Peripheral-type benzodiazepine receptor: structure and function of a cholesterol-binding protein in steroid and bile acid biosynthesis

Cholesterol transport from the outer to the inner mitochondrial membrane is the rate-determining step in steroid and bile acid biosyntheses. Biochemical, pharmacological and molecular studies have demonstrated that the peripheral-type benzodiazepine receptor (PBR) is a five transmembrane domain mitochondrial protein involved in the regulation of cholesterol transport. PBR gene disruption in Leydig cells completely blocked cholesterol transport into mitochondria and steroid formation, while PBR expression in bacteria, devoid of endogenous PBR and cholesterol, induced cholesterol uptake and transport. Molecular modeling of PBR suggested that cholesterol might cross the membrane through the five helices of the receptor and that synthetic and endogenous ligands might bind to common sites in the cytoplasmic loops. A cholesterol recognition/interaction amino acid consensus (CRAC) sequence in the cytoplasmic carboxy-terminus of the PBR was identified by mutagenesis studies. In vitro reconstitution of PBR into proteoliposomes demonstrated that PBR binds both drug ligands and cholesterol with high affinity. In vivo polymeric forms of PBR were observed and polymer formation was reproduced in vitro, using recombinant PBR protein reconstituted into proteoliposomes, associated with an increase in drug ligand binding and reduction of cholesterol-binding capacity. This suggests that the various polymeric states of PBR might be part of a cycle mediating cholesterol uptake and release into the mitochondria, with PBR functioning as a cholesterol exchanger against steroid product(s) arising from cytochrome P450 action. Taking into account the widespread presence of PBR in many tissues, a more general role of PBR in intracellular cholesterol transport and compartmentalization might be considered.     

Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding,activity, and sterol-sensing of the human ABCG2 multidrug transporter

Human ABCG2 is a plasma membrane glycoprotein causing multidrug resistance in cancer. Membrane cholesterol and bile acids are efficient regulators of ABCG2 function, while the molecular nature of the sterol-sensing sites has not been elucidated. The cholesterol recognition amino acid consensus (CRAC, L/V-(X)_(1–5)-Y-(X)_(1–5)-R/K) sequence is one of the conserved motifs involved in cholesterol binding in several proteins. We have identified five potential CRAC motifs in the transmembrane domain of the human ABCG2 protein. In order to define their roles in sterol-sensing, the central tyrosines of these CRACs (Y413, 459, 469, 570 and 645) were mutated to S or F and the mutants were expressed both in insect and mammalian cells. We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. We suggest that the Y413 residue within a putative CRAC motif has a role in sterol-sensing and the ATPase/drug transport coupling in the ABCG2 multidrug transporter.     

Peripheral-type benzodiazepine receptor-mediated action of steroidogenic acute regulatory protein on cholesterol entry into leydig cell mitochondria

Hormone-induced steroid biosynthesis begins with the transfer of cholesterol from intracellular stores into mitochondria. Steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptor (PBR) have been implicated in this rate-determining step of steroidogenesis. MA-10 mouse Leydig tumor cells were treated with and without oligodeoxynucleotides (ODNs) antisense to PBR and StAR followed by treatment with saturating concentrations of human choriogonadotropin. Treatment with ODNs antisense but not missense for both proteins inhibited the respective protein expression and the ability of the cells to synthesize steroids in response to human choriogonadotropin. Treatment of the cells with either ODNs antisense to PBR or a transducible peptide antagonist to PBR resulted in inhibition of the accumulation of the mature mitochondrial 30-kDa StAR protein, suggesting that the presence of PBR is required for StAR import into mitochondria. Addition of in vitro transcribed/translated 37-kDa StAR or a fusion protein of Tom20 (translocase of outer membrane) and StAR (Tom/StAR) to mitochondria isolated from control cells increased pregnenolone formation. Mitochondria isolated from cells treated with ODNs antisense, but not missense, to PBR failed to form pregnenolone and respond to either StAR or Tom/StAR proteins. Reincorporation of in vitro transcribed/translated PBR, but not PBR missing the cholesterol-binding domain, into MA-10 mitochondria rescued the ability of the mitochondria to form steroids and the ability of the mitochondria to respond to StAR and Tom/StAR proteins. These data suggest that both StAR and PBR proteins are indispensable elements of the steroidogenic machinery and function in a coordinated manner to transfer cholesterol into mitochondria.     

Secondary and tertiary structures of the transmembrane domains of the translocator protein TSPO determined by NMR. Stabilization of the TSPO tertiary fold upon ligand binding

Numerous biological functions are attributed to the peripheral-type benzodiazepine receptor (PBR) recently renamed translocator protein (TSPO). The best characterized function is the translocation of cholesterol from the outer to inner mitochondrial membrane, which is a rate-determining step in steroid biosynthesis. TSPO drug ligands have been shown to stimulate pregnenolone formation by inducing TSPO-mediated translocation of cholesterol. Until recently, no direct structural data on this membrane protein was available. In a previous paper, we showed that a part of the mouse TSPO (mTSPO) C-terminal region adopts a helical conformation, the side-chain distribution of which provides a groove able to fit a cholesterol molecule. We report here on the overall structural properties of mTSPO. This study was first undertaken by dissecting the protein sequence and studying the conformation of five peptides encompassing the five putative transmembrane domains from ¹H-NMR data. The secondary structure of the recombinant protein in micelles was then studied using CD spectroscopy. In parallel, the stability of its tertiary fold was probed using ¹H-¹⁵N NMR. This study provides the first experimental evidence for a five-helix fold of mTSPO and shows that the helical conformation of each transmembrane domain is mainly formed through local short-range interactions. Our data show that, in micelles, mTSPO exhibits helix content close to what is expected but an unstable tertiary fold. They reveal that the binding of a drug ligand that stimulates cholesterol translocation is able to stabilize the mTSPO tertiary structure.     

Cholesterol and the interaction of proteins with membrane domains

Cholesterol is not uniformly distributed in biological membranes. One of the factors influencing the formation of cholesterol-rich domains in membranes is the unequal lateral distribution of proteins in membranes. Certain proteins are found in cholesterol-rich domains. In some of these cases, it is as a consequence of the proteins interacting directly with cholesterol. There are several structural features of a protein that result in the protein preferentially associating with cholesterol-rich domains. One of the best documented of these is certain types of lipidations. In addition, however, there are segments of a protein that can preferentially sequester cholesterol. We discuss two examples of these cholesterol-recognition elements: the cholesterol recognition/interaction amino acid consensus (CRAC) domain and the sterol-sensing domain (SSD). The requirements for a CRAC motif are quite flexible and predict that a large number of sequences could recognize cholesterol. There are, however, certain proteins that are known to interact with cholesterol-rich domains of cell membranes that have CRAC motifs, and synthetic peptides corresponding to these segments also promote the formation of cholesterol-rich domains. Modeling studies have provided a rationale for certain requirements of the CRAC motif. The SSD is a larger protein segment comprising five transmembrane domains. The amino acid sequence YIYF is found in several SSD and in certain other proteins for which there is evidence that they interact with cholesterol-rich domains. The CRAC sequences as well as YIYF are generally found adjacent to a transmembrane helical segment. These regions appear to have a strong influence of the localization of certain proteins into domains in biological membranes. In addition to the SSD, there is also a domain found in soluble proteins, the START domain, that binds lipids. Certain proteins with START domains specifically bind cholesterol and are believed to function in intracellular cholesterol transport. One of these proteins is StAR-D1, that also has a mitochondrial targeting sequence and plays an important role in delivering cholesterol to the mitochondria of steroidogenic cells.     

In vitro studies on the role of the peripheral-type benzodiazepine receptor in steroidogenesis

In vitro studies using isolated cells, mitochondria and submitochondrial fractions demonstrated that in steroid synthesizing cells, the peripheral-type benzodiazepine receptor (PBR) is an outer mitochondrial membrane protein, preferentially located in the outer/inner membrane contact sites, involved in the regulation of cholesterol transport from the outer to the inner mitochondrial membrane, the rate-determining step in steroid biosynthesis. Mitochondrial PBR ligand binding characteristics and topography are sensitive to hormone treatment suggesting a role of PBR in the regulation of hormone-mediated steroidogenesis. Targeted disruption of the PBR gene in Leydig cells in vitro resulted in the arrest of cholesterol transport into mitochondria and steroid formation; transfection of the mutant cells with a PBR cDNA rescued steroidogenesis demonstrating an obligatory role for PBR in cholesterol transport. Molecular modeling of PBR suggested that it might function as a channel for cholesterol. This hypothesis was tested in a bacterial system devoid of PBR and cholesterol. Cholesterol uptake and transport by these cells was induced upon PBR expression. Amino acid deletion followed by site-directed mutagenesis studies and expression of mutant PBRs demonstrated the presence in the cytoplasmic carboxy-terminus of the receptor of a cholesterol recognition/interaction amino acid consensus sequence. This amino acid sequence may help for recruiting the cholesterol coming from intracellular sites to the mitochondria.     

In vivo and in vitro peripheral-type benzodiazepine receptor polymerization: functional significance in drug ligand and cholesterol binding

Peripheral-type benzodiazepine receptor (PBR) is an 18 kDa high-affinity drug ligand and cholesterol binding protein involved in various cell functions. Antisera for distinct PBR areas identified immunoreactive proteins of 18, 40, and 56 kDa and occasionally 72, 90, and 110 kDa in testicular Leydig and breast cancer cells. These sizes may correspond to PBR polymers and correlated to the levels of reactive oxygen species. Treatment of Leydig cells with human chorionic gonadotropin rapidly induced free radical, PBR polymer, and steroid formation. UV photoirradiation generates ROS species, which increased the size of intramembraneous particles of recombinant PBR reconstituted into proteoliposomes consistent with polymer formation, determined both by SDS-PAGE and by freeze-fracture electron microscopy. Spectroscopic analysis revealed the formation of dityrosines as the covalent cross-linker between PBR monomers. Moreover, photoirradiation increased PK 11195 drug ligand binding and reduced cholesterol binding capacity of proteoliposomes. Further addition of PK 11195 drug ligand to polymers increased the rate of cholesterol binding. These data indicate that reactive oxygen species induce in vivo and in vitro the formation of covalent PBR polymers. We propose that the PBR polymer might be the functional unit responsible for ligand-activated cholesterol binding and that PBR polymerization is a dynamic process modulating the function of this receptor in cholesterol transport and other cell-specific PBR-mediated functions.     

A CRAC-like motif in BAX sequence: relationship with protein insertion and pore activity in liposomes

Several proteins that interact with cholesterol have a highly conserved sequence, corresponding to the cholesterol recognition/interaction amino acid consensus. Since cholesterol has been proposed to modulate both oligomerization and insertion of the pro-apoptotic protein BAX, we investigated the existence of such a motif in the BAX sequence. Residues 113 to 119 of the recombinant BAX α5-helix, LFYFASK, correspond with the sequence motif described for the consensus pattern, -L/V-(X)(1-5)-Y-(X)(1-5)-R/K. Functional characterization of the point mutations, K119A, Y115F, and L113A in BAX, was performed in liposomes supplemented with cholesterol, comparing binding, integration, and pore forming activities. Our results show that the mutations Y115F and L113A changed the cholesterol-dependent insertion observed in the wild type protein. In addition, substitutions in the BAX sequence modified the concentration dependency of carboxyfluorescein release in liposomes, although neither pore activity of the wild type or of any of the mutants significantly increased in cholesterol-enriched liposomes. Thus, while it is likely that the putative CRAC motif in BAX accounts for its enhanced insertion in cholesterol-enriched liposomes; the pore forming properties of BAX did not depend on cholesterol content in the membranes, albeit those mutations changed the pore channeling activity of the protein.     

Cholesterol interaction attenuates scramblase activity of SCRM-1 in the artificial membrane

Phospholipid (PL) scramblases are single-pass transmembrane protein mediating bidirectional PL translocation. Previously in silico analysis of human PL scramblases, predicted the presence of an uncharacterized cholesterol-binding domain spanning partly in the transmembrane helix as well as in the adjacent extracellular coil. This domain was found to be universally conserved in diverse organisms like Caenorhabditis elegans. In this study, we investigated the saturable cholesterol-binding domain of SCRM-1 using fluorescence sterol binding assay, Stern-Volmer quenching, Förster resonance energy transfer, and CD spectroscopy. We observed high-affinity interaction between cholesterol and SCRM-1. Our results support a previous report, which showed that the cholesterol ordering effect reduced the scramblase activity of hPLSCR1. Considering the presence of a high-affinity binding sequence, we propose that the reduction in activity could partly be due to the cholesterol binding. To validate this, we generated a C-terminal helix (CTH) deletion construct (?CTH SCRM-1) and a point mutation in the putative cholesterol-binding domain I273D SCRM-1. Deletion construct greatly reduced cholesterol affinity along with loss of scramblase activity. In contrast to this, I273D SCRM-1 retained scrambling activity in proteoliposomes containing ~30 mol% cholesterol but lost sterol binding ability. These results suggest that C-terminal helix is crucial for membrane insertion and in the lipid bilayer the scrambling activity of SCRM-1 is modulated through its interaction with cholesterol.     

Deletion of the C-terminal domain of apolipoprotein A-I impairs cell surface binding and lipid efflux in macrophage.

The contribution of the amphipathic alpha-helices of apoA-I toward lipid efflux from human skin fibroblasts and macrophage was examined. Four apoA-I mutants were designed, each by deletion of a pair of predicted adjacent helices. Three mutants lacked two consecutive central alpha-helices [Delta(100-143), Delta(122-165), and Delta(144-186)], whereas the final mutant lacked the C-terminal domain [Delta(187-243)]. When compared to recombinant wild-type apoA-I and mutants with central domain deletions, Delta(187-243) exhibited a marked reduction in its ability to promote either cholesterol or phospholipid efflux from THP-1 macrophages. This mutant also demonstrated a decreased ability to bind lipids and to form lipoprotein complexes. In contrast, the four mutants and apoA-I equally supported cholesterol efflux from fibroblasts, albeit with a reduced capacity when compared to macrophages. Delta(187-243) bound poorly to the macrophage cell surface when compared to apoA-I, and competitive binding studies with the central domain and C-terminal deletions mutants showed that only Delta(187-243) did not compete effectively with [(125)I]apoA-I. Omission of PMA during cholesterol loading enhanced cholesterol efflux to both apoA-I (1.5-fold) and the C-terminal deletion mutant (2.5-fold). Inclusion of the Sandoz ACAT inhibitor (58-035) during loading and, in the absence of PMA, increased and equalized cholesterol efflux to apoA-I and Delta(187-243). Surprisingly, omission of PMA during cholesterol loading had minimal effects on the binding of apoA-I or Delta(187-243) to the THP-1 cell surface. Overall, these results show that cholesterol efflux from cells such as fibroblasts does not require any specific sequence between residues 100 and 243 of apoA-I. In contrast, optimal cholesterol efflux in macrophages requires binding of the C-terminal domain of apoA-I to a cell surface-binding site and the subsequent translocation of intracellular cholesterol to an efflux-competent pool.     

Secondary and tertiary structures of the transmembrane domains of the translocator protein TSPO determined by NMR. Stabilization of the TSPO tertiary fold upon ligand binding

Numerous biological functions are attributed to the peripheral-type benzodiazepine receptor (PBR) recently renamed translocator protein (TSPO). The best characterized function is the translocation of cholesterol from the outer to inner mitochondrial membrane, which is a rate-determining step in steroid biosynthesis. TSPO drug ligands have been shown to stimulate pregnenolone formation by inducing TSPO-mediated translocation of cholesterol. Until recently, no direct structural data on this membrane protein was available. In a previous paper, we showed that a part of the mouse TSPO (mTSPO) C-terminal region adopts a helical conformation, the side-chain distribution of which provides a groove able to fit a cholesterol molecule. We report here on the overall structural properties of mTSPO. This study was first undertaken by dissecting the protein sequence and studying the conformation of five peptides encompassing the five putative transmembrane domains from (1)H-NMR data. The secondary structure of the recombinant protein in micelles was then studied using CD spectroscopy. In parallel, the stability of its tertiary fold was probed using (1)H-(15)N NMR. This study provides the first experimental evidence for a five-helix fold of mTSPO and shows that the helical conformation of each transmembrane domain is mainly formed through local short-range interactions. Our data show that, in micelles, mTSPO exhibits helix content close to what is expected but an unstable tertiary fold. They reveal that the binding of a drug ligand that stimulates cholesterol translocation is able to stabilize the mTSPO tertiary structure.     

Peripheral-type benzodiazepine receptor (PBR) aggregation and absence of steroidogenic acute regulatory protein (StAR)/PBR association in the mitochondrial membrane as determined by bioluminescence resonance energy transfer (BRET)

The steroidogenic acute regulatory protein (StAR) is responsible for acute control of cholesterol transport across the mitochondrial membrane, however the mechanism of StAR-associated cholesterol transport is unknown and may involve the peripheral-type benzodiazepine receptor (PBR)/endozepine system. Several molecules of PBR may associate to form a channel through which cholesterol passes to the inner mitochondrial membrane, and endozepine is the natural ligand for PBR. Bioluminescence resonance energy transfer (BRET) was used to test StAR/PBR/endozepine interactions, PBR aggregation, and the effect of second messengers on interactions. There was no evidence of StAR/PBR, StAR/endozepine, or PBR/endozepine interactions. The StAR and PBR fusion proteins were trafficking to the mitochondria as expected, but the endozepine fusion protein was not localized to the mitochondria indicating that it was not biologically active. Data were obtained indicating that PBR forms aggregates in the mitochondrial membrane. Energy transfer between PBR fusion proteins was dose and time dependent, but there was no effect induced by PK11195 ligand binding or pharmacologic activation of PKA or PKC second messenger pathways. It appears that PBR aggregates in the mitochondrial membrane, however there was no evidence that PBR aggregation is regulated in the acute control of steroidogenesis, or that PBR and StAR interact.     

High-affinity peripheral benzodiazepine receptor ligand, PK11195, regulates protein phosphorylation in rat brain mitochondria under control of Ca(2+)

The effects of PK11195, a high-affinity peripheral benzodiazepine receptor (PBR) ligand, on protein phosphorylation in isolated purified rat brain mitochondria were investigated. The isoquinoline carboxamide ligand of PBR, PK11195, but not the benzodiazepine ligand Ro5-4864, in the nanomolar concentration range strongly increased the phosphorylation of 3.5 and 17 kDa polypeptides. The effect of PK11195 was seen in the presence of elevated Ca(2+) levels (3 x 10(-7) to 10(-6) m), but not at very low Ca(2+) levels (10(-8) to 3 x 10(-8) m). This indicates that PBR involves Ca(2+) as a second messenger in the regulation of protein phosphorylation. Staurosporine, an inhibitor of protein kinase activity was able to suppress the PK11195-promoted protein phosphorylation. When the permeability transition pore (PTP) was opened by threshold Ca(2+) load, phosphorylation of the 3.5-kDa polypeptide was diminished, but strong phosphorylation of the 43-kDa protein was revealed. The 43-kDa protein appears to be a PTP-specific phosphoprotein. If PTP was opened, PK11195 did not increase the phosphorylation of the 3.5 and 17-kDa proteins but suppressed the phosphorylation of the PTP-specific 43-kDa phosphoprotein. The ability of PK11195 to increase the protein phosphorylation, which was lost under Ca(2+)-induced PTP opening, was restored again in the presence of calmidazolium, an antagonist of calmodulin and inhibitor of protein phosphatase PP2B. These results show a tight interaction of PBR with the PTP complex in rat brain mitochondria. In conclusion, a novel function of PBR in brain mitochondria has been revealed, and the PBR-mediated protein phosphorylation has to be considered an important element of the PBR-associated signal transducing cascades in mitochondria and cells.     

Structure of the bacteriophage phi KZ lytic transglycosylase gp144

Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage phi KZ has been determined to 2.5-A resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. phi KZ gp144 is a 260-residue alpha-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G D-Ala-D-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu 115 in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the phi KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine)(4) has been determined to 2.6-A resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.     

Sensitization of differentiated PC12 cells to apoptosis by presenilin-2 is mediated by p38

Recombinant mouse 18 kDa peripheral-type benzodiazepine receptor (PBR) protein was overexpressed in Escherichia coli and isolated using a His. Bind metal chelation resin. Recombinant PBR protein was purified with sodium dodecyl sulfate and reincorporated into liposomes using Bio-Beads SM2 as a detergent removing agent. Negative staining of the reconstituted PBR samples, examined by electron microscopy, showed the formation of proteoliposomes. Freeze-fracture of these proteoliposomes revealed the presence of transmembranous particles of an average size of 3.5 +/- 0.25 nm, consistent with the presence of a monomeric form of the recombinant PBR protein. The reconstituted protein exhibited the ability to bind both the PBR drug ligand isoquinoline carboxamide PK 11195 and cholesterol with nanomolar affinities. These data suggest that a PBR monomer is the minimal functional unit, binding drug ligands and cholesterol.     

A Cholesterol Recognition Motif in Human Phospholipid Scramblase 1

Human phospholipid scramblase 1 (SCR) catalyzes phospholipid transmembrane (flip-flop) motion. This protein is assumed to bind the membrane hydrophobic core through a transmembrane domain (TMD) as well as via covalently bound palmitoyl residues. Here, we explore the possible interaction of the SCR TMD with cholesterol by using a variety of experimental and computational biophysical approaches. Our findings indicate that SCR contains an amino acid segment at the C-terminal region that shows a remarkable affinity for cholesterol, although it lacks the CRAC sequence. Other 3-OH sterols, but not steroids lacking the 3-OH group, also bind this region of the protein. The newly identified cholesterol-binding region is located partly at the C-terminal portion of the TMD and partly in the first amino acid residues in the SCR C-terminal extracellular coil. This finding could be related to the previously described affinity of SCR for cholesterol-rich domains in membranes.     

Control of hypercholesterolemia and atherosclerosis using the cholesterol recognition/interaction amino acid sequence of the translocator protein TSPO

The translocator protein (18-kDa) TSPO is an ubiquitous high affinity cholesterol-binding protein reported to be present in the endothelial and smooth muscle cells of the blood vessels; its expression dramatically increased in macrophages found in atherosclerotic plaques. A domain in the carboxy-terminus of TSPO was identified and characterized as the cholesterol recognition/interaction amino acid consensus (CRAC). The ability of the CRAC domain to bind to cholesterol led us to hypothesize that this peptide could be used as an hypocholesterolemic, with potential anti-atherogenic properties, agent. We report herein the therapeutic benefit that resulted for the administration of the VLNYYVWR human CRAC sequence to guinea pigs fed with a high cholesterol diet and ApoE knock-out B6.129P2-Apoetm1Unc/J mice. CRAC treatment (3 and 30 mg/kg once daily for 6 weeks) resulted in reduced circulating cholesterol levels in guinea pigs fed with 2% high cholesterol diet and ApoE knock-out B6.129P2-Apoetm1Unc/J mice. In high cholesterol fed guinea pigs, CRAC treatment administered once daily induced an increase in circulating HDL, decreased total, free and LDL cholesterol, and removed atheroma deposits in the aorta in a dose-dependent manner. The treatment also prevented the high cholesterol diet-induced increase in serum creatine kinase, total and isoforms, markers of neurological, cardiac and muscular damage. No toxicity was observed. Taken together these results support a role of TSPO in lipid homeostasis and atherosclerosis and indicate that CRAC may constitute a novel and safe treatment of hypercholesterolemia and atherosclerosis.