzymes (2,3). CYPs are a group of oxidative enzymes, all of which have about 500 amino acids and contain a prosthetic heme group, and they utilize electrons from NAD(P)H for enzymatic activity (9). Humans contain 57 CYPs, which can be further subclassified into mitochondrial and microsomal CYPs, each of which utilizes different enzymatic sources of electrons. Mitochondrial enzymes utilize a ferrodoxin reductase–ferrodoxin electron transport chain, whereas microsomal enzymes utilize a single P450 oxidoreductase (10). Six P450 enzymes are involved in steroidogenesis (Figure 2): the mitochondrial CYP11A1, Traffic 2014; 15: 895–914

CYP11B1 and CYP11B2, and the endoplasmic reticular (ER) CYP17A1, CYP19A1 and CYP21A1. The HSDs have molecular masses of approximately 40 kDa, do not have prosthetic heme groups and require nicotinamide adenine dinucleotide (phosphates) (NADH/NAD+ or NADPH/NADP+ ) as cofactors to either reduce or oxidize a steroid by two electrons via a hydride transfer mechanism (11). Most HSD reactions involve the conversion of an alcohol to a ketone (or vice versa), though in the case of 3β-HSDs, dehydrogenation is accompanied by the isomerization of the adjacent carbon–carbon double bond from the Δ5 position to the Δ4 position of the steroid backbone (12). Although steroidogenic enzymes are broadly distributed throughout the body, allowing multiple cells and tissues to locally metabolize steroids for particular needs (13), steroidogenic cells are able to synthesize steroids de novo and are defined by their expression of the cytochrome P450 enzyme, CYP11A1. Such steroidogenic cells include both the classical hormone-dependent steroidogenic cells of the adrenal cortex and the male and female gonad- and hormone-independent steroidogenic cells of the female placenta, as well as non-classical steroidogenic tissue, such as steroidogenic cells in the central nervous system (3,14). CYP11A1 is a member of the mitochondrial clade of cytochrome P450 enzymes, which contains six additional CYPs in humans (CYP11B1, CYP11B2, CYP24 and CYP27A1, as well as CYP27B1 and CYP27C1). This clade differs from microsomal cytochrome P450s in their cellular localization – mitochondrial CYPs are peripherally attached to the matrix side of the inner mitochondrial membrane, whereas microsomal CYPs are attached to the lumen face of the ER membranes – and their source of catalytic electrons – mitochondrial CYPs utilize an electron transport chain consisting of ferrodoxin reductase and ferrodoxin, in contrast to the single P450 oxidoreductase for microsomal CYPs. Interestingly, the mitochondrial CYP clade appears to share a preference for sterol-like substrates, as CYP11A1 and CYP27 hydroxylate the aliphatic tail of cholesterol (15,16), CYP11B1 and CYP11B2 hydroxylate the 11′ and 18′ carbons of steroid substrates (17) and CYP24 hydroxylates the secosteroid 1,25-dihydroxyvitamin D3 (18,19). This substrate preference may reflect a deep evolutionary relationship, 897

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Figure 2: Major steroidogenic and bile acid synthetic pathways. A simplified schematic diagram of the transformation of cholesterol to bioactive steroids and bile acids is shown; chemical intermediates have been removed for clarity. The mitochondria are shown centrally, as this is the rate-limiting site of cholesterol metabolism to steroid hormones, and a key site in the alternative pathway of bile acid synthesis. Human nomenclature of enzymes is used. Following scission of the aliphatic tail of cholesterol by the cytochrome P450 CYP11A1, the steroid pregnenolone is metabolized to further steroid products, which are dependent upon the expression of additional steroidogenic enzymes. Note that the majority of these enzymes are localized in the ER, though CYP11B1 and CYP11B2, facilitating the final conversion of glucocorticoid and mineralocorticoid steroid hormones, respectively, are mitochondrial enzymes, which necessitate the return of steroid metabolites. The intracellular trafficking of steroids after the synthesis of pregnenolone is believed to be due to diffusion, as steroids have increased hydrophobicity and no steroid transporters have been identified. In bile acid synthesis, the rate-limiting step in the majority of bile acid synthesis (∼75–90%) is the 7α-hydroxylation of the sterol B ring by cytochrome P450 CYP7A1 localized in the ER of hepatocytes; the 27-hydroxylation of cholesterol by mitochondrial CYP27A1 enzymes throughout the body contributes to circulating levels of 27-hydroxycholesterol, which can subsequently be 7α-hydroxylated by hepatic and extrahepatic CYP7B1. as the mitochondrial CYP family is highly conserved among metazoans (20,21), though any non-mammalian substrates are unknown at this time. As noted above, CYP11A1 is absolutely essential for the synthesis of all vertebrate steroids, and the proposed reaction mechanism of CYP11A1 involves three sequential modifications of cholesterol. First, cholesterol is hydroxylated at carbon 22; second, cholesterol is hydroxylated at carbon 20; and finally, oxidative scission of the C20–C22 bond of 20(3),22R-dihydroxycholesterol yields pregnenolone and isocaproaldehyde (22). These hypotheses have been supported by the crystal structures of bovine and human CYP11A1 (23,24), indicating that the prosthetic heme is in close proximity to the 20′ and 22′ carbons of cholesterol. 898

The transcriptional levels of CYP11A1 determine the cellular steroidogenic capacity of a cell (25), and as the expression of CYP11A1 is slow to rise and fall, it constitutes a key chronic mechanism of steroidogenic regulation. In the classical steroidogenic cells of the adrenal cortex and gonads, circulating pituitary hormones stimulate intracellular signaling cascades, leading to the elevation and maintenance of CYP11A1 expression (26,27) – processes that require the transcription factor, steroidogenic factor 1 (SF-1) (28). CYP11A1 regulation in other tissues is not as well characterized, but it appears to be constitutively maintained at physiological levels by different mechanisms (29). All steroidogenic cells metabolize cholesterol to steroids basally, and CYP11A1 expression dictates the level of basal Traffic 2014; 15: 895–914

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steroidogenesis. The mechanisms dictating basal steroidogenesis are not clear at this time, though they remain an important area for research, as this form of steroidogenesis is performed by the placenta and steroidogenic cells in the central nervous system, which are not stimulated by known hormones and factors. Although basal steroidogenesis may be a consequence of low levels of sterol diffusion to CYP11A1 in the mitochondrial matrix, sterol-binding proteins discussed below appear critical in facilitating the process, suggesting a role for protein machinery in the process. Basal steroidogenesis is insufficient to maintain the necessary levels of steroid hormones; however, the pituitary hormones that stimulate the adrenal cortex and gonads not only promote chronic expression of steroidogenic enzymes but also acutely stimulate a rapid and dramatic rise in steroid hormone synthesis. This acute phase of steroidogenesis is characterized by significant morphological changes (30) and intramitochondrial cholesterol transport and delivery to CYP11A1 – a process that is rate limiting in the synthesis of steroids (31,32). Given that intramitochondrial cholesterol transport constitutes the primary locus of control for hormone-stimulated steroidogenesis, this acute step has become a key area of research into the synthesis of steroids as well as a proxy for steroidogenesis in general (25). In the rest of this review, we will examine our current knowledge of how sterols move between membranes and then proceed to a discussion of the protein motifs that help facilitate cholesterol movement.

Biophysical Concepts and Sterol Chemical Activity Although the theoretical considerations of mitochondrial cholesterol transport hinge on the large difference in the inner and outer mitochondrial membrane cholesterol levels, it is important to note that this difference is only part of the larger compartmentalization of cholesterol that cells undertake as part of their homeostatic physiology. Cholesterol concentrations are quite divergent throughout the cellular organelles (33,34) and the following sections describe both this distribution and the models that describe lipid’s contribution to this distribution, before transitioning to a discussion on the models that underline the contribution of protein. Traffic 2014; 15: 895–914

The plasma membrane contains the highest levels of cholesterol in the cell, followed by compartments intimately associated with the plasma membrane, such as the Golgi apparatus and the endosomal recycling compartment (ERC). There appears to be a gradient of cholesterol along the secretory pathway of cells, with the highest levels found at the plasma membrane, intermediate levels in the Golgi apparatus and low levels in the ER (35,36). For this reason, the delivery of cholesterol via vesicular transport, utilizing cellular secretory machinery, is proposed to play a major role in cellular cholesterol homeostasis, though mechanisms exist to transfer cholesterol to organelles outside of the secretory pathway, such as the mitochondria (37). The fact that there are low levels of cholesterol in the ER is interesting, as the ER is the site of cholesterol synthesis, and cholesterol that is added to the cell, either directly or in endocytosed lipoproteins, is quickly acetylated by the ER-localized acyl-CoA cholesterol acyltransferase (ACAT) enzyme and delivered to lipid droplets (themselves considered to be lipid-rich buds of the ER) (38). Collectively, these observations indicate that the ER serves as a central cholesterol distribution center for the cell, though it retains low levels of cholesterol. These low levels of cholesterol make the ER an excellent sterol-sensing organelle, and it has been shown that the transcriptional machinery regulating sterol synthesis and metabolism, governed by the transcription factor SREBP, is especially sensitive to sterol concentrations (36,39). This genetic machinery, operating on the time scale of hours to days, does not, however, explain how cells rapidly control cellular sterol distribution within seconds to minutes (40) – mechanisms that we are only recently beginning to understand. Much of the work on the cellular distribution and movement of cholesterol has been performed in non-steroidogenic mammalian and yeast cells. With regard to steroidogenic cells, the source of cholesterol transported to the mitochondria has long been considered to be the lipid droplets, beginning with ultrastructural work indicating that lipid droplet volume decreased upon hormonal stimulation of the adrenocortical cells (41). More recently, non-perturbational imaging of mouse Y-1 adrenocortical tumor cells demonstrated cytoskeletal trafficking of lipid droplets to the mitochondria (42), suggesting the direct delivery of lipid droplet cholesterol for steroidogenesis. Such a system was supported 899

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by knockout of the intermediate filament vimentin, in which reduction of lipid droplet cholesterol delivery along the cytoskeleton disturbed adrenal and ovarian, but not testicular, steroidogenesis (43). Interestingly, work by Freeman utilizing a Leydig tumor cell model suggested that steroidogenic cells were opportunistic, deriving cholesterol both from stores in the plasma membrane as well as diverting newly synthesized cholesterol from the ER (44–46). The precise contribution of the subcellular compartments to steroidogenesis remains to be determined, but it is plausible that steroidogenic cells obtain cholesterol from any source they can, with the actively metabolizing steroidogenic mitochondria serving as a cholesterol ‘drain’, drawing cholesterol from multiple sources along the sterol trafficking pathways (Figure 3). A key theoretical model in understanding sterol distribution within lipid membranes and organelles is the concept of sterol chemical ‘activity’, which refers to the sterol capacity to carry out chemical or physical processes (47). Molecules in a membrane can be viewed as moving laterally within the membrane, traversing the membrane or escaping the membrane (Figure 4A) (47,48). The last mode of molecular movement corresponds to sterol chemical activity, making the sterol accessible to other molecular agents outside of the membrane (49). This accessibility was demonstrated in cellular systems utilizing the cholesterol-modifying cholesterol oxidase enzyme (50) and the cholesterol cyclodextrin polysaccharide (MCD) (51,52), which showed that increasing the local concentrations of cholesterol in membranes promoted the escape of cholesterol from the membranes and increased cholesterol’s association with aqueous protein acceptors (Figure 4B). Two biophysical models of cholesterol interactions with phospholipids provide useful frameworks through which to understand cellular cholesterol activity. Each of these models addresses separate, but not exclusive, aspects of cholesterol interaction with neighboring phospholipids. The condensed complex model of cholesterol–phospholipid interaction concentrates upon cholesterol’s interaction with the fatty acyl chains of phospholipids (53,54). In this model, stoichiometric hydrophobic associations between cholesterol and phospholipids with long saturated acyl chains form compact 900

Figure 3: Cholesterol trafficking pathways in mammalian cells. Cholesterol transport in mammalian cells can proceed along ‘inside-out’ or ‘outside-in’ traffic patterns, based on cellular needs. Sterol can be transported from its site of synthesis to the plasma membrane along the secretory pathway, involving the Golgi apparatus, or directly to the plasma membrane, presumably involving sterol carrier proteins, with the latter comprising the bulk of sterol transfer. Cholesterol from the plasma membrane, or absorbed from circulating lipoproteins, proceeds from the PM or the ERC to the ER, where the cholesterol is rapidly acetylated by the resident ER protein, ACAT. Acetylated cholesterol is incorporated in lipid droplets where it is stored for cellular needs. Cholesterol concentrations exceeding the carrying capacity of the ER activate the SREBP cholesterol genetic regulatory machinery, enacting the cellular homeostatic response to lower cholesterol levels. Mitochondria are commonly cholesterol-poor organelles, primarily due to extremely low inner mitochondrial membrane levels. Hormonal activation of steroidogenesis promotes the intramitochondrial transfer of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane, and it appears to traffic cholesterol along multiple cellular pathways to maintain steroidogenesis. Abbreviations: PM, plasma membrane; ERC, endosomal recycling compartment; Golgi, Golgi apparatus; ER, endoplasmic reticulum; LD, lipid droplet; Mito, mitochondria. complexes of low free energy and small lateral areas (Figure 4C). Conversely, cholesterol associates less well with unsaturated and short-chain fatty acyl chains, minimizing the formation of condensed complexes (55). Cholesterol within these complexes has lower accessibility and activity than cholesterol outside of these complexes and, consequently, cholesterol activity within, and escape Traffic 2014; 15: 895–914

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Figure 4: Schematics of cholesterol behavior within the membranes and their interactions with phospholipids and proteins. A) Schematic depiction of cholesterol lipid’s lateral and transverse movement within a phospholipid membrane. Cholesterol has a high degree of mobility within the lipid environment of the membrane, with lateral diffusion times on the millisecond timescale observed in model and cellular membranes. Transverse movement within the membrane (flip-flop) is also rapid, with theoretical and experimental measurements on the millisecond to minute timescale. However, transverse movement out of the membrane (escape activity) is quite slow, as it takes place on the timescale of hours to days in the absence of proximal acceptors, whether they are proteins or other membranes. B) Stepwise schematic of LBP-mediated sterol transfer. Cholesterol in the membranes (yellow rectangles; red circle denotes 3′ OH group) is recognized (1) by a soluble LBP, which binds to the sterol and (2) extracts it from the donor membrane. The association of the LBP with an acceptor membrane (3) results in the release of the sterol, which begins the cycle anew (4). C) Simplified illustration of a condensed complex model of the cholesterol–phospholipid association and sterol escape activity. At low sterol-to-phospholipid ratios, cholesterol is able to associate stoichiometrically with the acyl chains of the surrounding phospholipids. When local cholesterol concentration exceeds the capacity of the membrane to form stoichiometric complexes, the excess cholesterol looks to move (either laterally or transversely), increasing its activity and possible association with molecules outside of its membrane. D) Simplified illustration of the umbrella model of the cholesterol–phospholipid association in sterol escape activity. Phospholipids with a high degree of acyl chain unsaturation (left , unsaturated acyl chains in orange) will sterically clash with cholesterol in the membranes, which is in contrast to phospholipids with a high degree of acyl chain saturation (right , saturated acyl chains in purple). Moreover, phospholipids with small headgroups (left ) will insufficiently shield the hydrophobic cholesterol molecule from the aqueous media, making the escape of the cholesterol molecule to a more hydrophobic environment energetically favorable. In contrast, phospholipids with large headgroups (right ) shield the smaller cholesterol molecule from the aqueous surroundings, reducing the degree of accessibility and activity of the cholesterol. Traffic 2014; 15: 895–914


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from, the membranes is greater when its concentration exceeds the stoichiometric capacity of the membrane (56). This model well addresses the high sterol distribution in the plasma membrane, which contains high levels of phospholipids with long saturated acyl chains (e.g. sphingomyelin), as well as the low sterol distribution in the ER and mitochondria, which contains proportionally greater levels of unsaturated phospholipids (57). The condensed complex model also fits well with cellular work that shows that amphipathic molecules capable of breaking cholesterol–phospholipid interactions sharply facilitate cellular cholesterol movement (58,59). Cholesterol–phospholipid interactions can be influenced not only by the fatty acyl tails of phospholipids but also by the headgroups at the membrane interface. This aspect of sterol chemistry is addressed by the umbrella model (60). In this model, the hydrophilic 3′ hydroxyl group of cholesterol is insufficient for shielding the hydrophobic bulk of the molecule from the aqueous media. Consequently, cholesterol ‘snorkels’ in the phospholipid membrane shielded from water by the amphipathic headgroups of the surrounding phospholipids (Figure 4D). The model predicts that phospholipids with larger headgroups, such as sphingomyelin and phosphatidylcholine, will better shield and reduce the activity of cholesterol than phospholipids with smaller headgroups, such as phosphatidylethanolamine and cardiolipin. This model also addresses acyl chain dynamics, as unsaturated acyl tails sterically clash with cholesterol, whereas saturated chains fit well with the rigid ringed structure of cholesterol. The umbrella model has received theoretical and experimental support in model membranes (61), and has interesting implications for steroidogenesis. The inner mitochondrial membranes in rat liver (62,63) and adrenocortical cells (64,65) contain proportionally greater levels of small-headed than large-headed phospholipids, which is consistent with the low levels of cholesterol observed. However, the ratio of large-headed to small-headed phospholipids in the outer mitochondrial membrane approaches that of the plasma membrane and the ERC, possibly contributing to the lack of equilibration noted between the inner and outer mitochondrial membranes. Moreover, work with CYP11A1 in reconstituted liposomal systems supports the importance of the enzyme’s phospholipid microenvironment and cholesterol’s escape activity 902

in steroidogenesis, as CYP11A1 itself could be viewed as a sterol-acceptor protein akin to cholesterol oxidase (66). A series of articles from Kimura and coworkers well delineated the positive correlation between increasing phospholipid acyl chain unsaturation and increasing CYP11A1 activity (67–70). This work also supported the importance of the effects that phospholipid headgroup exerted in steroidogenesis, as cardiolipin with its minimal headgroup was highly effective at stimulating CYP11A1 activity (69). Our discussion of sterol movement thus far has remained focused upon the lipid membrane environment. However, while cholesterol moves rapidly within lipid environments, its movement in aqueous media is energetically highly unfavorable, and the transfer between lipid membranes across the aqueous space is very slow (71), especially in comparison to the rate at which cholesterol is transported intramitochondrially in steroidogenic cells. For this reason, candidate proteins have been proposed to facilitate intermembrane cholesterol transport (72), extracting cholesterol from donor membranes (much akin to cyclodextrin) and delivering it to acceptor membranes (Figure 4B). Consequently, sterol activity plays a strong theoretical role in the discussion of lipid-binding and transfer proteins. Many of these proteins, which include members of the steroidogenic acute regulatory protein (STAR)-related lipid transfer (START) domain (73) and oxysterol-binding protein-related domain (ORD) (74), among others, contain hydrophobic cavities that facilitate sterol binding. In addition to the large hydrophobic cavities of these proposed lipid transport proteins, smaller sterol-binding domains have been identified which, instead of binding and transporting sterol, may be involved in binding and targeting sterol for cellular processes. In the following sections, we discuss work investigating the mechanisms of these sterol-binding domains in the context of steroid biosynthesis.

The STAR and the START Domain In humans, the START domain family of proteins contains 16 members, of which the founding member, STAR, was initially discovered in the investigation of the acute mitochondrial production of steroids (75). The remaining 15 members of the START family were identified in the human genome by homologous sequence alignment Traffic 2014; 15: 895–914

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(76), and the proteins can be mapped to several distinct subfamilies – many with a characterized ligand, but several with no known ligand. The protein family has also been shown to be quite ancient, with members distributed across multiple phyla of life (77), although the ligands for these proteins, if any, are largely unknown. The closest mammalian homologs to STAR – STARD3, STARD4, STARD5 and STARD6 – constitute a proposed family of sterol-binding proteins, and the majority of research into this family has aimed at validating this hypothesis. As noted above, STAR was originally identified in the search for a protein factor that mediated the acute mitochondrial transport of cholesterol and steroidogenesis in hormone-responsive cells. Orme-Johnson and coworkers (75,78), using two-dimensional polyacrylamide gel electrophoresis, identified a mitochondrial phosphoprotein that was rapidly upregulated by hormonal stimulation in steroidogenic cells. The subsequent isolation and characterization of this protein, named STAR, revealed a 285-amino acid protein characterized by an approximately 65-amino acid mitochondrial targeting sequence attached to an approximately 220-amino acid START domain (79). Research into the human condition of congenital adrenal lipoid hyperplasia (CAH), characterized by cellular lipid accumulation and the inability to synthesize steroids, with early death due to a lack of glucocorticoids (80), revealed that these patients contained missense and deletion mutations in STAR (81–83). The subsequent knockout of Star in mice confirmed that this protein was essential for adrenal steroidogenesis (84). It is interesting to note that STAR expression appears limited to classical hormone-dependent steroidogenic tissues, and does not seem to contribute to the basal steroid production of these tissues or the steroidogenesis of hormone-independent and non-classical tissues (1), suggesting that it is essential for stimulation of high-flux mitochondrial cholesterol movement, a critical, albeit specialized, form of cholesterol movement. Because STAR was identified as a mitochondrial protein, original models of its action involved its import into the mitochondria, possibly forming contact sites through which cholesterol could traverse from the outer to the inner mitochondrial membrane (85). However, it was found that the expression of STAR lacking the mitochondrial targeting Traffic 2014; 15: 895–914

sequence in steroidogenic cells stimulated steroidogenesis to the same levels as the expression of full-length STAR. This suggests that the import of STAR is not necessary for its action, and that the protein functions on the outside of the mitochondria (86). This hypothesis was confirmed by Miller and coworkers who showed that tethering of STAR to the outer mitochondrial membrane increased its activity, while tethering it to the mitochondrial intermembrane space or the matrix abolished its activity, suggesting that the mitochondrial import of STAR serves as an ‘off’ switch for its steroidogenic activity (87). That the mitochondrial targeting sequence of STAR is unnecessary is questioned, however, by mouse models in which full-length STAR has been replaced by STAR that lacks the mitochondrial targeting sequence, and which stochastically exhibits the CAH phenotype of an inability to synthesize steroids, indicating a more complex scenario (88). The fact that STAR facilitated mitochondrial cholesterol transport led to the hypothesis that STAR directly facilitates cholesterol transport. The crystal structure of the homologous MLN64/STARD3 was shown to consist of a helix–fold–helix motif forming a hydrophobic cavity capable of fitting a cholesterol molecule, and both STAR and MLN64/STARD3 were shown to physically associate with cholesterol (89). This initial START domain structure carries over to subsequently obtained crystal structures of the cholesterol-associated STAR, STARD4 and STARD5 proteins (90,91). Molecular modeling and dynamics studies have been used to support a model of STAR as a sterol transporter, with its C-terminal α-helix serving as a lid over its hydrophobic cavity (92,93). The model of STAR as a cholesterol transporter was initially supported by biochemical work showing the transfer of radiolabeled cholesterol and fluorescent sterol from donor to acceptor membranes by wild-type, but not mutated, STAR (94,95). Furthermore, reconstitution of the CYP11A1 system in a minimal liposomal system revealed that STAR was able to facilitate the transfer of cholesterol from donor vesicles to acceptor vesicles containing CYP11A1, where it was metabolized to pregnenolone (96). The finding that STAR mutants that were incapable of stimulating steroid production also possessed diminished capacity to facilitate sterol transfer in vitro further supported a model for STAR-mediated mitochondrial sterol transport. However, the stoichiometry of STAR 903

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sterol transport in vitro, calculated to be approximately two sterol molecules per minute (96), was several orders of magnitude less than that observed in a cellular system, which was calculated to be ∼400 sterol molecules per minute (97). In addition, the inclusion of cardiolipin in donor vesicles increased the escape activity of cholesterol, which is consistent with the umbrella model of membrane sterol dynamics discussed above, but this had no effect on STAR sterol transfer activity, indicating that STAR was not acting on active cholesterol (96). Moreover, the findings that a CAH STAR mutation reduces STAR activity, but is functional in terms of sterol binding and transport (98), in conjunction with the observation of STAR’s extramitochondrial site of action, suggest that STAR does not serve as a cellular sterol transporter; rather, it acts as a cellular sterol sensor. Moreover, the findings that STAR associates physically and functionally with the outer mitochondrial membrane proteins, such as the voltage-dependent anion channel (VDAC) (99), indicate that additional molecular interactions contribute to STAR’s action on the outer mitochondrial membrane and that reductionist models of STAR acting in isolation are overly simplistic. Moreover, a large body of work illustrates that STAR requires conformational flexibility for its action, which is consistent with both transporter and sensor hypotheses; as such, further work will be required to reconcile models of STAR action (100–102).

The ERC: Niemann–Pick Type C Proteins and STARD3 The ERC contains among the highest cholesterol content in the cell, in many cases rivaling that of the plasma membrane. Moreover, interest in ERC cholesterol trafficking has been motivated by the study of the human disease of Niemann–Pick type C (NPC), a condition in which the ERC pathologically collects cholesterol and glycosphingolipids leading to progressive neurodegeneration and death (103). Genetic linkage analysis has revealed mutations in two genes, termed NPC1 and NPC2, that underlie the pathology of NPC disease, with NPC1 accounting for approximately 95% of cases (104) and NPC2 accounting for the remaining 5% (105). NPC1 is a 1278-residue endosomal integral membrane glycoprotein containing 13 transmembrane domains, and NPC2 is a soluble 151-residue glycoprotein found in the lysosomal lumen. Both proteins 904

are cholesterol binding (106,107), with NPC2 enveloping the aliphatic end of the molecule, leaving the 3β-OH group exposed, while NPC1 binds the 3β-OH end of the molecule at its N-terminus located in the lysosomal lumen. The reverse orientation of cholesterol binding by these two molecules has led to the model that NPC1 and NPC2 act in tandem transporting cholesterol out the ERC (108). In this model, NPC2, which appears to function as a soluble lipid-binding and transfer protein (109), delivers cholesterol from the endosomal lipoproteins to NPC1, which promotes cholesterol egress from the organelle. Interestingly, the BALB/c mouse characterized by a spontaneous mutation that assisted in identifying NPC mutations in humans (110) was identified as hypogonadal (though still fertile), with diminished capacity to synthesize testosterone (111). Whether NPC-mediated endosomal cholesterol directly contributes to steroidogenesis, or whether these observations were due to non-specific toxicity, remains unclear. In addition to the NPC proteins, the sterol-associated STARD3 protein has been linked to ERC cholesterol efflux. STARD3 is the most closely related sterol-associated START domain protein to STAR, and it was originally termed MLN64 for metastatic lymph node, clone 64. STARD3 was initially identified in a screen of clones prepared from the metastatic lymph nodes of women with ductal breast carcinoma (112). In humans, the STARD3 gene is located on chromosome 17q12-21, a region termed the HER2 amplicon, containing ERBB2, TOP2 and MED1, among others, and heterogeneously amplified in approximately 15% of breast cancers (113,114). Whether STARD3 plays a key role in the initiation and/or progression of breast cancer remains unclear, but co-silencing HER2 and STARD3 led to the additive inhibition of cell viability, and also induced apoptosis in breast cancer cells, suggesting that STARD3 may be a potential pharmacological target for breast cancer therapy (115,116). Human STARD3 contains 445-amino acid residues and is divided into two regions. The N-terminus of the protein contains four predicted transmembrane helices and is homologous to the 234-amino acid STARD3NL/MENTHO protein located on chromosome 7 (117). The C-terminus of STARD3 contains the START domain, which is structurally similar to STAR (89,118), and similar to the START domain of STAR, it Traffic 2014; 15: 895–914

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is able to stimulate mitochondrial cholesterol movement (119). STARD3 is integrated in the endosomal membrane through its STARD3NL-like motif (120), with its START domain pointing into the cytoplasm. This cytoplasmically facing topology, in conjunction with the finding that the STAR3NL-like motif is able to bind cholesterol (121), makes STARD3 an attractive candidate for cholesterol efflux from the ERC. Indeed, there is evidence that STARD3 is able to mediate cholesterol egress from the endosomes to the mitochondria (122), partnering with NPC2 in the absence of NPC1 (123). Moreover, STARD3 is proposed to play a role in steroidogenic cells that do not express STAR, such as in the placenta (124), because STARD3 START domain fragments have been found in these cells (118). Moreover, the STARD3 START domain possesses approximately 75% of the steroidogenic activity as the STAR START domain (125). However, the role that STARD3 plays in mitochondrial cholesterol delivery or in intramitochondrial cholesterol transport remains controversial, as STARD3 expression in hepatocytes negates the steroidogenic activity of exogenously expressed STAR (126). Furthermore, the targeted deletion of the STARD3 START domain in mice causes little to no alterations in markers of cellular and physiological sterol metabolism (127). It is plausible that STARD3 does not play a role as a cholesterol transporter per se, but rather as a sterol sensor facilitating sterol movement, as it is enriched in late endosomes (128) that are also enriched in cholesterol (129). In addition, the STARD3NL-like domain facilitates a tethering interaction with the resident ER vesicle-associated protein (130). Interestingly, STARD3 was recently identified as a high-affinity lutein-binding protein in primate retina (131,132). The STARD3 homolog in the moth Bombyx mori was also discovered to bind lutein, (133) suggesting that STARD3 may be evolutionarily promiscuous in the binding of hydrophobic molecules.

The Cytosolic START Domain Proteins, STARD4, STARD5 and STARD6 Three additional START domain proteins are associated with cholesterol, though their role in steroidogenesis, if any, is unclear. These proteins, STARD4, STARD5 and STARD6, lack any domains outside of the START domain (134), and as they lack any targeting sequences, they are generally Traffic 2014; 15: 895–914

considered cytosolic (135). Evidence for STARD4 from hepatocytes and macrophages supports this hypothesis, though the minor association of STARD4 with the ER has been observed (136). STARD5 has also been observed in a number of compartments, including the Golgi apparatus and the nucleus (137), though the role of these localizations is unclear. Of these proteins, STARD4’s role in cellular cholesterol trafficking is the best supported. As noted above, the crystal structure of STARD4 consists of the basic helix–loop–helix domain, which is characteristic of the START domain (89). STARD4 expression is regulated by the sterol regulatory element-binding protein-2 (SREBP-2) sterol synthetic machinery (138), the only putative cellular sterol transporter to be regulated by the SREBP-2 genetic network (139). Moreover, STARD4 was found to bind cholesterol using a cholesterol overlay assay, and it increased cholesteryl ester formation in mouse hepatocytes when overexpressed, suggesting that STARD4 plays a role in ER cholesterol delivery and in ACAT sterol esterification (140). Conversely, STARD4 knockdown in the hepatocyte HepG2 cell line resulted in the retention of sterol in the plasma membrane, with reduced trafficking to the ERC and ER (140). Elegant work by Mesmin et al. provided strong evidence that STARD4 functions as a theoretical lipid-binding and transport protein, as STARD4 knockdown in U2OS osteosarcoma cells was able to be compensated for by the polysaccharide MCD (141). However, similar to other START proteins, e.g. STARD3, knockout animals deleted for STARD4 show minimal phenotype (142). Comparatively less is known about the START domain proteins, STARD5 and STARD6. Both consist solely of a START domain with no other features, much akin to STARD4 (134). STARD5 has been shown to be expressed in macrophages (138), testicular Sertoli cells (143) and in the kidney (137) and liver (144), though its role in these tissues is unknown. STARD5 has been shown to biochemically bind both radiolabeled cholesterol and 25-hydroxycholesterol, but not 27-hydroxycholesterol (145), suggesting that STARD5 is involved in intracellular cholesterol and oxysterol homeostasis, possibly in the context of ER stress (146). However, a structural analysis of STARD5 using circular dichroism and nuclear magnetic resonance indicated that STARD5 is not able to bind cholesterol or 25-hydroxycholesterol; rather, it binds the bile acids, cholate and chenocholate, indicating that it plays 905

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a role in bile acid signaling and transport (147,148). The discordant results observed between the methodologies used remain to be resolved. STARD6 has been observed in rat male germ cells (149) as well as in the rat brain (150), though again its exact function is unclear. Most recently, STARD6 expression was detected in the steroidogenic cells of the porcine corpus luteum, peaking during the mid-luteal phase (151). The observation that STARD6 is capable of promoting intramitochondrial cholesterol movement and steroidogenesis in a COS-1 cell system transfected with the CYP11A1 enzymatic system, and it does so to the same levels as STAR (125,151), suggests that STARD6 may be involved in ovarian cholesterol trafficking and steroidogenesis.

ORD Family of Sterol Transporters The ORD family of proteins is another family of soluble protein factors implicated in cellular sterol movement. The founding member of the family, oxysterol-binding protein (OSBP), was originally identified during a search for the mechanisms that provide acute control of sterol synthesis by oxysterols (152), though OSBP does not appear to be involved in this process (153). In humans, there are 12 members of the ORD family (154,155), and in addition to the ORD, many of the proteins contain additional proteinand lipid-binding domains that have been hypothesized to target them to subcellular spaces. ORD proteins have attracted a significant amount of research into cellular cholesterol trafficking, especially in yeast, where they comprise the largest family of sterol-binding proteins, as yeast lacks the START domain family (156). However, there is no evidence that any of the ORD proteins play any role in steroidogenesis or in the trafficking of cholesterol to or within the mitochondria, and so they will not be reviewed here; more general recent reviews are available (74,157,158). It should be noted that one recent finding is of interest to the present discussion, as molecular promiscuity has arisen when discussing STARD3 and STARD5. In this context, it has been observed that the ORD of the yeast Osh4p, as well as the mammalian OSBP, is able to bind and transport the lipid phosphatidylinositol 4-phosphate using their ORDs – a process that competes with their cholesterol-binding and transport capabilities at the same domains (159,160). This ligand promiscuity has been 906

tied to cellular function by observing that these proteins localize to the ER–Golgi interface, where they appear to regulate sterol distribution between the organelles, as well as in ER cholesterol sensing. It is interesting to speculate whether such a mechanism translates to the START domain proteins discussed above.

The Translocator Protein (18 kDa) and the Cholesterol Recognition Amino Acid Consensus Motif The finding that benzodiazepines were able to stimulate steroidogenesis in a variety of steroidogenic systems, including classical and non-classical steroidogenic cells (161,162), led to the proposal that a peripheral benzodiazepine receptor, later renamed the 18-kDa translocator protein (TSPO) (163), is involved in mitochondrial cholesterol transport and steroidogenesis. This hypothesis was supported by findings that other pharmacological TSPO ligands, as well as the endogenous protein diazepam-binding inhibitor (164) [also known as acyl-CoA-binding domain protein 1 (ACBD1)], were able to stimulate steroid production in a TSPO-associated manner. The involvement of TSPO in natural steroidogenesis was further supported by pharmacological work showing that the TSPO ligand, flunitrazepam, was able to inhibit steroidogenesis in Leydig and adrenal cell lines (165), and in genetic work in the transformed rat R2C Leydig cell line. Homologous recombination-based TSPO deletion in R2C cells eliminated the ability of cells to synthesize steroids, and this lesion was corrected by transfection of a plasmid containing wild-type TSPO and by the subsequent expression of the protein (166). The absolute necessity of TSPO has been recently questioned; however, Testis-specific knockout of TSPO in a mouse model did not exhibit any steroidogenic phenotype (167). Whether this lack of phenotype was due to TSPO not being a component of the natural steroidogenic machinery, or due to compensation by another factor, remains to be determined (168). Regardless, TSPO’s ability to stimulate steroidogenesis and mitochondrial cholesterol movement in both classical steroidogenic cells as well as in non-classical tissues makes it an attractive target of study, especially as it is a proven druggable target (169–174). Traffic 2014; 15: 895–914

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TSPO’s involvement in drug-mediated steroidogenesis and the associated mitochondrial cholesterol transport has led to an investigation of this protein’s association with cholesterol. Deletion mutation analysis of TSPO revealed that it was able to increase the uptake of cholesterol in Escherichia coli, which naturally lacks cholesterol, and that the deletion of the C-terminus of the protein abolished this cholesterol uptake. Comparison of this region of TSPO with other proteins associated with cholesterol revealed a consensus sequence consisting of -L/V-X(1–5) -Y-X(1–5) -R/K-, where X(1–5) corresponds to one to five of any amino acid (175). Cholesterol inhibited the cross-linking of radioactive promegestone to a recombinant peptide containing the cholesterol recognition amino acid consensus (CRAC) motif of TSPO (176), and recombinant mouse TSPO reconstituted in liposomes was found to bind radioactive cholesterol with nanomolar affinity (177). Furthermore, the mutation of the central tyrosine in the recombinant protein abolished cholesterol binding (178). The involvement of TSPO and its CRAC motif was further supported by work showing that the pharmacological targeting of CRAC with the steroid 5-androsten-3β,17,19-triol was able to inhibit acute hormone-mediated steroidogenesis (179). Recently, the crystal structure of mouse TSPO was solved in complex with the TSPO drug ligand, PK 11195, showing that drug ligand binding to TSPO distorts the CRAC motif, possibly explaining how TSPO ligands stimulate steroid synthesis by releasing TSPO-bound cholesterol (180). The definition of the CRAC motif has also been recently expanded to include its palindromic sequence following the discovery that the left-to-right CRAC motif was insufficient in detecting cholesterol-binding portions of the human nicotinic acetylcholine receptor, and it was further delineated as a CARC motif (181,182). The cholesterol specificity of the CRAC motif is also of great interest, in light of observations that TSPO’s CRAC motif is also able to bind the steroids promegestone (175), 5-androsten-3β,17,19-triol and 5-androsten-3β,19-diol-17-one (but not 5-androsten3β,7β,19-triol or 5-androsten-3β,11β,19-triol; 183), and steroids are able to induce structural changes (184). The role of such promiscuity remains to be determined. Although TSPO has been hypothesized to be a cholesterol translocator (185), whether it facilitates cholesterol movement on its own, or whether it facilitates cholesterol Traffic 2014; 15: 895–914

movement through its association with other proteins remains unclear. TSPO has been known to closely interact with mitochondrial VDAC (186). VDAC itself is a potential regulator of mitochondrial cellular cholesterol movement (187) and a potential functional partner for STAR (99). TSPO and STAR appear to be functionally complementary in steroidogenic model systems (188), though the physical association between these proteins has been less than conclusive, with FRET and protein cross-linking (189,190) suggesting that a physical association exists, but bioluminescence resonance energy transfer (BRET) analysis remains inconclusive (191). Moreover, the hormonal stimulation of steroidogenic cells triggers the assembly of a multiprotein complex at the outer mitochondrial membrane, termed the transduceosome (190), containing VDAC, protein kinase A (PKA), the PKA-anchoring protein ACBD3 (192) and the inner mitochondrial membrane/membrane contact site protein ATAD3 (193). In this complex, TSPO may serve as a cholesterol reservoir, specifically targeting cholesterol to contact sites for delivery to CYP11A1.

Conclusions and Perspectives We have described the current understanding of steroid biosynthesis in relation to cholesterol’s physical properties and its transport via cellular protein machinery. Biophysical models have begun to explain the molecular details of cholesterol’s dynamics within phospholipid membranes. Genome sequencing has revealed the numerous families of lipid-binding proteins (LBPs) that have been conserved throughout many domains of life. Biochemical and cell biology experiments have begun to demonstrate the roles of these LBPs in cellular cholesterol homeostasis. Proteomic technologies have begun to elucidate the protein interactions that drive cellular processes. With regard to steroidogenesis, the convergence of technologies and investigations has painted a picture of macromolecular protein complexes with components that work in concert to transport cholesterol into the mitochondria. However, the molecular details of cholesterol movement between the lipid and protein environments involved are still lacking. Further work incorporating the models and concepts outlined here will be required to better understand this fundamental process of steroid biosynthesis. 907

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Acknowledgments This work was supported by grants from the Canadian Institutes of Health Research (MOP102647 and MOP125983), a Canada Research Chair in Biochemical Pharmacology to V. P. and a postdoctoral fellowship from the Fonds de recherche du Québec – Santé to A. M. The authors have no conflict of interest to declare.

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Binding domain-driven intracellular trafficking of sterols for synthesis of steroid hormones, bile acids and oxysterols.

Steroid hormones, bioactive oxysterols and bile acids are all derived from the biological metabolism of lipid cholesterol. The enzymatic pathways gene...
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