Biochimica et Biophysica Acta 1791 (2009) 646–658

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Review

Cholesterol transport in steroid biosynthesis: Role of protein–protein interactionsand implications in disease states

Malena B. Rone a,b, Jinjiang Fan a, Vassilios Papadopoulos a,b,⁎a The Research Institute of the McGill University Health Centre and Department of Medicine, McGill University, 1650 Cedar Avenue, Montreal, Quebec, Canada H3G 1A4b Department of Biochemistry, Molecular and Cellular Biology, Georgetown University Medical Center, Washington DC 20007, USA

⁎ Corresponding author. The Research Institute ofCenter, 1650 Cedar Avenue, C10-148, Montreal, Quebec,934 1934x44580; fax: +1 514 934 8439.

E-mail address: vassilios.papadopoulos@mcgill.ca (V

1388-1981/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.bbalip.2009.03.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 30 December 2008Received in revised form 28 February 2009Accepted 3 March 2009Available online 12 March 2009

Keywords:Steroid biosynthesisTranslocator proteinSteroidogenic acute regulatory proteinPeripheral benzodiazepine receptor

The transfer of cholesterol from the outer to the inner mitochondrial membrane is the rate-limiting step inhormone-induced steroid formation. To ensure that this step is achieved efficiently, free cholesterol mustaccumulate in excess at the outer mitochondrial membrane and then be transferred to the inner membrane.This is accomplished through a series of steps that involve various intracellular organelles, includinglysosomes and lipid droplets, and proteins such as the translocator protein (18 kDa, TSPO) and steroidogenicacute regulatory (StAR) proteins. TSPO, previously known as the peripheral-type benzodiazepine receptor, isa high-affinity drug- and cholesterol-binding mitochondrial protein. StAR is a hormone-inducedmitochondria-targeted protein that has been shown to initiate cholesterol transfer into mitochondria.Through the assistance of proteins such as the cAMP-dependent protein kinase regulatory subunit Iα (PKA-RIα) and the PKA-RIα- and TSPO-associated acyl-coenzyme A binding domain containing 3 (ACBD3) protein,PAP7, cholesterol is transferred to and docked at the outer mitochondrial membrane. The TSPO-dependentimport of StAR into mitochondria, and the association of TSPO with the outer/inner mitochondrialmembrane contact sites, drives the intramitochondrial cholesterol transfer and subsequent steroid formation.The focus of this review is on (i) the intracellular pathways and protein–protein interactions involved incholesterol transport and steroid biosynthesis and (ii) the roles and interactions of these proteins inendocrine pathologies and neurological diseases where steroid synthesis plays a critical role.

© 2009 Elsevier B.V. All rights reserved.

1. Cholesterol and steroid synthesis

Cholesterol is the sole precursor of steroids. Steroid synthesis isinitiated at the inner mitochondrial membrane (IMM), where thecytochrome P450 cholesterol side chain cleavage enzyme (CYP11A1)catalyzes the conversion of cholesterol to pregnenolone [1]. Pregne-nolone then enters the endoplasmic reticulum (ER) where furtherenzymatic reactions occur to produce the final steroid products. It hasbeen shown that the translocation of cholesterol from the outermitochondrial membrane (OMM) to the IMM is the rate-limiting stepin the production of all steroids [2,3]. Therefore, the ability ofcholesterol to move into mitochondria to be available to CYP11A1determines the efficiency of steroid production.

The production of steroids is regulated by trophic hormones,specifically, the adrenocorticotropic hormone (ACTH) in adrenocor-tical cells and luteinizing hormone (LH) in testicular Leydig andovarian cells [2,3]. The presence of these hormones activates the Gprotein-coupled receptors, which release the stimulatory subunit,resulting in the activation of adenylyl cyclase and a rise in intracellular

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cAMP [4]. This increase in cAMP leads to an increase in lipid synthesis,protein synthesis, and protein phosphorylation. All these processeshave been shown to play a role in steroidogenesis and assist withcholesterol trafficking to the mitochondria.

Mitochondria are relatively cholesterol-poor organelles, with themajority of cholesterol located in the OMM. In the mitochondria ofsteroidogenic cells the pool of cholesterol available for steroidogenesisis segregated from the structural membrane cholesterol and is boundto the cholesterol-binding domain of the translocator protein (18-kDa,TSPO), formerly called the peripheral-type benzodiazepine receptor(PBR) [5]. It is from this site that cholesterol is released underhormonal stimulation to move to the matrix side of the IMM, wherethe cholesterol side chain cleavage enzyme CYP11A1, which willmetabolize cholesterol to pregnenolone, is located. This pathwaywill be discussed in detail later.

As the initial translocation of cholesterol from TSPO is notsufficient to sustain the continuous production of elevated concentra-tions of steroids, additional free cholesterol must be moved fromintracellular stores to the mitochondria. This intracellular cholesterolis known to come from three sources: i) de novo synthesis ofcholesterol in the endoplasmic reticulum (ER) ii) mobilization ofcholesterol in the plasmamembranewith further uptake of circulatingcholesterol esters from receptors found on the plasma membrane andiii) mobilization of cholesterol in lipid droplets (LD) (Fig. 1).

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2. Cholesterol sources in the cell

With 65% to 80% of the total cellular cholesterol located in theplasma membrane, comprising 20% to 25% of the total lipids present,cholesterol plays a significant role in the structure and function of theplasma membrane. These interactions affect the organization ofproteins and lipids in the membrane, alter the permeability of themembrane, and initiate the formation of lipid rafts [6]. The secondhighest concentration of cholesterol is found in the endosomalpathway, with the majority found in the endosomal to trans-Golgicompartment [6]. While the majority of total cholesterol found in thecell is located in the plasma membrane, the ER contains only 1% to 2%of the total cell cholesterol [7]. This gradient provides a mechanism fortransport of cholesterol inside the cell from the ER to the plasmamembrane and allows it to be recycled back [8].

3. ER cholesterol

The ER functions as the cholesterol sensing organelle of the cell,regulating endogenous cholesterol production primarily through thesterol regulatory element binding protein (SREPB) complex [9,10]. TheSREBP proteins are translocated to the Golgi upon a decrease incholesterol and upon arrival SREBP is cleaved by two proteases. Theresulting N-terminus is an active transcription factor that translocatesto the nucleus. This results in an increase in the activity of cholesteroltranscription genes, including HMG-CoA reductase (HMGR) [11].HMGR is the rate-limiting enzyme in cholesterol synthesis and iseasily degraded under the high sterol conditions. HMGR is upregu-lated in the presence of hormones, resulting in an increase incholesterol production under hormonal stimulation [9]. The increaseof cholesterol via this pathway though has not been shown to play aprimary role in steroid production [12].

As cholesterol synthesis is tightly regulated in the ER duringhormonal stimulation, cholesterol transport out of the ER is tightlycontrolled as well. Cholesterol flux from the ER can occur through

Fig. 1. Trafficking of cholesterol to the mitochondria for steroidogenesis. Pathway 1: Cholestethe mitochondria via the PAP7 protein for steroidogenesis. Passive diffusion from the ER to tboth organelles, is another possible pathway for steroidogenic cholesterol to be transferredbinds to the LDL receptor and is trafficked through the endosomal pathway. MLN-64 assislysosomes for use in steroidogenesis. NPC1 and NPC2 associate with MLN-64 for cholesterosteroidogenesis. Pathway 3: Cholesterol is transferred from high density lipoprotein (HDL) toesterified cholesterol from the plasma membrane to free cholesterol, which can be used forlipid droplets (LD), which converts esterified cholesterol to free cholesterol for use in steroipresent in the cytosol for delivery to the mitochondria.

many pathways, including cytosolic lipid transfer proteins, throughintracellular compartments or passive diffusion through contact sites.Contact sites are common between the ER and other intracellularorganelles, facilitating cholesterol flux out of the ER. ER-mitochondrialcontact sites have been identified in which mitochondria-associatedmembranes (MAM) cluster with stacks of ER [13]. The cholesterolgenerated in the ER could potentially use this pathway for receivingsteroidogenic cholesterol (Fig. 1, Pathway 1). Recently an ER protein,acyl CoA:diacylglycerol acyltransferase 2 (DGAT2) was found asso-ciated with both LD and themitochondriawhile still present in the ER.As DGAT2 functions in the final stage of triglycerol synthesis it ispossible that this is a pathway for lipid transfer [14]. Currently it isunknown whether an interaction between the ER and mitochondriaoccurs in steroidogenic cells; therefore further studies will benecessary to investigate the presence and function of such aninteraction in steroidogenesis.

4. Plasma membrane

Cholesterol is primarily stored in the plasma membrane. Uponhormonal stimulation there is increased cholesterol absorptionthrough the plasma membrane. When cholesterol is imported intothe cell via the plasma membrane it greatly increases the cholesterolcontent stored elsewhere in the cell. This was observed when anincrease of 50% in cellular cholesterol absorbed via the plasmamembrane resulted in a 10-fold increase in ER cholesterol [15].Currently, there are two known pathways for this cholesterolabsorption and import into the cell: a non-selective endocyticpathway and a “selective” absorption pathway. In the non-selectivepathway LDLmolecules are specifically bound and internalized via theLDL receptors. Once the receptor has been internalized it fuses withthe endosomal pathway for distribution of the lipoproteins (Fig. 1,Pathway 2). The “selective” pathway uses scavenger receptors class Btype I (SR-BI) located at the plasma membrane to bind both LDL andHDL. Through local bindingmechanisms the cholesterol present in the

rol synthesized in the ER is trafficked to the Golgi apparatus where it can be targeted tohe mitochondria, shown here with the corresponding cholesterol molecules present onto the mitochondria. Pathway 2: Low density lipoprotein (LDL), containing cholesterol,ts with the transfer of cholesterol to the mitochondria from the late endosomes andl transfer out of the lysosomes, although it is not known if this cholesterol is used forthe plasma membrane by the SR-BI receptor. Hormone-sensitive lipase (HSL) converts

steroidogenesis. Pathway 4: HSL also interacts with esterified cholesterol present in thedogenesis as well. Free cholesterol from the LD can interact with lipid-binding proteins

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lipoproteins is transferred directly to the cell membrane withoutabsorption of the lipoprotein particles [16] (Fig. 1, Pathway 3). Furtheranalysis of these two pathways has shown that adrenal steroidogen-esis is dependent primarily on HDL cholesterol absorbed from theplasma membrane, primarily via the SR-BI pathway [17].

The non-selective vesicular pathway is initiated primarily by LDLparticles binding to the LDL receptor, followed by the endocytosis andbudding of clathrin-coated pits into the cytoplasm [18] (Fig. 1,Pathway 2). These vesicles fuse with early endosomes, releasingtheir clathrin coats and allowing the LDL receptors to cycle back to themembrane. This endosomal fusion and trafficking occur throughinteractions with microtubules which are controlled through Rabs[19]. Rabs are small GTPases that regulate membrane traffic throughbinding at their active site, currently there have been more then 60proteins identified in mammalian cells [20,21]. The early endosomesbind to recycling endosomes coordinated by Rab5, and then to lateendosomes via Rab7, to further distribute cholesterol throughout thecell [20,22]. The endosomes also undergo a decrease in pH from 7.4 atthe plasma membrane to 5.5–6 at the late endosome [23]. Thisdecrease in pH helps further dissolve the absorbed lipoprotein andprepares the late endosomes to fuse with the lysosomes.

It has been shown that the LDL receptor is not necessary for acuteadrenal steroidogenesis, suggesting that cholesterol absorbed via thispathway is not necessarily used for steroidogenesis [24]. However, inFSH- or FSH plus androstenedione-treated granulosa cells the rate ofLDL receptor absorption increases while the time needed for the LDLto reach the lysosome decreases compared to non-hormone-treatedcells suggesting this pathway is used for steroidogenesis [25]. Asendosomes contain a large percentage of the cytosolic cholesterolpresent in the cell; they can function in the trafficking of intracellularcholesterol to the mitochondria without first absorbing cholesterolfrom the plasma membrane. This pathway could occur specificallythrough the cholesterol-rich late endosomes, shown to fuse with thelysosomes and the Golgi apparatus and transiently interacting withthe mitochondria, thus allowing for multiple sources of cholesterol tobe available to the mitochondria. Endosomal trafficking in the cell hasalso been shown to be altered by cholesterol concentrations,specifically via Rab7, suggesting a mechanism by which traffickingto the mitochondria could be regulated [26].

The second pathway identified for cholesterol absorption andtrafficking in steroidogenic cells occurs through the action of the SR-BIreceptor [27] (Fig. 1, Pathway 3). The SR-BI receptor is found in manytissues such as intestines, macrophages, and endothelial cells, thoughit is expressed in highest concentrations in steroidogenic tissues suchas the adrenals, ovary, and testis [16]. Unlike the LDL receptor inwhichthe apoprotein is absorbed, the SR-BI receptor forms a non-aqueouschannel that allows a large influx of cholesterol directly into theplasma membrane [16]. This non-aqueous channel has also beenshown to be regulated in the intestines through other proteins foundin the plasma membrane, such as CD36 and other proteins yet to beidentified [28]. Suggesting this pathway might require a complex,multiprotein interaction to regulate cholesterol absorption into theplasma membrane. Because SR-BI absorbs free cholesterol from HDLand stores it in the plasma membrane, this cholesterol can movespontaneously between bilayers and membranes in the cell withoutthe assistance of any proteins. This process is slow and therefore notsuggested as a pathway involved in the acute stimulation ofsteroidogenesis [29]. The esterified cholesterol absorbed from theSR-BI receptor must be converted to free cholesterol before it can beused for steroidogenesis via a cholesterol ester hydrolase [30]. Insteroidogenic cells cholesterol ester hydrolysis is performed throughhormone-sensitive lipase (HSL) [31]. HSL becomes activated whenphosphorylated by cAMP; inhibition of HSL results in decreasedsteroidogenesis in the adrenals and inhibits sperm production in thetestis [32–34]. The cholesterol absorbed via the plasmamembrane hasbeen shown to be hydrolyzed rapidly, presumably close to the plasma

membrane, to form free cholesterol. Once the cholesterol esters haveundergone hydrolysis, the HSL can interact with various cholesterol-binding proteins to direct the cholesterol to the OMM for steroidogen-esis [35]. This pathway involving cholesterol-binding proteins will bediscussed later in detail.

5. Lipid droplets

Lipid droplets (LD) are bounded by a phospholipid membrane andfunction as a repository of cholesterol esters and triglycerides in thecell. It has been proposed that LD form from the ER when excessneutral lipids bud off, although there is no direct evidence to supportthis model [36]. The cholesterol esters found in the LD are theproducts of the ER enzyme acyl-coenzyme A:cholesterol acyltransfer-ase (ACAT), which becomes active in the presence of high levels ofcholesterol. This enzyme attaches an ester to the free cholesterolfound in the ER, increasing the cholesterol ester content present in thecell [37]. Because lipids in the LD can be used for various biologicalactivities their size fluctuates depending upon the cell's activity. Insteroidogenic cells, LDs are small to increase the surface area for lipidretrieval [36].

The cholesterol esters present in the LD are also converted to freecholesterol in the same manner as cholesterol absorbed via the SR-BIreceptor, i.e., through HSL. Transfer of steroidogenic cholesterol fromintracellular organelles to the mitochondria is thought to occurthrough cholesterol-binding proteins found in the cytosol (Fig. 1,Pathway 4) [38]. Other mechanisms for lipid transport from the LDhave been demonstrated. Rab5, which localizes to early endosomes,has been shown to interact with LD, suggesting a mechanism forcholesterol transfer from the LD to the endosome and vice versa,allowing for an increase in cholesterol in the endosomal pathway [39].Rab18 also associates with LD, regulating the contact between thelipid droplet and the ER, which controls the flux of cholesterol duringlipolysis [40]. Since LDs play an important role in regulatingintracellular cholesterol through storage, trafficking, and esterifica-tion, further studies are needed to determine the endosome/LDinteractionwhich would allow for transfer or fusion of the cholesterolfrom the LDs into the early endosomal pathway for steroidogenesis.

6. Targeting cholesterol to the mitochondria

The primary pathway for targeting cholesterol to the mitochondriahas not been definitively identified. Two pathways have beenproposed: (i) the non-vesicular pathway involving cholesterol-binding proteins transferring cholesterol through the cytosol to themitochondria, and (ii) a vesicular pathway characterized by anincrease in the fusion of vesicular membranes, such as endosomesand lysosomes, which results in an increase in cholesterol targeted tothe mitochondria [41]. An overlap between these two pathways ishighly likely as cholesterol-binding proteins have been found onendosomes; this suggests that an increase in cholesterol targeting toendosomes could also have a direct effect on cholesterol-bindingproteins which target cholesterol for transfer to the mitochondria.

7. Sterol carrier protein-2

Sterol carrier protein-2 (SCP-2) was one of the initial cholesterol-binding proteins identified; SCP-2 was shown to play a role in theintracellular transfer of cholesterol, including from the lysosomal tomitochondrial membranes [42,43]. SCP-2 is found in tissues involvedin cholesterol trafficking and oxidation, such as the liver, intestines,adrenal, testis, and ovary, suggesting it could play a role insteroidogenesis [44]. Further studies showed SCP-2 increases choles-terol uptake and transport throughout the cell while inhibiting theefflux of cholesterol from the cell through the HDL receptor [45,46].Because no alteration in steroidogenesis was observed in SCP-2 knock-

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out mice, it has been assumed that SCP-2 does not play a primary rolein steroidogenesis in vivo, though other interactions are still possible[47]. This focused attention on other identified cholesterol-binding/transfer proteins.

8. START domain proteins

The steroidogenic acute regulatory (StAR)-related lipid transfer(START) domain is an amino acid motif that has been proposed to playa role in cholesterol and lipid binding [48] (Fig. 2A). Themotif first wasidentified in the StAR protein, discussed below, which has been shownto play a role in cholesterol transport to the mitochondria forsteroidogenesis. The 210-amino acid sequence forms a beta sheetcore surrounded by two alpha helices, resulting in a hydrophobicchannel that can hold a sterol molecule capped by the C-terminalalpha helix [38]. The structure becomes stable upon binding of thecholesterol molecule, which has been shown to bind at a 1:1 ratio [49].

MLN64, the second identified START domain protein, was shown tobe upregulated in breast and ovarian cancers and found to have a C-terminal domain with 37% amino acid identity and 50% amino acidsimilarity to the C-terminal domain of StAR [50,51]. Further studiesconfirmed that this was a START domain that could bind cholesteroland transfer free cholesterol from sterol-rich vesicles to acceptormembranes. MLN64 was found to be localized primarily on lateendosomes, integrating at the plasma membrane and functioning inthe vesicular trafficking of LDL cholesterol [52]. When the STARTdomain was removed from MLN64, cholesterol accumulated in thelysosomes and altered late endosome trafficking [53]. In COS-F2 cells,this accumulation of cholesterol suppressed steroidogenesis, presum-ably by limiting the efflux of free cholesterol from the late endosomesand lysosomes to the mitochondria.

MLN64 was also found to be associated with Niemann–Pick type Cdisease protein 1 (NPC1) in the late endosomes [54]. Niemann–Picktype C disease (NPC) is a disorder characterized by the accumulationof LDL-derived unesterified cholesterol in the late endosomes andlysosomes caused by mutations in either NPC1 or NPC2 [55]. NPC1gene expression has been shown to be responsive to cAMP andessential for the normal development of the adrenals [56]. NPC1 isfound bound to late endosomal membranes while NPC2 is foundinside the late endosomes, early lysosomes, and in the cytosol. Thepresence of NPC2 accelerates the transfer of free cholesterol from lateendosomes to lysosomes and cholesterol efflux to the plasma

Fig. 2. Cholesterol-binding domains of StARand TSPO. Cholesterol-binding domains of StARanbinding domain (PDB ID code: 2I93). Homologymodels are deduced via the Swiss-model servcode: 1EM2) as template (http://swissmodel.expasy.org/SWISS-MODEL.html). (B) Moleculaaccommodating a cholesterol molecule. Homology modeling of mouse TSPO was performe(C) Ribbon diagramofmTSPO, showing thefive helices aswell as the CRACdomain, consistingthe TSPO CRAC domain. The accessible surface of the peptide and cholesterol molecules are

membrane [57,58]. NPC1 is necessary for cholesterol efflux from thelate lysosome for use in the cell [23]. When steroidogenic humangranulosa-lutein cells deficient in NPC1 protein were used forstudying cholesterol trafficking, steroidogenesis levels decreased tolevels seen with LDL-deficient media [59]. This observation suggeststhat the NPC1/late endosome pathway is used primarily for LDL-derived steroidogenesis. As NPC1 and NPC2 function in the efflux ofcholesterol from the endosome, this pathway could also interact withMLN-64 for steroidogenic purposes.

Steroidogenic acute regulatory (StAR) protein was first identifieddue to its rapid phosphorylation and protein expression soon after theaddition of hormones and cAMP in steroidogenic cells [60–62].Expression was confirmed in the adrenal cortex, testis, and ovaryand later in the brain and placenta, suggesting a connection betweenStAR protein expression and steroid production [63,64]. Transfectionof StAR expression vectors in both mouse Leydig MA-10 cells and COSF2 cells, which contain the components of CYP11A1, was found toincrease steroidogenesis [65,66].

There have beenmanymodels proposed for the function of StAR insteroidogenesis [67]. It was suggested early that StAR assists with thetransfer of cholesterol to the mitochondria, although no clearmechanism was identified. Because StAR contains an N-terminalmitochondrial-targeting sequence, which is cleaved from its activemolecular weight of 37 kDa to its inactive weight of 30 kDa uponimport into the mitochondrion, it was proposed that this targetingsequence could assist with the formation of a mitochondrial “contactsite”. This would be accomplished by the interaction of StAR withmitochondrial protein import complexes found on both the OMM andIMM and allow the N-terminus to form a linker connecting themembranes. Cholesterol would then be able to flow from the OMM tothe IMM and interact with CYP11A1. However, removal of the N-terminus targeting sequence in the construct N-62 StAR andtransfection into steroidogenic cells had no effect on steroidogenesis,suggesting this is not the mechanism by which StAR facilitatescholesterol transfer [68]. It was later observed that N-62 StARwas ableto insert cholesterol into cytosolic membranes other than themitochondria, suggesting that the primary function of the N-terminalsequence is not to form mitochondrial contact sites but to limit StAR'scholesterol targeting abilities solely to the OMM [69].

The effect of StAR on mitochondrial steroidogenesis was furtherconfirmed by fusing mitochondrial translocases to the N-62 StARconstruct. Fusion of N-62 StAR with Tim9, a mitochondrial inner

d TSPO (A)Human StAR proteinmodeledwith cholesterol (brown) present in the sterol-ices under project (optimize)mode using the crystal structures of humanMLN64 (PDB IDr model of TSPO's five alpha helices in the presence of cholesterol, demonstrating a pored using apolipophorin-III from Manduca sexta as template (PDB ID code: 1EQ1) [157].of Leu/Tyr/Arg residues, and cholesterol (top view). (D)Dockingmodel of cholesterol torepresented in red and blue, respectively (reprinted with permission from [158]).

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membrane space protein, or Tim40, an IMM protein, resulted in noincrease in steroidogenesis while fusion of N-62 StAR with Tom20, anOMM protein, resulted in maximal production of steroids [70]. Thisobservation demonstrated that StAR's site of action was confined tothe OMM and that, once imported, StAR does not stimulatesteroidogenesis. Because StAR interacts only transiently with theOMM before being imported, the amount of time StAR spends at theOMM would be able to alter the rate of steroid production. This wasshown when a StAR/StAR construct, which is imported at a slowerrate than wild-type StAR, was shown to increase pregnenoloneproduction over both wild-type and N-62 StAR construct levels inCOS-F2 cells [70]. This finding shows that StAR functions primarily atthe OMM, possibly activating a pathway of cholesterol transport forsteroidogenesis.

Another proposed model built to explain StAR's activity suggestedthat it could function as an intermembrane cholesterol shuttle,moving cholesterol from the outer to the inner mitochondriamembrane one molecule at a time during StAR's import [49].Tsujishita and Hurley proposed that during StAR's import into themitochondria several molecules of cholesterol could be transferredthrough the import of one StAR molecule through transient openings.Several issues were raised with the model, including the lack ofunderstanding of how StAR could both bind cholesterol and thenrelease it in the IMM space. It was also uncertain how StAR wouldreside in the IMM space as it does not have a mitochondria targetingsequence for the inner mitochondria membrane space.

One of the more current proposed models is the “molten globule”model identified through the studying of the StAR's C-terminus.Removal of the last 10 C-terminal amino acids resulted in decreasedsteroidogenesis, while removal of the 28 C-terminal amino acidsresulted in a biologically inactive protein, suggesting that StAR's modeof actionwas occurring at the C-terminus. Further studies showed thatthe C-terminus forms a sterol-binding domain (SBD) with which acholesterol molecule is proposed to interact. The SBD forms a pocketthat prevents the release of the bound cholesterol molecule, whichcan occur only following a conformational change. This is proposed tooccur through the “molten globule” configuration in which tertiarystructures are removed, allowing the remaining secondary structuresto undergo a conformational shift and allow the cholesterol moleculeto enter the mitochondria. The molten globule model has beenproposed to occur through the protonation of the C-terminus at theOMM. Since binding of cholesterol to the SBD in the StAR protein hasnot been definitively demonstrated, it is still not clear if this is themechanism by which cholesterol is transferred to the OMM. It shouldbe noted that the cholesterol-binding activity of StAR is independentof its activity at the OMM, because StAR mutant R182L can stillfunction in the binding and transfer of cholesterol in isolatedliposomes, although this results in an increase in cholesterol presentin the cell [71]. This suggests that cholesterol binding is necessary butnot sufficient for StAR's function on the OMM.

Further homology modeling and biophysical studies recentlyindicated the existence of a two-state model [72]. The first and openstate of the model proposes that the C-terminal alpha-helix 4 of StAR,acting primarily as a gatingmechanism to the cholesterol-binding site,undergoes partial unfolding allowing cholesterol to bind. Thischolesterol bound state, in theory, would lead to the stabilizationand the refolding of alpha-helix 4; resulting in a well-defined tertiarystructure [72]. This stable tertiary structure could be necessary forprotein–protein interactions formed for cholesterol transfer at thesurface of the mitochondria, suggesting that both StAR conformationand cholesterol binding are necessary for its proper steroidogenicactivity.

These models of StAR's function have been able to provide manydescriptions of cholesterol transport to the mitochondria which canrelate to the function of START proteins. As six other START domainfamilies have been identified, such as StarD4, StarD5, and StarD6, the

understanding of how the START domains function can be applied tothese proteins as well [73]. This can have far reaching effects, ascompared to StAR and MLN64; StarD4, StarD5, and StarD6 lack anorganelle-targeting sequence and therefore are thought to becytoplasmic. StarD4 and StarD5 are widely expressed, while StarD6is found primarily in the testis [48]. This ability of the START domain totarget and transport cholesterol from multiple sources, including theplasma membrane, ER, and endosomes, could ensure a large source ofcholesterol for steroidogenesis.

9. Importing cholesterol into the mitochondria

Cholesterol successfully imported via the plasma membrane oraccessed in LDs and transported to the OMM remains segregated inthe OMMuntil translocation to the IMM. This, the rate-limiting step insteroidogenesis, has been suggested to occur primarily through TSPO.

10. TSPO and cholesterol

TSPO was first identified by the presence of radiolabeled diazepambinding in the kidney [74] and it was later found to be present in mosttissues of the body [75]. It was proposed that TSPO plays a role insteroidogenesis when ligand-binding studies revealed increasedexpression of TSPO in steroidogenic tissues and subcellular localiza-tion studies indicated that it was primarily localized to the OMM[75–78].

Because the benzodiazepine diazepam is awidely used drug ligandspecific for the GABAA receptor in the central nervous system thesubsequent identification of TSPO-specific ligands allowed for thepharmacological differentiation between TSPO and the GABAA

receptor. This was accomplished by use of the isoquinoline carbox-amide PK 11195, which binds with nanomolar affinity to TSPO [79] buthas no affinity for the GABAA receptor. Many endogenous TSPO ligandsexist in the cell, with porphyrins being able to bind TSPO with highnanomolar affinity [80]. The diazepam binding inhibitor (DBI) isanother endogenous ligand. This 10-kDa protein, which also binds theGABAA receptor with low affinity, is expressed in many tissues but isprimarily expressed in steroidogenic tissues where it is localized in thecytosol in contact with the OMM [81]. Naturally processed peptides ofDBI, octadecaneuropeptide (ODN, DBI33–50) and triakontatetraneur-opetide (TNN, DBI17–50), expressed in a hormone-dependent manner,were functional in binding TSPO in the brain, adrenal, and testis[74,82]. DBI and its peptides stimulated mitochondrial pregnenoloneformation. When DBI expression was suppressed in the presence ofantisense oligonucleotides MA-10 Leydig cells failed to respond tohormonal stimulation and steroid production was inhibited.

Early experiments in multiple steroidogenic cell systems showedthat pregnenolone production was stimulated upon exposure of thecells to TSPO ligands [83,84]. When these experiments were repeatedin isolated mitochondria incubated with TSPO ligands a similarincrease in pregnenolone was observed [76,84]. This increase was notseen in mitoplasts, mitochondria devoid of their OMM and thereforedeficient in TSPO. To determine the effect of TSPO ligands on themitochondria, cholesterol content in the OMM and the IMM wasmeasured both before and after TSPO ligand treatment [85]. This studyrevealed that ligand binding to TSPO induced the translocation ofcholesterol from the OMM to the IMM and confirmed that TSPOparticipates in the binding and release of cholesterol at the OMM, aninitial step in the production of steroids.

To further confirm TSPO's role in cholesterol binding andtranslocation a bacterial expression system was devised becausebacteria contain no endogenous cholesterol. E. coli were transformedwith an inducible mouse cDNA TSPO vector, resulting in fullyexpressed TSPO. Ligand-binding experiments were performed toverify that the bacterial TSPO possessed the same pharmacologicalbinding properties as native TSPO; this was confirmed when bacterial

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TSPO bound both cholesterol and PK 11195 with nanomolar affinities[86]. When bacterial TSPO was incubated with radiolabeled steroids,time- and temperature-dependent uptake of cholesterol was seen inthe protoplasts although no uptake of other steroids was seen. Whenthe bacterial cholesterol-loaded membranes were treated with PK11195, the cholesterol was released [86]. These findings confirm thatTSPO functions as a cholesterol translocator and suggest that TSPOmight further function as a cholesterol sink, holding cholesterol untilit is released by the binding of a ligand.

To identify the basis of the interaction of TSPO with cholesterol,molecular modeling and site-directed mutagenesis were used toidentify potential binding sites. Previous studies had shown that TSPOspans the OMM in five alpha helices, composed of approximately 21amino acids each. The 3-D models produced suggested that the fivealpha helices come together to form a channel with a hydrophilic butuncharged interior surface [86] (Fig. 2B). It was shown that theinterior of the channel could bind a cholesterol molecule that had notbeen significantly modified, suggesting that TSPO could function as atransporter of cholesterol to the IMM. To identify the cholesterol-binding domain several deletion constructs were generated. A regionon the C-terminus (Δ153–169) was identified as necessary forcholesterol binding by virtue of the mutant's reduced ability to takeup cholesterol, although PK 11195 binding was unaltered [87]. Furthersite-directed mutagenesis experiments identified the specific aminoacids necessary for cholesterol binding, yielding a CRAC (cholesterol-recognition amino acid consensus) domain (Fig. 2C and D). The CRACdomain showed the high nanomolar affinity for cholesterol that hadbeen observed in other proteins interacting with cholesterol [87].These data suggest that the C-terminus of TSPO plays an importantrole in the uptake and translocation of cholesterol into the IMM.

To confirm the role of TSPO in ligand-binding and cholesteroltranslocation, a peptide antagonist was developed using a randomseven-mer peptide library attached to an HIV-TAT domain [88]. TheTAT domain allows receptor-independent entry of the protein orpeptide attached to the sequence [89]. The random seven-merpeptides attached to the TAT domain were incubated with MA-10Leydig cells and peptides eluted with ligand Ro5-4864. It was shownthat the domain STXXXXP, specifically STPHSTP, competed with thehighest efficiency for the ligand-binding domain [90]. This was furtherconfirmed when hormone-induced steroidogenesis was shown to beinhibited by this peptide in a dose-dependent fashion. The CRACdomain was then also fused to the TAT domain, allowing entry intoMA-10 cells in a dose-dependent manner [87]. This was shown toinhibit steroidogenesis through a dominate-negative effect by alteringthe translocation of cholesterol from the mitochondria to the TAT-CRAC domain. These data confirmed the importance of the C-terminusin the binding and translocation of cholesterol. In both cases theproduction of steroids from 22R-hydroxycholesterol was not altered,demonstrating that the peptide affected only TSPO and its ability tobind cholesterol and endogenous ligands [87,90].

From these experiments it was shown that TSPO is a high-affinitycholesterol- and drug ligand-binding protein. It functions in thetranslocation of cholesterol from the OMM to the IMM in the presenceof its ligands. It should be mentioned that due to the important role ofcholesterol in mammalian cells and the diverse localization of TSPO inmany tissues, TSPO may play a more extensive role in the cell,participating in targeting cholesterol to mitochondria for membranebiogenesis and also cholesterol transport in the cell. This idea gainedfurther support when treatment of both steroidogenic and non-steroidogenic cells with TSPO ligands resulted in a redistribution ofcholesterol from the plasma membrane to LD [91], suggesting apossible role for TSPO in the intracellular regulation and trafficking ofcholesterol, independent of cell type.

The next step was to determine if TSPO was solely responsible or ifother proteins could be assisting with this translocation of cholesterolin the mitochondria. To do this the R2C rat Leydig cell line, derived

from rat Leydig tumors and shown to constitutively produce steroids[92], was used. The TSPO gene was disrupted by homologousrecombination, resulting in a dramatic decrease in steroid productionto 10% of control values [93]. However, when 22R-hydroxycholesterol,a hydrophilic CYP11A1 substrate that can pass directly into the IMM,was added the levels of steroid production returned to normal [93].The role of TSPO in steroidogenesis was further verified when a TSPOknock-out mouse model proved to be embryonic lethal [94],demonstrating that TSPO is not only necessary for steroidogenesisbut it also plays a critical role in early embryonic development.

Hormonal stimulation in steroidogenic cells initiates the transfer ofcholesterol from the OMM to the IMM. As TSPO is suggested to play arole in this process, the effect of hormones on TSPO activity wasexamined. Upon the addition of the gonadotropin hCG in MA-10Leydig cells, TSPO was shown to cluster in groups of four to sixmolecules [95,96]. This clustering results in increased ligand binding,cholesterol dispersal into the IMM, and steroid production and hasbeen shown to increase the formation of contact sites. The clusteringof TSPO caused by hCG can be inhibited by the addition of a cAMP-dependent protein kinase (PKA) inhibitor, suggesting that localizationand clustering of TSPO is a cAMP-inducible event [97]. Becauseantibodies against TSPO recognize immunoreactive proteins ofmolecular weight greater than 18 kDa, it has been suggested thesewere polymers of TSPO formed through the hormonally inducedclustering. The clusters of TSPO have been shown to be due to theformation of permanent dityrosine bonds [98]. Bond formation isachieved through the generation of reactive oxygen species (ROS)triggered by the presence of hormones in Leydig cells. This has beenfurther confirmed to follow the pathway of induction of cAMP-induced and PKA-dependent ROS formation via the mitochondrialrespiration complex I [99].

To better understand the role of drug ligands in cholesteroltransport in the mitochondria NMR analyses were performed. Theresults showed that the alpha helical structure of TSPO was present inthemonomer form, while the overall tertiary structure was somewhatless structured [100]. The presence of the drug ligand PK 11195stabilized TSPO, providing a more stable environment for thetranslocation of cholesterol to the IMM.

11. Interactions of TSPO and mitochondrial proteins

Once cholesterol has been bound to TSPO it is committed to use insteroidogenesis. Because TSPO is located primarily at mitochondrialcontact sites [101], it has been suggested that TSPO does not functionalone in the OMM. Native TSPO in digitonin solubilized mitochondrialextracts elutes on gel filtration column chromatography in a digitonincontaining buffer as a 200- to 240-kDa complex (unpublished results)while cross-linked TSPO solubilized with digitonin elutes at 170 to210 kDa [102,103]. Studies identifying proteins in these complexesshowed TSPO to be eluting at 18, 36, and 54 kDa, presumablyrepresenting the monomer and polymers of TSPO induced by hCG.Other proteins eluted were identified as voltage-dependent anionchannel (VDAC), adenine nucleotide transporter (ANT), and uniden-tified proteins at 60 kDa (Fig. 3A) [104,105].

VDAC is an OMM channel-forming protein that regulates thepassage of ions and small molecules through the OMM. This functiondetermines membrane potential, thus assisting with the regulation ofapoptosis and cell metabolism [106]. VDAC's interaction with ANTforms the mitochondrial permeability transition pore (MPTP), whichis located primarily at mitochondrial contact sites and regulates thecell's response to apoptotic events. ANT's role in the MPTP has beenshown not to be essential though it is believed that it might functionin a regulatory manner [107]. Its interactions with TSPO are currentlyunknown though it has recently been shown that ANTcan also bind anidentified TSPO ligand, protoporphyrin IX, and transport it into themitochondrial matrix [108]. It has been suggested that interactions of

Fig. 3. Protein–protein interactions at the OMM. (A) Proposed basal-state protein–protein interactions of TSPO, VDAC, and ANT. (B) Transduceosome complex formed after acutehormonal stimulation. The TSPO, PAP7 (ACBD3), and VDAC complex recruits PKA-RIα, which is shown binding to PAP7 (ACBD3). The accumulation of cAMP activates PKA-RIα,releasing the catalytic subunits and phosphorylating StAR present at the OMM. StAR interacts with both TSPO and VDAC and is imported into the IMM. Cholesterol is imported intothe IMMwith the assistance of the transduceosome complex and the presence of DBI; there the cholesterol interacts with the CYP11A1 side chain cleavage enzyme to be converted topregnenolone. ANT is represented in the IMM as identified from previous isolated complexes with TSPO.

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TSPO, VDAC, and ANT might modulate the cell's response to apoptoticsignals. Because TSPO has been shown to be involved in ROSproduction, it has also been suggested that TSPO might function inthe apoptotic response [109]. It has also been suggested that MPTP canalter steroidogenic rates as it is known that Leydig cell mitochondrianeed to be fully functional for steroidogenesis and that the openingand closing of the MPTP alters the cells’ ability to produce steroids(unpublished data). As the number of contact sites can be modulated(increased) by hormone treatment; which in theory, could increasecholesterol transport between the OMM and the IMM, the perme-ability of themitochondria could also affect steroidogenesis, regulatedin part by TSPO [110].

Modeling has shown that VDAC binds cholesterol and it has beenindependently demonstrated that VDAC influences cholesterol dis-tribution in the mitochondria [105,111,112]. It is also known that PK11195 affects TSPO tertiary structure by stabilizing the protein in thecell [100]. TPSO's loss of flexibility as a result of binding to VDAC couldtranslate into a more rigid VDAC, altering the respiratory state andpossibly cholesterol distribution in the mitochondria as well.

12. TSPO and cytosolic cholesterol import

12.1. TSPO and StAR

Steroidogenic cholesterol is targeted to the mitochondria thoughproteins containing the START domain – StAR and MLN64 – asmentioned earlier. For this cholesterol to be used effectively forsteroidogenesis it must interact with TSPO. Because TSPO and StARhave been shown to interact by FRET [113], but not BRET [114] analysis,this might be a pathway throughwhich cholesterol can be transferred.

To determine how TSPO and StAR interact at the OMM, antisenseoligodeoxynucleotides (ODNs) were used to reduce expression of thetwo proteins. When StAR expression was reduced, hCG-stimulatedMA-10 Leydig cells stopped producing progesterone after 20 min,while in TSPO-depleted cells steroidogenesis was inhibited after10 min [115]. Together these results show that both TSPO and StARfunction in steroidogenesis; the difference in time of arrest of

steroidogenesis was attributed to the presence of cholesterol on theOMMavailable to be used for steroidogenesis. Sincewewere unable todemonstrate a direct physical StAR/TSPO interactionwe searched for afunctional one. Thus, StAR expressionwas examined in TSPO-depletedcells. It was shown that StAR was not processed from the 37-kDacytosolic protein to the mature intramitochondrial 30-kDa proteinthat is normally seen under hormonal stimulation [115]. This wasfurther confirmed when a peptide antagonist shown to bind to thecholesterol-binding domain of TSPO also inhibited the intramitochon-drial formation of the 30-kDa StAR [90,115]. Based on these results itwas suggested that TSPO plays a direct role in the import of StAR intothe IMM and that StAR is dependent upon TSPO for its activity.

To test this hypothesis, TSPO-depleted mitochondria were trans-fected with a Tom/StAR construct that would be targeted to andimported into the OMM. Previously it had been shown that thisconstruct increased progesterone production two-fold in MA-10 cells;however, no effect was seen on steroid production in TSPO-depletedmitochondria [70,115]. TSPOwas then reincorporated into the isolatedmitochondria, restoring the ability of the mitochondria to producepregnenolone. This effect was seen with both Tom/StAR and StARconstructs. Based on separate analysis and reintroduction of these twoproteins into the cell system, it was proposed that StAR's primaryfunction is in the hormone-induced transport of cholesterol to theOMM while TSPO regulates the translocation of cholesterol intothe IMM.

Acute stimulation of steroidogenesis in vivo results in measurablehormone productionwithinminutes. However, in vitro this effect is notobserved until 10–20 min, which is interesting because StAR proteinsynthesis shows a lag of 20–30min after hormone stimulation [116]. Aspreviously mentioned, steroidogenic cholesterol can be found boundto TSPO in the OMM, suggesting that this time frame would depleteTSPO of cholesterol and allow StAR to then replenish cholesterolconcentrations. Limiting StAR protein expression and, therefore, itsactivity in the cell, would allow for cholesterol to be specificallytargeted to the mitochondria for steroidogenesis and inhibit excesscholesterol transport. This delayed protein expression also confirmsthat StAR does not act alone on the OMM in steroidogenesis.

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StAR has been shown to cycle sufficiently rapidly to transfer 400molecules of cholesterol per minute into adrenal cells [117]. However,experimental observations have demonstrated that the stoichiometry ofcholesterol transfer is 1.82 molecules of cholesterol per minute inisolated mitochondria [118], suggesting that further modification in thecell is needed for maximal StAR activity. One proposed mechanism forthis modification is cholesterol binding to the SBD, which has beenshown to be necessary for StAR's function [119]. StAR binds cholesterolwith an affinity of 32 nM [120], although binding at an affinity of 95 μMhas recently been reported [121]. Because StAR rapidly shuttlescholesterol through the OMM, high-affinity binding of cholesterol toStAR would not favor this transfer. It is also important to note that thisaffinity is substantially lower than the affinity of 5 nM of TSPO forcholesterol [122], suggesting a possible mechanism by which TSPOremoves cholesterol fromStARwhen it is in closeproximity to theOMM.

12.2. TSPO and PAP7

To determine if other proteins are necessary for importingcholesterol into the mitochondria a yeast-two hybrid screen wasperformed with TSPO as the bait. This approach demonstrated theassociation of several PBR-associated proteins (PAPs) with TSPO, withPAP7 demonstrating the most compelling interaction [123]. PAP7 wasfound to have an expressionpattern similar toTSPO andwas localized tothe Golgi and mitochondria. Interestingly, in another yeast-two hybridscreenwith the regulatory subunit RIα of PKA as the bait, PAP7was alsoidentified [123]. These data provided useful insight into the function ofPAP7 because PKA phosphorylates proteins in a hormone-specificmanner through the activation of cAMP. When cAMP levels rise theproteins bind to the two regulatory subunits of PKA, RI and RII and theirisoforms (Iα and Iβ, IIα and IIβ), which release the two catalyticsubunits; these are then able to phosphorylate specific serine andthreonine residues, activating select proteins (Fig. 3B). It was thenconfirmed that PAP7 binds both TSPO and PKA-RIα in vitro in MA-10mouse Leydig cells [123]. Overexpression of PAP7 was shown tostimulate progesterone production in MA-10 cells and transfection ofthe TSPO- and PKA-Riα-binding domain of PAP7 (a.a. 228–445) signi-ficantly inhibits steroidogenesis [123,124]. These results further confirmPAP7′s role in steroidogenesis via interactionwith TSPO and PKA-RIα. Inthese studies we identified an acyl-coA bindingmotif in PAP7, similar tothe one identified in DBI [124]. More recently, DBI and PAP7 wererenamed acyl-coenzyme A binding domain containing 1 (ACBD1) and 3(ACBD3) proteins, respectively (Fan et al., manuscript submitted).

Since PAP7 is known to bind PKA-RIα, it was suggested that PAP7functions as an A Kinase Anchoring Protein (AKAP). AKAPs are afamily of proteins known to recruit the PKA holoenzyme intoproximity to its substrate, confining its activity [125]. In this process,PAP7 is presumed to bring PKA-RIα into closer proximity to theproteins mediating cholesterol transport and thus steroidogenesis,playing a role in regulating their activity via phosphorylation. Thismechanismwould allow the signaling mechanism for steroidogenesisto be localized to certain areas on the mitochondria, limiting theconcentration of protein needed for maximal stimulation. Thus, as theconcentration of trophic hormone needed to stimulate maximal cAMPproduction is about 15 times higher than that needed to maximallystimulate testosterone secretion [126] such a mechanismwould allowmaximal steroid formation in the presence of submaximal cAMPaccumulation. Targeting of PKA-RIα close to mitochondrial TSPOwould result in the localization and amplification of its ability tophosphorylate proteins involved in steroidogenesis.

It is known that cloned rat, bovine, and murine TSPO arephosphorylated in the C-terminal domain, although a phosphoryla-tion site has not been identified in human TSPO [127]. StAR becomesrapidly phosphorylated upon the addition of trophic hormones in allspecies investigated to date [128]. Based on these observations it wasproposed that PAP7 anchors PKA-RIα, facilitating the phosphorylation

of StAR and possibly TSPO, in a cAMP-dependent manner (Fig. 3B).This mechanism would allow the activity of StAR to be regulated andactivated by proteins localized to the OMM, controlled by proteinsknown to anchor to TSPO.

To determine if theproteinsunder investigation interact at theOMM,COS-F2-130 and MA-10 cells were transfected with TSPO, StAR, PAP7,and PKA-RIα. The transfection of the four proteins together in the non-steroidogenic cells induced an increase in steroid production greaterthan that inducedbyeach individual protein alone, suggesting that theseproteins form a complex that performs a necessary role in steroidogen-esis. Hormonal stimulation of non-transfected Leydig cells induced agreater increase in steroid synthesis, suggesting that the complex ofproteins is still dependent upon stimulation through cAMP and not onthe amount of proteins present. The interactions between TSPO, StAR,and PKA-RIαwere subsequently analyzed by microscopy. These studiesindicated that, upon hormonal stimulation, PAP7 translocated from theGolgi to the mitochondria [129] and StAR translocates to themitochondria, where PKA-RIα and PAP7 colocalize with TSPO [129].

To further confirm that these proteins interact at the OMM, photo-activatable amino acids, specifically leucine and methionine, wereused. These amino acids are functionally incorporated in the proteinsand then can be cross-linked by exposure to UV light, allowingidentification of protein–protein interactions [130]. Since hormonalstimulation of Leydig cells results in an acute response, the immediatecross-linking of proteins permits the identification of transientprotein–protein interactions, which normally could not be observedusing classical procedures such as immunoprecipitation. Upontransfection of TSPO, StAR, PAP7, and PKA-RIα in the presence ofphoto-activatable amino acids in COS-F2-130 cells, cells were exposedto UV light. Immunoblot analysis identified a 210-kDa complexcontaining immunoreactive TSPO and StAR proteins [129]. MA-10Leydig cells treated with hCG under the same conditions as describedabove were cross-linked under UV light. Immunoblot analysisidentified a 240-kDa protein complex in which TSPO, PAP7, PKA-Riα,and VDAC were observed to be associated in a time-dependentmanner following hCG exposure. StAR levels peaked after a 30-mintreatment with hCG and decreased after 2 h of stimulation, whileVDAC levels increased slightly and then also decreased at 2 h [129].Because StAR is dependent upon VDAC for import [131], it isinteresting to note that VDAC also undergoes depletion during thehCG response [129]. This finding suggests that VDAC could play a rolein the acute response to hormonal stimulation, assisting in the bindingof StAR to the OMM. StAR would then be phosphorylated through theidentified protein complex; TSPO, PAP7, and PKA-RIα, would increaseimport. These experiments demonstrated that the inducible hormonecomplex of steroidogenic proteins, the “transduceosome,” interact tofacilitate the transfer of cholesterol from the OMM to the IMM. Thiscomplex occurs at the OMM, not at the IMM, as confirmed by presenceof VDAC and the absence of ANT in the cross-linked complex [129].

13. TSPO, StAR and cholesterol transport in disease

Improper storage and targeting of cholesterol can be toxic to cells,as seen in cells affected with NPC disease, mentioned previously.There are many opportunities for inappropriate storage and targetingto occur in the multiple steps required to target cholesterol to themitochondria. Moreover, limiting or accelerating the transfer andaccess of cholesterol to CYP11A1 would result in changes in the levelsof steroids formed that could affect the target cell function as well astissue and body homeostasis. We will discuss examples of diseasesthat arise when cholesterol targeting is altered below.

14. Endocrine pathologies

Lipoid congenital adrenal hyperplasia (Lipoid CAH) was initiallyproposed to be caused by deficiency of a steroidogenic enzyme but

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further study identified point mutations or stop codon insertion intothe C-terminus of the StAR protein [63,132]. Patients with lipoid CAHsuffer from a decrease in both adrenal and gonadal steroidogenesis,resulting in an increase in cholesterol accumulating in steroidogeniccells [67]. Patients affected by Lipoid CAH are still be able to producelow levels of steroids, approximately 14% of normal, which wassuggested to occur through cholesterol present on the mitochondria[67]. Since the steroidogenic cells are unable to correctly process thecholesterol esters, inhibition of the non-StAR-mediated steroidogen-esis occurs. This results in a further increase in cell damage andsometimes death. This process has been confirmed in the StAR-knock-out mouse model, which displays the same phenotype as humanLipoid CAH [133]. The mice could be kept alive if they were givencorticosteroid replacement, although they still suffered from anincrease in lipids with aging; this was not observed in females untilafter puberty [134]. Interestingly, the Tspo gene was found to be intactin Lipoid CAH [67]. This is not surprising, considering that knock-outof Tspo results in an embryonic lethal phenotype, as mentioned above.

Primary pigmented nodular adrenocortical disease (PPNAD) is acomponent of the Carney complex (CNC), in which the patient carriesa PKA-RIα-inactivating mutation [135]. This mutation causes anadrenal disorder characterized by an ACTH-independent hypercorti-solism, which is stimulated by exogenous steroids [136]. Analysis ofthe adrenal cortical nodular cells showed either very weak or nostaining at all for PKA-RIα. Previously it had been proposed that PKA-RIα is a tumor suppressor [135], confirming the profile commonlyseen in tumor cells, in which one allele contains an inactivatingmutation and the other allele is missing. On further observation it wasshown that the area around the nodular cells expressed PKA-RIα atlevels higher than those seen in control cells, suggesting this area ofthe cortex could be compensating for the lack of expression of PKA-RIα [135].

Because it is known that PAP7 interacts with PKA-RIα insteroidogenesis, PAP7 expressionwas investigated in patients affectedwith PPNAD. In these patients PAP7 protein expression levels mirroredPKA-RIα expression levels, with very low expression in the adrenalcortical nodular cells and higher than wild-type expression of PAP7 inthe area surrounding the nodular cells [137]; PKA-RIIα expressionwasnot affected. It was already known that PAP7 protein expression is notaltered in adrenal hyperplasia or in brain, breast, or colon tumormRNA levels, so this provided further evidence that PKA-RIαexpression correlated strongly with PAP7 protein expression levels[137].

Previous data have shown that CNC tumors with PKA-RIαmutations respond to cAMP stimulation with a larger increase inPKA activity compared to cells lacking the mutations. The observationthat PKA-RIα and PAP7 expression is higher in the adrenal corticalcells surrounding the nodules provided an explanation for theincrease in hormones produced following even a low level of cAMPstimulation, compared to wild type. These findings further supportthe hypothesis that PKA-RIα and PAP7 interact in a cAMP-dependentmanner to control steroidogenesis.

15. Neurological disorders

Steroids are able to diffuse across the blood–brain barrier, althoughconcentrations present in the brain have been shown to be differentfrom those found in the peripheral nervous system, suggesting thatthe brain is capable of locally synthesizing steroids [138,139]. Inaddition, the brain is a major site of cholesterol synthesis, withcholesterol itself unable to pass through the blood–brain barrier. Theterm “neurosteroids” was first introduced to represent the steroidintermediates present in the brain; however, today it is accepted thatthe brain is a steroidogenic organ and the definition has evolved torepresent steroids produced and metabolized in the brain [140].Neurosteroids required to induce changes in neuronal activity are

present and act at extremely low (nanomolar) concentrations, incontrast to the high concentrations of gonadal and adrenal steroidsproduced for distribution through the circulation and action at distantsites throughout the body. This may explain why no activator ofneurosteroid biosynthesis has been identified to date.

TSPO is present in the brain and is primarily expressed in thesteroid-producing glial cells, although it is also present at low levels inthe neurons [141,142]. Since StAR and MLN-64 have also beenidentified in glial cells, it has been suggested that the steroidogenicpathway in the brain is similar to that in the periphery [143,144]. Thiswas confirmed when activation of glial TSPO and isolated glial cellmitochondria with drug ligands of TSPO was shown to result in theproduction of pregnenolone and neurosteroids [145,146]. Neuroster-oids have been shown to have an immediate, specific, and local effecton neural development; therefore, neural development would besignificantly impacted if the regulation of neurosteroids were altered.

Examination of TSPO expression showed upregulation in manyneurolopathologies, neurodegenerative disorders, and during braininjury and inflammation. Upregulation of TSPO was confirmed ingliomas [147], multiple sclerosis [148], Parkinson's and Huntington'sdisease [149,150], and epilepsy [151]. TSPO expressionwas also shownto be increased after nerve degeneration and during regeneration,although once regeneration was complete the levels of TSPOdecreased [152]. Microglia are more active in many of theseneurolopathologies and neurodegenerative diseases and, as TSPO isprimarily localized in glial cells, it has been possible to use TSPO as amarker to diagnose and determine the rate of progression of manydisease and injuries.

TSPO is also up regulated in Alzheimer's disease (AD) with aresulting increase in pregnenolone levels in the hippocampal region ofthe brain [149,153]. Interestingly, it has been shown that a steroidintermediate in the conversion of cholesterol to pregnenolone, 22R-hydroxycholesterol, was found at lower levels in AD brain comparedto control [154]. It was also later shown that 22R-hydroxycholesterolexerts neuroprotective effects against the neurotoxicant β-amyloid(Aβ) peptide, functioning through binding and inactivation of thepeptide. This neuroprotectant is decreased in AD patients, allowing Aβto exert its toxic effect on the cells. Because Aβ is an intermediate inthe steroidogenic pathway, overexpressed TSPO would presumablycompensate for the decrease in 22R-hydroxycholesterol. Because thisdoes not occur, it has been suggested that TSPO does not functionnormally in Alzheimer's patients. It will be necessary to furtherexplore the role of TSPO in neurosteroid production and neuropro-tective effects in AD.

TSPO has been shown to be altered in anxiety and mood disordersas well. The concentration of TSPO determined through assays ofradiolabeled PK 11195 binding to platelets showed a decrease inanxiety, panic, and post-traumatic stress disorders. These data suggestthat TSPO may exert an anti-anxiety effect through the production ofneurosteroids, if the same reduction in TSPO is seen in the CNS. Inagreement with these findings it was shown that many drugs used inthe treatment of psychiatric disorders act on specific enzymes of theneurosteroidogenic pathway, resulting in normal levels of theneurosteroid allopregnanolone [155]. This was further confirmedwhen it was shown that DBI also has an effect on patients withschizophrenia [156] and suggests that TSPO ligands could play a role inthe prevention and treatment of psychiatric disorders.

16. Conclusion

In the past few yearsmany advances have beenmade in identifyingthe protein–protein interactions necessary for steroidogenesis. Fromthe initial stages of cholesterol import into the plasma membrane,through lipoprotein binding to receptors, to the actual translocation ofcholesterol into the IMM for pregnenolone production each step hasbeen shown to be tightly controlled and regulated though these

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interactions. Newly identified proteins, such as PAP7 (ACBD3),contribute to a more complete picture of how steroidogenesis isregulated in each cell model and of the protein interactions involvedin steroidogenesis. These discoveries have direct effects on theunderstanding of the bases of several neurological and endocrinediseases.

Despite recent progress in understanding the cellular andmolecular mechanisms underlying the hormonal regulation ofcholesterol transport into mitochondria and, thus, of steroidogenesis,there are several issues that remain to be addressed. These includeacquiring a better understanding of the spatiotemporal and structuraldependence of the mechanisms of both membrane and cholesterolmovement in the cell under hormonal stimulation. Mitochondria ingeneral are cholesterol-poor organelles; however the mitochondria ofsteroidogenic cells contain a relatively high amount of cholesterol.Since TSPO also occurs at higher levels in steroidogenic cells, thischolesterol could be bound to the excess TSPO present in the OMM.Due to the location of the CRAC domain at the interface of the OMMand the cytosol, it is unclear where this excess cholesterol in themitochondria is binding; it could be present in the interior of the poreformed by the alpha helices of TSPO or in the membrane. In theabsence of the crystal structure of TSPO, this question remains to beanswered. Moreover, the clustering of TSPO upon hormonal stimula-tion would increase the occurrence of cholesterol aggregation andpossible cholesterol concentration in the OMM. This effect wouldcause alterations in both membrane permeability and ROS signaling,as is seen following hormonal stimulation. This would also alloweasier access of cholesterol into the OMM since free cholesteroldiffuses more readily into and out of areas of high cholesterolconcentration. The clustering of TSPO could be conceived of as areservoir of cholesterol molecules, which would be available fortransfer to the IMM and use in steroidogenesis. The creation of amolten globule structure by StAR on the OMM is proposed to alter theOMM. This StAR-OMM interaction could cooperate with the clusteringof TSPO, facilitating the formation of the “active” TSPO structures inwhich cholesterol could be transferred to the IMM and allowing StARto directly transfer cholesterol to a cholesterol-rich area. However, thishypothesis remains to be tested. Ultimately, it is unclear howcholesterol locates and binds to CYP11A1 after it moves into the IMM.

In addition to these questions, it must be taken into considerationthat steroidogenic cells have been shown to rapidly undergomorphological changes in response to hormone treatment. Thiswell-established phenomenon further suggests that additional pro-tein–protein interactions could be necessary for trafficking ofcholesterol and for the reorganization of the mitochondria inpreparation for optimal steroidogenesis.

Acknowledgments

This work was supported by National Institutes of Health grantsES07747 and HD37031. V.P. was also supported by a Canada ResearchChair in Biochemical Pharmacology.Wewould like to thankMr. DanielMartinez-Arguelles for his assistance with the graphics.

References

[1] P.F. Hall, Role of cytochromes P-450 in the biosynthesis of steroid hormones,Vitam. Horm. 42 (1985) 315–368.

[2] E.R. Simpson, M.R. Waterman, Regulation by ACTH of steroid hormonebiosynthesis in the adrenal cortex, Can. J. Biochem. Cell Biol. 61 (1983) 692–707.

[3] C. Jefcoate, High-fluxmitochondrial cholesterol trafficking, a specialized functionof the adrenal cortex, J. Clin. Invest 110 (2002) 881–890.

[4] L.D. Garren, G.N. Gill, H. Masui, G.M. Walton, On the mechanism of action ofACTH, Recent Prog. Horm. Res. 27 (1971) 433–478.

[5] V. Papadopoulos, M. Baraldi, T.R. Guilarte, T.B. Knudsen, J.J. Lacapere, P.Lindemann, M.D. Norenberg, D. Nutt, A. Weizman, M.R. Zhang, M. Gavish,Translocator protein (18 kDa): new nomenclature for the peripheral-typebenzodiazepine receptor based on its structure and molecular function, TrendsPharmacol. Sci. 27 (2006) 402–409.

[6] E. Ikonen, Cellular cholesterol trafficking and compartmentalization, Nat. Rev.,Mol. Cell Biol. 9 (2008) 125–138.

[7] D.E. Warnock, C. Roberts, M.S. Lutz, W.A. Blackburn, W.W. Young Jr., J.U.Baenziger, Determination of plasma membrane lipid mass and composition incultured Chinese hamster ovary cells using high gradient magnetic affinitychromatography, J. Biol. Chem. 268 (1993) 10145–10153.

[8] S. Mukherjee, X. Zha, I. Tabas, F.R. Maxfield, Cholesterol distribution in livingcells: fluorescence imaging using dehydroergosterol as a fluorescent cholesterolanalog, Biophys. J. 75 (1998) 1915–1925.

[9] J.L. Goldstein, R.A. Bose-Boyd, M.S. Brown, Protein sensors for membrane sterols,Cell 124 (2006) 35–46.

[10] R. Raghow, C. Yellaturu, X. Deng, E.A. Park, M.B. Elam, SREBPs: the crossroads ofphysiological and pathological lipid homeostasis, Trends Endocrinol. Metab. 19(2008) 65–73.

[11] G.C. Ness, C.M. Chambers, Feedback and hormonal regulation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase: the concept of cholesterolbuffering capacity, Proc. Soc. Exp. Biol. Med. 224 (2000) 8–19.

[12] A.S. Plump, S.K. Erickson, W. Weng, J.S. Partin, J.L. Breslow, D.L. Williams,Apolipoprotein A-I is required for cholesteryl ester accumulation in steroido-genic cells and for normal adrenal steroid production, J. Clin. Invest 97 (1996)2660–2671.

[13] C.A. Mannella, K. Buttle, B.K. Rath, M.Marko, Electronmicroscopic tomography ofrat-liver mitochondria and their interaction with the endoplasmic reticulum,Biofactors 8 (1998) 225–228.

[14] S.S. Stone, M.C. Levin, P. Zhou, J. Han, T.C. Walther, R.V. Farese Jr., The endoplasmicreticulum enzyme, DGAT2, is found in mitochondria-associated membranes andhas a mitochondrial targeting signal that promotes its association withmitochondria, J. Biol. Chem. 284 (8) (2008) 5352–5361.

[15] Y. Lange, J. Ye, M. Rigney, T.L. Steck, Regulation of endoplasmic reticulumcholesterol by plasma membrane cholesterol, J. Lipid Res. 40 (1999) 2264–2270.

[16] M.A. Connelly, D.L. Williams, SR-BI and cholesterol uptake into steroidogeniccells, Trends Endocrinol. Metab. 14 (2003) 467–472.

[17] A.H. Verschoor-Klootwyk, L. Verschoor, S. Azhar, G.M. Reaven, Role of exogenouscholesterol in regulation of adrenal steroidogenesis in the rat, J. Biol. Chem. 257(1982) 7666–7671.

[18] E.J. Ungewickell, L. Hinrichsen, Endocytosis: clathrin-mediated membranebudding, Curr. Opin. Cell Biol. 19 (2007) 417–425.

[19] J.W. Murray, A.W. Wolkoff, Roles of the cytoskeleton and motor proteins inendocytic sorting, Adv. Drug Deliv. Rev. 55 (2003) 1385–1403.

[20] I. Jordens, M. Marsman, C. Kuijl, J. Neefjes, Rab proteins, connecting transport andvesicle fusion, Traffic 6 (2005) 1070–1077.

[21] J. Schultz, T. Doerks, C.P. Ponting, R.R. Copley, P. Bork, More than 1,000 putativenew human signalling proteins revealed by EST data mining, Nat. Genet. 25(2000) 201–204.

[22] B.L. Grosshans, D. Ortiz, P. Novick, Rabs and their effectors: achieving specificityin membrane traffic, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 11821–11827.

[23] S. Mukherjee, F.R. Maxfield, Lipid and cholesterol trafficking in NPC, Biochim.Biophys. Acta 1685 (2004) 28–37.

[24] F.B. Kraemer, W.J. Shen, S. Patel, J. Osuga, S. Ishibashi, S. Azhar, The LDL receptor isnot necessary for acute adrenal steroidogenesis in mouse adrenocortical cells,Am. J. Physiol Endocrinol. Metab. 292 (2007) E408–E412.

[25] J.D. Foster, J.F. Strauss III, L.G. Paavola, Cellular events involved in hormonalcontrol of receptor-mediated endocytosis: regulation occurs at multiple sites inthe low density lipoprotein pathway, including steps beyond the receptor,Endocrinology 132 (1993) 337–350.

[26] H. Chen, J. Yang, P.S. Low, J.X. Cheng, Cholesterol level regulates endosomemotility via Rab proteins, Biophys. J. 94 (2008) 1508–1520.

[27] S. Acton, A. Rigotti, K.T. Landschulz, S. Xu, H.H. Hobbs, M. Krieger, Identification ofscavenger receptor SR-BI as a high density lipoprotein receptor, Science 271(1996) 518–520.

[28] M. Werder, C.H. Han, E. Wehrli, D. Bimmler, G. Schulthess, H. Hauser, Role ofscavenger receptors SR-BI and CD36 in selective sterol uptake in the smallintestine, Biochemistry 40 (2001) 11643–11650.

[29] J.A. Hamilton, Fast flip-flop of cholesterol and fatty acids in membranes:implications for membrane transport proteins, Curr. Opin. Lipidol. 14 (2003)263–271.

[30] M.A. Connelly, G. Kellner-Weibel, G.H. Rothblat, D.L. Williams, SR-BI-directedHDL-cholesteryl ester hydrolysis, J. Lipid Res. 44 (2003) 331–341.

[31] F.B. Kraemer, W.J. Shen, Hormone-sensitive lipase: control of intracellular tri-(di-)acylglycerol and cholesteryl ester hydrolysis, J. Lipid Res. 43 (2002)1585–1594.

[32] S.J. Yeaman, Hormone-sensitive lipase—new roles for an old enzyme, Biochem. J.379 (2004) 11–22.

[33] F.B. Kraemer, W.J. Shen, K. Harada, S. Patel, J. Osuga, S. Ishibashi, S. Azhar,Hormone-sensitive lipase is required for high-density lipoprotein cholesterylester-supported adrenal steroidogenesis, Mol. Endocrinol. 18 (2004) 549–557.

[34] J. Osuga, S. Ishibashi, T. Oka, H. Yagyu, R. Tozawa, A. Fujimoto, F. Shionoiri, N.Yahagi, F.B. Kraemer, O. Tsutsumi, N. Yamada, Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not inobesity, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 787–792.

[35] W.J. Shen, S. Patel, V. Natu, R. Hong, J. Wang, S. Azhar, F.B. Kraemer, Interactionof hormone-sensitive lipase with steroidogenic acute regulatory protein:facilitation of cholesterol transfer in adrenal, J. Biol. Chem. 278 (2003)43870–43876.

[36] S. Murphy, S. Martin, R.G. Parton, Lipid droplet-organelle interactions; sharingthe fats, Biochim. Biophys. Acta (2008), doi:10.1016/j.bbalip.2008.07.004.

656 M.B. Rone et al. / Biochimica et Biophysica Acta 1791 (2009) 646–658

[37] T.Y. Chang, C.C. Chang, D. Cheng, Acyl-coenzyme A:cholesterol acyltransferase,Annu. Rev. Biochem. 66 (1997) 613–638.

[38] J.F. Strauss III, T. Kishida, L.K. Christenson, T. Fujimoto, H. Hiroi, START domainproteins and the intracellular trafficking of cholesterol in steroidogenic cells, Mol.Cell Endocrinol. 202 (2003) 59–65.

[39] P. Liu, R. Bartz, J.K. Zehmer, Y.S. Ying, M. Zhu, G. Serrero, R.G. Anderson, Rab-regulated interaction of early endosomes with lipid droplets, Biochim. Biophys.Acta 1773 (2007) 784–793.

[40] S. Martin, K. Driessen, S.J. Nixon, M. Zerial, R.G. Parton, Regulated localization ofRab18 to lipid droplets: effects of lipolytic stimulation and inhibition of lipiddroplet catabolism, J. Biol. Chem. 280 (2005) 42325–42335.

[41] R.E. Soccio, J.L. Breslow, Intracellular cholesterol transport, Arterioscler. Thromb.Vasc. Biol. 24 (2004) 1150–1160.

[42] F. Schroeder, B.P. Atshaves, A.L. McIntosh, A.M. Gallegos, S.M. Storey, R.D. Parr, J.R.Jefferson, J.M. Ball, A.B. Kier, Sterol carrier protein-2: new roles in regulating lipidrafts and signaling, Biochim. Biophys. Acta 1771 (2007) 700–718.

[43] A.M. Gallegos, J.K. Schoer, O. Starodub, A.B. Kier, J.T. Billheimer, F. Schroeder, Apotential role for sterol carrier protein-2 in cholesterol transfer to mitochondria,Chem. Phys. Lipids 105 (2000) 9–29.

[44] A.M. Gallegos, B.P. Atshaves, S.M. Storey, O. Starodub, A.D. Petrescu, H. Huang, A.L.McIntosh, G.G. Martin, H. Chao, A.B. Kier, F. Schroeder, Gene structure,intracellular localization, and functional roles of sterol carrier protein-2, Prog.Lipid Res. 40 (2001) 498–563.

[45] C.L. Baum, E.J. Reschly, A.K. Gayen, M.E. Groh, K. Schadick, Sterol carrier protein-2overexpression enhances sterol cycling and inhibits cholesterol ester synthesisand high density lipoprotein cholesterol secretion, J. Biol. Chem. 272 (1997)6490–6498.

[46] D. Moncecchi, E.J. Murphy, D.R. Prows, F. Schroeder, Sterol carrier protein-2expression in mouse L-cell fibroblasts alters cholesterol uptake, Biochim.Biophys. Acta 1302 (1996) 110–116.

[47] U. Seedorf, M. Raabe, P. Ellinghaus, F. Kannenberg, M. Fobker, T. Engel, S. Denis,F. Wouters, K.W. Wirtz, R.J. Wanders, N. Maeda, G. Assmann, Defectiveperoxisomal catabolism of branched fatty acyl coenzyme A in mice lackingthe sterol carrier protein-2/sterol carrier protein-x gene function, Genes Dev. 12(1998) 1189–1201.

[48] F. Alpy, C. Tomasetto, Give lipids a START: the StAR-related lipid transfer (START)domain in mammals, J. Cell. Sci. 118 (2005) 2791–2801.

[49] Y. Tsujishita, J.H. Hurley, Structure and lipid transport mechanism of a StAR-related domain, Nat. Struct. Biol. 7 (2000) 408–414.

[50] C. Tomasetto, C. Regnier, C. Moog-Lutz, M.G. Mattei, M.P. Chenard, R. Lidereau, P.Basset, M.C. Rio, Identification of four novel human genes amplified andoverexpressed in breast carcinoma and localized to the q11–q21.3 region ofchromosome 17, Genomics 28 (1995) 367–376.

[51] C. Moog-Lutz, C. Tomasetto, C.H. Regnier, C. Wendling, Y. Lutz, D. Muller, M.P.Chenard, P. Basset, M.C. Rio, MLN64 exhibits homology with the steroidogenicacute regulatory protein (STAR) and is over-expressed in human breastcarcinomas, Int. J. Cancer 71 (1997) 183–191.

[52] F. Alpy, M.E. Stoeckel, A. Dierich, J.M. Escola, C. Wendling, M.P. Chenard, M.T.Vanier, J. Gruenberg, C. Tomasetto, M.C. Rio, The steroidogenic acute regulatoryprotein homolog MLN64, a late endosomal cholesterol-binding protein, J. Biol.Chem. 276 (2001) 4261–4269.

[53] M. Zhang, P. Liu, N.K. Dwyer, L.K. Christenson, T. Fujimoto, F. Martinez, M. Comly,J.A. Hanover, E.J. Blanchette-Mackie, J.F. Strauss III, MLN64 mediates mobilizationof lysosomal cholesterol to steroidogenic mitochondria, J. Biol. Chem. 277 (2002)33300–33310.

[54] J.F. Strauss III, P. Liu, L.K. Christenson, H.Watari, Sterols and intracellular vesiculartrafficking: lessons from the study of NPC1, Steroids 67 (2002) 947–951.

[55] S.L. Sturley, M.C. Patterson, W. Balch, L. Liscum, The pathophysiology andmechanisms of NP-C disease, Biochim. Biophys. Acta 1685 (2004) 83–87.

[56] N.Y. Gevry, B.D. Murphy, The role and regulation of the Niemann–Pick C1 gene inadrenal steroidogenesis, Endocr. Res. 28 (2002) 403–412.

[57] Z. Xu, W. Farver, S. Kodukula, J. Storch, Regulation of sterol transport betweenmembranes and NPC2, Biochemistry 47 (2008) 11134–11143.

[58] R.E. Infante, M.L. Wang, A. Radhakrishnan, H.J. Kwon, M.S. Brown, J.L. Goldstein,NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipidbilayers, a step in cholesterol egress from lysosomes, Proc. Natl. Acad. Sci. U. S. A.105 (2008) 15287–15292.

[59] H. Watari, E.J. Blanchette-Mackie, N.K. Dwyer, G. Sun, J.M. Glick, S. Patel, E.B.Neufeld, P.G. Pentchev, J.F. Strauss III, NPC1-containing compartment of humangranulosa-lutein cells: a role in the intracellular trafficking of cholesterolsupporting steroidogenesis, Exp. Cell Res. 255 (2000) 56–66.

[60] P.J. Espenshade, A.L. Hughes, Regulation of sterol synthesis in eukaryotes, Annu.Rev. Genet. 41 (2007) 401–427.

[61] L.A. Pon, N.R. Orme-Johnson, Acute stimulation of steroidogenesis in corpusluteum and adrenal cortex by peptide hormones. Rapid induction of a similarprotein in both tissues, J. Biol. Chem. 261 (1986) 6594–6599.

[62] L.A. Pon, J.A. Hartigan, N.R. Orme-Johnson, Acute ACTH regulation of adrenalcorticosteroid biosynthesis. Rapid accumulation of a phosphoprotein, J. Biol.Chem. 261 (1986) 13309–13316.

[63] D. Lin, T. Sugawara, J.F. Strauss III, B.J. Clark, D.M. Stocco, P. Saenger, A. Rogol, W.L.Miller, Role of steroidogenic acute regulatory protein in adrenal and gonadalsteroidogenesis, Science 267 (1995) 1828–1831.

[64] A. Sierra, Neurosteroids: the StAR protein in the brain, J. Neuroendocrinol. 16(2004) 787–793.

[65] B.J. Clark, J. Wells, S.R. King, D.M. Stocco, The purification, cloning, and expressionof a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse

Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein(StAR), J. Biol. Chem. 269 (1994) 28314–28322.

[66] T. Sugawara, J.A. Holt, D. Driscoll, J.F. Strauss III, D. Lin, W.L. Miller, D. Patterson,K.P. Clancy, I.M. Hart, B.J. Clark, Human steroidogenic acute regulatory protein:functional activity in COS-1 cells, tissue-specific expression, and mapping of thestructural gene to 8p11.2 and a pseudogene to chromosome 13, Proc. Natl. Acad.Sci. U. S. A. 92 (1995) 4778–4782.

[67] W.L. Miller, Steroidogenic acute regulatory protein (StAR), a novel mitochondrialcholesterol transporter, Biochim. Biophys. Acta 1771 (2007) 663–676.

[68] F. Arakane, T. Sugawara, H. Nishino, Z. Liu, J.A. Holt, D. Pain, D.M. Stocco, W.L.Miller, J.F. Strauss III, Steroidogenic acute regulatory protein (StAR) retainsactivity in the absence of its mitochondrial import sequence: implicationsfor the mechanism of StAR action, Proc. Natl. Acad. Sci. U. S. A. 93 (1996)13731–13736.

[69] C.B. Kallen, J.T. Billheimer, S.A. Summers, S.E. Stayrook, M. Lewis, J.F. Strauss III,Steroidogenic acute regulatory protein (StAR) is a sterol transfer protein, J. Biol.Chem. 273 (1998) 26285–26288.

[70] H.S. Bose, V.R. Lingappa, W.L. Miller, The steroidogenic acute regulatory protein,StAR, works only at the outer mitochondrial membrane, Endocr. Res. 28 (2002)295–308.

[71] B.Y. Baker, R.F. Epand, R.M. Epand, W.L. Miller, Cholesterol binding does notpredict activity of the steroidogenic acute regulatory protein, StAR, J. Biol. Chem.282 (2007) 10223–10232.

[72] E. Barbar, J.G. LeHoux, P. Lavigne, Toward the NMR structure of StAR, Mol. CellEndocrinol. 300 (2009) 89–93.

[73] R.E. Soccio, R.M. Adams, M.J. Romanowski, E. Sehayek, S.K. Burley, J.L. Breslow,The cholesterol-regulated StarD4 gene encodes a StAR-related lipid transferproteinwith two closely related homologues, StarD5 and StarD6, Proc. Natl. Acad.Sci. U. S. A. 99 (2002) 6943–6948.

[74] V. Papadopoulos, Peripheral-type benzodiazepine/diazepam binding inhibitorreceptor: biological role in steroidogenic cell function, Endocr. Rev. 14 (1993)222–240.

[75] R.R. Anholt, E.B. De Souza, M.L. Oster-Granite, S.H. Snyder, Peripheral-typebenzodiazepine receptors: autoradiographic localization in whole-body sectionsof neonatal rats, J. Pharmacol. Exp. Ther. 233 (1985) 517–526.

[76] V. Papadopoulos, A.G. Mukhin, E. Costa, K.E. Krueger, The peripheral-typebenzodiazepine receptor is functionally linked to Leydig cell steroidogenesis,J. Biol. Chem. 265 (1990) 3772–3779.

[77] R.R. Anholt, U. Aebi, P.L. Pedersen, S.H. Snyder, Solubilization and reassembly ofthe mitochondrial benzodiazepine receptor, Biochemistry 25 (1986) 2120–2125.

[78] M.J. Woods, D.M. Zisterer, D.C. Williams, Two cellular and subcellular locationsfor the peripheral-type benzodiazepine receptor in rat liver, Biochem. Pharmacol.51 (1996) 1283–1292.

[79] F.G. Le, M.L. Perrier, N. Vaucher, F. Imbault, A. Flamier, J. Benavides, A. Uzan, C.Renault, M.C. Dubroeucq, C. Gueremy, Peripheral benzodiazepine binding sites:effect of PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoqui-nolinecarboxamide. I. In vitro studies, Life Sci. 32 (1983) 1839–1847.

[80] A. Verma, J.S. Nye, S.H. Snyder, Porphyrins are endogenous ligands for themitochondrial (peripheral-type) benzodiazepine receptor, Proc. Natl. Acad. Sci.U. S. A. 84 (1987) 2256–2260.

[81] P. Bovolin, J. Schlichting, M. Miyata, C. Ferrarese, A. Guidotti, H. Alho, Distributionand characterization of diazepam binding inhibitor (DBI) in peripheral tissues ofrat, Regul. Pept. 29 (1990) 267–281.

[82] V. Papadopoulos, A. Berkovich, K.E. Krueger, The role of diazepam bindinginhibitor and its processing products at mitochondrial benzodiazepine receptors:regulation of steroid biosynthesis, Neuropharmacology 30 (1991) 1417–1423.

[83] A.G. Mukhin, V. Papadopoulos, E. Costa, K.E. Krueger, Mitochondrial benzodia-zepine receptors regulate steroid biosynthesis, Proc. Natl. Acad. Sci. U. S. A. 86(1989) 9813–9816.

[84] E.R. Barnea, F. Fares, M. Gavish, Modulatory action of benzodiazepines on humanterm placental steroidogenesis in vitro, Mol. Cell Endocrinol. 64 (1989) 155–159.

[85] K.E. Krueger, V. Papadopoulos, Peripheral-type benzodiazepine receptorsmediate translocation of cholesterol from outer to inner mitochondrialmembranes in adrenocortical cells, J. Biol. Chem. 265 (1990) 15015–15022.

[86] J.J. Lacapere, F. Delavoie, H. Li, G. Peranzi, J. Maccario, V. Papadopoulos, B. Vidic,Structural and functional study of reconstituted peripheral benzodiazepinereceptor, Biochem. Biophys. Res. Commun. 284 (2001) 536–541.

[87] H. Li, Z. Yao, B. Degenhardt, G. Teper, V. Papadopoulos, Cholesterol binding at thecholesterol recognition/interaction amino acid consensus (CRAC) of theperipheral-type benzodiazepine receptor and inhibition of steroidogenesis byan HIV TAT-CRAC peptide, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 1267–1272.

[88] J.K. Scott, G.P. Smith, Searching for peptide ligands with an epitope library,Science 249 (1990) 386–390.

[89] S. Fawell, J. Seery, Y. Daikh, C. Moore, L.L. Chen, B. Pepinsky, J. Barsoum, Tat-mediated delivery of heterologous proteins into cells, Proc. Natl. Acad. Sci. U. S. A.91 (1994) 664–668.

[90] M. Gazouli, Z. Han, V. Papadopoulos, Identification of a peptide antagonist to theperipheral-type benzodiazepine receptor that inhibits hormone-stimulatedLeydig cell steroid formation, J. Pharmacol. Exp. Ther. 303 (2002) 627–632.

[91] A.M. Falchi, B. Battetta, F. Sanna, M. Piludu, V. Sogos, M. Serra, M. Melis, M.Putzolu, G. Diaz, Intracellular cholesterol changes induced by translocatorprotein (18 kDa) TSPO/PBR ligands, Neuropharmacology 53 (2007) 318–329.

[92] D.A. Freeman, Constitutive steroidogenesis in the R2C Leydig tumor cell line ismaintained by the adenosine 3′,5′-cyclic monophosphate-independent produc-tion of a cycloheximide-sensitive factor that enhances mitochondrial pregneno-lone biosynthesis, Endocrinology 120 (1987) 124–132.

657M.B. Rone et al. / Biochimica et Biophysica Acta 1791 (2009) 646–658

[93] V. Papadopoulos, H. Amri, H. Li, N. Boujrad, B. Vidic, M. Garnier, Targeteddisruption of the peripheral-type benzodiazepine receptor gene inhibitssteroidogenesis in the R2C Leydig tumor cell line, J. Biol. Chem. 272 (1997)32129–32135.

[94] V. Papadopoulos, H. Amri, N. Boujrad, C. Cascio, M. Culty, M. Garnier, M.Hardwick, H. Li, B. Vidic, A.S. Brown, J.L. Reversa, J.M. Bernassau, K. Drieu,Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis,Steroids 62 (1997) 21–28.

[95] N. Boujrad, B. Vidic, V. Papadopoulos, Acute action of choriogonadotropin onLeydig tumor cells: changes in the topography of the mitochondrial peripheral-type benzodiazepine receptor, Endocrinology 137 (1996) 5727–5730.

[96] V. Papadopoulos, N. Boujrad, M.D. Ikonomovic, P. Ferrara, B. Vidic, Topography ofthe Leydig cell mitochondrial peripheral-type benzodiazepine receptor, Mol. CellEndocrinol. 104 (1994) R5–R9.

[97] C.T. Privalle, J.F. Crivello, C.R. Jefcoate, Regulation of intramitochondrialcholesterol transfer to side-chain cleavage cytochrome P-450 in rat adrenalgland, Proc. Natl. Acad. Sci. U. S. A. 80 (1983) 702–706.

[98] F. Delavoie, H. Li, M. Hardwick, J.C. Robert, C. Giatzakis, G. Peranzi, Z.X. Yao, J.Maccario, J.J. Lacapere, V. Papadopoulos, In vivo and in vitro peripheral-typebenzodiazepine receptor polymerization: functional significance in drug ligandand cholesterol binding, Biochemistry 42 (2003) 4506–4519.

[99] S. Raha, A.T. Myint, L. Johnstone, B.H. Robinson, Control of oxygen free radicalformation frommitochondrial complex I: roles for protein kinase A and pyruvatedehydrogenase kinase, Free Radic. Biol. Med. 32 (2002) 421–430.

[100] S. Murail, J.C. Robert, Y.M. Coic, J.M. Neumann, M.A. Ostuni, Z.X. Yao, V.Papadopoulos, N. Jamin, J.J. Lacapere, Secondary and tertiary structures of thetransmembrane domains of the translocator protein TSPO determined by NMR.Stabilization of the TSPO tertiary fold upon ligand binding, Biochim. Biophys.Acta 1778 (2008) 1375–1381.

[101] M. Culty, H. Li, N. Boujrad, H. Amri, B. Vidic, J.M. Bernassau, J.L. Reversat, V.Papadopoulos, In vitro studies on the role of the peripheral-type benzodiazepinereceptor in steroidogenesis, J. Steroid Biochem. Mol. Biol. 69 (1999) 123–130.

[102] A. Doble, O. Ferris, M.C. Burgevin, J. Menager, A. Uzan, M.C. Dubroeucq, C. Renault,C. Gueremy, G. Le Fur, Photoaffinity labeling of peripheral-type benzodiazepine-binding sites, Mol. Pharmacol. 31 (1987) 42–49.

[103] J. Riond, N. Vita, G. Le Fur, P. Ferrara, Characterization of a peripheral-typebenzodiazepine-binding site in the mitochondria of Chinese hamster ovary cells,FEBS Lett. 245 (1989) 238–244.

[104] M.W. McEnery, A.M. Snowman, R.R. Trifiletti, S.H. Snyder, Isolation of themitochondrial benzodiazepine receptor: associationwith the voltage-dependentanion channel and the adenine nucleotide carrier, Proc. Natl. Acad. Sci. U. S. A 89(1992) 3170–3174.

[105] M. Garnier, A.B. Dimchev, N. Boujrad, J.M. Price, N.A. Musto, V. Papadopoulos, Invitro reconstitution of a functional peripheral-type benzodiazepine receptorfrom mouse Leydig tumor cells, Mol. Pharmacol. 45 (1994) 201–211.

[106] V. Shoshan-Barmatz, N. Keinan, H. Zaid, Uncovering the role of VDAC in theregulation of cell life and death, J. Bioenerg. Biomembr. 40 (2008) 183–191.

[107] J.E. Kokoszka, K.G. Waymire, S.E. Levy, J.E. Sligh, J. Cai, D.P. Jones, G.R. MacGregor,D.C. Wallace, The ADP/ATP translocator is not essential for the mitochondrialpermeability transition pore, Nature 427 (2004) 461–465.

[108] M. Azuma, Y. Kabe, C. Kuramori, M. Kondo, Y. Yamaguchi, H. Handa, Adeninenucleotide translocator transports haem precursors intomitochondria, PLoS ONE3 (2008) e3070.

[109] L. Veenman, Y. Shandalov, M. Gavish, VDAC activation by the 18 kDa translocatorprotein (TSPO), implications for apoptosis, J. Bioenerg. Biomembr. 40 (2008)199–205.

[110] I. Golani, A. Weizman, S. Leschiner, I. Spanier, N. Eckstein, R. Limor, J. Yanai, K.Maaser, H. Scherubl, G. Weisinger, M. Gavish, Hormonal regulation of peripheralbenzodiazepine receptor binding properties is mediated by subunit interaction,Biochemistry 40 (2001) 10213–10222.

[111] A.M. Campbell, S.H. Chan, The voltage dependent anion channel affectsmitochondrial cholesterol distribution and function, Arch. Biochem. Biophys.466 (2007) 203–210.

[112] S. Hiller, R.G. Garces, T.J. Malia, V.Y. Orekhov, M. Colombini, G. Wagner, Solutionstructure of the integral human membrane protein VDAC-1 in detergentmicelles, Science 321 (2008) 1206–1210.

[113] L.A. West, R.D. Horvat, D.A. Roess, B.G. Barisas, J.L. Juengel, G.D. Niswender,Steroidogenic acute regulatory protein and peripheral-type benzodiazepinereceptor associate at the mitochondrial membrane, Endocrinology 142 (2001)502–505.

[114] R.L. Bogan, T.L. Davis, G.D. Niswender, Peripheral-type benzodiazepine receptor(PBR) aggregation and absence of steroidogenic acute regulatory protein (StAR)/PBR association in themitochondrialmembrane asdetermined by bioluminescenceresonance energy transfer (BRET), J. Steroid Biochem. Mol. Biol. 104 (2007) 61–67.

[115] T. Hauet, Z.X. Yao, H.S. Bose, C.T. Wall, Z. Han, W. Li, D.B. Hales, W.L. Miller, M.Culty, V. Papadopoulos, Peripheral-type benzodiazepine receptor-mediatedaction of steroidogenic acute regulatory protein on cholesterol entry into Leydigcell mitochondria, Mol. Endocrinol. 19 (2005) 540–554.

[116] N. Ariyoshi, Y.C. Kim, I. Artemenko, K.K. Bhattacharyya, C.R. Jefcoate, Character-ization of the rat Star gene that encodes the predominant 3.5-kilobase pairmRNA. ACTH stimulation of adrenal steroids in vivo precedes elevation of StarmRNA and protein, J. Biol. Chem. 273 (1998) 7610–7619.

[117] I.P. Artemenko, D. Zhao, D.B. Hales, K.H. Hales, C.R. Jefcoate, Mitochondrialprocessing of newly synthesized steroidogenic acute regulatory protein (StAR),but not total StAR, mediates cholesterol transfer to cytochrome P450 side chaincleavage enzyme in adrenal cells, J. Biol. Chem. 276 (2001) 46583–46596.

[118] R.C. Tuckey, M.J. Headlam, H.S. Bose, W.L. Miller, Transfer of cholesterol betweenphospholipid vesicles mediated by the steroidogenic acute regulatory protein(StAR), J. Biol. Chem. 277 (2002) 47123–47128.

[119] A. Roostaee, E. Barbar, J.G. LeHoux, P. Lavigne, Cholesterol binding is aprerequisite for the activity of the steroidogenic acute regulatory protein(StAR), Biochem. J. 412 (2008) 553–562.

[120] A.D. Petrescu, A.M. Gallegos, Y. Okamura, J.F. Strauss III, F. Schroeder,Steroidogenic acute regulatory protein binds cholesterol and modulatesmitochondrial membrane sterol domain dynamics, J. Biol. Chem. 276 (2001)36970–36982.

[121] A.P. Mathieu, A. Fleury, L. Ducharme, P. Lavigne, J.G. LeHoux, Insights intosteroidogenic acute regulatory protein (StAR)-dependent cholesterol transfer inmitochondria: evidence from molecular modeling and structure-based thermo-dynamics supporting the existence of partially unfolded states of StAR, J. Mol.Endocrinol. 29 (2002) 327–345.

[122] J.J. Lacapere, V. Papadopoulos, Peripheral-type benzodiazepine receptor: struc-ture and function of a cholesterol-binding protein in steroid and bile acidbiosynthesis, Steroids 68 (2003) 569–585.

[123] H. Li, B. Degenhardt, D. Tobin, Z.X. Yao, K. Tasken, V. Papadopoulos, Identification,localization, and function in steroidogenesis of PAP7: a peripheral-typebenzodiazepine receptor- and PKA (RIalpha)-associated protein,Mol. Endocrinol.15 (2001) 2211–2228.

[124] J. Liu, H. Li, V. Papadopoulos, PAP7, a PBR/PKA-RIalpha-associated protein: a newelement in the relay of the hormonal induction of steroidogenesis, J. SteroidBiochem. Mol. Biol. 85 (2003) 275–283.

[125] K.L. Dodge-Kafka, A. Bauman, M.S. Kapiloff, A-kinase anchoring proteins as thebasis for cAMP signaling, Handb. Exp. Pharmacol. (2008) 3–14.

[126] K.J. Catt, J.P. Harwood, R.N. Clayton, T.F. Davies, V. Chan, M. Katikineni, K. Nozu,M.L. Dufau, Regulation of peptide hormone receptors and gonadal steroidogen-esis, Recent Prog. Horm. Res. 36 (1980) 557–662.

[127] M.E. Whalin, N. Boujrad, V. Papadopoulos, K.E. Krueger, Studies on thephosphorylation of the 18 kDa mitochondrial benzodiazepine receptor protein,J. Recept. Res. 14 (1994) 217–228.

[128] C. Poderoso, P. Maloberti, A. Duarte, I. Neuman, C. Paz, F.C. Maciel, E.J. Podesta,Hormonal activation of a kinase cascade localized at themitochondria is requiredfor StAR protein activity, Mol. Cell Endocrinol. 300 (1–2) (2009) 37–42.

[129] J. Liu, M.B. Rone, V. Papadopoulos, Protein–protein interactions mediatemitochondrial cholesterol transport and steroid biosynthesis, J. Biol. Chem. 281(2006) 38879–38893.

[130] M. Suchanek, A. Radzikowska, C. Thiele, Photo-leucine and photo-methionineallow identification of protein–protein interactions in living cells, Nat. Methods 2(2005) 261–267.

[131] M. Bose, R.M. Whittal, W.L. Miller, H.S. Bose, Steroidogenic activity of StARrequires contact withmitochondrial VDAC1 and phosphate carrier protein, J. Biol.Chem. 283 (2008) 8837–8845.

[132] H.S. Bose, T. Sugawara, J.F. Strauss III, W.L. Miller, The pathophysiology andgenetics of congenital lipoid adrenal hyperplasia. International Congenital LipoidAdrenal Hyperplasia Consortium, N. Engl. J. Med. 335 (1996) 1870–1878.

[133] K.M. Caron, S.C. Soo, W.C. Wetsel, D.M. Stocco, B.J. Clark, K.L. Parker, Targeteddisruption of the mouse gene encoding steroidogenic acute regulatory proteinprovides insights into congenital lipoid adrenal hyperplasia, Proc. Natl. Acad. Sci.U. S. A. 94 (1997) 11540–11545.

[134] T. Hasegawa, L. Zhao, K.M. Caron, G. Majdic, T. Suzuki, S. Shizawa, H. Sasano, K.L.Parker, Developmental roles of the steroidogenic acute regulatory protein (StAR)as revealed by StAR knockout mice, Mol. Endocrinol. 14 (2000) 1462–1471.

[135] L.S. Kirschner, J.A. Carney, S.D. Pack, S.E. Taymans, C. Giatzakis, Y.S. Cho, Y.S.Cho-Chung, C.A. Stratakis, Mutations of the gene encoding the protein kinase Atype I-alpha regulatory subunit in patients with the Carney complex, Nat. Genet.26 (2000) 89–92.

[136] N.J. Sarlis, G.P. Chrousos, J.L. Doppman, J.A. Carney, C.A. Stratakis, Primarypigmented nodular adrenocortical disease: reevaluation of a patient with Carneycomplex 27 years after unilateral adrenalectomy, J. Clin. Endocrinol. Metab. 82(1997) 1274–1278.

[137] J. Liu, L. Matyakhina, Z. Han, F. Sandrini, T. Bei, C.A. Stratakis, V. Papadopoulos,Molecular cloning, chromosomal localization of human peripheral-type benzo-diazepine receptor and PKA regulatory subunit type 1A (PRKAR1A)-associatedprotein PAP7, and studies in PRKAR1A mutant cells and tissues, FASEB J. 17(2003) 1189–1191.

[138] C. Corpechot, P. Robel, M. Axelson, J. Sjovall, E.E. Baulieu, Characterization andmeasurement of dehydroepiandrosterone sulfate in rat brain, Proc. Natl. Acad.Sci. U. S. A. 78 (1981) 4704–4707.

[139] C. Corpechot, M. Synguelakis, S. Talha, M. Axelson, J. Sjovall, R. Vihko, E.E. Baulieu,P. Robel, Pregnenolone and its sulfate ester in the rat brain, Brain Res. 270 (1983)119–125.

[140] E.E. Baulieu, P. Robel, Neurosteroids: a new brain function? J. Steroid Biochem.Mol. Biol. 37 (1990) 395–403.

[141] A.C. Kuhlmann, T.R. Guilarte, Cellular and subcellular localization of peripheralbenzodiazepine receptors after trimethyltin neurotoxicity, J. Neurochem. 74(2000) 1694–1704.

[142] P. Casellas, S. Galiegue, A.S. Basile, Peripheral benzodiazepine receptors andmitochondrial function, Neurochem. Int. 40 (2002) 475–486.

[143] S.R. King, S.D. Ginsberg, T. Ishii, R.G. Smith, K.L. Parker, D.J. Lamb, Thesteroidogenic acute regulatory protein is expressed in steroidogenic cells ofthe day-old brain, Endocrinology 145 (2004) 4775–4780.

[144] E. Lavaque, A. Sierra, I. Azcoitia, L.M. Garcia-Segura, Steroidogenic acuteregulatory protein in the brain, Neuroscience 138 (2006) 741–747.

658 M.B. Rone et al. / Biochimica et Biophysica Acta 1791 (2009) 646–658

[145] E.E. Baulieu,Neurosteroids: anovel functionof the brain, Psychoneuroendocrinology23 (1998) 963–987.

[146] V. Papadopoulos, P. Guarneri, K.E. Kreuger, A. Guidotti, E. Costa, Pregnenolonebiosynthesis in C6-2B glioma cell mitochondria: regulation by a mitochondrialdiazepam binding inhibitor receptor, Proc. Natl. Acad. Sci. U. S. A. 89 (1992)5113–5117.

[147] P. Cornu, J. Benavides, B. Scatton, J.J. Hauw, J. Philippon, Increase in omega 3(peripheral-type benzodiazepine) binding site densities in different types ofhuman brain tumours. A quantitative autoradiography study, Acta Neurochir.(Wien.) 119 (1992) 146–152.

[148] E. Vowinckel, D. Reutens, B. Becher, G. Verge, A. Evans, T. Owens, J.P. Antel,PK11195 binding to the peripheral benzodiazepine receptor as a marker ofmicroglia activation in multiple sclerosis and experimental autoimmuneencephalomyelitis, J. Neurosci. Res. 50 (1997) 345–353.

[149] E.G. McGeer, E.A. Singh, P.L. McGeer, Peripheral-type benzodiazepine binding inAlzheimer disease, Alzheimer Dis. Assoc. Disord. 2 (1988) 331–336.

[150] H. Schoemaker, M. Morelli, P. Deshmukh, H.I. Yamamura, [3H]Ro5-4864benzodiazepine binding in the kainate lesioned striatum and Huntington'sdiseased basal ganglia, Brain Res. 248 (1982) 396–401.

[151] Minireview. J.V. Nadler, Kainic acid as a tool for the study of temporal lobeepilepsy, Life Sci. 29 (1981) 2031–2042.

[152] P. Lacor, P. Gandolfo, M.C. Tonon, E. Brault, I. Dalibert, M. Schumacher, J.Benavides, B. Ferzaz, Regulation of the expression of peripheral benzodiazepine

receptors and their endogenous ligands during rat sciatic nerve degenerationand regeneration: a role for PBR in neurosteroidogenesis, Brain Res. 815 (1999)70–80.

[153] R.C. Brown, Z. Han, C. Cascio, V. Papadopoulos, Oxidative stress-mediatedDHEA formation in Alzheimer's disease pathology, Neurobiol. Aging 24 (2003)57–65.

[154] L. Lecanu, W. Yao, G.L. Teper, Z.X. Yao, J. Greeson, V. Papadopoulos, Identificationof naturally occurring spirostenols preventing beta-amyloid-induced neurotoxicity,Steroids 69 (2004) 1–16.

[155] A. Guidotti, E. Dong, K. Matsumoto, G. Pinna, A.M. Rasmusson, E. Costa, Thesocially-isolated mouse: a model to study the putative role of allopregnanoloneand 5alpha-dihydroprogesterone in psychiatric disorders, Brain Res. Brain Res.Rev. 37 (2001) 110–115.

[156] D.P. van Kammen, A. Guidotti, M.E. Kelley, J. Gurklis, P. Guarneri, M.W. Gilbertson,J.K. Yao, J. Peters, E. Costa, CSF diazepam binding inhibitor and schizophrenia:clinical and biochemical relationships, Biol. Psychiatry 34 (1993) 515–522.

[157] J. Wang, B.D. Sykes, R.O. Ryan, Structural basis for the conformationaladaptability of apolipophorin III, a helix-bundle exchangeable apolipoprotein,Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 1188–1193.

[158] N. Jamin, J.M. Neumann, M.A. Ostuni, T.K. Vu, Z.X. Yao, S. Murail, J.C. Robert, C.Giatzakis, V. Papadopoulos, J.J. Lacapere, Characterization of the cholesterolrecognition amino acid consensus sequence of the peripheral-type benzodiaze-pine receptor, Mol. Endocrinol. 19 (2005) 588–594.