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.     

Differential dynamics of the serotonin1A receptor in membrane bilayers of varying cholesterol content revealed by all atom molecular dynamics simulation

Abstract

The serotonin_1A receptor belongs to the superfamily of G protein-coupled receptors (GPCRs) and is a potential drug target in neuropsychiatric disorders. The receptor has been shown to require membrane cholesterol for its organization, dynamics and function. Although recent work suggests a close interaction of cholesterol with the receptor, the structural integrity of the serotonin_1A receptor in the presence of cholesterol has not been explored. In this work, we have carried out all atom molecular dynamics simulations, totaling to 3?μs, to analyze the effect of cholesterol on the structure and dynamics of the serotonin_1A receptor. Our results show that the presence of physiologically relevant concentration of membrane cholesterol alters conformational dynamics of the serotonin_1A receptor and, on an average lowers conformational fluctuations. Our results show that, in general, transmembrane helix VII is most affected by the absence of membrane cholesterol. These results are in overall agreement with experimental data showing enhancement of GPCR stability in the presence of membrane cholesterol. Our results constitute a molecular level understanding of GPCR-cholesterol interaction, and represent an important step in our overall understanding of GPCR function in health and disease.     

Role of the peripheral-type benzodiazepine receptor and the polypeptide diazepam binding inhibitor in steroidogenesis

Steroidogenesis begins with the metabolism of cholesterol to pregnenolone by the inner mitochondrial membrane cytochrome P450 side-chain cleavage (P450scc) enzyme. The rate of steroid formation, however, depends on the rate of (i) cholesterol transport from intracellular stores to the inner mitochondrial membrane and (ii) loading of P450scc with cholesterol. We demonstrated that a key element in the regulation of cholesterol transport is the mitochondrial peripheral-type benzodiazepine receptor (PBR) and that the presence of the polypeptide diazepam binding inhibitor (DBI) was vital for steroidogenesis. We also showed that DBI, as the endogenous PBR ligand, stimulates cholesterol transport. In addition, DBI directly promotes loading of cholesterol to P450scc. We review herein our studies on the structure, function, topography and hormonal regulation of PBR and DBI in steroidogenic cells. Based on these data we propose a model where the interaction of DBI with PBR, at the outer/inner membrane contact sites, is the signal transducer of hormone-stimulated and constitutive steroidogenesis at the mitochondrial level. Hormone-induced changes in PBR microenvironment/structure regulate the affinity of the receptor. PBR ligand binding to a higher affinity receptor results in increased cholesterol transport. In addition, hormone-induced release (processing?) of a 30,000 M_W DBI-immunoreactive protein from the inner mitochondrial membrane may result to the intramitochondrial production of DBI which directly stimulates loading of P450scc with cholesterol. Thus, in vivo, hormonal activation of these two mechanisms results in efficient cholesterol delivery and utilization and thus high levels of steroid synthesis.     

Molecular dynamics simulations of GPCR–cholesterol interaction: An emerging paradigm

G protein-coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across cell membranes and represent major targets in the development of novel drug candidates. Membrane cholesterol plays an important role in GPCR structure and function. Molecular dynamics simulations have been successful in exploring the effect of cholesterol on the receptor and a general consensus molecular view is emerging. We review here recent molecular dynamics studies at multiple resolutions highlighting the main features of cholesterol-GPCR interaction. Several cholesterol interaction sites have been identified on the receptor that are reminiscent of nonannular sites. These cholesterol hot-spots are highly dynamic and have a microsecond time scale of exchange with the bulk lipids. A few consensus sites (such as the CRAC site) have been identified that correspond to higher cholesterol interaction. Interestingly, high plasticity is observed in the modes of cholesterol interaction and several sites have been suggested to have high cholesterol occupancy. We therefore believe that these cholesterol hot-spots are indicative of ‘high occupancy sites’ rather than ‘binding sites’. The results suggest that the energy landscape of cholesterol association with GPCRs corresponds to a series of shallow minima interconnected by low barriers. These specific interactions, along with general membrane effects, have been observed to modulate GPCR organization. Membrane cholesterol effects on receptor structure and organization, that in turn influences receptor cross-talk and drug efficacy, represent a new frontier in GPCR research. This article is part of a Special Issue entitled: Lipid-protein interactions. Guest Editors: Amitabha Chattopadhyay and Jean-Marie Ruysschaert.     

Noninvasive cellular internalization of silver molecules by chitosan nanoneedles: a novel nanocarrier

We explore with molecular modeling, dynamics simulations, and a statistical model the ability of chitosan nanoneedles (CNNs) to be internalized into a model lipid bilayer as a function of their length, keeping in view of their applications in the field of biomedicine for advanced targeted drug delivery. In this study, we have computationally modeled and studied the structural geometry and the stability of CNNs formed by 4, 6, and 8 subunits. We reported the molecular surface analysis of the modeled CNNs along with molecular dynamic (MD) simulations studies toward revealing the noninvasive cellular internalization potential of these CNNs and a case study has been carried to study the ability of CNNs to translocate silver nanoparticles across membrane. The present results are strongly in support of further exploration of 8 subunits based CNNs for their application as target drug delivery vehicles. The hydrophilicity of the CNNs has been attributed as one of the key factors responsible for the internalization process. Moreover, our MD simulation studies marched the ability of CNNs to translocate silver nanoparticles through biological membrane in a similar manner that resembles cell-penetrating peptides.     

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.     

Hormonal regulation of peripheral-type benzodiazepine receptors

Central benzodiazepine (BZ) receptors are located only in the central nervous system and mediate the clinical effects obtained by various BZs. In addition, there is another receptor that binds BZs with different drug specificities, which is located mainly on the outer mitochondrial membrane of various peripheral tissues. Peripheral BZ receptors (PBR) are composed of three subunits: an isoquinoline binding site, a voltage-dependent anion channel, and an adenine nucleotide carrier, with molecular weights of 18, 32, and 30 kDa, respectively. Complementary DNA of the isoquinoline binding subunit has been cloned in rat, calf, and human. The major role of PBR is in the regulation of steroid biosynthesis. Various PBR ligands stimulate the conversion of cholesterol into pregnenolone and the production of steroid hormones. The naturally occurring diazepam-binding inhibitor stimulates in vivo steroidogenesis via binding to PBR. In the female, PBR density is increased in rat and human ovary proportional with greater cell maturation and differentiation. In the male, testosterone modulates PBR density in the genital tract. These results show the strong relationship between PBR and the endocrine system.     

Lateral organization in lipid-cholesterol mixed bilayers

Interactions between lipid and cholesterol molecules in membranes play an important role in the structural and functional properties of cell membranes. Although structural properties of lipid-cholesterol mixtures have been extensively studied, an understanding of the role of cholesterol in the lateral organization of bilayers has been elusive. In this article, we propose a simple yet powerful model, based on self-consistent mean-field theory and molecular dynamics simulations, for lipid bilayers containing cholesterol. Properties predicted by our model are shown to be in excellent agreement with experimental data. Our model predicts that cholesterol induces structural changes in the bilayer through the formation of regions of ordered lipids surrounding each cholesterol molecule. We find that the "smooth" and "rough" sides of cholesterol play crucial roles in formation and distribution of the ordered regions. Our model is predictive in that input parameters are obtained from independent atomistic molecular dynamics simulations. The model and method are general enough to describe other heterogeneous lipid bilayers, including lipid rafts.     

Peripheral benzodiazepine receptor agonists exhibit potent antiapoptotic activities

The peripheral benzodiazepine receptor (PBR) has been implicated in several mitochondrial functions but the exact physiological role of this receptor is still under debate. Since the mitochondria have been attributed a central role in cell death, we have determined the effects of various PBR agonists and antagonists on the apoptosis of the human lymphoblastoid cell line U937. On this cell type, the PBR agonist Ro5-4864 was found to strongly protect the cells against apoptosis induced by TNFalpha. The antiapoptotic effect of PBR agonists was due to a selective interaction with the PBR as demonstrated by: (1) a close correlation between the antiapoptotic activity of various PBR agonists and their respective affinity for the PBR determined on the same cells, (2) a lack of effect of central benzodiazepine receptors agonists such as clonazepam on cell survival, (3) the lack of an antiapoptotic activity of Ro5-4864 on wild-type Jurkat cells (lacking the PBR receptor) and the reappearance of this effect on PBR-transfected Jurkat cells, and (4) the blockade of the antiapoptotic effect of PBR agonists by a selective PBR antagonist. The present results therefore indicate that PBR agonists are potent antiapoptotic compounds and show that this effect might represent a major function for this enigmatic receptor.     

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.     

Cloning and expression of a pharmacologically unique bovine peripheral-type benzodiazepine receptor isoquinoline binding protein.

High affinity binding of isoquinolines, such as PK 11195, is a conserved feature of peripheral-type benzodiazepine receptors (PBR) across species. However, species differences in PBR ligand binding have been described based on the affinity for N1-alkyl-1,4-benzodiazepines, such as Ro5-4864. Ro5-4864 binds with high affinity to the rat receptor but has low affinity for the bovine PBR. Photolabeling with an isoquinoline ligand, [3H]PK 14105, identifies a 17-kDa protein, the PBR isoquinoline binding protein (PBR/IBP), in both species. To further elucidate the role of the PBR/IBP in determining PBR benzodiazepine and isoquinoline binding characteristics, the bovine PBR/IBP was cloned and expressed. Using a cDNA encoding a rat PBR/IBP to screen a fetal bovine adrenal cDNA library, a bovine cDNA encoding a polypeptide of 169 residues was cloned. The bovine and rat PBR/IBPs had similar hydropathy profiles exhibiting five potential transmembrane domains. Transfecting the cloned bovine PBR/IBP cDNA into COS-7 cells resulted in an 11-fold increase in the density of high affinity [3H]PK 11195 binding sites which had only low affinity for Ro5-4864. Expression of the bovine PBR/IBP yields a receptor which is pharmacologically distinct from both endogenous COS-7 PBR and the rat PBR based on the affinity for several N1-alkyl-1,4-benzodiazepine ligands. These results suggest the PBR/IBP is the minimal functional component required for PBR ligand binding characteristics and the different protein sequences account for the species differences in PBR benzodiazepine ligand binding.     

State-dependent protein-lipid interactions of a pentameric ligand-gated ion channel in a neuronal membrane

Pentameric ligand-gated ion channels (pLGICs) are receptor proteins that are sensitive to their membrane environment, but the mechanism for how lipids modulate function under physiological conditions in a state dependent manner is not known. The glycine receptor is a pLGIC whose structure has been resolved in different functional states. Using a realistic model of a neuronal membrane coupled with coarse-grained molecular dynamics simulations, we demonstrate that some key lipid-protein interactions are dependent on the receptor state, suggesting that lipids may regulate the receptor’s conformational dynamics. Comparison with existing structural data confirms known lipid binding sites, but we also predict further protein-lipid interactions including a site at the communication interface between the extracellular and transmembrane domain. Moreover, in the active state, cholesterol can bind to the binding site of the positive allosteric modulator ivermectin. These protein-lipid interaction sites could in future be exploited for the rational design of lipid-like allosteric drugs.     

Targeted molecular imaging agents for cellular-scale bimodal imaging

Molecular imaging is a powerful tool that has the ability to elucidate biochemical mechanisms and signal the early onset of disease. Overexpression of the peripheral benzodiazepine receptor (PBR) has been observed in a variety disease states, including glioblastoma, breast cancer, and Alzheimer's disease. Thus, the PBR could be an attractive target for molecular imaging. In this paper, the authors report cellular uptake and multimodal (MRI and fluorescence) imaging of PBR-overexpressing C6 glioblastoma (brain cancer) cells using a cocktail administration approach and a new PBR targeted lanthanide chelate molecular imaging agent.     

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.     

Characterization of the outer membrane protein OprF of Pseudomonas aeruginosa in a lipopolysaccharide membrane by computer simulation

The N-terminal domain of outer membrane protein OprF of Pseudomonas aeruginosa forms a membrane spanning eight-stranded antiparallel beta-barrel domain that folds into a membrane channel with low conductance. The structure of this protein has been modeled after the crystal structure of the homologous protein OmpA of Escherichia coli. A number of molecular dynamics simulations have been carried out for the homology modeled structure of OprF in an explicit molecular model for the rough lipopolysaccharide (LPS) outer membrane of P. aeruginosa. The structural stability of the outer membrane model as a result of the strong electrostatic interactions compared with simple lipid bilayers is restricting both the conformational flexibility and the lateral diffusion of the porin in the membrane. Constricting side-chain interactions within the pore are similar to those found in reported simulations of the protein in a solvated lipid bilayer membrane. Because of the strong interactions between the loop regions of OprF and functional groups in the saccharide core of the LPS, the entrance to the channel from the extracellular space is widened compared with the lipid bilayer simulations in which the loops are extruding in the solvent. The specific electrostatic signature of the LPS membrane, which results in a net intrinsic dipole across the membrane, is found to be altered by the presence of OprF, resulting in a small electrically positive patch at the position of the channel.     

Molecular dynamics simulations of mixed micelles modeling human bile

Extensive molecular dynamics (MD) simulations of binary systems of phospholipids and bile salts, a model for human bile, have been performed. Recent progress in hardware and software development allows simulation of the spontaneous aggregation of the constituents into small mixed micelles, in agreement with experimental observations. The MD simulations reveal the structure of these micelles at atomic detail. The phospholipids are packed radially with their headgroups at the surface and the hydrophobic tails pointing toward the micellar center. The bile salts act as wedges between the phospholipid headgroups, with their hydrophilic sides exposed to the aqueous environment. The structure of the micelles strongly resembles the previously proposed radial shell model. Simulations including small fractions of cholesterol reveal how cholesterol is solubilized inside these mixed micelles without changing their overall structure.     

人外周型苯二氮卓受体及其突变受体cDNA克隆和序列分析

用ＲＴ－ＰＣＲ方法扩增并克隆了三种人外周型苯二氮卓受体ＰＢＲｃＤＮＡ,测序表明,４４２ｂｐ片段与文献报道相比缺失８４ｂｐ编码序列,其转录水平高于正常ＰＢＲ．该序列编码一个与ＰＢＲ结构相关但缺失了２８个氨基酸残基的突变受体蛋白．这一异常转录本可能是通过选择性剪接方式转录产生并只存在于中国人肝癌ＢＥＬ７４０２细胞系,表明ＰＢＲ基因表达具有细胞特异性和异质性．突变受体的发现为研究ＰＢＲ的结构和功能提供了理想的分子和细胞模型     

Molecular modeling of the human serotonin(1A) receptor: role of membrane cholesterol in ligand binding of the receptor

Serotonin(1A) receptors are important neurotransmitter receptors and belong to the superfamily of G-protein coupled receptors (GPCRs). Although it is an important drug target, the crystal structure of the serotonin(1A) receptor has not been solved yet. Earlier homology models of the serotonin(1A) receptor were generated using rhodopsin as a template. We have used two recent crystal structures of the human β(2)-adrenergic receptor, one of which shows specific cholesterol binding site(s), as templates to model the human serotonin(1A) receptor. Since the sequence similarity between the serotonin(1A) receptor and β(2)-adrenergic receptor is considerably higher than the similarity between the serotonin(1A) receptor and rhodopsin, our model is more reliable. Based on these templates, we generated models of the serotonin(1A) receptor in the absence and presence of cholesterol. The receptor model appears more compact in the presence of cholesterol. We validated the stability of 'compactness' using coarse-grain MD simulation. Importantly, all ligands exhibit higher binding energies when docked to the receptor in the presence of cholesterol, thereby implying that membrane cholesterol facilitates ligand binding to the serotonin(1A) receptor. To the best of our knowledge, this is one of the first reports in which lipid-specific receptor conformations have been modeled by homology modeling.