Peripheral-type Benzodiazepine Receptor Research Articles

Exploring Translocator Protein (TSPO) Medicinal Chemistry: An Approach for Targeting Radionuclides and Boron Atoms to Mitochondria

Translocator protein 18 kDa [TSPO or peripheral-type benzodiazepine receptor (PBR)] was identified in the search of binding sites for benzodiazepine anxiolytic drugs in peripheral regions. In these areas, binding sites for TSPO ligands were recognized in steroid-producing tissues. TSPO plays an important role in many cellular functions, and its coding sequence is highly conserved across species. TSPO is located predominantly on the membrane of mitochondria and is overexpressed in several solid cancers. TSPO basal expression in the CNS is low, but it becomes high in neurodegenerative conditions. Thus, TSPO constitutes not only as an outstanding drug target but also as a valuable marker for the diagnosis of a number of diseases. The aim of the present article is to show the lesson we have learned from our activity in TSPO medicinal chemistry and in approaching the targeted delivery to mitochondria by means of TSPO ligands.

TSPO ligand etifoxine attenuates LPS-induced cognitive dysfunction in mice

The translocator protein (TSPO), once known as peripheral-type benzodiazepine receptor, was reported to be related with several physiological functions. Etifoxine is a clinically available anxiolytic drug, and has recently shown neuroprotective effects as a TSPO ligand. The aim of the present study was to investigate the influence of etifoxine on LPS-induced neuroinflammation and cognitive dysfunction. C57/BL6 male mice were injected with etifoxine (50 mg/kg, i.p.) three days before lipopolysaccharide (LPS, 500 μg/kg, i.p.) administration. Etifoxine pretreatment alleviated hippocampal inflammation, increased brain levels of progesterone, allopregnanolone and attenuated cognitive dysfunction in LPS-injected mice. While LPS increased expression of caspase-3 and decreased p-Akt/Akt, etifoxine returned caspase-3 and p-Akt/Akt to control levels. Finasteride, a 5α-reductase inhibitor that blocked allopregnanolone production, partially reversed the effects of etifoxine. We concluded that etifoxine exerted neuroprotective effects in LPS-induced neuroinflammation and the neuroprotection may be related with increase of neurosteroids synthesis and decrease of apoptosis.

Screening of Bombyx mori brain proteins interacting with protein tyrosine phosphatase of BmNPV.

In this study, glutathione-S-transferase pull-down combined with mass spectrometry techniques were used to identify the candidate proteins interacting with protein tyrosine phosphatase of theBombyx Mori nucleopolyhedrovirus in theB. mori (BmNPV-PTP) brain. A total of 36 proteins were identified from BmNPV-PTP coprecipitate samples by searching the NCBI_Bombyx Mori database with the original mass spectrum data. Among those proteins, the interaction between BmNPV-PTP and B. mori cyclophilin A may acceleratethe apoptosis of certain nerve cells involvedin regulating behavior, and thus may be an inducer of enhanced locomotor activity (ELA). After the BmNPV invasion, BmNPV-PTP binding to peripheral-type benzodiazepine receptors may initiate a series of abnormal cascades of the nervous system, which resultsin abnormal hyperactive behavior in B. mori. Besides this, vacuolar ATP synthase catalytic subunit A, annexin, and several enzymes for energy conversion were identified, which may play a rolein enhancing viral entry and infectivity and provide energy for enhancing the locomotor activity of B. mori. In general, the results of this study will facilitate the understanding of the molecular mechanisms underlying the ELA of B. mori larva induced by BmNPV.

Translocator protein mediates olfactory repulsion.

Translocator protein (TSPO, 18kDa), which was previously known as a peripheral-type benzodiazepine receptor, is associated with psychiatric disorders and acts as a neuroimaging biomarker. However, its function and mechanism in modulating behaviors are less well-known. Herein, we found that TSPO in migratory locusts shows conserved protein traits and is expressed at high levels in the brains. The expression levels of tspo mRNA and protein were higher in brains of solitary locusts than those in gregarious locusts, whereas the mRNA and protein expression levels remained stable during crowding and isolation, suggesting that the expression level of TSPO is potentially associated with behavioral phenotype of solitary locusts. Moreover, tspo RNAi knockdown in the brains of solitary locusts decreased their olfactory repulsion. After RNAi knockdown of tyramine receptor (TyR) in the brains of solitary locusts, RNA-seq analysis identified that a functional class of receptors, which included tspo, was downregulated significantly. Moreover, tspo mRNA and protein expression levels were downregulated and upregulated after TyR RNAi knockdown and activation, respectively. tspo RNAi knockdown in the brains of solitary locusts induced the attractive response and inhibited the function of tyramine (TA)-TyR in inducing olfactory repulsion. In gregarious locusts, tspo RNAi knockdown inhibited the function of TA-TyR inducing olfactory repulsion. This study confirms that TSPO acts as a crucial effector protein in TA-TyR signaling to modulate olfactory repulsion. Furthermore, this study provides a novel mechanism by which TSPO functionally connects a G-protein-coupled receptor and a mitochondria membrane protein in modulating olfactory repulsion.

Regulation of Mitochondrial, Cellular, and Organismal Functions by TSPO.

In 1999, the enigma of the 18kDa mitochondrial translocator protein (TSPO), also known as the peripheral-type benzodiazepine receptor, was the seeming disparity of the many functions attributed to TSPO, ranging from the potential of TSPO acting as a housekeeping gene at molecular biological levels to adaptations to stress, and even involvement in higher emotional and cognitive functioning, such as anxiety and depression. In the years since then, knowledge regarding the many functions modulated by TSPO has expanded, and understanding has deepened. In addition, new functions could be firmly associated with TSPO, such as regulation of programmed cell death and modulation of gene expression. Interestingly, control by the mitochondrial TSPO over both of these life and death functions appears to include Ca++ homeostasis, generation of reactive oxygen species (ROS), and ATP production. Other mitochondrial functions under TSPO control are considered to be steroidogenesis and tetrapyrrole metabolism. As TSPO effects on gene expression and on programmed cell death can be related to the wide range of functions that can be associated with TSPO, several of these five elements of Ca++, ROS, ATP, steroids, and tetrapyrroles may indeed form the basis of TSPO's capability to operate as a multifunctional housekeeping gene to maintain homeostasis of the cell and of the whole multicellular organism.

TSPO IMMUNOSTAINING IN AD CASES WITH/WITHOUT AN AD-ASSOCIATED TREM2 VARIANT

The Peripheral-Type Benzodiazepine Receptor (TSPO) is a well-established target for the Positron Emission Tomography (PET) ligands [11C]-PK11195 and [11C]-PBR28. TSPO is believed to translocate cholesterol across the mitochondrial intermembrane space and is highly expressed in activated glia. TSPO PET ligands thus enable sensitive in vivo monitoring of neuroinflammation, a phenomenon closely associated with neuronal cell death in Alzheimer's disease (AD). AD patients have recently been identified with rare disease-associated variants in the TREM2 gene (1, 2). Disease vulnerability in these people appears to be linked to inappropriate microglial function and immune response. TREM2 is a plasma membrane receptor highly expressed in brain microglia (3) where it is believed to regulate phagocytic and inflammatory microglial responses to brain pathology. We therefore sought to establish if TSPO levels are impaired in AD patients who have a disease-associated TREM2 variant and establish which glial cells impairment is associated. Hippocampal sections (5–7μM) from age and gender matched AD (N=37) and control (N=18) donors were analysed by immunohistochemistry using published TSPO antibodies (4, 5) (PAB7095 and NP155). The AD cases consisted of Braak-matched cases (≥IV) with TREM2+ (N=16) and without TREM2-(N=21) disease-associated variants in TREM2. Sections were also immunostained for p-tau (AT8), Aβ (4G8) and by co-immunoflurescence with markers of microglia (Iba-1, CD68 and HLA-DR, DP, DQ) and astrocytes (GFAP) to ascertain cell types expressing TSPO. We will report results of immunohistochemistry analyses using two previously reported TSPO antibodies in human post mortem brain sections. As reported previously, these antibodies stain macrophages, astrocytes and microglia in AD brains with more limited staining in non-AD cases. We used astrocyte and microglia specific markers to determine which cells were differentially stained between TREM2+ and TREM2- AD cases and determine differences in cells adjacent to amyloid or Tau which stain for TSPO. The biomarker TSPO has been used extensively to explore immune activation in vivo in the brain. Levels are increased in AD, particularly associated with the emergence of abnormal Tau and neuronal cell loss. Our results will facilitate interpretation of future PET imaging in this group of patients using the TSPO tracers [11C]-PK11195 or [11C]-PBR28.

4'-Chlorodiazepam Protects Mitochondria in T98G Astrocyte Cell Line from Glucose Deprivation.

The translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor (PBR), is considered an important regulator of steroidogenesis and a potential therapeutic target in neurological disorders. Previous evidence suggests that TSPO ligands can protect cells during injury and prevent apoptosis in central nervous system (CNS) cells. However, its actions on astrocytic cells under metabolic injury are not well understood. In this study, we explored whether 4'-chlorodiazepam (Ro5-4864), a TSPO ligand, might protect astrocyte mitochondria under glucose deprivation. Our results showed that 4'-chlorodiazepam preserved cell viability and reduced nuclear fragmentation in glucose-deprived cells. These effects were accompanied by a reduced production of free radicals and maintenance of mitochondrial functions in cells treated with 4'-chlorodiazepam. Finally, our findings suggest that TSPO might be involved in reducing oxidative stress by preserving mitochondrial functions in astrocytic cells exposed to glucose withdrawal.

Protoporphyrin IX regulates peripheral benzodiazepine receptor associated protein 7 (PAP7) and divalent metal transporter 1 (DMT1) in K562 cells

BackgroundProtoporphyrin IX (PP IX), the immediate precursor to heme, combines with ferrous iron to make this product. The effects of exogenous PP IX on iron metabolism remain to be elucidated. Peripheral-type benzodiazepine receptor (PBR) is implicated in the transport of coproporphyrinogen into the mitochondria for conversion to PP IX. We have demonstrated that PBR-Associated Protein 7 (PAP7) bound to the Iron Responsive Element (IRE) isoform of divalent metal transporter 1 (DMT1). PP IX and PAP7 are ligands for PBR, thus, we hypothesized that PAP7 interact with PP IX via PBR. MethodsWe have examined in K562 cells, which can be induced to undergo erythroid differentiation by PP IX and hemin, the effects of PP IX on the expression of PAP7 and other proteins involved in cellular iron metabolism, transferrin receptor 1 (TfR1), DMT1, ferritin heavy chain (FTH), c-Myc and C/EBPα by western blot and quantitative real time PCR analyses. ResultsPP IX significantly decreased mRNA levels of DMT1 (IRE) and (non-IRE) from 4h. PP IX markedly decreased protein levels of C/EBPα, PAP7 and DMT1. In contrast, hemin, which like PP IX also induces K562 cell differentiation, had no effect on PAP7 or DMT1 expression. ConclusionWe hypothesize that PP IX binds to PBR displacing PAP7 protein, which is then degraded, decreasing the interaction of PAP7 with DMT1 (IRE) and resulting in increased turnover of DMT1. General significanceThese results suggest that exogenous PP IX disrupts iron metabolism by decreasing the protein expression levels of PAP7, DMT1 and C/EBPα.

Interlink between cholesterol & cell cycle in prostate carcinoma

Background & objectives:Earlier reports have shown hypocholesterolaemia in cancer patients and high number of lipid rafts in cancer cells. The primary objective of this study was to compare the intracellular cholesterol turnover in non-cancerous (benign) prostatic hyperplasia (BPH) and carcinoma prostate (CAP) with normal prostate cells obtained from patients undergoing radical cystectomy for carcinoma bladder (sham control).Methods:ELISA-based estimation of prostate-specific antigen (PSA), evaluation of expression of low-density lipoprotein receptor (LDLR), peripheral-type benzodiazepine receptor (PBR) and cyclin E, immunohistochemistry and confocal microscopy, measurement of integrated optical density of the diaminobenzidine (DAB)-stained immunohistograms, isolation of nucleus and cell cytoplasm from prostate tissue by ultracentrifugation followed by estimation of cholesterol spectrophotometrically in isolated nuclear and cytoplasmic fractions were performed.Results:Seventy five individuals, 25 for each group (BPH n=25; CAP n=25 and sham control n=25), were included in the study. Cholesterol was increased in the cytoplasm and nucleus of the prostate cancer cells along with elevated expression of LDLR. Increased cholesterol concentration in the cell nucleus was found comparable with the increased expression of cholesterol transporter viz. PBR in the prostate tumour tissues as compared to its expression in normal prostate cells obtained from individuals undergoing radical cystectomy for carcinoma bladder. Cell cycle protein cyclin E was also highly expressed in cancer tissues.Interpretation & conclusions:The present findings along with increased expression of cell cycle protein cyclin E in the cell nucleus of the tumour tissue suggested the possibility of an intriguing role of cholesterol in the mechanism of cell cycle process of prostate cell proliferation.

Differential Expressions of p53, p53R2, hRRM2 and PBR in Chronic Lymphocytic Leukemia: A Correlation with Intracellular Cholesterol.

Regulation of intracellular cholesterol homeostasis exists under balance between intracellular biosynthesis and uptake from extracellular origin by cell surface transport proteins. Expected role of cholesterol on either tumor suppressor gene and/or DNA synthesis has been aimed in the present study to explore intracellular cholesterol homeostasis in CLL subjects. Higher expressions of p53R2 (p53 dependent subunit of ribonucleotide reductase) and p53 were found in lymphocytes of chronic human lymphocytic leukemia as comparison to their normal counterparts. Inverse relation was found with p53 independent R2 subunit (in human hRRM2) of ribonucleotide reductase, which was found to be decreased from its control group. More expression of peripheral type benzodiazepine receptor, a cholesterol transporter, was noticed in isolated nuclear fraction with simultaneous increase of cholesterol concentration in cytoplasmic and nuclear compartments. A parallel increase of cholesterol in cell nucleus with increased p53R2 expression shows priority of the involvement of cholesterol in the process of cell replication.

Expression of the translocator protein (TSPO) from Pseudomonas fluorescens Pf0-1 requires the stress regulatory sigma factors AlgU and RpoH.

The translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is an evolutionary conserved protein that is found in many Eukarya, Archae, and Bacteria, in which it plays several important functions including for example membrane biogenesis, signaling, and stress response. A tspo homolog gene has been identified in several members of the Pseudomonas genus, among which the soil bacterium P. fluorescens Pf0-1. In this bacterium, the tspo gene is located in the vicinity of a putative hybrid histidine kinase-encoding gene. Since tspo has been involved in water stress related response in plants, we explored the effects of hyperosmolarity and temperature on P. fluorescens Pf0-1 tspo expression using a strategy based on lux-reporter fusions. We show that the two genes Pfl01_2810 and tspo are co-transcribed forming a transcription unit. The expression of this operon is growth phase-dependent and is increased in response to high concentrations of NaCl, sucrose and to a D-cycloserine treatment, which are conditions leading to activity of the major cell wall stress responsive extracytoplasmic sigma factor AlgU. Interestingly, the promoter region activity is strongly lowered in a P. aeruginosa algU mutant, suggesting that AlgU may be involved at least partly in the molecular mechanism leading to Pfl01_2810-tspo expression. In silico analysis of this promoter region failed to detect an AlgU consensus binding site; however, a putative binding site for the heat shock response RpoH sigma factor was detected. Accordingly, the promoter activity of the region containing this sequence is increased in response to high growth temperature and slightly lowered in a P. aeruginosa rpoH mutant strain. Taken together, our data suggest that P. fluorescens tspo gene may belong at least partly to the cell wall stress response.

Imaging Neuroinflammation in Ischemic Stroke and in the Atherosclerotic Vascular Disease

Neuroinflammation is a complex biological response to any injury occurring to the central nervous system. It is mainly characterized by the recruitment of immune system cells, namely the microglial cells, in the site of injury. Once activated, microglia expresses a cholesterol transporter protein (TSPO), previously also known as peripheral type benzodiazepine receptor. PK11195 is a ligand for TSPO and, labelled with a positron emitter, it is also the most used tracer for PET molecular imaging to in vivo map the microglia activation in various neurological disorders, including ischemic stroke. Recent [(11)C]PK11195 PET studies proved activated microglia both locally in the area of the infarct and at distance along the affected fibre tracts, suggesting the presence of two different microglia subtypes with peculiar functions in disease progression. The aim of this review is to discuss the most recent knowledge about imaging neuroinflammation in ischemic stroke and in the atherosclerotic and vascular inflammatory disorders, trying to elucidate the interplay between the clinical course and the activation of a microglial response.

Changes of peripheral-type benzodiazepine receptors in the penumbra area after cerebral ischemia-reperfusion injury and effects of astragaloside IV on rats.

This study investigated the changes in peripheral benzodiazepine receptors (PBRs) in the penumbra after cerebral ischemia-reperfusion injury, and examined the effects of astragaloside IV (AST) on PBRs in rats. Sixty Sprague-Dawley rats were divided into a sham operation group, a model group, and three AST treatment groups. Cerebral ischemic models were induced by the clue-blocked method. Neurological deficits were examined. The animals were sacrificed after 2 h of ischemia and 24 h of reperfusion, and mitochondria from the penumbra were purified. PBR density (Bmax) and affinity were measured by radioligand assays. Mitochondrial [(3)H]PK11195 binding was correlated with neurological deficits in rats. Compared to the model group, the 10 mg/kg AST group, 40 mg/kg AST group, and 100 mg/kg AST group had fewer neurological deficits. The effects in the 40 mg/ kg group did not significantly differ from the effects in the 100 mg/ kg group. Compared to the model group, the 10 mg/kg AST group, 40 mg/kg group, and 100 mg/kg group had a decreased Bmax in the penumbra. The Bmax decreased in the 40 mg/kg AST group and in the 100 mg/kg AST group compared with the 10 mg/kg group. The Bmax and neurological deficits in the 40 mg/kg did not significantly differ from those in the 100 mg/kg group. By contrast, the AST-treated rats showed no significant changes in the binding parameter equilibrium dissociation constant compared with those in the sham operation group and the model group. AST protects ischemic brain tissue by inhibiting PBR expression after cerebral ischemia.

Structure-to-function relationships of bacterial translocator protein (TSPO): a focus on Pseudomonas.

The translocator protein (TSPO), which was previously designated as the peripheral-type benzodiazepine receptor, is a 3.5 billion year-old evolutionarily conserved protein expressed by most Eukarya, Archae and Bacteria, but its organization and functions differ remarkably. By taking advantage of the genomic data available on TSPO, we focused on bacterial TSPO and attempted to define functions of TSPO in Pseudomonas via in silico approaches. A tspo ortholog has been identified in several fluorescent Pseudomonas. This protein presents putative binding motifs for cholesterol and PK 11195, which is a specific drug ligand of mitochondrial TSPO. While it is a common surface distribution, the sense of insertion and membrane localization differ between α- and γ-proteobacteria. Experimental published data and STRING analysis of common TSPO partners in fluorescent Pseudomonas indicate a potential role of TSPO in the oxidative stress response, iron homeostasis and virulence expression. In these bacteria, TSPO could also take part in signal transduction and in the preservation of membrane integrity.

Peptide Targeting of Mitochondria Elicits Testosterone Formation

Testosterone (T) is the principal sex hormone responsible for growth and development of the reproductive system in male vertebrates.1 It has historically received much attention not only from the medical and scientific communities2 but also from the general public, owing to its psychological and behavioral effects.3 T drives the asymmetry of several biological processes spanning virilization to anabolism, disease prevention,1 and aging.4 In this issue of Molecular Therapy, Aghazadeh et al.5 describe a novel fusion peptide, TVS167, that can induce T formation in rat testes and increase its serum level as well as rescue its synthesis in adult male rats exposed to antagonists of the gonadotropin-releasing hormone, which constitutes the initial step in the hypothalamic–pituitary–gonadal axis governing T production. This peptide acts by exploiting the interaction between the voltage-dependent anion channel 1 (VDAC1) and the 18-kDa translocator protein (TSPO) within the mitochondrial transduceosome, a multicomponent molecular machine that controls lipid import and steroidogenesis. This peptide reveals a regulatory mechanism in the mitochondrial pathway of steroid anabolism that could provide a new target for therapeutic intervention (see model in Figure 1).

Effects of icariin on reproductive functions in male rats.

The present study investigated the effects and potential mechanism(s) of action of icariin on the reproductive functions of male rats. Adult rats were treated orally with icariin at doses of 0 (control), 50, 100, or 200 mg/kg body weight for 35 consecutive days. The results show that icariin had virtually no effect on the body weight or organ coefficients of the testes or epididymides. However, 100 mg/kg icariin significantly increased epididymal sperm counts. In addition, 50 and 100 mg/kg icariin significantly increased testosterone levels. Real-time PCR suggests icariin may be involved in testosterone production via mRNA expression regulation of genes such as peripheral type benzodiazepine receptor (PBR) and steroidogenic acute regulatory protein (StAR). Furthermore, 100 mg/kg icariin treatment also affected follicle stimulating hormone receptor (FSHR) and claudin-11 mRNA expression in Sertoli cells. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels were measured in the testes; 50 and 100 mg/kg icariin treatment improved antioxidative capacity, while 200 mg/kg icariin treatment upregulated oxidative stress. These results collectively suggest that icariin within a certain dose range is beneficial to male reproductive functions; meanwhile, higher doses of icariin may damage reproductive functions by increasing oxidative stress in the testes.

Sleep bruxism is affected by peripheral benzodiazepine receptor gene polymorphisms

Background There is increasing interest in genetic effects on sleep bruxism, as well as on psychological stress. Peripheral type benzodiazepine receptor (PBR) gene polymorphisms are associated with stress sensitivity but it is not yet clear whether polymorphisms in this gene have any influence on psychological stress before and after sleep bruxism. The present study was performed to examine the relationship between sleep bruxism and PBR gene polymorphisms.

On the Role of the Translocator Protein (18-kDa) TSPO in Steroid Hormone Biosynthesis

Morohaku et al (1) report that the translocator protein (18-kDa) TSPO, previously known as peripheraltype benzodiazepine receptor, is not required for steroid hormone biosynthesis. The study was based on analysis of a conditional knockout of TSPO in Leydig and Sertoli cells. The findings were surprising and were coupled to a definitive statement implying that what was suggested for Leydig cells is the case for all steroid-synthesizing cells in the body (1). To understand the reported findings and place them within the context of the TSPO and steroidogenesis literature, I will try to address five questions: 1) What are the complexities of cholesterol mobilization and transport? 2) What are the data indicating that TSPO is integrally involved in steroid formation? 3) Are there limitations of the Morohaku paper? 4) Where do the discrepancies lie? and 5) Where do we go from here?

Stress sensitivity in patients with atopic dermatitis in relation to the translocator protein 18 kDa (TSPO).

Atopic dermatitis (AD) is a chronic inflammatory skin disease, characterized by pruritic and eczematous skin lesions and dermatitis that worsens under stressful conditions. However, the relation of these symptoms to an individual's stress sensitivity is not well understood. On the other hand, expression of the translocator protein (18 kDa) (TSPO), formerly known as the peripheral-type benzodiazepine receptor, has been used as a biological marker of trait anxiety and stress sensitivity. The present study was designed to address this issue by examining TSPO in patients with AD. Fifty-two patients with AD (30 male and 22 female) and 163 healthy volunteers (89 male and 74 female) participated in this study. State-Trait Anxiety Inventory (STAI) scores were significantly higher in patients with AD, especially male patients, than in healthy subjects. The expression of platelet TSPO, as determined with a binding assay with [(3)H] PK11195, was also significantly higher in patients with AD, indicating that AD is a stress-responsive disease. In genomic analysis using lymphocytes, a single-nucleotide polymorphism of the human TSPO gene at exon 4 (485G>A), which is presumably associated with an individual's stress sensitivity, showed significantly lower frequencies of G/G and higher frequencies of G/A in patients with AD than in healthy subjects. The severity of AD, as determined with the Scoring of Atopic Dermatitis index, was correlated with TSPO expression in male patients with the G/A phenotype. In conclusion, the present study provides new evidence that variation in the TSPO gene affects susceptibility to AD.

Characterization and Modeling of the Oligomeric State and Ligand Binding Behavior of Purified Translocator Protein 18 kDa from Rhodobacter sphaeroides

Translocator Protein 18 kDa (TSPO), previously known as the peripheral-type benzodiazepine receptor (PBR), is a mitochondrial outer membrane protein that has been identified as a key player in cholesterol and porphyrin transport, apoptotic signaling, and cancer development, as well as neurological inflammation and disease. Despite a number of TSPO ligands whose effects have been studied with respect to these varied biological activities, the nature of their interactions with TSPO and the molecular mechanism of their effects remain controversial, in part because of the lack of an atomic-resolution structure. We expressed and purified the homologue of mammalian TSPO from Rhodobacter sphaeroides (RsTSPO), as well as a mutant form in a proposed drug binding loop, RsTSPOW38C. We characterized their binding behaviors with endogenous ligands and a series of compounds that affect apoptosis by using a sensitive tryptophan fluorescence quenching assay. Our results show that RsTSPO behaves as a dimer in the purified state and binds with low micromolar affinity to many of these ligands, including retinoic acid, curcumin, and a known Bcl-2 inhibitor, gossypol, suggesting a possible direct role for TSPO in their regulation of apoptosis. A computational model of the RsTSPO dimer is constructed using EM-Fold, Rosetta, and a cryo-electron microscopy density map. Binding behaviors of known ligands are discussed in the context of the model with respect to regions that may be involved in binding.