Translocator Protein 2 Is Involved in Cholesterol Redistribution during Erythropoiesis

Translocator protein (TSPO) is an 18-kDa cholesterol- and drug-binding protein conserved from bacteria to humans. While surveying for Tspo-like genes, we identified its paralogous gene, Tspo2, encoding an evolutionarily conserved family of proteins that arose by gene duplications before the divergence of avians and mammals. Comparative analysis of Tspo1 and Tspo2 functions suggested that Tspo2 has become subfunctionalized, typical of duplicated genes, characterized by the loss of diagnostic drug ligand-binding but retention of cholesterol-binding properties, hematopoietic tissue- and erythroid cell-specific distribution, and subcellular endoplasmic reticulum and nuclear membrane localization. Expression of Tspo2 in erythroblasts is strongly correlated with the down-regulation of the enzymes involved in cholesterol biosynthesis. Overexpression of TSPO2 in erythroid cells resulted in the redistribution of intracellular free cholesterol, an essential step in nucleus expulsion during erythrocyte maturation. Taken together, these data identify the TSPO2 family of proteins as mediators of cholesterol redistribution-dependent erythroblast maturation during mammalian erythropoiesis. Translocator protein (TSPO) is an 18-kDa cholesterol- and drug-binding protein conserved from bacteria to humans. While surveying for Tspo-like genes, we identified its paralogous gene, Tspo2, encoding an evolutionarily conserved family of proteins that arose by gene duplications before the divergence of avians and mammals. Comparative analysis of Tspo1 and Tspo2 functions suggested that Tspo2 has become subfunctionalized, typical of duplicated genes, characterized by the loss of diagnostic drug ligand-binding but retention of cholesterol-binding properties, hematopoietic tissue- and erythroid cell-specific distribution, and subcellular endoplasmic reticulum and nuclear membrane localization. Expression of Tspo2 in erythroblasts is strongly correlated with the down-regulation of the enzymes involved in cholesterol biosynthesis. Overexpression of TSPO2 in erythroid cells resulted in the redistribution of intracellular free cholesterol, an essential step in nucleus expulsion during erythrocyte maturation. Taken together, these data identify the TSPO2 family of proteins as mediators of cholesterol redistribution-dependent erythroblast maturation during mammalian erythropoiesis. Translocator protein (TSPO) 2The abbreviations used are: TSPOtranslocator proteinNJneighbor-joiningORFopen reading frameNBD-cholesterol22-(N-7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-23,24-bisnor-5-cholen-3-β-olWTwild-typeGFPgreen fluorescent proteinRFPred fluorescent proteinERendoplasmic reticulumISHin situ hybridizationPBSphosphate-buffered salineRTreverse transcriptionOMMouter mitochondria membrane. is an 18-kDa protein that was previously known as PBR (peripheral type benzodiazepine receptor) and represents a gene family evolutionarily conserved from bacteria to humans (1Papadopoulos V. Baraldi M. Guilarte T.R. Knudsen T.B. Lacapere J.J. Lindemann P. Norenberg M.D. Nutt D. Weizman A. Zhang M.R. Gavish M. Trends Pharmacol. Sci. 2006; 27: 402-409Abstract Full Text Full Text PDF PubMed Scopus (1159) Google Scholar). In bacteria, TSPO is the tryptophan-rich sensory protein, an integral membrane protein that acts as a negative regulator of the expression of specific photosynthesis genes in response to oxygen and light (2Yeliseev A.A. Kaplan S. J. Biol. Chem. 1995; 270: 21167-21175Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). It is involved in the efflux of porphyrin intermediates from the cell, and several conserved aromatic residues within TSPO are thought to be involved in binding porphyrin intermediates (2Yeliseev A.A. Kaplan S. J. Biol. Chem. 1995; 270: 21167-21175Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). TSPO of bacterial origin has been shown to have the same ligand binding properties as mammalian TSPO proteins (3Donohue T.J. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 4821-4822Crossref PubMed Scopus (3) Google Scholar). In addition to the binding of porphyrin and heme, mammalian TSPO can replace the activity of its bacterial homologs (2Yeliseev A.A. Kaplan S. J. Biol. Chem. 1995; 270: 21167-21175Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 4Verma A. Nye J.S. Snyder S.H. Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 2256-2260Crossref PubMed Scopus (291) Google Scholar, 5Taketani S. Kohno H. Okuda M. Furukawa T. Tokunaga R. J. Biol. Chem. 1994; 269: 7527-7531Abstract Full Text PDF PubMed Google Scholar). Rat TSPO was shown to retain its structure within the bacterial outer membrane, to functionally substitute for the bacterial homolog, and to act in a manner similar to TSPO in the outer mitochondrial membrane (6Yeliseev A.A. Krueger K.E. Kaplan S. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 5101-5106Crossref PubMed Scopus (113) Google Scholar). Therefore, it is conceivable that some conserved functions of the Tspo genes within a cell are maintained from bacteria to plants and to mammals. translocator protein neighbor-joining open reading frame 22-(N-7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-23,24-bisnor-5-cholen-3-β-ol wild-type green fluorescent protein red fluorescent protein endoplasmic reticulum in situ hybridization phosphate-buffered saline reverse transcription outer mitochondria membrane. In mammals, the biological significance of TSPO has been studied for decades, and TSPO has been shown to be involved in a variety of cellular functions, including cholesterol transport and steroid hormone synthesis, mitochondrial respiration, permeability transition pore opening, apoptosis, and proliferation (7Gavish M. Bachman I. Shoukrun R. Katz Y. Veenman L. Weisinger G. Weizman A. Pharmacol. Rev. 1999; 51: 629-650PubMed Google Scholar, 8Papadopoulos V. Lecanu L. Brown R.C. Han Z. Yao Z.X. Neuroscience. 2006; 138: 749-756Crossref PubMed Scopus (198) Google Scholar, 9Azarashvili T. Grachev D. Krestinina O. Evtodienko Y. Yurkov I. Papadopoulos V. Reiser G. Cell Calcium. 2007; 42: 27-39Crossref PubMed Scopus (100) Google Scholar, 10Li W. Hardwick M.J. Rosenthal D. Culty M. Papadopoulos V. Biochem. Pharmacol. 2007; 73: 491-503Crossref PubMed Scopus (39) Google Scholar). Moreover, its expression correlates with certain pathological conditions such as cancer and endocrine and neurological diseases (8Papadopoulos V. Lecanu L. Brown R.C. Han Z. Yao Z.X. Neuroscience. 2006; 138: 749-756Crossref PubMed Scopus (198) Google Scholar). Although some conserved cellular functions of Tspo are shared from bacteria to mammals, such as cholesterol-binding and transport, their biological significance seems to have adapted to serve specific functions critical for each organism. For instance, cholesterol transport into mitochondria is the rate-determining step in steroidogenesis (8Papadopoulos V. Lecanu L. Brown R.C. Han Z. Yao Z.X. Neuroscience. 2006; 138: 749-756Crossref PubMed Scopus (198) Google Scholar, 11Rone M.B. Fan J. Papadopoulos V. BBA-Molecular and Cell Biology of Lipids. 2009; 1791: 646-658Crossref PubMed Scopus (285) Google Scholar). TSPO serves the similar function in plants (12Lindemann P. Koch A. Degenhardt B. Hause G. Grimm B. Papadopoulos V. Plant Cell Physiol. 2004; 45: 723-733Crossref PubMed Scopus (52) Google Scholar), insects (13Snyder M.J. Van Antwerpen R. Cell Tissue Res. 1998; 294: 161-168Crossref PubMed Scopus (36) Google Scholar), and mammals (14Papadopoulos V. Mukhin A.G. Costa E. Krueger K.E. J. Biol. Chem. 1990; 265: 3772-3779Abstract Full Text PDF PubMed Google Scholar). However, the appearance of the drug, such as the benzodiazepine diazepam, binding sites on TSPO evolved later than the brain-specific γ-aminobutyric acid A receptor benzodiazepine binding sites (15Bolger G.T. Weissman B.A. Lueddens H. Basile A.S. Mantione C.R. Barrett J.E. Witkin J.M. Paul S.M. Skolnick P. Brain Res. 1985; 338: 366-370Crossref PubMed Scopus (31) Google Scholar), although drug binding was observed in both the plant and insect TSPOs (12Lindemann P. Koch A. Degenhardt B. Hause G. Grimm B. Papadopoulos V. Plant Cell Physiol. 2004; 45: 723-733Crossref PubMed Scopus (52) Google Scholar, 13Snyder M.J. Van Antwerpen R. Cell Tissue Res. 1998; 294: 161-168Crossref PubMed Scopus (36) Google Scholar). Thus, throughout evolution, mammalian Tspo genes have exhibited extraordinary plasticity, a valuable trait to be further exploited. We sought to reveal the mechanisms controlling the molecular evolution of Tspo and Tspo-like genes and the ligand binding sites in recently sequenced mammalian and other eukaryotic genomes and characterize the relationships and potential functional similarities in cholesterol synthesis, trafficking, and cholesterol-supported steroidogenesis between different Tspo genes. During these studies, we identified a new family of Tspo-like genes involved in cholesterol trafficking and redistribution, which is linked to erythropoiesis and probably to a new mechanism of erythroblast maturation. The TSPO homologous sequences were retrieved from the non-redundant GenBankTM data base through BLAST searches (supplemental Table S1). Neighbor-joining (NJ) trees and maximum parsimony analyses were performed using the MEGA3 software with options of pairwise deletion and Dayhoff PAM matrix model (16Kumar S. Tamura K. Nei M. Brief. Bioinform. 2004; 5: 150-163Crossref PubMed Scopus (10647) Google Scholar). Maximum likelihood analyses were performed using PHYLIP, Version 3.6 (17Felsenstein J. PHYLIP (Phylogeny Inference Package). University of Washington, Seattle, WA2004Google Scholar). Bootstrap support values for NJ and maximum parsimony trees were obtained from 1000 replicates, and support for maximum likelihood trees was from 100 replicates. Saccharomyces cerevisiae (INVSc1; his3 Δ1/his3 Δ1 leu2/leu2 trp1–289/trp1–289 ura3–52/ura3–52) was grown in YPD medium (1% yeast extract, 2% peptone, 2% glucose) or on YPD medium plates (containing 2% agar); Minimal medium plates contained 0.67% yeast nitrogen base, 2% agar, auxotrophic supplements, and a carbon source at a concentration of 2%, as follows: glucose for SD medium and galactose for SG medium. Heterologous expression of TSPO genes was performed using transformation of plasmids containing the open reading frame (ORF) of mouse Tspo2 (mTspo2), human TSPO2 (hTSPO2), or mouse Tspo1 (mTspo1) in pYES3/CT (supplemental Table as a was used to in yeast cells the of the TSPO in yeast was using pYES3/CT containing the of each using the plates were with SD medium 2% cells were grown in medium at for and by Cell were into 100 containing were for each the cells on for cell was at to binding of specific using of was performed as we previously (14Papadopoulos V. Mukhin A.G. Costa E. Krueger K.E. J. Biol. Chem. 1990; 265: 3772-3779Abstract Full Text PDF PubMed Google Scholar). binding were performed on as previously H. Papadopoulos V. 1998; PubMed Google Scholar). cholesterol was as further the of cholesterol to the expression of we used 22-(N-7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-23,24-bisnor-5-cholen-3-β-ol to the cholesterol The wild-type cells and cells were with a of cholesterol and in were a at with with medium containing as an and by using a on an with a were with to and using Version of was performed using and as shown with some A. L. Brown J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). wild-type and proteins with an were of the of into the using the and (supplemental Table The plasmids were into expression and were with to the proteins at to the of the the and analysis were by and with of the was the and with of binding A at on the were with at for cholesterol was with of A containing with A and by cells were maintained in medium with and T. Yao Z.X. Han Z. W. Culty M. Papadopoulos V. PubMed Scopus Google Scholar). cells were grown in medium with cells were grown in medium with TSPO expression we the of both the mouse and human Tspo2 genes into to and The used are in Table The was from the from J. M.B. Papadopoulos V. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google with and into For of we mouse and human cells on a The cells were with and or with or and using cells were with to the were observed a with the green red and from the were for the of the was into using in Table and was with or on to the in transcription from the The were in to the and with was performed using containing mouse from different or from each were into on in and with and at in yeast and The was to at in and by in before with A at for in and for at the were to for and in nuclear and for in with at were by with to receptor gene expression were by both and were or were into and as data are shown at as are at as by on a The of cell used was with In and human were obtained from and was by using the for was performed with of for for for and used of and (supplemental Table for and were sequenced using the cells were from by the with using a were with a in the of and through a cell with or cells were at in 2% in the of mouse to were with and in the for were with to cells from the cells were with 2% and and into to a concentration of cell and erythroblasts or were to a M. H. M.D. PubMed Scopus Google Scholar, I. M. J. M.R. Van T.J. Van M. Full Text Full Text PDF PubMed Scopus Google Scholar). The different cell are in the and the cells were into on a University Expression of Tspo2 in each of the cell was by using the in Table erythroid cells were maintained in medium with at in a cell a encoding was into the mammalian expression and sites using the specific (supplemental Table the expression of TSPO2 the gene The was into cells using of for of cells were for in medium containing were and in the same medium. the were in an and used as a negative cell cells and cell which and which was with were grown in The of medium containing in concentration was and cells were for at For cells were with with for and with at were in medium and into a the of TSPO2 is for cholesterol trafficking in we proteins protein its and a wild-type protein, using and the in Table was used as cells grown in a were with the cells were with and as were using a with an was with the of an and was with the of a the intracellular free cholesterol, fluorescent was performed by using a cholesterol cells were a cells were with a concentration of an of The cells were with in saline and with for cells were using a with an with a at were with software and using and the relationships within the TSPO we performed a analysis using sequences (supplemental and Table S1). The of the acid sequences were on the TSPO protein, and the sequences were an NJ the relationships the TSPO for the was from 1000 replicates, and in than of the are The relationships of Tspo genes are in with evolution, which is with the that Tspo1 is a gene conserved from bacteria to humans (1Papadopoulos V. Baraldi M. Guilarte T.R. Knudsen T.B. Lacapere J.J. Lindemann P. Norenberg M.D. Nutt D. Weizman A. Zhang M.R. Gavish M. Trends Pharmacol. Sci. 2006; 27: 402-409Abstract Full Text Full Text PDF PubMed Scopus (1159) Google Scholar). However, Tspo2 in the and mammalian A and The values for the between Tspo2 and Tspo1 are strongly that the is and that the Tspo2 arose from Tspo1 through gene before the divergence of avians and mammals. In these genes are involved in different gene that their different functions evolved during the evolution of the TSPO gene family (supplemental and and A of the of the acid sequences of human and mouse is the of between of the TSPO family in both and and The by the K. W. Biol. Chem. Scholar). is on the outer mitochondria membrane although in cancer it is in and the nucleus M. D. Culty M. H. B. Papadopoulos V. Res. 1999; Google Scholar). the of we the mouse and human cell and cells with and were with to these proteins that with the but with which was in mitochondria J. M.B. Papadopoulos V. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google and of an fluorescent protein with retention using with and (supplemental In using with each we that the performed the of TSPO2 (supplemental Thus, that TSPO2 is to the and nuclear on the we that TSPO2 functional properties have from of and it probably has the ligand binding we and the as as in has to be an to the ligand binding properties, yeast has the gene E. R. P. P. G. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of proteins were using and A and The conditions for TSPO2 were in the of SG medium. The the which was to as previously for H. Hardwick M. G. Yao Z.X. J. Lacapere J.J. Papadopoulos V. 42: PubMed Scopus Google Scholar). The of TSPO2 was using and as negative and expression as a the diagnostic drug that the functional protein and In TSPO2 to specific binding for that TSPO2 the drug binding the which suggested a loss of drug binding to the of residues and of the for to (supplemental and TSPO2 the conserved cholesterol binding known as the cholesterol-binding cholesterol acid in at its H. Yao Z. Degenhardt B. G. Papadopoulos V. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google and we that TSPO2 probably functions in the intracellular cholesterol transport yeast cholesterol, the cell acts as a S. D. R. Biochem. PubMed Google Scholar). For that we yeast and with cholesterol and of shown in the yeast cholesterol than yeast with further the of TSPO2 in cholesterol we used as a fluorescent to its cells with the cholesterol conditions and we cells to the of and However, that the protein the cholesterol conditions with of yeast cells were to cholesterol, that TSPO2 the cholesterol and Although the mechanism of TSPO2 function in these data that TSPO2 is involved in cholesterol synthesis, further that TSPO2 has a functional cholesterol binding at its we the and the of into to and of the proteins was by and proteins were in the of the cell but in the in of the we performed to the proteins cholesterol A. L. Brown J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The obtained that wild-type of the from the of the protein the of the protein to cholesterol, that cholesterol on the is to the of the is with the of as the of cholesterol binding to protein H. Hardwick M. G. Yao Z.X. J. Lacapere J.J. Papadopoulos V. 42: PubMed Scopus Google Scholar, H. Yao Z. Degenhardt B. G. Papadopoulos V. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, J.J. H. G. J. Papadopoulos V. B. Biochem. Res. PubMed Scopus Google Scholar). We the expression of Tspo2 by in mouse throughout A of Tspo2 was observed in and and during Tspo2 was at the but was in the the hematopoietic sites from the to the to E. P. S.H. Cell Biol. 1990; PubMed Scopus Google Scholar). of Tspo2 expression were on by a throughout and and to the of cell of the erythroid Tspo2 expression in the the hematopoietic cells in the in hematopoietic function with and the hematopoietic cells from the to the and I. Res. Google that Tspo2 expression is to hematopoietic cells in the during is the hematopoietic in such as and and to expression in the Tspo2 expression in the the and Thus, in the of the a of Tspo2 the cells are and and during Tspo2 was in the red cells hybridization in the which was in other genes in the In and Tspo2 was observed in the and The red which are of erythropoiesis and K. G. T. 45: Scopus Google Scholar), were but was in the which of for and that Tspo2 is in the erythroid cells in hematopoietic hematopoietic expression of the Tspo2 gene in as as in humans was by (supplemental further the expression of Tspo2, we erythroid cell from During erythroid the before into are and and erythroblasts A and Tspo2 expression was in the and However, Tspo2 was in the erythroblasts and and erythroblasts during erythroid maturation and was in or erythroblasts in was in from serve as a cell erythroblasts some of during erythroid maturation J. Cell Biol. PubMed Scopus Google Scholar). Tspo1 and as to the of from each of cell can the maturation of mammalian the cholesterol of erythroid we used the human erythroid cell as a model and cell and which were with a expression or the as a The expression of in the cell was by The cell and cells were with and the fluorescent that to the of was in the cell However, the of the was observed in the cell as as in the cells and data that a in the intracellular trafficking of cholesterol and in the and of free cholesterol and cell membrane for and In cellular cholesterol has been shown to erythroid maturation A. M. M. PubMed Scopus Google Scholar). of the gene intracellular free cholesterol redistribution, we cholesterol by by The that cholesterol in the The cholesterol in cells was similar to the observed of a known which cholesterol transport 2006; PubMed Scopus Google The of TSPO2 was shown to be for cholesterol binding in The function of the in cholesterol was in cells with plasmids containing wild-type and the to (supplemental The protein the a subcellular similar to that of the wild-type protein, although the protein (supplemental We recently the of such in cells with a in the M.B. J. J. Papadopoulos V. 2009; PubMed Scopus Google Scholar). the and of TSPO2 the of intracellular (supplemental and by the cholesterol (supplemental In wild-type TSPO2 in the and nuclear and and with (supplemental data that the cholesterol binding of TSPO2 an essential in cholesterol as as the intracellular of Moreover, the appearance of Tspo2 in erythroblasts strongly correlates with the down-regulation of other enzymes in the cholesterol that TSPO2 cholesterol trafficking and redistribution but during of the TSPO family before the divergence of and mammals have some specific biological function to the TSPO2 The Tspo gene is evolutionarily conserved and from bacteria to plants and humans (7Gavish M. Bachman I. Shoukrun R. Katz Y. Veenman L. Weisinger G. Weizman A. Pharmacol. Rev. 1999; 51: 629-650PubMed Google Scholar, 8Papadopoulos V. Lecanu L. Brown R.C. Han Z. Yao Z.X. Neuroscience. 2006; 138: 749-756Crossref PubMed Scopus (198) Google Scholar). TSPO is in the and to the it is involved in cellular However, the TSPO protein has been shown to be in the membrane of red cell, a cell that has both the nucleus and mitochondria J.M. J. Pharmacol. PubMed Scopus Google Scholar), as as in the membrane of by and it was shown to of these cells A. L. P. B. Biochem. Res. PubMed Scopus Google Scholar). The membrane and mitochondrial of TSPO have some for its including mitochondrial respiration, cell and M.J. Biochem. Pharmacol. PubMed Scopus Google Scholar). The of the TSPO2 of proteins in hematopoietic has that and mammals are the Tspo gene in these have an biological The of the acid sequences of human and mouse a of between of the TSPO family in both and as in the identified Moreover, the of both proteins the to the that specific with that these proteins in the the between TSPO2 and Thus, performed with hematopoietic using be with these the of TSPO2 a of which the intracellular on and nuclear loss of drug ligand binding properties functional of and the of TSPO2 in hematopoietic The erythroid cell expression of Tspo2 further of which identified an Tspo in mouse from and as a or for erythrocyte and S.M. A.A. O. Cell 2007; Full Text Full Text PDF PubMed Scopus Google and analysis of human cells during the same gene was to be in erythropoiesis M. S. D. Cell Scopus Google Scholar, S. R. B. K. H. S. Physiol. 2006; PubMed Scopus Google Scholar). it seems that Tspo2 to the erythroid be used as a for erythroid a genes previously to be erythroid are in other such as erythroid and in E. T. H. H. L. M. J. M. PubMed Scopus Google Scholar, Biol. 1995; PubMed Scopus Google Scholar, J. K. O. K. S. K. M. PubMed Scopus Google Scholar). Therefore, TSPO2 serve as in cell studies, in with other genes, such as gene, and erythroid erythroid cell expression of Tspo2 have in erythropoiesis to is the of red cell in which hematopoietic from cells and through a of that the of and and G. and and Google Scholar, J. Physiol. 1997; Google Scholar). In the such as endoplasmic and are The during is that the nucleus is from the cell to that to the expulsion is by cellular cholesterol A. M. M. PubMed Scopus Google Scholar), and the of cholesterol during has been linked to H. J. A. H. H. J. 2007; PubMed Scopus Google Scholar). of intracellular cholesterol to the nuclear membrane to erythrocyte maturation is a have shown that membrane cholesterol during maturation of erythroblasts to A. Lipids. PubMed Scopus Google Scholar). in a data analysis of the proteins involved in cholesterol we observed that the cholesterol during the during the hematopoietic characterized by the expression of from erythroid to which to to and erythroblasts and the nucleus to However, the mechanism is we the that TSPO2 free cholesterol, from or that TSPO2 an during erythrocyte maturation. During the of a the gene (peripheral benzodiazepine which to Tspo2, in erythropoiesis was M. Y. G. 2009; PubMed Scopus Google Scholar). Although it is to the in the of of expression and functional data in was Moreover, the data for the in that a mitochondrial similar to that of are in to the as as to a that identified an 18-kDa protein acid similarities to mitochondrial in the erythrocyte nuclear a with nuclear and the receptor G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). on the data by M. Y. G. 2009; PubMed Scopus Google to be We for with for the and for with with

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.