Translocator Protein 2 Is Involved in Cholesterol Redistribution during Erythropoiesis

Abstract

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 ligandbinding 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) 2 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 (1). 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 (2). 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 (2). TSPO of bacterial origin has been shown to have the same ligand binding properties as mammalian TSPO proteins (3). In addition to the binding of porphyrin and heme, mammalian TSPO can replace the activity of its bacterial homologs (2, 4, 5). 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 (6). Therefore, it is conceivable that some conserved functions of the Tspo genes within a cell are maintained from bacteria to plants and to mammals.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 (7-10). Moreover, its expression correlates with certain pathological conditions such as cancer and endocrine and neurological diseases (8). Although some conserved cellular functions of Tspo are shared from bacteria to mammals, such as cholesterolbinding and transport, their biological significance seems to have adapted to serve specific functions critica...

Current status and future perspectives: TSPO in steroid neuroendocrinology

“…This led to the conclusion that TSPO2 may be involved in heme availability for the assembly of hemoglobin (Nakazawa et al 2009). In contrast, the study with mouse and human TSPO2 was an extrapolation of previous work on TSPO and steroidogenesis from the same authors, and suggested that TSPO2 is involved in cholesterol uptake and trafficking in erythroid cell types (Fan et al 2009). At the present time, there are no gene-knockout models to study loss-of-function resulting from TSPO2 deletion, and understanding of its function remains rudimentary.…”

“…Unlike TSPO expression that is observed at high levels in cells active in lipid storage/metabolism (Fig. 2), TSPO2 expression appears restricted to the bone marrow (Fan et al 2009, Nakazawa et al 2009), and has not been detected in steroidogenic tissues (confirmed in Morohaku et al 2014, Tu et al 2014. Subcellular localization of TSPO2 using specific antibodies has not been performed; localization based on overexpression studies using C-terminal GFP/DsRed-TSPO2 fusion proteins (chicken, mouse and human) in cell lines have led to conflicting proposals.…”

“…Findings that in the same Leydig cells a complete TSPO deletion using CRISPR/Cas9-mediated targeting had no effect on steroid hormone production (Tu et al 2015) suggest that this is not a case of functional redundancy, but a core problem with reproducibility, and needs to be considered as such. The TSPO homolog, TSPO2, is expressed almost exclusively in hematopoietic tissues, and does not present a case for functional redundancy in these studies (Fan et al 2009) (discussed later in this review). If there is indeed a redundant protein or mechanism, it is not clear why these mechanisms did not become apparent in earlier studies that asserted an 'indispensable' role for TSPO in the steroidogenic machinery.…”

“…Subcellular localization of TSPO2 using specific antibodies has not been performed; localization based on overexpression studies using C-terminal GFP/DsRed-TSPO2 fusion proteins (chicken, mouse and human) in cell lines have led to conflicting proposals. The group who studied chicken TSPO2 demonstrated mitochondrial localization (Nakazawa et al 2009), and the group who studied mouse and human TSPO2 proposed that TSPO2 is localized to the endoplasmic reticulum and the nuclear membrane (Fan et al 2009). These studies have demonstrated functional disparities as well.…”

Characterization of fluorescent NBD-cholesterol efflux in THP-1-derived macrophages

“…1C). When 0.1 µM of NBD-cholesterol was used, which represents a concentration reported in several previous studies (10,19,20), for up to 12 h of incubation, real-time imaging yielded dim fluorescent images. This finding was confirmed by quantitative FI detection, in which the FI of the cell lysate was undetectable following 12 h incubation.…”