Mitochondrial Translocator Protein (TSPO) Function Is Not Essential for Heme Biosynthesis

Abstract

Function of the mammalian translocator protein (TSPO; previously known as the peripheral benzodiazepine receptor) remains unclear because its presumed role in steroidogenesis and mitochondrial permeability transition established using pharmacological methods has been refuted in recent genetic studies. Protoporphyrin IX (PPIX) is considered a conserved endogenous ligand for TSPO. In bacteria, TSPO was identified to regulate tetrapyrrole metabolism and chemical catalysis of PPIX in the presence of light, and in vertebrates, TSPO function has been linked to porphyrin transport and heme biosynthesis. Positive correlation between high TSPO expression in cancer cells and susceptibility to photodynamic therapy based on their increased ability to convert the precursor 5-aminolevulinic acid (ALA) to PPIX appeared to reinforce this mechanism. In this study, we used TSPO knock-out (Tspo ؊/؊ ) mice, primary cells, and different tumor cell lines to examine the role of TSPO in erythropoiesis, heme levels, PPIX biosynthesis, phototoxic cell death, and mitochondrial bioenergetic homeostasis. In contrast to expectations, our results demonstrate that TSPO deficiency does not adversely affect erythropoiesis, heme biosynthesis, bioconversion of ALA to PPIX, and porphyrin-mediated phototoxic cell death. TSPO expression levels in cancer cells do not correlate with their ability to convert ALA to PPIX. In fibroblasts, we observed that TSPO deficiency decreased the oxygen consumption rate and mitochondrial membrane potential (⌬⌿m) indicative of a cellular metabolic shift, without a negative impact on porphyrin biosynthetic capability. Based on these findings, we conclude that mammalian TSPO does not have a critical physiological function related to PPIX and heme biosynthesis.Mammalian translocator protein (TSPO), 2 previously known as the peripheral benzodiazepine receptor (1), is a highly conserved protein enriched in the outer mitochondrial membrane (2). Despite extensive efforts to characterize TSPO, its precise physiological function remains elusive (3, 4). High levels of TSPO expression in steroidogenic cells, its localization to the outer mitochondrial membrane, and increased steroid production upon pharmacological binding led to the primary prospective model that TSPO was a mitochondrial cholesterol transporter essential for steroidogenesis (5). In recent studies using precise genetic tools, we and others have systematically refuted the involvement of TSPO in this process (6 -10). Similarly, copurification of TSPO with putative members of the mitochondrial permeability transition pore (MPTP) (11) and effects mediated by TSPO binding drugs on modulating apoptosis (12, 13) resulted in a secondary model that TSPO was associated with MPTP function and cell death (14). Again, recent discovery of the molecular identity of MPTP (15) Binding of porphyrins to TSPO has been a consistent property reported in bacteria (18), plants (19), and animals (17). In Rhodobacter sphaeroides, TSPO was found localized to the outer membrane (18) and pl...

Global Deletion of TSPO Does Not Affect the Viability and Gene Expression Profile

“…The link between TSPO and mitochondrial energy homeostasis is inconsistent and perhaps dependent on cell type. Studies have reported that TSPO deletion results in a decreased oxygen consumption rate (OCR) in microglia[4] and fibroblasts[40], but no change in OCR was observed in hepatocytes[13] and Leydig cells[41]. An increase in mitochondrial fatty acid oxidation was observed in steroidogenic cells and tissues[41].…”

“…An increase in mitochondrial fatty acid oxidation was observed in steroidogenic cells and tissues[41]. But TSPO is not required for steroid hormone biosynthesis as previously suggested[4,5,12,17,40]. The loss of TSPO in experimental autoimmune encephalomyelitis (EAE) mice, an animal model of multiple sclerosis (MS), caused mild astrogliosis and less EAE clinical scoring[42].…”

Translocator Protein 18 kDa (TSPO): An Old Protein with New Functions?

“…70–72 The seemingly definitive TSPO knockout experiments have also led to conflicting results. 31,61,62 However, one consistent finding from knockout experiments appears to be altered mitochondrial energy metabolism, including lower oxygen consumption, membrane potential, and ATP levels. 40,41,61,73 This observation fits with the additional finding of an increased level of fatty acid oxidation in the absence of TSPO, 74 but how these observations relate to another emerging common theme, sensing or responding to stress, and how they may be associated with oxygen radical production and regulatory phenomena, remains to be determined.…”

“…29–31 Several other TSPO −/− mouse constructs have yielded conflicting reports regarding lethality and other characteristics. 31,41,42,58,61 These differences have been discussed in detail, 39,62,75 clarifying some of the complex issues involved in the methodology that may explain the discrepancies. Recent results from conditional, cell-targeted TSPO deletion show altered development and hormone-driven steroid synthesis, 62 in keeping with the role of TSPO as a stress response player in steroid metabolism.…”

“…19 Interestingly, TSPO is reported to play a similar role in regulating the accumulation of porphyrin in mammalian cell lines 95 when challenged with protoporphyrin IX, although recent studies in the TSPO knockout mouse do not confirm this role. 61 In plants, 37,57,96 a role for At TSPO in scavenging of heme and porphyrins is reported, involving degradation of TSPO via an autophagy mechanism. Recent data in mouse also indicate that TSPO is involved in regulating mitochondrial ROS levels and mitophagy, dependent on the ratio of TSPO to VDAC1.…”