Structural and functional evolution of the translocator protein (18 kDa)

Current Molecular Medicine 2012, 12, 369-386 369

Structural and Functional Evolution of the Translocator Protein (18 kDa)

J. Fan1, P. Lindemann2, M.G.J. Feuilloley3 and V. Papadopoulos*,1,4

1The Research Institute of the McGill University Health Center, Montréal, Québec H3A 1A4, Canada 2Institut für Pharmazie, AG Molekulare Biotechnologie, Martin-Luther--Universität, Hoher Weg 8, 06120, Halle (Saale), Germany 3Laboratory of Cold Microbiology UPRES EA4312, University of Rouen, Evreux, France 4Departments of Medicine, Biochemistry, and Pharmacology & Therapeutics, McGill University, Montréal, Québec H3A 1A4, Canada

Abstract: Translocator proteins (TSPO) are the products of a family of genes that is evolutionarily conserved from bacteria to humans and expressed in most mammalian tissues and cells. Human TSPO (18 kDa) is expressed at high levels in steroid synthesizing endocrine tissues where it localizes to mitochondria and functions in the first step of steroid formation, the transport of cholesterol into the mitochondria. TSPO expression is elevated in cancerous tissues and during tissue injury, which has lead to the hypothesis that TSPO has roles in apoptosis and the maintenance of mitochondrial integrity. We recently identified a new paralog of Tspo in both the human and mouse. This paralog arose from an ancient gene duplication event before the divergence of the classes aves and mammals, and appears to have specialized tissue-, cell-, and organelle-specific functions. Evidence from the study of TSPO homologs in mammals, bacteria, and plants supports the conclusion that the TSPO family of proteins regulates specialized functions related to oxygen-mediated metabolism. In this review, we provide a comprehensive overview of the divergent function and evolutionary origin of Tspo genes in Bacteria, Archaea, and Eukarya domains.

Keywords: Cholesterol transport/binding, evolutionary origin, gene family, oxygen sensor, peripheral benzodiazepine receptor, steroidogenesis, translocator protein (18 kDa).

INTRODUCTION Translocator protein (18 kDa; TSPO), previously

known as the peripheral-type benzodiazepine receptor (PBR), was first described in 1977 as a binding target for the benzodiazepine diazepam in peripheral tissues. However, it exhibited features distinct from the classical benzodiazepine binding site on the central nervous system GABAA receptor [1]. Subsequently, TSPO was shown to be a widely expressed and primarily localized to mitochondria. In addition, TSPO protein levels were found to be elevated in endocrine steroid synthesizing organs, rapidly proliferating tissues like tumor tissue, and regenerating tissues following tissue injury [2-6]. Moreover, TSPO was shown to bind with high affinity to cholesterol and a wide range of drug ligands, in addition to benzodiazepines [7]. TSPO ligand binding is of diagnostic value for various pathological features in humans [1]. Pharmacological activation and inhibition is a common approach to the study of TSPO function in cells, tissues, and animal models of disease [1]. The wealth of information published in the TSPO field has demonstrated that this protein plays fundamental role in (i) general mitochondrial functions including

*Address correspondence to this author at the Research Institute of the McGill University Health Centre, Montreal General Hospital, 1650 Cedar Avenue, Room C10-148, Montréal, Québec H3G 1A4, Canada; Tel: +1 514 934 1934, Ext. 44580; Fax: +1 514 934 8439; E-mail: [email protected]

mitochondrial membrane biogenesis, respiration, permeability transition pore opening, and protein import; and (ii) specialized mitochondrial functions such as cholesterol import into mitochondria, the rate-limiting step in steroid biosynthesis [2, 7]. It is not yet clear if the specialized functions are related to the general mitochondrial functions, or if they represent an evolutionary gain-of-function adaptation to tissue specific needs.

Recently, we identified a new member of the Tspo gene family, Tspo2, which emerged from a gene duplication event before the divergence of the classes aves and mammals about 300 million years ago [8]. Comparative analysis of TSPO1 and TSPO2 functions demonstrated that TSPO2 lost its drug ligand-binding ability, but retained cholesterol-binding properties, hematopoietic tissue- and erythroid cell-specific distribution, and subcellular ER and nuclear membrane localization. In addition, we found that Tspo2 regulated cholesterol biosynthesis and redistribution during mammalian erythropoiesis. This function for Tspo genes in erythropoiesis is supported by experimental evidence from avian and zebrafish models [9-11]. TSPO homologs have also been identified in plants and bacteria, where they have developed specialized functions [12, 13]. In this review, we will summarize the evolutionary divergence of TSPO structure and its function(s).

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370 Current Molecular Medicine, 2012, Vol. 12, No. 4 Fan et al.

STRUCTURAL EVOLUTION OF TSPOs TSPO genes are present in almost all organisms

from the three main evolutionary lines or phylogenetic domains: Bacteria (eubacteria), Archaea (formally archaebacteria), and Eukarya (eukaryotes) [14] (Fig. 1A). However, some organisms, such as the yeast Saccharomyces cerevisiae, apparently lack a TSPO homolog [15]. In contrast, animals and plants have

more than one Tspo gene, namely Tspo1 and Tspo2 [8]. The TSPO superfamily proteins are enriched with tryptophan amino acids and share five conserved transmembrane domains (Fig. 1B). Moreover, their biological functions as cellular membrane components that contribute to tetrapyrrole biosynthesis and/or sterol metabolism are conserved in humans, animals, plants, and bacteria.

Fig. (1). The distribution and structure of translocator proteins in a Neighbor-joining tree was analyzed in three life domains: Bacteria, Archaea, and Eukaryota, where different kinds of sterols used in each kingdom were illustrated. A. Phylogeny of TSPO/TspO gene family. B. Comparison of the hydropathy plots of the predicted amino acid sequences of TSPO/TspO family members. The hydrophobicity indices were determined as described [128]. Shaded bars below the plots indicate transmembrane domains predicted by the TMpred algorithm [129]. Hydrophobic residues are positive.

Position25024524023523022522021521020520019519018518017517016516015515014514013513012512011511010510095908580757065605550454035302520151050

Me

an

Hyd

roph

obi

city

3.6

2.7

1.8

0.9

0

-0.9

-1.8

-2.7

-3.6TM1 TM2 TM3 TM4 TM5

B

TS

PO

1-C

RA

-a-H

uman

s .

TSPO2-Monodelphis .

0.0

0.5

1.0

Animals(Cholesterol)

Fungi(Ergosterol)

Bacteria(Hopanoids )

Archaea

Plants(24-alkylsteroids/cycloartenol)

A

Structural and Functional Evolution of the Translocator Protein Current Molecular Medicine, 2012, Vol. 12, No. 4 371

TSPO in Bacteria and Archaea The bacterial TspO (formerly crtK, ORF160) gene

encodes tryptophan-rich sensory protein (TSPO), an integral membrane protein that acts as a negative regulator photosynthesis gene expression in response to oxygen and light in the photosynthetic proteobacterium Rhodobacter sphaeroides [16]. Early studies suggested that TSPO was likely to be involved in the carotenoid biosynthetic pathway because it was located in the carotenoid biosynthesis gene (crt) cluster, and the rare codon usage in this gene indicated it had foreign origin and/or rapid evolution in R. capsulatus [17, 18]. Despite this evidence, deletion of the TspO did not adversely affect carotenoid biosynthesis, but instead increased the production of carotenoids and bacteriochlorophyll [19]. In this model, TSPO was shown to function as a transcriptional regulator of genes involved in photopigment biosynthesis, likely controlling the efflux of an intermediate in the heme biosynthesis pathway [20, 21]. A similar mechanism was proposed for the regulation of a nutrient deprivation-induced (ndi) locus in the endosymbiotic soil bacteria Sinorhizobium meliloti [22]. Interestingly, mammalian TSPO can functionally replace bacterial TSPO as an “oxygen sensor”, even though both TSPOs share less than 30% in protein sequence identity, indicating an evolutionary functional conservation [16]. In a recent study, the TSPO of the non-photosynthetic eubacterium Pseudomonas fluorescens was shown to share structural and functional characteristics with mammalian TSPO. The shared characteristics include binding for the high affinity diagnostic isoquinoline carboxamide ligand PK 11195, and involvement in bacterial adhesion and virulence [12]. Adhesion and expression of virulence factors are closely regulated by iron availability. Gram negative bacteria, such as Pseudomonas, have developed different strategies to acquire iron including secretion of hemolysins and import of heme through ABC-type permeases [23]. Bacterial TSPOs have very limited structural homology with bacterial hemophores; yet in Pseudomonads, iron-protoporphyrin export and resistance to oxidative stress are correlated [24] and the diversity of iron uptake systems is far from completely characterized [25]. Considering that bacterial TSPOs are involved in tetrapyrrole metabolism, we postulate that the binding of the tetrapyrrole metabolite protoporphyrin to plant and mammalian TSPOs may have originated from this ancient function. Rhodobacter sphaeroides, higher plant and mouse TSPOs share similar affinities for protoporphyin IX [26-28].

In silico screening of databases demonstrated that TSPOs are also present in Archaea (Fig. 1A) [12, 29]. As more prokaryotic genome sequences become available, we can further clarify the TSPO distribution among different species. Nevertheless from currently available data it can be concluded that some prokaryotes such as Escherichia coli or Pseudomonas aeruginosa lack this gene in their genomes [12, 30]. In P. fluorescens and P. syringae, the presence of

transposases encoded in the vicinity of TspO that lack the usual CG enrichment relative to neighboring genes suggests that if eubacterial TspO resulted from a transfer, then the transfer event was ancient [12]. It is not clear whether E. coli and other species that lack a Tspo gene lost Tspo during evolution or never had a Tspo gene.

TSPO in Yeasts and Fungi Very few reports of Tspo genes in fungi exist. TSPO

from Cryptococcus neoformansat is expressed at 37°C and is assumed to facilitate adaptation to host temperatures. It was not associated with sterol biosynthesis because the sterol synthesis-related enzymes are expressed only at 25°C [31]. Considering that this organism is an opportunistic human pathogenic fungus, we believe that TSPO expression is likely to be related to the pathogenesis and/or pathogen survival in the human host. The budding yeast Saccharomyces cerevisiae is known to lack Tspo, which may be due to the loss of almost 90% of duplicated genes in this organisms [32]. This characteristic makes S. cerevisiae well-suited for heteroexpression studies and an ideal model system for studying the biochemical and pharmacological properties of TSPO [15]. However, this lack of TSPO in S. cerevisiae also shows that TSPO is not essential for yeast viability and therefore other pathways perform the functions attributed to the TSPO of other organisms.

By screening the fungi genomes available from the NCBI, we identified Tspo genes in several groups of fungi including Basidiomycota, Schizosaccharo-mycetes, Prezizomycotina, and Saccharomycotina. Interestingly, the TSPO ortholog in Schizosaccharo-myces pombe is one of the core environmental stress response genes that responds to excess copper [33]. Also of interest, the only organism within the Saccharomycotina sequenced genome group that had an identifiable Tspo gene is Kluyveromyces lactis. Gene structure analysis showed that the Tspo from K. lactis is likely to be an alien gene from bacteria, because it had different GC content than the up- and down-stream genes, and incongruent placement in the phylogenetic trees (Figs. 1A and 2). This suggests that the Tspo gene from K. lactis is one of the few cases of horizontal gene transfer from prokaryotes to fungi [34]. It is not yet clear how this occurred or what the function of this gene is in K. lactis. Gut fungi, as well as bacteria, synthesize benzodiazepine precursors, and this activity is likely to be related to TSPO expression [35, 36]. Moreover, TSPO may also function in the tetrapyrrole biosynthetic pathway [37], which is conserved in fungi and other animals, as well as the α-proteobacteria.

TSPO in Plants In recent years, there have been several reports of

plant Tspo genes. TSPO from the potato plant


Fig. (2). The TSPO in yeast Kluyveromyces lactis was acquired by a bacterial-fungi horizontal gene transfer event. A. Genomic location and GC content of the TSPO gene and its upstream and downstream genomic sequences. TSPO, Thi4, KIPHO3, and Gtt1 genes are shown. The GC contents were calculated and plotted using cpgplot in the EMBOSS package [130]. The ratio of the observed to expected number (Obs/Exp) of GC dinucleotides was calculated over a window (sequence region) which was moved along the sequence (base number): NC 006037 (GenBank, NCBI). The high and low GC contents are indicated by arrows. B. Phylogentic tree illustrating the clustering of a K. lactis TSPO with bacteria TSPOs. The tree was constructed using the neighbor-joining method of the MEGA 4 package with options of pair-wise deletion and Dayhoff PAM matrix model [131]. Only bootstrap values larger than 50%, the percentage of bootstrap replications out of 1000 replications, are shown, in which the given note was observed. The bold letters indicate the main branches. The estimated genetic distance indicated as the number of substitutions per amino acid site is proportional to the horizontal length of each branch. GenBank accession numbers and other abbreviations used are indicated. The circle view of the phylogenetic topology shows the relationship of the top 500 TSPO genes from the GenBank, NCBI. The scale bar represents the number of substitutions observed per site.

A

NC 006037 11237 bp

TSPO KlPHO3/acid phosphatase Gtt1/glutathione transferase

Ato2/acetate transporter Thi4/thiamine metabolism-related protein

Schizosaccharomycetes

Saccharomycotina

EEU48511Nectria .

XP 381207Gibberella .

EEY18812Verticillium .

XP 363659Magnaporthe .

XP 001558937Botryotinia .

XP 001597624Sclerotinia .

XP 001794921Phaeosphaeria .

XP 001937862Pyrenophora .

XP 001913022Podospora .

XP 964457Neurospora .

XP 001399328Aspergillus .







XP 002479604Talaromyces .

XP 002143301Penicillium .

XP 002171969Schizosaccharomyces .

NP 595490Schizosaccharomyces .

XP 001877141Laccaria .

XP 001837230Coprinopsis .

XP 568678Cryptococcus .

XP 762553Ustilago .

ZP 01733475Flavobacteria .

XP 001619759Nematostella .

YP 001193741Flavobacterium .

YP 001295240Flavobacterium .

XP 451009 Kluyveromyces . YP 003322820Thermobaculum .

NP 070304Archaeoglobus .

YP 001818571Opitutus .

99

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74

94

79

68

78

100

100

100

100

100

100

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99

53

64

99

60

75

87

100

93

85

54

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51

99

0.2

Bacteria

Basidiomycota

Prezizomycotina

B

0.0

0.2

0.4

0.6

Fung

i


Fig. (3). Alignment of the deduced amino acid sequence of TSPO and TSPO-like genes from plants. The TSPO-like domains are indicated as follows: TM1-TM5, transmembrane domains. The red bar indicates the potential conserved PK11195 binding motif (see below). Residues that show 100% conservation, 80% or greater conservation, and 60% or greater conservation are highlighted in black, dark grey, and light grey, respectively. Similar amino acids are defined by Higgins [132] as being grouped in the same class as identity. Dashes indicate gaps and were introduced to facilitate alignment.

(Solanum tuberosum) was found to bind the benzodiazepine RO 5-4864 with an affinity similar to mammalian TSPO [38]. Also similar to the mammalian ortholog, recombinant TSPO protein from Arabidopsis thaliana also exhibits high affinity benzodiazepine binding properties and elevated the uptake of cholesterol and protoporphyrin IX by transfected E. coli protoplasts in response to benzodiazepine [27]. In addition, TSPO from both S. tubuerosum (StTSPO) and A. thaliana (AtTSPO) bind benzodiazepines and protophorphyrin IX, and are implicated in porphyrin synthesis and metabolism. TSPO has been implicated in porphyrin metabolism in other plants, such as the moss Physcomitrella patens. Null TSPO mutants of P. patens that lack PpTSPO1, one of the three genes

encoding TSPOs (Fig. 3), have shown that TSPO regulates the transport of porphyrin intermediates into mitochondria for heme biosynthesis, and removal of photoreactive tetrapyrrole intermediates from the cytosol. Additionally, this null mutant produced elevated levels of H2O2, implying that TSPO1 prevents reactive oxygen species (ROS) formation or scavenges ROS [13]. PpTSPO1 has 193 amino acids and a longer amino-terminus than bacterial and animal TSPOs. In contrast, PpTSPO2 and 3 contain only 173. Thus, the amino-terminal elongation may be acquired by the bryophyta (Fig. 3).

The presence of two other Tspo paralogs in this organism suggests that the three TSPO gene products

* 20 * 40 * 60 * 80TSPOiso1Physcomitrella : MNSEGLQKRSRDTSEFVHDNDPTQQKYVAKAYRKTTEVAKKP---------------GVPSLIVACALPLAAGFLVSMFATSPOiso2Physcomitrella : ------------------------------------MWSLW----------------SIGCLVVAVFVPVAAGFSGTAFGTSPOiso3Physcomitrella : ------------------------------------MWNLW----------------DVGSLVIAVGLPVGVGFAGTAFGTSPOiso1Vitis : MAST-NLKQRIKDGPSTP------FPTNNTT----KREKKM-----------AMAKRGLRSLTTAVSIPLSLTLVTIYFSTSPOiso2Vitis : MELK-HG------------------PEDQ---APTHQHKIS-----------------LPKLAVAVGVPVCLTMAIIFLFTSPOiso1Populus : MDSQ--NVKQRTRDTVDEP---NTIVGKNNSKNVRFRREKRV----------AMAQRGIRSLAIAVALPLSLALCNVYFFTSPOiso2Populus : MDSQ--NAKQRIRDTDYDPNISNPTKSKDNSKTVRLKREKRV----------AMAKRGIRSLAIAVALPLSLTISNLYFFTSPOiso3Populus : MASR-TLKNL--------------AEKDQPVLAKPKTRKTSN------------TKGSLWSLFMVLVAPPSLSLTIIYLFTSPOiso1Ricinus : MDSQ--SIKHRTKDPDQSD------TPKETDMRSYSRRDKRM----------AMAKRGIRSLAIAVAFPLSLTLFNIYFFTSPOiso2Ricinus : MAVQ--KSKTR-------------SKKDQFTAGAEAKAKLL----------------SFCSLFLITLVPLCLTMTITYLFTSPOSolanum : MASQQDELKHRITTKSQQNEPQQTEQHTKSAHDNDSKTNKNINK----STRKQIAKRGLKSLTIALTIPLLLTLIDISLFTSPOPicea : --------------------------------------MQLV---------------NAVSLILSVALPVGLGFLGSLAGTSPOSorghum : MAAAAHEGMTHRVATSRNDGGGSDAAAVSGPNKKPGGGGGGGGGGRVSSSSSSSSRRGLRSLAAAVSLSAALTALSFFFATSPOZea : MATAAHEGVTQRVAASGSRDDGASGGIAAVSGPNKKPGGG-------------RRRRGLRSVAAAVSIPAALVALS-FFATSPOArabidopsis : MDSQ-DIRYRGGDDRDAATTAMAETERKSADDNKGKRDQKR-----------AMAKRGLKSLTVAVAAPVLVTLFATYFL * 100 * 120 * 140 * 160TSPOiso1Physcomitrella : S--PDQWYKNLNK---PSWTPPGPLFGLIWTFIYPVMGLASWLVWADGGFQRNGFALGA-YFVQLGLNLLWSVLFFKF--TSPOiso2Physcomitrella : GGGDTEWYKELNK---PSWTPPDWVFPVMWTTLYILMGISSWLVWKEGGFAAQGYPLGA-YIFQLALNFLWTPIFFGM--TSPOiso3Physcomitrella : GGGDSEWYKELNK---PPWTPPGWVFPIMWTTLYILMGVSSWLVYKEGGFSAQGYPLGA-YIFQLALNFLWTPIFFGM--TSPOiso1Vitis : GA--TDRYRTLPK---PFWFPPLWAVHATSMASSLLMGLSAWLVWAEGGFHKKPTALPL-FVAQLALSLIRDPIVFGY--TSPOiso2Vitis : GS--SHKYRAIAK---PFWFPPLWVMHVGSLVCSGLMGVSAWAVWSEGGFRGESDALPL-YVAHISLGIVWEPLVVVM--TSPOiso1Populus : GSTKGYGTSSSRSISKPFWFPPLWALHMTCVTSSFLMGLSAWLVWAEGGFHRNPTALYL-YLTQLGLSLAWDPIVFRM--TSPOiso2Populus : GTTRGYGTSTGSISM-PFWFPPPWALHLTCMTSSFLMGLSAWLVWAEGGFHRNPAALYL-YLAQLGLSLAWEPIVFRM--TSPOiso3Populus : GS--GRRYRALAK---PSWFPSLTIIHLGSVGSTFLMSLAAWLVWTNDGFHVDSDALPL-YIAQISLSMVWDPLVLRI--TSPOiso1Ricinus : GS-RNGYGPLSTK---PFWFPPMWALHFTCLASSFLMGLSAWLVWAEGGFHKKPAALSF-YLAQLGLGLIWDPIVFRM--TSPOiso2Ricinus : GSSKKYQALDR-----PFWFPSLTLIHTASVGSALLMSLAACYVWADGGFRLDSDALPL-YISQVSLSIVWDPLVLKI--TSPOSolanum : GS--SYQYVSMEK---PFWFPRLWALHLACLGSSLLMGLSAWLVWAEGGFHRKPMAMLL-YLSQLGLSLAWDPVVFKS--TSPOPicea : DGGNSDWYKQLKK---PPWTPPNWAFPVMWTILYVMMGVAAWYVWLHGGFEKQGVVLGV-YLLQLFLNLLWTPLFFGM--TSPOSorghum : AGGGHSSSSPP-----SASTTTVAMVRAGSVASEAVLALAAWMAWAEGGVHARPAATLLPYAAQLGAALAWAPLVLGQGHTSPOZea : AG--HSTP--------PPSSATVAVVRAGSVASEAVLALAAWMAWAEGGLHARPAATLLPYAARLGAALAWAPLVLGR--TSPOArabidopsis : GT--SDGYGNRAKS--SSWIPPLWLLHTTCLASSGLMGLAAWLVWVDGGFHKKPNALYL-YLAQFLLCLVWDPVTFRV-- * 180 * 200 * 220 * TSPOiso1Physcomitrella : -HSVTLAFVDILALGAAVFTTIGAFQPVNHIAANLMKIYFGWVVFASVLTASILMKNSRGGH-----------------TSPOiso2Physcomitrella : -HRPGYALVEIVILWLAITVTIFLFYPVNPIAAYLLIPYIAWVTVATSLNWYIWLYTVARTSPNLCSPREVHTPSDPSETSPOiso3Physcomitrella : -HFVGYGLIEIIILWFAIALTIYLFLPVNPIAAYLLIPYIGWVTIATSLNWYIWMYN---GSSEVTQP--LQSGSSAYNTSPOiso1Vitis : -GGTRVGLVVCMGLFGALVGCARMFREVNPIAGDLVKPCLAWTAYVGVVTLKLLFA-----------------------TSPOiso2Vitis : -GAAWMGLGFCVVHFGTLVACYSAFRNVNPVVGELVKPCLAWVAILTFLTFKLIYL-----------------------TSPOiso1Populus : -SAPWVGLLVCLATFGALVGCSRQFKEVNPIAGNLVMPCLAWASFLAFVNLKLLFL-----------------------TSPOiso2Populus : -AAPWVGLLVCLATFAVLVGCSRQFKEVNPIAGDLVKPCLAWASFLAIVNLKLLFL-----------------------TSPOiso3Populus : -GAVWLGFLFSMLNLGTLLACYWAFGKVNPLSKKFVKPCLTWVAYLTLVTFDLMFL-----------------------TSPOiso1Ricinus : -DATWVGLVVCLAMFGSLVGCSRQFKEVNPIAGDLVKPSLTWAAFLAIVNLKLVFL-----------------------TSPOiso2Ricinus : -GEAWLGVVFSLVNLGTLFGCYCMFGKKGDFGLFSERALKCYYIRVLKVKAKVAKPAGLDCKNSSELNKPFPRRHM---TSPOSolanum : -GATRIGLVLCMALFGVLIACFRAFKNVNPIAGDLVKPCFGWAGF----------------------------------TSPOPicea : -HNPGAAFADIMLLWFTIAANIYLFWHVEPVAAYLLVPYIIWVTLASTINLYVWIHYVNDSQYHVTNRPDLHSKSS---TSPOSorghum : AAAPRAGLACCAAMAAAAVACARGFGAVNPVAGDLAKPAVAWAVILAVVNYKML-------------------------TSPOZea : HAAARAGLACCAAMAVGAVACARGFGAVNPVAGDLAKPAVAWAVILAVVNYKML-------------------------TSPOArabidopsis : -GSGVAGLAVWLGQSAALFGCYKAFNEISPVAGNLVKPCLAWAAFVAAVNVKLAVA-----------------------

LxKPS/FW

TM1

TM2 TM3

TM4 TM5


have specialized functions. Sequences encoding multiple TSPOs in other plants have also been identified, suggesting that multiple TSPO genes arose from gene duplication events, similar to the origin of Tspo2 genes in vertebrates [8].

Affymetrix ATH1 gene arrays indicated that AtTSPO is expressed in most cell types at low levels. The highest level of expression was found in germinating seeds [39]. The intracellular distribution, however, is contradictory; PpTSPO1-GFP constructs is localized to mitochondria [13], whereas AtTSPO-GFP was localized to the Golgi and ER [40]. However, A. thaliana, Digitalis lanata (foxglove), and S. tuberosum mitochondria had high affinity Ro5-4864 binding [27].

Plant TSPO proteins have physiological functions in the adaptation to adverse environmental conditions, such as cold, osmotic and salt stress. Specifically, PpTSPO, AtTSPO, and StTSPO have roles in abiotic stress adaptation [13, 40-43], which is mediated by ABA. The ABA-dependent induction of AtTSPO is apparently linked to tetrapyrrole metabolism [13, 40, 44]. Interestingly, magnesium deficiency also induces expression of AtTSPO (At2g47770), by a mechanism involving ABA signaling [45]. Another clue to the regulation of AtTSPO expression was found in the T-DNA-tagged double mutant srk2cf, which lacks two SnRK2-related protein kinases. This mutant had depleted AtTSPO expression. The authors concluded that subclass II SnRK2s might be involved in a parallel pathway for activation of AREB/ABF bZIP-type transcription factors in response to drought stress [46].

Taken together, these studies show that plant TSPOs possess drug ligand binding properties, which may be relevant to the natural benzodiazepines or benzodiazepine-derivatives commonly found in plants [47-49]. Additionally, TSPO-mediated tetrapyrrole/ porphyrin metabolism, which uses glutamate as the starting point in plants, may have the same mechanisms in plants, bacteria, and archaea [37, 40]. This pathway may also be similar in the animal pathways, which utilize glycine and succinyl-coenzyme A, instead of glutamate, as the starting materials to synthesize the universal precursor of tetrapyrrole biosynthesis [50].

Plant tetrapyrrole biosynthesis is complex and branched, and consists of plastidal and mitochondrial pathways [50]. The proposed plastidal and mitochondrial colocalization of coproporphyrinogen III oxidase, protoporphyrinogen IX oxidase [51], and ferrochelatase [52] is still controversial and may vary between species. In addition, duplicity of the pathways may have facilitated the loss of the mitochondrial pathway in some systems. The direct involvement of plant TSPOs in steroidogenesis remains hypothetical. C17 and C21 steroids are common in plants [53-55] and may have function as hormones in concert with the brassinosteroids [56, 57]. The biosynthesis of these steroids requires cleavage of the side-chain. Most of the studies in plants have been performed in cardiac glycoside containing Digitalis species [58, 59]. The

side-chain cleavage in plants may be catalyzed by a P450 monooxygenase [60]. The constituents of the accompanying redox system, like ferredoxin and ferredoxin reductase, have been reported to be present in higher plant mitochondria [61, 62] but little is known about mitochondrial-localized cytochrome P450s [63]. Indeed, there are reports indicating that recombinant tobacco plants expressing bovine side-chain cleavage CYP11A1 import this enzyme into mitochondria and synthesize elevated progesterone levels [64-66]. Moreover, mitochondrial pregnenolone formation has been reported for D. purpurea and D. lanata mitochondria [59, 60, 67].

TSPO in Animals The most extensive studies of TSPO structure and

function have been performed with mouse and human TSPO proteins. These studies have focused on the drugs and endogenous ligand binding properties of TSPOs, and the roles of TSPOs in tetrapyrrole/ porphyrin metabolism, cholesterol transport, steroido-genesis, mitochondrial function, cell proliferation, apoptosis, and erythropoiesis. Few reports on the TSPOs in other animals are available. However, the Genbank (www.ncbi.nlm.nih.gov) sequence survey performed herein clearly indicates that TSPO is widely distributed throughout most animal phyla (Fig. 1).

Of the invertebrates, nematodes and insects possess a single gene encoding TSPO. In addition, Brugia malayi, Caenorhabditis elegans, Caenorhabditis briggsae, and Drosophila melanogaster TSPOs share a TSPO-related protein domain [68]. Photoaffinity labeling experiments demonstrated that TSPO is expressed in the insect Manduca sexta, a finding that led to the speculation that TSPO-mediated ecdysteroidogenesis exists in insects [69]. While the low expression of TSPO in this species weakens this hypothesis [70], the fact that the synthesis of insect ecdysteroids requires mitochondrial cytochrome P450 monooxygenases (CYPs) implies that the precursor of ecdysteroids must move inside the mitochondria [71]. CYP12A1 from the housefly is considered to be the closest structural CYP enzyme homolog of vertebrate mitochondrial CYP11A1 [72], even though they are of polyphyletic origins [73]. The sequence divergence might be caused by rapid positive selection following CYP12-like gene formation [74]. Several CYP11s have evolved in insects to metabolize a variety of xenobiotics, but not insect ecdysteroids; despite this, CYP11 is likely to have an ancestral role in steroidogenesis [75]. What is the relationship between steroidogenesis and xenobiotic metabolism and what is the ancestral role of TSPO in steroidogenesis are questions that remain to be addressed.

While no other research on insect Tspo genes has been reported, genome-wide protein-protein interaction analysis of D. melanogaster revealed that TSPO is likely to interact with seven proteins: α-crystallin-type small(s) heat shock protein (GC4167, Hsp67Ba), zinc finger protein (CG9060, Zpr1), protein with N-terminal


ubiquitin-like domain (UbL) and C-terminal ubiquitin-associated domain(s) (UBA), Rad23 (CG10694, Rad23), GIY-YIG domain containing protein (CG18271, Gi-Yi), tubulin folding cofactor B (CG11242, Tbcb), integrator complex subunit 1 (CG3173, Ints1), and 26S proteasome (prosome, macropain) non-ATPase 5 subunit (CG12096, PSMD5) (Fig. 4) [76]. TSPO also indirectly interacts with 26S proteasome ATP-dependent regulatory subunit 1 (CG1341, Rpt1). This information suggests that in D. Melanogaster, TSPO may function in an ATP/ubiquitin-dependent "26S" proteasome complex. Attenuation of the 26S proteasome has a strong influence on the aging process because of the intracellular accumulation of unfolded, misfolded, and/or aggregated proteins, which can result in aging or age-related neurodegenerative diseases [77] In addition, TSPOs from both D. melanogaster and C. elegans play a significant role in longevity [78]. Thus, we hypothesize that TSPO plays a role in aging by regulating 26S proteasome function.

A Tspo gene has been discovered in the genome of the blood fluke Schistosoma mansoni [79] Like other TSPO family members, S. mansoni TSPO exhibited high affinity binding to 3H-PK 11195 [80]. In contrast, the Asian blood fluke Schistosoma japonicum seems to have lost this gene, which may explain why S. japonicum is insensitive to the benzodiazepine Ro11-3128 [81]. The binding sites of benzodiazepine and praziquantel are different in schistosomes, suggesting that TSPO is a potential drug target for the human

pathogenic worm S. mansoni. Mollusks that act as the intermediate hosts of schistosomes harbor TSPO in soft tissues. In this environment, TSPO exhibits pharmacological properties comparable to those of lower vertebrates such as the trout [82, 83].

In vertebrates, TSPO has multiple cellular functions in cell proliferation, apoptosis, regulation of mitochondrial function, porphyrin transport, heme synthesis, cholesterol transport, and steroidogenesis [7]. Tspo is expressed in zebrafish as a maternal transcript that is mainly distributed in the hematopoietic intermediate cell mass. In this context, TSPO contributes to primitive erythropoiesis by a mechanisms independent of its cholesterol binding ability [11, 84]. In zebrafish, whether TSPO is involved in steroidogenesis is not known, even though Cyp11a1 is found in the embryonic interrenal primordia (adrenocortical equivalent in zebrafish) through adulthood [85].

TSPO, as well as the TSPO endogenous polypeptide ligand diazepam-binding inhibitor (DBI or ACBD1)-related peptide, is present in the frog adrenal gland [86]. While benzodiazepine derivatives stimulate active sodium transport in frog skin, it is not known if TSPO plays a role in this process [87]. In birds, there are two genes encoding TSPO-like proteins, TSPO1 (the ortholog of the well characterized mammalian TSPO) and TSPO2 (also known as p18 and/or PBRL), a new member of the TSPO family [8-10]. Chicken Tspo1 is weakly expressed in the developing central

Fig. (4). Genome-wide protein-protein interactions in D. melanogaster revealed that TSPO (CG2789) interacts with seven proteins: α-crystallin-type small(s) heat shock protein (GC4167, Hsp67Ba), zinc finger protein (CG9060, Zpr1), one member of a family of proteins that possesses an N-terminal ubiquitin-like domain (UbL) and a C-terminal ubiquitin-associated domain(s) (UBA), UV excision repair protein RAD23 homolog A (CG10694, RAD23), GIY-YIG domain containing protein (CG18271, GIYI), tubulin folding cofactor B (CG11242, TBCB), integrator complex subunit 1 (CG3173, INTS1), and proteasome (prosome, macropain) 26S subunit, non-ATPase, 5 (CG12096, PSMD5) (http://www.thebiogrid.org). Rpt1, ATP-dependent 26S proteasome regulatory subunit.

26S Proteasome Complex

Zinc finger

protein

-crystallin-type small(s) heat shock protein

UV excision repair

protein RAD23

homolog A

GIY-YIG domain containing protein (excinuclease)

ATP-dependent 26S proteasome regulatory subunit


nervous system, whereas chicken Tspo2 is expressed exclusively in differentiating primitive erythrocytes and its expression is tightly correlated hemoglobin gene expression [10]. A similar TSPO2 expression pattern has been observed in the turkey, where TSPO2 can form a multimeric erythrocyte nuclear complexes with lamin B receptor (LBR or p58), the nuclear lamins, an LBR-specific kinase, and a 34-kDa protein [9].

In mammals, TSPO1 is primarily localized in the outer mitochondrial membrane of eukaryotic cells and mainly functions in porphyrin synthesis, cholesterol transport, and steroidogenesis. In contrast, TSPO2 is localized to the endoplasmic reticulum (ER) and nuclear membranes, and functions in the re-distribution of cholesterol during erythrocyte maturation [8]. Several publications have reviewed the function and properties of TSPO1 [7, 88-94]. However, the discovery of the presence of TSPO2 in 2009 opened new avenues of investigation into the evolution of TSPO family member functions [8]. The observation that TSPO can compensate for the loss of function of homologs in some bacterial species demonstrates that TSPO functions are, at least in part, evolutionarily conserved [16]. While other TSPO functions such as cholesterol binding and trafficking have been gained during speciation and evolution, the conserved functions have been maintained from bacteria to plants and to mammals. This knowledge assists us in understanding the biological function of TSPOs in pathological conditions such as cancer, endocrine, and neurological diseases. It also allows us to design additional studies that further characterize the extraordinary plasticity of this family of proteins and their involvement in multiple pathways that control cellular metabolism and/or signal transduction.

PHYSIOLOGICAL RELEVANCE OF TSPO STRUCTURE

All members of the TSPO protein family possess five evolutionarily conserved transmembrane domains, two less conserved cytoplasmic loops, and either N- or cytoplasmic C- termini extending into the mitochondrial lumen (Figs. 1B and 5). With the exception of the portions of the protein buried within the hydrophobic phospholipid bilayer, the hydrophilic portions are proposed to be the essential functional segments that interact with other intracellular proteins and/or native ligands. Site-directed mutagenesis of the human, bovine, and mouse TSPOs was used to identify conserved sites within TSPOs form multiple species with high binding affinity for isoquinoline carboxamides such as PK11195 [30, 95, 96]. In contrast, the sites with binding affinity for benzodiazepines such as Ro5-4864 are not conserved between species. TSPOs also have multiple binding sites for benzodiazepines [97], even though TSPO binding to benzodiazepines is dependent upon the presence of other partners such as the voltage-dependent anion channel (VDAC) [91, 98, 99]. A functional relationship between TSPO and the major porin OprF exists in the bacterium P.

fluorescens [12]. Gram-negative bacteria have two membranes, with OprF located in the outer membrane, and TSPO located in the cytoplasmic (or inner) membrane. This organization could explain why P. fluorescens is not sensitive to diazepam. It is also important to note that benzodiazepine binding sites are present in other proteins, such as GABAA receptors [100] in mammals, and VDAC/porin and adenine nucleotide translocator (ANT) in lower unicellular organisms [101].

PK 11195 seems to bind selectively to the TSPO with high affinity in all the species ranging from bacteria to humans that have been tested to date. TSPO in the bacterium P. fluorescens has a Kd for PK 11195 binding that is similar to the eukaryotic Kd, and consistent with this, treatment with PK 11195 affects the adhesion and virulence of the bacterium [12]. Arabidopsis TSPO also has high affinity for PK 11195 [27]. Other plants with TSPOs that have similar PK 11195 binding include Matricaria chamomilla and Ceratonia siliqua (carobe tree) [48, 102]. The PK 11195 binding ability of TSPO is conserved back to bacteria, and PK11195 is therefore referred to as the TSPO diagnostic ligand. Site directed mutagenesis of mouse TSPO has narrowed down the PK 11195 binding sites to amino acids 41–51 in the amino-terminus of the receptor, since the deletion of this region resulted decreases PK 11195 binding by 30–45% [30]. The theoretical PK 11195 and Ro5-4864 binding sites have been predicted using a synthetic-computational approach [103]. However, the sites responsible for PK 11195 binding in human TSPO1 have been experimentally confirmed to be R24, E29, L31, L37, P40, S41, W42, W107, and W161. In addition, the Ro5-4864 binding sites were experimentally confirmed to be E29, R32, K39, and V154 [96]. These ligand binding residues are not mutually exclusive, since disruption of benzodiazepine binding sites does not abolish the PK 11195 binding properties. In bovine TSPO1, V154 is essential for benzodiazepine binding [96], but based on sequence alignments the most conserved PK 11195 binding residue is W107. Despite this, zebrafish TSPO can bind PK 11195 even though it does not contain the corresponding conserved W107 [11]. Similarly the bacterial TSPOs from Pseudomonas shares amino acid 53.8% identity with human TSPO, lacks the conserved W107 and W161 residues, but is well conserved from L37 to G50 [12]. Therefore, the identification of binding sites in TSPO from a single species does not necessarily reflect the structural properties of the whole protein family.

Some members of the TSPO family do not bind to PK 11195. TSPO2, the newly described subfamily of TSPO proteins, does not bind PK 11195 [8] and the same is true for the R. sphaeroides bacterial TSPO [16]. Analysis sequence comparisons were used to identify a conserved LxKPsW/F motif for PK 11195 binding within the first loop of each member of the TSPO protein family. The designation of LxKPsW/F as a binding motif for PK 11195 is supported by experimental evidence from the studies of the binding


Fig. (5). Schematic structure of human and mouse TSPO1/TSPO2 models. The primary sequence shown is that of human TSPO1 and TSPO2 (AO, and mouse TSPO1 and TSPO2 (B) to illustrate the five transmembrane domains and cytoplasmic domains. The lines indicate the mitochondrial membrane. c1 and c2 refer to extramitochondrial cytoplasmic loops, m1 and m2 to intramitochondrial loops, n1 and n2 to intra-nuclear membrane loops, and Ct and Nt to the carboxyl- and amino-terminal domains, respectively. The position of the unique sites under positive selection in the TSPO2 lineage is shown by a red arrow. Dark arrowheads indicate potentially important amino acid residues for binding benzodiazepine. Also noted as arrows are the conserved motif of L.Y….D, corresponding to the previously reported cholesterol recognition/interaction amino acid consensus (CRAC) domain [30]. The open circles are the omitted amino acid sites. The snake-like diagram was constructed by using residue-based diagram editor RbDe [133]. This research was originally published in the Journal of Biological Chemistry as supplementary supporting data [134]; ©2009 by the American Society for Biochemistry and Molecular Biology.

Human TPSO1

Human TPSO2

Cholesterol binding

Cholesterol binding

Nt

Ct

C1

C2

n1 n2

AA substitution(gray arrow)

C1

C2

PK11195 (green arrow)Ro5-4864 (red arrow)

Nt

m1 m2

Ct

A

Sites under divergence

Mt location

Mouse TPSO1

Mouse TPSO2

Cholesterol binding

Cholesterolbinding

Ct

C2

n2

C2

m2

Ct

Nt

C1

n1

C1

Ntm1

B

Sites under divergence

Mt location

PK11195 (green arrow)Ro5-4864 (red arrow)

AA substitution(gray arrow)


properties of several TSPO proteins (Fig. 6A). Confirmation of this motif awaits systematic experimentation.

Within the C-termini of mammalian TSPOs, a conserved cholesterol recognition/ interaction amino acid consensus (CRAC) was identified to be –L/V/I-(X)1–5-Y-(X)1–5-R/K- (30). All TSPOs of animal origin have this motif and therefore presumably bind cholesterol, whereas TSPOs from bacteria, fungi, and plants do not possess this motif, suggesting that TSPOs do not participate in cholesterol transport and metabolism in these organisms (Fig. 6B-D). In most prokaryotes, sterols are replaced by bacterioho-panepolyols, generally referred as hopanoids, which have a similar pentacyclic structure [104] and function in the preservation of membrane integrity [105]. Interestingly, CRAC domains are rarely found in bacterial and archaeal proteins. Rare examples include proteins from the marine bacterium Fulvimarina pelagi (ZP_01438239), the photosynthetic bacterium Rhodobacter (ZP_05842938), and the thermophilic obligately-aceticlastic methane-producing archaeon Methanosaeta thermophila (YP_842785). Interactions between hopanes and bacterial TSPO deserve further investigation, but support the view that TSPOs have an ancient role in the transport of sterol-like structures which may be conserved in the early development of higher organisms when cholesterol is used as the precursor for steroid synthesis, including mammalian steroid hormones and insect ecdysteroids. In the nematode C. elegans, the role of cholesterol is mainly believed to be in signaling to control molting and induce a specialized non-feeding larval stage [106]. No evidence exists that cholesterol-derived steroids act as signaling molecules in this species. Thus, the function of nematode TSPO is not clear. Knockdown of the one known mitochondrial P450 in C. elegans, CYP44A1, did not cause obvious developmental abnormalities [107]. Taken together, these data argue against a role for TSPO and CYP44A1 in the endocrinology and/or development of C. elegans.

A similar but more complicated situation exists in insects, where TSPO with a CRAC domain and two mitochondria CYP enzymes, CYP12A1 and CYP301 have been identified [75]. One of the CYP genes is essential while the other generates a variable number of taxon-specific paralogous CYPs that are rapidly evolving and are involved in xenobiotic metabolism [75]. Interestingly, prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis, involves three hydroxylations of the precursor molecule in the mitochondria [108]. The early steps in the regulation of ecdysteroid synthesis remain unknown, but are likely to occur in the mitochondria and have been characterized as the rate-limiting “black box” in ecdysteroid biosynthesis [109]. These characteristics appear to parallel the process of rate-limiting cholesterol transport into mammalian mitochondria. Studies with the ecdysteroid-deficient mutant ecd1 suggest that some corresponding gene products may play critical roles in this proposed steroid precursor translocation event. For

example, expression of the steroidogenic acute regulatory protein gene Start1 in the ecd-1 mutant was reduced and may be responsible for the reduced ecdysone levels [70, 110]. The presence of TSPO in insects suggests that TSPO may be involved in the transport of ecdysteroid precursors into mitochondria [69]. Moreover, insect TSPO interacts with the 26S proteasome complex, which is located on the mitochondria. This interaction occurs via the 38-kDa FK506 binding protein 8 (FKBP8), and in mammalian cells plays a role in the turnover of the mitochondrial steroidogenic acute regulatory protein STAR by a Lon protease [111-113]. The role of insect and mammalian TSPO in 26S proteasome complex formation and STAR degradation warrants further investigation.

Zebrafish TSPO has a maternal and zygotic contributions to the hematopoietic intermediate cell mass, but the role of TSPO during erythropoiesis was recently shown to be independent of its cholesterol binding ability [11]. Avian TSPOs are encoded by two genes, Tspo1 and Tspo2 (8), which evolved by an ancient gene duplication event, one of the main mechanisms for the evolution of new gene functions [114]. While little is known about the avian TSPO1, turkey and chicken TSPO2 is known to be involved in primitive erythropoiesis [9, 10]. Whether the CRAC domain plays a role in this process remains unclear. However, the CRAC domain of mammalian TSPO2 does play a role in the re-distribution of cholesterol in the nuclear and ER membranes during erythropoiesis, which is necessary for erythrocyte maturation [8]. The reasons why the CRAC domain of TSPOs is involved in erythropoiesis in mammals and possibly in birds, but not in zebrafish, are probably related to the differences in the mechanisms of erythrocyte maturation in these organisms. For example, mature erythrocytes of fish, amphibians, and birds are nucleated, whereas mammalian erythrocytes are enucleated. Based on the limited information available, it appears that TSPO1 regulates erythrocyte maturation in lower vertebrates in a cholesterol-independent manner. In contrast, TSPOs of higher mammals with the CRAC domains confer porphyrin transport and heme biosynthesis functions to TSPOs. However, the CRAC domain of TSPO2 in higher mammals is exclusively used for the re-distribution of cholesterol that is essential for erythrocyte enucleation.

The most extensive studies of mammalian TSPOs are in steroid hormone biosynthesis, which has been linked to cholesterol binding function of the C-terminal CRAC domain. Deletion of the CRAC domain results in the loss of TSPO cholesterol binding ability and attenuation of steroidogenesis in various cell model systems [115]. Deletion of the CRAC domain in TSPO2 caused dramatic uneven distribution of NBD-cholesterol in the ER and nuclear membranes (Fig. 7), indicating that the TSPO2 CRAC domain plays a role in the redistribution of cholesterol during erythropoietic maturation [8]. The distinctive functions between these two paralogous proteins are purely spatial specialized because of the hematopoietic tissue-, erythrocyte-, and


Fig. (6). Sequence logo of the most conserved drug PK11195 binding sites among the 500 TSPO predicted amino acid sequences and the CRAC domain sequences (GenBank, NCBI). A. The potential drug binding motif, LxKPsW/F, is located in the first loop of TSPOs and highlighted with arrows. Sequence alignment is illustrated below the sequence logo and the red bars indicate the conserved sequences. B. Sequence logo of the defined cholesterol binding domain, CRAC, is uniquely distributed within the vertebrate TSPOs. The transmembrane domains and CRAC domain are highlighted. C. Sequence logo of the invertebrate CRACs. D. Sequence logo of the C-termini of plant TSPOs. Stars indicate the conserved cholesterol binding sites.


Fig. (7). The effect of the TSPO2 C-terminal CRAC-domain on intracellular cholesterol distribution. HeLa cells were transfected with pDsRed monomer C1 vector control or plasmids encoding wild-type hTSPO2 or mutant hTSPO2 with deletion of its C-terminal CRAC domain. The cells were then treated with NBD-cholesterol. A-C. Mutant DsRed-hTSPO2 with deletion of its C-terminal CRAC domain; D-F, Wild-type DsRed-hTSPO2; G-I, pDsRed monomer C1 vector. A, D, and G. DsRed-fusion proteins (Red). B, E, and H. NBD-cholesterol (Green); C, F, and I. Superimposed images with Hoechst 33342 nuclear staining (Blue). J-O. Intensity surface plots of the images as shown in A-C and D-F. Unevenly distributed cholesterol in HeLa cells transfected with the hTSPO2 mutant is highlighted (K). This research was originally published in the Journal of Biological Chemistry as supplementary supporting data [134]; ©2009 by the American Society for Biochemistry and Molecular Biology.

A B C

D E F

G H I

hTSPO2-crac

hTSPO2-wt

DsRed

J K L

M N OhTSPO2-wt

hTSPO2-crac NBD-cholesterol

NBD-cholesterol

Co-localization

Co-localization


Fig. (8). Comparison of three conserved amino acids in the TM2- TM4 regions for animal TSPOs. A. A 3D model of mouse TSPO docking with a single cholesterol molecule. The internal and external negative patches are shown herein [135]. B. Amino acid sequence alignment of the partial regions of animal TSPOs. The corresponding amino acids are indicated by red arrows.

organelle-specific expression of TSPO2. The structural features of the protein that determine these spatial differences remain elusive. However, the divergent sequences between TSPO2- and TSPO1- subfamilies, which may be responsible for subfunctionalization of the TSPO gene family, were predicted by using DIVERGE [116]. Mapping of these areas onto the secondary protein structures showed that the areas of divergence are within the TM3 and TM4 domains (Fig. 8). It is noteworthy that the second cytoplasmic domain is likely to be responsible for the insertion of the TSPO protein into the mitochondria [117]. Clearly, the corresponding domain in TSPO2 may not be able to drive the protein to the mitochondria but rather cause it

to be retained in the ER and nuclear membranes (Figs. 5 and 7).

Other biological functions of TSPO including protein import, mitochondrial respiration, oxidative processes, ion transport, cell growth and differentiation, and immunomodulation are well documented. Yet little is known about the relationship between the TSPO structure to these functions [7, 118]. Although TSPO-mediated changes in cholesterol levels in mitochondrial membranes could affect most of the above mentioned processes, clear experimental evidence linking this mechanism to those processes is lacking. TSPO three-dimensional (3D) modeling using molecular dynamics simulations showed that TSPO has a channel-like

Internal negative charged surface(D111)

Top Bottom

External negative charged surface(E71,E76,D77)

A

Cholesterol

* 20 * 40 * 60TSPO1-CRA-b-Humans : SYLVWKELGG-FTEKAVVPLGLYTGQLALNWAWPPIFFGARQMGW-ALVDLLLVSGAAAATSPO1-CRA-a-Humans : SYLVWKELGG-FTEKAVVPLGLYTGQLALNWAWPPIFFGARQMGW-ALVDLLLVSGAAAATSPO1-isox-Human : SYLVWKELGG-FTEKAVGSPGPLHWAAGPELGMAPHLLGARQMGW-ALVDLLLVSGAAAATSPO1-iso1-Pan : SYLVWKELGG-FTEEAVVPLGLYTGQLALNWAWPPIFFGARQMGW-ALVDLLLVSGAAAATSPO1-iso2-Pan : SYLVWKELGG-FTEEAVVPLGLYTGQLALNWAWPPIFFGARQMGW-ALVDLLLVSGAAAATSPO1-iso1-Macaca : SYLVWKELGG-FTEEAVVPLGLYTGQLALNWAWPPIFFGARQMGW-ALVDLLLVSGAAAATSPO1-iso2-Macaca : SYLVWKELGG-FTEEAVVPLGLYTGQLALNWAWPPIFFGARQMGW-ALVDLLLVSGAAAATSPO1-Bovine : SYMIWKELGG-FSKEAVVPLGLYAGQLALNWAWPPLFFGTRQMGW-ALVDLLLTGGMAAATSPO1-Sheep : SYLIWKELGG-FSKEAVVPLGLYAGQLALNWAWPPLFFGARQMGW-AFVDLLLTGGMAAATSPO1-Pig : SYMIWKELGG-FSEEAVVPLGLYAGQLALNWAWPPLFFGARQMGW-ALVDLVLTGGVAAATSPO1-Dog : SYMVWKELGG-FSEEAVVPLGLYAGQLALNWAWPPLFFGTRQMGW-ALVDLLLTGGLAGATSPO1-Mouse : SYIVWKELGG-FTEDAMVPLGLYTGQLALNWAWPPIFFGARQMGW-ALADLLLVSGVATATSPO1-Rat : SYIIWKELGG-FTEEAMVPLGLYTGQLALNWAWPPIFFGARQMGW-ALVDLMLVSGVATATSPO1-Monodelphis : SYLVWKELGG-FTEQALVPLGLYAGQLALNWAWPPLFFGAHQMGW-GLVEIVLTSGAAVATSPO1-Gallus : SYLVWKELGG-FTEKAAVPLGLYAGQLALNWAWTPIFFGAHKMGW-GLATLLLTTGTATATSPO-Oncorhychus : SYLVWKECGG-FTEDAVVPLGFYGLQLALNWAWTPIFFGAHKLKM-ALIEIVMLTGAVGATSPO-Danio : SYLVWKELGG-FTQDAMVPLGLYGLQLALNWAWTPIFFGAHKIQL-ALIELLLMSGTVAATSPO-Tetraodon : SYLVYEELGG-FTEDAVVPLGLYGLQLALNWSWPLLFFGAHKLKW-AFVDVILVAVTAVATSPO-Xeopus : SYLIYKELGG-LNEKAVVPLGLYAGQLALNWAWTPIFFGAHKIGW-GLVDLVFLWGTAVATSPO-Bombyx : SYLIWEECDG-FTEDAVLPLTLYGVQLLLNWSWTPIFFGLKDFKL-AFIEISVLSGAAVATSPO-Drosophola : SYLVWRDGGGFAGEAAKLPLIAYGTQLALNWAWTPIFFGQHNIKG-GLIDIVALTAAASATSPO-Apis : SYLVWRDGDGFRN--AILPLSIYGTNLILNWSWSPLFFGLHKIKW-ALYEIILLWGSTAATSPO-Anopheles : SYLVWKTGGGFGG-PAQLPLALYGTQLALNWAWTPIFFGLHQLKW-SVVEILALTGSVAATSPO-Schistosoma : SYLVWRDTPH---EKVMVPLAVYGAQLLLNWSWTPVFFGQHKIKYGAMINLGILGGAVT-TSPO-briggsae : SYLVYKNGGGFDYNDTKLALGLYGASVTLAIATIPIVKKRE-LGCLWKNTAVVSLTATG-TSPO-elegans : SYLVYKNGGGFDYNDTKLALGLYGASVTLAVATIPIVKKKE-LGCLWKNTTVVSLTAAA-TSPO-Trichoplax : SYLVWRDGGG-FEGPALKALQAYELNLVFNGIWTPLFFGAKRMGL-AGIDIVATWASIVYTSPO2-Humans : SYLVWKDLGGGLGWPLALPLGLYAVQLTISWTVLVLFFTVHNPGL-ALLHLLLLYGLVVSTSPO2-Gallus : SYLIWNDLGG-CSSKAIIPLGLYGAQLAFNWAWPPFFFSARNLKM-ALIDILCLDSLAIGTSPO2-Mouse : SYLVWKELGGGFRWPLALPLGLYSFQLALSWTFLVLFLAADSPGL-ALLDLLLLYGLVASTSPO2-Rat : SYLVWKDLGGGFRWSLALPLGLYSFQLALSWTFLMLFLVVDSPGL-ALLDLLLLYGLVASTSPO2-Pig : SYLVWKELGGGLGWPLALPLGLYSVQLAVSWTVLILFFVAHSAGL-ALLHLLLLFGLVVSTSPO2-Dog : SYLVWKDLGGGFGRPLALPLGLYAVQLAVSWAVLIFFFAAHAHGL-ALLHMLLLYGLVVSTSPO2-Monodelphis : SYLIWKDLGGSLGRLLALPLGLYAVQLILNWIILLLVFGAHNLGL-ALCHLVLLFGLVLG

B


structure and channel-like activity in membranes [93, 119, 120]. Using on this model, TSPO was shown to be capable of accommodating a cholesterol molecule within the five alpha-helices transmembrane domains. Molecular docking supports the molecular dynamic model (Fig. 8A). Mapping of negative charges on the model shows that there are two negative patches inside and outside the molecule, which are distinctive features that may be responsible for some of the physiological functions attributed to TSPO (Fig. 8B). In addition, the topology of the C-termini of bacterial TSPOs and mammalian mitochondrial TSPOs all face the cytoplasmic compartment, which would allow them to function as a sensor of intracellular oxygen levels [121-123]. This includes the functionally interchangeable bacterial and mammalians TSPOs and the transport of heme-related metabolites. As a consequence of this topology, TSPOs are appear to be inserted in the opposite way in bacterial and mitochondrial membranes, with the protein being inserted from the inside in bacteria and from the outside (since encoded in the nucleus) in mitochondria [12]. The heme-related metabolite functions can also be traced from mammals to bacteria, whereas other functions like steroidogenesis evolved later by natural selection (Fig. 9). Further support for this model comes from the observations that TSPO monomer polymerization is driven by dityrosine formation, occurs in response to the sensing of oxygen species, and reduces the ability of the protein to bind cholesterol [124]. Since many monooxygenases can produce ROS during their catalytic cycle [125], it is likely that the polymerization of TSPO is a feed-back regulatory mechanism in the steroidogenesis pathway that senses and responds to intracellular oxygen levels.

CONCLUSIONS AND PERSPECTIVES In this review, we focused on the recent

developments in our understanding of TSPO sequence divergence during evolutionary selection and its physiological consequences. In general, the available information indicates that the Tspo gene family has been expanded during evolution from an environmental sensor or signal transducer to a functional bioregulator adapted to organism-, tissue-, cell-, and organelle- specific needs. A key observation is that bacterial and mammalian TSPOs are interchangeable, meaning that one compensates for the loss of oxygen sensing function that occurs when the other is depleted. In addition, TSPO has established functions in tetrapyrrole biosynthesis, porphyrin transport, heme metabolism, the protection of mitochondria from free radical damage, erythrocyte maturation, and self-polymerization in response to ROS. Thus, we believe that the TSPO function in oxygen-mediated metabolism is the central role of this protein during evolutionary history with diversified specific roles in other cell signaling, metabolism, and hormone synthesis pathways, including cholesterol trafficking and oxygen-mediated steroidogenesis.

The biosynthesis of steroids that require oxygen-utilizing enzymes was previously thought to be highly conserved in eukaryotic domains [126]. In mammals, steroid hormones synthesis from cholesterol requires multiple monooxygenases, which produce ROS as byproducts. In our recent studies of the effects of phthalate plasticizers on Leydig cell steroidogenesis, we observed that increased ROS can reduced steroid formation [127]. Further studies of the function of TSPO in oxygen sensing could further clarify the role of TSPO

Fig. (9). Illustration of TSPO/TspO protein family functions. Porphyrin transport via TSPOs in mammals is conserved in bacteria. Although TSPO’s role in sterol substrate import for steroidogenesis may have evolved later in mammals, it is likely that TSPO may function in hopanoid transport in bacteria and plant pregnenolone synthesis from cholesterol/24-alkylsteroids.

Animals

Cholesterol

Pregnenolone

TSPO

Tetrapyrrole

Porphyrin

TSPO

Plants

Porphyrin

Bacteria

Tetrapyrrole

Porphyrin

TSPO

TSPO

Hopanoids? Pregnenolone?

Cholesterol/24-alkylsteroids


in mitochondrial function, including the cholesterol transport into mitochondria, the TSPO-mediated rate limiting step of steroid hormone biosynthesis.

ACKNOWLEDGEMENTS This work was supported by Grants from the

National Institutes of Health (R01 ES07747) and Canadian Institutes of Health Research (MOP 102647). V.P. was also supported by a Canada Research Chair in Biochemical Pharmacology.

ABBREVIATIONS ANT = adenine nucleotide translocator CRAC = cholesterol recognition/interaction amino

acid consensus domain CYPs = cytochrome P450 monooxygenases DBI = diazepam binding inhibitor PK11195 = 1-(2-chlorophenyl)-N- methyl-N-(1-

methyl-propyl)-3-isoquinolinecarboxamide

STAR = mitochondrial steroidogenic acute regulatory protein

TSPO or = translocator protein (18 kDa) TSPO1 TSPO2 = translocator protein 2 VDAC = voltage-dependent anion channel

REFERENCES [1] Braestrup C, Squires RF. Specific benzodiazepine receptors

in rat brain characterized by high-affinity (3H)diazepam binding. Proc Natl Acad Sci USA 1977; 74: 3805-9.

[2] Batarseh A, Papadopoulos V. Regulation of translocator protein 18 kDa (TSPO) expression in health and disease states. Mol Cell Endocrinol 2010; 327: 1-12.

[3] Hardwick M, Fertikh D, Culty M, Li H, Vidic B, Papadopoulos V. Peripheral-type benzodiazepine receptor (PBR) in human breast cancer: correlation of breast cancer cell aggressive phenotype with PBR expression, nuclear localization, and PBR-mediated cell proliferation and nuclear transport of cholesterol. Cancer Res 1999; 59: 831-42.

[4] Hardwick M, Rone J, Han Z, Haddad B, Papadopoulos V. Peripheral-type benzodiazepine receptor levels correlate with the ability of human breast cancer MDA-MB-231 cell line to grow in SCID mice. Int J Cancer 2001; 94: 322-7.

[5] Hardwick M, Cavalli LR, Barlow KD, Haddad BR, Papadopoulos V. Peripheral-type benzodiazepine receptor (PBR) gene amplification in MDA-MB-231 aggressive breast cancer cells. Cancer Genet Cytogenet 2002; 139: 48-51.

[6] Papadopoulos V, Kapsis A, Li H, et al. Drug-induced inhibition of the peripheral-type benzodiazepine receptor expression and cell proliferation in human breast cancer cells. Anticancer Res 2000; 20: 2835-47.

[7] Papadopoulos V, Baraldi M, Guilarte TR, et al. Translocator protein (18kDa): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function. Trends Pharmacol Sci 2006; 27: 402-9.

[8] Fan J, Rone MB, Papadopoulos V. Translocator protein 2 is involved in cholesterol redistribution during erythropoiesis. J Biol Chem 2009; 284: 30484-97.

[9] Simos G, Maison C, Georgatos SD. Characterization of p18, a component of the lamin B receptor complex and a new integral membrane protein of the avian erythrocyte nuclear envelope. J Biol Chem 1996; 271: 12617-25.

[10] Nakazawa F, Alev C, Shin M, Nakaya Y, Jakt LM, Sheng G. PBRL, a putative peripheral benzodiazepine receptor, in primitive erythropoiesis. Gene Expr Patterns 2009; 9: 114-21.

[11] Rampon C, Bouzaffour M, Ostuni MA, et al. Translocator protein (18 kDa) is involved in primitive erythropoiesis in zebrafish. FASEB J 2009; 23: 4181-92.

[12] Chapalain A, Chevalier S, Orange N, Murillo L, Papadopoulos V, Herman C. Bacterial Ortholog of Mammalian Translocator Protein (TSPO) with Virulence. PLoS ONE 2009; 4: e6096.

[13] Frank W, Baar KM, Qudeimat E, et al. A mitochondrial protein homologous to the mammalian peripheral-type benzodiazepine receptor is essential for stress adaptation in plants. Plant J 2007; 51: 1004-18.

[14] Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 1990; 87: 4576-9.

[15] Riond J, Leplatois P, Laurent P, et al. Expression and pharmacological characterization of the human peripheral-type benzodiazepine receptor in yeast. Eur J Pharmacol 1991; 208: 307-12.

[16] Yeliseev AA, Krueger KE, Kaplan S. A mammalian mitochondrial drug receptor functions as a bacterial "oxygen" sensor. Proc Natl Acad Sci USA 1997; 94: 5101-6.

[17] Armstrong GA, Alberti M, Leach F, Hearst JE. Nucleotide sequence, organization, and nature of the protein products of the carotenoid biosynthesis gene cluster of Rhodobacter capsulatus. Mol Gen Genet 1989; 216: 254-68.

[18] Phadwal K. Carotenoid biosynthetic pathway: molecular phylogenies and evolutionary behavior of crt genes in eubacteria. Gene 2005; 345: 35-43.

[19] Yeliseev AA, Kaplan S. A sensory transducer homologous to the mammalian peripheral-type benzodiazepine receptor regulates photosynthetic membrane complex formation in Rhodobacter sphaeroides 2.4.1. J Biol Chem 1995; 270: 21167-75.

[20] Yeliseev AA, Kaplan S. TspO of rhodobacter sphaeroides. A structural and functional model for the mammalian peripheral benzodiazepine receptor. J Biol Chem 2000; 275: 5657-67.

[21] Zeng X, Kaplan S. TspO as a modulator of the repressor/antirepressor (PpsR/AppA) regulatory system in Rhodobacter sphaeroides 2.4. 1. J Bacteriol 2001; 183: 6355-64.

[22] Davey ME, de Bruijn FJ. A homologue of the tryptophan-rich sensory protein TspO and FixL regulate a novel nutrient deprivation-induced Sinorhizobium meliloti locus. Appl Environ Microbiol 2000; 66: 5353-9.

[23] Tong Y, Guo M. Bacterial heme-transport proteins and their heme-coordination modes. Arch Biochem Biophys 2009; 481: 1-15.

[24] Baysse C, Matthijs S, Schobert M, Layer G, Jahn D, Cornelis P. Co-ordination of iron acquisition, iron porphyrin chelation and iron-protoporphyrin export via the cytochrome c biogenesis protein CcmC in Pseudomonas fluorescens. Microbiology 2003; 149: 3543-52.

[25] Cornelis P, Matthijs S. Diversity of siderophore-mediated iron uptake systems in fluorescent pseudomonads: not only pyoverdines. Environ Microbiol 2002; 4: 787-98.

[26] Korkhov VM, Sachse C, Short JM, Tate CG. Three-dimensional structure of TspO by electron cryomicroscopy of helical crystals. Structure 2010; 18: 677-87.

[27] Lindemann P, Koch A, Degenhardt B, Hause G, Grimm B, Papadopoulos V. A novel Arabidopsis thaliana protein is a functional peripheral-type benzodiazepine receptor. Plant Cell Physiol 2004; 45: 723-33.

[28] Wendler G, Lindemann P, Lacapere JJ, Papadopoulos V. Protoporphyrin IX binding and transport by recombinant mouse PBR. Biochem Biophys Res Commun 2003; 311: 847-52.


[29] Donohue TJ. Eubacterial signal transduction by ligands of the mammalian peripheral benzodiazepine receptor complex. Proc Natl Acad Sci USA 1997; 94: 4821-2.

[30] Li H, Papadopoulos V. Peripheral-type benzodiazepine receptor function in cholesterol transport. Identification of a putative cholesterol recognition/interaction amino acid sequence and consensus pattern. Endocrinology 1998; 139: 4991-7.

[31] Steen BR, Lian T, Zuyderduyn S, et al. Temperature-regulated transcription in the pathogenic fungus Cryptococcus neoformans. Genome Res 2002; 12: 1386-400.

[32] Kellis M, Birren BW, Lander ES. Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae. Nature 2004; 428: 617-24.

[33] Chen D, Toone WM, Mata J, et al. Global transcriptional responses of fission yeast to environmental stress. Mol Biol Cell 2003; 14: 214-29.

[34] Dujon B, Sherman D, Fischer G, et al. Genome evolution in yeasts. Nature 2004; 430: 35-44.

[35] Luckner M. Secondary metabolism in microorganisms, plants and animals. Springer: Berlin 1990.

[36] Yurdaydin C, Walsh TJ, Engler HD, et al. Gut bacteria provide precursors of benzodiazepine receptor ligands in a rat model of hepatic encephalopathy. Brain Res 1995; 679: 42-8.

[37] Weinstein JD, Beale SI. Separate physiological roles and subcellular compartments for two tetrapyrrole biosynthetic pathways in Euglena gracilis. J Biol Chem 1983; 258: 6799-807.

[38] Corsi L, Avallone R, Geminiani E, Cosenza F, Venturini I, Baraldi M. Peripheral benzodiazepine receptors in potatoes (Solanum tuberosum). Biochem Biophys Res Commun 2004; 313: 62-6.

[39] Schmid M, Davison TS, Henz SR, et al. A gene expression map of Arabidopsis thaliana development. Nat Genet 2005; 37: 501-6.

[40] Guillaumot D, Guillon S, Morsomme P, Batoko H. ABA, porphyrins and plant TSPO-related protein. Plant Signal Behav 2009; 4: 1087-90.

[41] Sun M, Li L, Xie H, Ma R, He Y. Differentially expressed genes under cold acclimation in Physcomitrella patens. J Biochem Mol Biol 2007; 40: 986-1001.

[42] Kreps JA, Wu Y, Chang HS, Zhu T, Wang X, Harper JF. Transcriptome changes for Arabidopsis in response to salt, osmotic, and cold stress. Plant Physiol 2002; 130: 2129-41.

[43] Flinn B, Rothwell C, Griffiths R, et al. Potato expressed sequence tag generation and analysis using standard and unique cDNA libraries. Plant Mol Biol 2005; 59: 407-33.

[44] Guillaumot D, Guillon S, Dëplanque T, et al. The Arabidopsis TSPO-related protein is a stress and abscisic acid-regulated, endoplasmic reticulum-Golgi-localized membrane protein. Plant J 2009; 60: 242-56.

[45] Hermans C, Vuylsteke M, Coppens F, Craciun A, Inzé D, Verbruggen N. Early transcriptomic changes induced by magnesium deficiency in Arabidopsis thaliana reveal the alteration of circadian clock gene expression in roots and the triggering of abscisic acid-responsive genes. New Phytol 2010; 187: 119-31.

[46] Mizoguchi M, Umezawa T, Nakashima K, et al. Two closely related subclass II SnRK2 protein kinases cooperatively regulate drought-inducible gene expression. Plant Cell Physiol 2010; 51: 842-7.

[47] Medina JH, Pena C, Levi SM, Wolfman C, Paladini AC. Benzodiazepine-like molecules, as well as other ligands for the brain benzodiazepine receptors, are relatively common constituents of plants. Biochem Biophys Res Commun 1989; 165: 547-53.

[48] Avallone R, Zanoli P, Corsi L, Cannazza G, Baraldi M. Benzodiazepine-like compounds and GABA in flower heads of Matricaria chamomilla. Phytother Res 1996; 10: S177-S179.

[49] Wildmann J. Increase of natural benzodiazepines in wheat and potato during germination. Biochem Biophys Res Commun 1988; 157: 1436-43.

[50] Tanaka R, Tanaka A. Tetrapyrrole biosynthesis in higher plants. Annu Rev Plant Biol 2007; 58: 321-46.

[51] Jung S, Lee HJ, Lee Y, et al. Toxic tetrapyrrole accumulation in protoporphyrinogen IX oxidase-overexpressing transgenic rice plants. Plant Mol Biol 2008; 67: 535-46.

[52] Masuda T, Suzuki T, Shimada H, Ohta H, Takamiya K. Subcellular localization of two types of ferrochelatase in cucumber. Planta 2003; 217: 602-9.

[53] Janeczko A, Skoczowski A. Mammalian sex hormones in plants. Fol Histoch Cytochem 2005; 43: 71-9.

[54] Iino M, Nomura T, Tamaki Y, et al. Progesterone: its occurrence in plants and involvement in plant growth. Phytochemistry 2007; 68: 1664-73.

[55] Simersky R, Novák O, Morris DA, Pouzar V, Strnad M. Identification and quantification of several mammalian steroid hormones in plants by UPLC-MS/MS. J Plant Growth Regul 2009; 28: 125-36.

[56] Janeczko A, Budziszewska B, Skoczowski A, Dybala M. Specific binding sites for progesterone and 17 -estradiol in cells of Triticum aestivum L. Acta Biochim Pol 2008; 55: 707-11.

[57] Janeczko A, Filek W, Biesaga-Kocielniak J, Marciska I, Janeczko Z. The influence of animal sex hormones on the induction of flowering in Arabidopsis thaliana: comparison with the effect of 24-epibrassinolide. Plant Cell, Tissue Organ Cult 2003; 72: 147-51.

[58] Bennett RD, Heftmann E, Winter BJ. A function of sitosterol. Phytochemistry 1969; 8: 2325-8.

[59] Lindemann P, Luckner M. Biosynthesis of pregnane derivatives in somatic embryos of Digitalis lanata. Phytochemistry 1997; 46: 507-13.

[60] Palazon J, Bonfill M, Cusido RM, Pinol MT, Morales C. Effects of auxin and phenobarbital on morphogenesis and production of digitoxin in Digitalis callus. Plant Cell Physiol 1995; 36: 247-52.

[61] Grinberg AV, Hannemann F, Schiffler B, Müller J, Heinemann U, Bernhardt R. Adrenodoxin: structure, stability, and electron transfer properties. Proteins: Structure, Function, and Bioinformatics 2000; 40: 590-612.

[62] Ohta D, Mizutani M. Redundancy or flexibility: molecular diversity of the electron transfer components for P450 monooxygenases in higher plants. Front Biosci 2004; 9: 1587-97.

[63] Millar AH, Heazlewood JL, Kristensen BK, Braun HP, Moller IM. The plant mitochondrial proteome. Trends Plant Sci 2005; 10: 36-43.

[64] Luzikov VN, Novikova LA, Spiridonova VA, et al. Design of heterologous mitochondria: import of cattle cytochrome P-450SCC precursors in plant mitochondria. Biokhimiia (Moscow, Russia) 1994; 59: 1098-101.

[65] Spivak SG, Berdichevets IN, Yarmolinsky DG, Maneshina TV, Shpakovski GV, Kartel NA. Construction and characteristics of transgenic tobacco Nicotiana tabacum L. plants expressing CYP11A1 cDNA encoding cytochrome P450 SCC. Russ J Genet 2009; 45: 1067-73.

[66] Spivak SG, Berdichevets IN, Litvinovskaya RP, Drach SV, Kartel NA, Shpakovski GV. Some peculiarities of steroid metabolism in transgenic Nicotiana tabacum plants bearing the CYP11A1 cDNA of cytochrome P450 SCC from the bovine adrenal cortex. Russ J Org Chem 2010; 36: 224-32.

[67] Pilgrim H. 'Cholesterol side-chain cleaving enzyme' aktivitat in keimlingen und in vitro kultivierten geweben von Digitalis purpurea. Phytochemistry 1972; 11: 1725-8.

[68] Ghedin E, Wang S, Spiro D, et al. Draft genome of the filarial nematode parasite Brugia malayi. Science 2007; 317: 1756-60.

[69] Snyder MJ, Van Antwerpen R. Evidence for a diazepam-binding inhibitor (DBI) benzodiazepine receptor-like mechanism in ecdysteroidogenesis by the insect prothoracic gland. Cell Tissue Res 1998; 294: 161-8.


[70] Roth GE, Gierl MS, Vollborn L, Meise M, Lintermann R, Korge G. The Drosophila gene Start1: a putative cholesterol transporter and key regulator of ecdysteroid synthesis. Proc Natl Acad Sci USA 2004; 101: 1601-6.

[71] Warren JT, Petryk A, Marqués G, et al. Molecular and biochemical characterization of two P450 enzymes in the ecdysteroidogenic pathway of Drosophila melanogaster. Proc Natl Acad Sci USA 2002; 99: 11043-8.

[72] Guzov VM, Unnithan GC, Chernogolov AA, Feyereisen R. CYP12A1, a mitochondrial cytochrome P450 from the house fly. Arch Biochem Biophys 1998; 359: 231-40.

[73] Markov GV, Tavares R, Dauphin-Villemant C, Demeneix BA, Baker ME, Laudet V. Independent elaboration of steroid hormone signaling pathways in metazoans. Proc Natl Acad Sci USA 2009; 106: 11913-8.

[74] Ranson H, Claudianos C, Ortelli F, et al. Evolution of supergene families associated with insecticide resistance. Science 2002; 298: 179-81.

[75] Feyereisen R. Evolution of insect P450. Biochem Soc T 2006; 34: 1252-5.

[76] Giot L, Bader JS, Brouwer C, et al. A protein interaction map of Drosophila melanogaster. Science 2003; 302: 1727-36.

[77] Tonoki A, Kuranaga E, Tomioka T, et al. Genetic evidence linking age-dependent attenuation of the 26S proteasome with the aging process. Mol Cell Biol 2009; 29: 1095-106.

[78] Curtis C, Landis G, Folk D, et al. Transcriptional profiling of MnSOD-mediated lifespan extension in Drosophila reveals a species-general network of aging and metabolic genes. Genome Biol 2007; 8: R262.

[79] Santos TM, Johnston DA, Azevedo V, et al. Analysis of the gene expression profile of Schistosoma mansoni cercariae using the expressed sequence tag approach. Mol Biochem Parasitol 1999; 103: 79-98.

[80] Noel F, Mendonca-Silva DL, Thibaut JP, Lopes DV. Characterization of two classes of benzodiazepine binding sites in Schistosoma mansoni. Parasitology 2007; 134: 1003-12.

[81] Pica-Mattoccia L, Ruppel A, Xia CM, Cioli D. Praziquantel and the benzodiazepine Ro11-3128 do not compete for the same binding sites in schistosomes. Parasitology 2007; 135: 47-54.

[82] Betti L, Giannaccini G, Nigro M, Dianda S, Gremigni V, Lucacchini A. Studies of peripheral benzodiazepine receptors in mussels: comparison between a polluted and a nonpolluted site. Ecotox Environ Safe 2003; 54: 36-42.

[83] Eshleman AJ, Murray TF. Differential binding properties of the peripheral-type benzodiazepine ligands [3H] PK 11195 and [3H] Ro 5-4864 in trout and mouse brain membranes. J Neurochem 1989; 53: 494-502.

[84] Hsu HJ, Hsiao P, Kuo MW, Chung B. Expression of zebrafish cyp11a1 as a maternal transcript and in yolk syncytial layer. Gene Expr Patterns 2002; 2: 219-22.

[85] Hsu HJ, Lin G, Chung B. Parallel early development of zebrafish interrenal glands and pronephros: differential control by wt1 and ff1b. Development 2003; 130: 2107-16.

[86] Lesouhaitier O, Feuilloley M, Lihrmann I, et al. Localization of diazepam-binding inhibitor-related peptides and peripheral type benzodiazepine receptors in the frog adrenal gland. Cell Tissue Res 1996; 283: 403-12.

[87] Ziyadeh FN, Kelepouris E, Agus ZS. Benzodiazepines stimulate sodium ion transport in frog skin epithelium. BBA-Biomembranes 1988; 940: 93-8.

[88] Papadopoulos V. Peripheral-type benzodiazepine/diazepam binding inhibitor receptor: biological role in steroidogenic cell function. Endocr Rev 1993; 14: 222-40.

[89] Rone MB, Fan J, Papadopoulos V. Cholesterol transport in steroid biosynthesis: Role of proteinûprotein interactions and implications in disease states. BBA-Mol Cell Biol L 2009; 1791: 646-58.

[90] Papadopoulos V. In search of the function of the peripheral-type benzodiazepine receptor. Endocr Res 2004; 30: 677-84.

[91] Lacapere JJ, Delavoie F, Li H, et al. Structural and functional study of reconstituted peripheral benzodiazepine receptor. Biochem Biophys Res Commun 2001; 284: 536-41.

[92] Beurdeley-Thomas A, Miccoli L, Oudard S, Dutrillaux B, Poupon MF. The peripheral benzodiazepine receptors: a review. J Neuro-Oncol 2000; 46: 45-56.

[93] Culty M, Li H, Boujrad N, et al. In vitro studies on the role of the peripheral-type benzodiazepine receptor in steroidogenesis. J Steroid Biochem Mol Biol 1999; 69: 123-30.

[94] Gavish M, Bachman I, Shoukrun R, et al. Enigma of the Peripheral Benzodiazepine Receptor. Pharmacol Rev 1999; 51: 629-50.

[95] Farges R, Joseph-Liauzun E, Shire D, et al. Molecular basis for the different binding properties of benzodiazepines to human and bovine peripheral-type benzodiazepine receptors. FEBS Lett 1993; 335: 305-8.

[96] Farges R, Joseph-Liauzun E, Shire D, Caput D, Le Fur G, Ferrara P. Site-directed mutagenesis of the peripheral benzodiazepine receptor: identification of amino acids implicated in the binding site of Ro5-4864. Mol Pharmacol 1994; 46: 1160-7.

[97] Joseph-Liauzun E, Farges R, Delmas P, Ferrara P, Loison G. The Mr 18,000 subunit of the peripheral-type benzodiazepine receptor exhibits both benzodiazepine and isoquinoline carboxamide binding sites in the absence of the voltage-dependent anion channel or of the adenine nucleotide carrier. J Biol Chem 1997; 272: 28102-6.

[98] McEnery MW, Snowman AM, Trifiletti RR, Snyder SH. Isolation of the mitochondrial benzodiazepine receptor: association with the voltage-dependent anion channel and the adenine nucleotide carrier. Proc Natl Acad Sci USA 1992; 89: 3170-4.

[99] Garnier M, Dimchev AB, Boujrad N, Price JM, Musto NA, Papadopoulos V. In vitro reconstitution of a functional peripheral-type benzodiazepine receptor from mouse Leydig tumor cells. Mol Pharmacol 1994; 45: 201-11.

[100] Sigel E, Buhr A. The benzodiazepine binding site of GABAA receptors. Trends Pharmacol Sci 1997; 18: 425-9.

[101] Slocinska M, Szewczyk A, Hryniewiecka L, Kmita H. Benzodiazepine binding to mitochondrial membranes of the amoeba Acanthamoeba castellanii and the yeast Saccharomyces cerevisiae. Acta Biochim Pol 2004; 51: 953-62.

[102] Avallone R, Cosenza F, Farina F, Baraldi C, Baraldi M. Extraction and purification from Ceratonia siliqua of compounds acting on central and peripheral benzodiazepine receptors. Fitoterapia 2002; 73: 390-6.

[103] Anzini M, Cappelli A, Vomero S, et al. Mapping and fitting the peripheral benzodiazepine receptor binding site by carboxamide derivatives. Comparison of different approaches to quantitative ligand-receptor interaction modeling. J Med Chem 2001; 44: 1134-50.

[104] Ourisson G, Rohmer M, Poralla K. Prokaryotic hopanoids and other polyterpenoid sterol surrogates. Annu Rev Microbiol 1987; 41: 301-33.

[105] Welander PV, Hunter RC, Zhang L, Sessions AL, Summons RE, Newman DK. Hopanoids play a role in membrane integrity and pH homeostasis in Rhodopseudomonas palustris TIE-1. J Bacteriol 2009; 191: 6145-56.

[106] Kurzchalia TV, Ward S. Why do worms need cholesterol? Nat Cell Biol 2003; 5: 684-8.

[107] Kamath RS, Fraser AG, Dong Y, et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 2003; 421: 231-7.

[108] Lafont R. Understanding insect endocrine systems: molecular approaches. Entomol Exp Appl 2000; 97: 123-36.

[109] Gilbert LI, Rybczynski R, Warren JT. Control and biochemical nature of the ecdysteroidogenic pathway. Annu Rev Entomol 2002; 47: 883-916.

[110] Warren JT, Bachmann JS, Dai JD, Gilbert LI. Differential incorporation of cholesterol and cholesterol derivatives into ecdysteroids by the larval ring glands and adult ovaries of Drosophila melanogaster: a putative explanation for the l(3) ecd1 mutation. Insect Biochem Mol Biol 1996; 26: 931-43.

[111] Granot Z, Kobiler O, Melamed-Book N, et al. Turnover of mitochondrial steroidogenic acute regulatory (StAR) protein


by Lon protease: the unexpected effect of proteasome inhibitors. Mol Endocrinol 2007; 21: 2164-77.

[112] Nakagawa T, Shirane M, Iemura S, Natsume T, Nakayama KI. Anchoring of the 26S proteasome to the organellar membrane by FKBP38. Genes Cells 2007; 12: 709-19.

[113] Soccio RE, Breslow JL. StAR-related lipid transfer (START) proteins: mediators of intracellular lipid metabolism. J Biol Chem 2003; 278: 22183-6.

[114] Ohno S. Evolution by gene duplication. New York: Springer-Verlag 1970.

[115] Jamin N, Neumann JM, Ostuni MA, et al. Characterization of the cholesterol recognition amino acid consensus sequence of the peripheral-type benzodiazepine receptor. Mol Endocrinol 2005; 19: 588-94.

[116] Gu X, Vander VK. DIVERGE: phylogeny-based analysis for functional-structural divergence of a protein family. Bioinformatics 2002; 18: 500-1.

[117] Rone MB, Liu J, Blonder J, et al. Targeting and insertion of the cholesterol-binding translocator protein into the outer mitochondrial membrane. Biochemistry 2009; 48: 6909-20.

[118] Parola AL, Yamamura HI, rd HE. Peripheral-type benzodiazepine receptors. Life Sci 1993; 52: 1329-42.

[119] Bernassau JM, Reversat JL, Ferrara P, Caput D, Lefur G. A 3D model of the peripheral benzodiazepine receptor and its implication in intra mitochondrial cholesterol transport. J Mol Graphics 1993; 11: 236-44.

[120] Veenman L, Papadopoulos V, Gavish M. Channel-like functions of the 18-kDa translocator protein (TSPO): regulation of apoptosis and steroidogenesis as part of the host-defense response. Curr Pharm Des 2007; 13: 2385-405.

[121] Carayon P, Portier M, Dussossoy D, et al. Involvement of peripheral benzodiazepine receptors in the protection of hematopoietic cells against oxygen radical damage. Blood 1996; 87: 3170-8.

[122] O'Hara MF, Nibbio BJ, Craig RC, Nemeth KR, Charlap JH, Knudsen TB. Mitochondrial benzodiazepine receptors regulate oxygen homeostasis in the early mouse embryo. Reprod Toxicol 2003; 17: 365-75.

[123] Knudsen TB, Green ML. Response characteristics of the mitochondrial DNA genome in developmental health and disease. Birth Defects Res C Embryo Today 2004; 72: 313-29.

[124] Delavoie F, Li H, Hardwick M, et al. In vivo and in vitro peripheral-type benzodiazepine receptor polymerization: functional significance in drug ligand and cholesterol binding. Biochemistry 2003; 42: 4506-19.

[125] Hanukoglu I. Antioxidant protective mechanisms against reactive oxygen species (ROS) generated by mitochondrial P450 systems in steroidogenic cells. Drug Metab Rev 2006; 38: 171-96.

[126] Summons RE, Bradley AS, Jahnke LL, Waldbauer JR. Steroids, triterpenoids and molecular oxygen. Philos T Roy Soc B 2006; 361: 951-68.

[127] Fan J, Traore K, Li W, et al. Molecular mechanisms mediating the effect of mono-(2-ethylhexyl) phthalate (MEHP) on hormone-stimulated steroidogenesis in MA-10 mouse tumor Leydig cells. Endocrinology 2010; 151: 3348-62.

[128] Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol 1982; 157: 105-32.

[129] Hofmann K, Stoffel W. TMbase: a database of membrane spanning proteins segments. Biol Chem Hoppe-Seyler 1993; 347: 166-70.

[130] Rice P, Longden I, Bleasby A. EMBOSS: the European molecular biology open software suite. Trends Genet 2000; 16: 276-7.

[131] Kumar S, Tamura K, Nei M. MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 2004; 5: 150-63.

[132] Higgins DG, Bleasby AJ, Fuchs R. CLUSTAL V: improved software for multiple sequence alignment. Comput Appl Biosci 1992; 8: 189-91.

[133] Konvicka K, Campagne F, Weinstein H. Interactive construction of residue-based diagrams of proteins: the RbDe web service. Protein Eng 2000; 13: 395-6.

[134] Rupprecht R, Papadopoulos V, Rammes G, et al. Translocator protein (18 kDa)(TSPO) as a therapeutic target for neurological and psychiatric disorders. Nat Rev Drug Discov 2010; 9: 971-88.

[135] Fan J, Papadopoulos V. Translocator protein 2 (TSPO2), a new family of proteins involved in cholesterol redistribution in erythropoiesis. FASEB J 2009; 23(1_MeetingAbstracts) 521. 1.

Received: August 10, 2011 Revised: January 09, 2012 Accepted: February 02, 2012

Structural and functional evolution of the translocator protein (18 kDa)

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