9.A.24 The Mitochondrial Cholesterol/Porphyrin/5-aminolevulinic acid Uptake Translocator Protein (TSPO) Family

The central channel Tom40 of the preprotein translocase of the outer mitochondrial membrane (TOM of the OMM) complex is thought to be responsible for the import of virtually all preproteins synthesized outside the mitochondria. Otera et al. (2007) analyzed the topogenesis of the peripheral benzodiazepine receptor (PBR), which integrates into the mitochondrial outer membrane (MOM) through five hydrophobic TMSs and functions in cholesterol and porphyrin import into the inner membrane. Analyses of in vitro and in vivo import into TOM component-depleted mitochondria revealed that PBR import (1) depends on the import receptor Tom70 but requires neither the Tom20 nor Tom22 import receptors, nor the import channel Tom40, (2) shares the post-Tom70 pathway with  C-tail-anchored proteins, and (3) requires factors in the mitochondrial intermembrane space. Furthermore, membrane integration of mitofusins and mitochondrial ubiquitin ligase, MOM proteins with two and four TMSs, respectively, proceeds through the same initial pathway (Otera et al., 2007). TSPO, a 5 TMS protein localized to the outer mitochondria membrane, is a high affinity cholesterol-binding protein (Georges et al. 2021). TSPO ligands modulate neuroplasticity and elicit antidepressant and anxiolytic therapeutic effects in animals and humans (Rupprecht et al. 2022). TSPO ligands are promising candidates for post-ischaemic recovery, exerting neuroprotection, in contrast to midazolam, without detrimental effects on synaptic plasticity (Puig-Bosch et al. 2023).

TSPO or PBR is an 18 kDa high affinity cholesterol, porphyrin uptake and drug-binding protein. Although TSPO is found in many tissue types, it is expressed at the highest levels under normal conditions in tissues that synthesize steroids (Batarseh and Papadopoulos, 2010). TSPO, the 3-d structure of which is known (PDB# 2MGY) has been associated with cholesterol import into mitochondria, a key function in steroidogenesis, and directly or indirectly with multiple other cellular functions including apoptosis, cell proliferation, differentiation, anion transport, porphyrin transport, heme synthesis, and regulation of mitochondrial function (Jaremko et al. 2014). Aberrant expression of TSPO has been linked to multiple diseases, including cancer, brain injury, neurodegeneration, Parkinson''s and Alheimer''s diseases, and ischemia-reperfusion injury.  It forms a large complex that includes VDAC-1, TSPO-associated protein-7 (PAP7; ACBD3), a protein kinase regulatory subunit, PKAR1A, and the StAR regluatory protein (Miller 2013).  TSPO is conformationally flexible (Jaremko et al. 2015). More recent studies have led to the conclusion that TSPO2 transports 5-aminolevulinic acid (see 9.A.24.1.17) (Manceau et al. 2020). The interplay of cholesterol and ligand binding in human TSPO (TC# 9.A.24.1.1) has been studied using classical molecular dynamics simulations (Lai et al. 2021).

Neurosteroids are able to rapidly control the excitability of the central nervous system, acting as regulators of type A receptors for GABA. Neurosteroid level alterations occur in psychiatric disorders, including anxiety disorders. Investigators have manipulated neurosteroidogenesis in an effort to correct neuronal excitation and inhibition imbalances, which may lie at the root of neuropsychiatric conditions. A promising target for therapy of anxiety disorders is the Translocator Protein (TSPO). TSPO is expressed predominantly in steroid-synthesizing tissues and is localized to contact sites between the outer and inner mitochondrial membranes.  It may mediate the rate-limiting step of neurosteroidogenesis. Brain concentrations of neurosteroids can be affected by selective TSPO activation. Indeed, TSPO drug ligands are able to stimulate primary neurosteroid formation that enhances GABAA receptor activity, pregnenolone and allopregnenalone, in both in vitro steroidogenic cells and in vivo animal models. A spectrum of TSPO ligands has been shown to exert anxiolytic actions when administered in rodents. The selective TSPO ligand, XBD173 (AC-5216, Emapunil), exerts anxiolytic effects not only in animal models, but also in humans. (Costa et al., 2012) reviewed the literature regarding the central nervous system biology of TSPO.

The TSPO (18 kDa translocator protein) is involved in cholesterol transport in organs that synthesize steroids and bile salts. Different natural and synthetic high-affinity TSPO ligands have been characterized through their ability to stimulate cholesterol transport, but they also stimulate other physiological processes including cell proliferation, apoptosis and calcium-dependent transepithelial ion secretion. TSPO is present in enterocyte mitochondria but not rat intestinal goblet cells (Ostuni et al. 2009). Enterocyte cytoplasm also contains the endogenous TSPO ligand, polypeptide DBI (diazepam-binding inhibitor). Whereas intestinal TSPO had high affinity for the synthetic ligand PK 11195, the pharmacological profile of TSPO in the duodenum was distinct from that in the jejunum and ileum. Specifically, benzodiazepine Ro5-4864 and protoporphyrin IX showed 5-13-fold lower affinity for duodenal TSPO. PK 11195 stimulated calcium-dependent chloride secretion in the duodenum and calcium-dependent chloride absorption in the ileum, but did not affect jejunum ion transport. Thus, the functional differences in subpopulations of TSPO in different regions of the intestine could be related to the structural organization of mitochondrial protein complexes that mediate the ability of TSPO to modulate either chloride secretion or absorption in the duodenum and ileum, respectively.

A 5 TMS bacterial homologue called the tryptophan-rich sensory protein of Rhodobacter spheroides binds retinoic acid, cucumin and an inhibitor of Bcl-2 actioin called gossypol (Li et al. 2013).  It appears to function in porphyrin degredation in a light- and oxygen-dependent process (Ginter et al. 2013). The protein and its function(s) have been reviewed. TSPO is believed to be involved either directly or indirectly in numerous biological functions, including mitochondrial cholesterol transport and steroid hormone biosynthesis, porphyrin transport and heme synthesis, apoptosis, cell proliferation, and anion transport. Localized to the outer mitochondrial membrane of steroidogenic cells, TSPO has been shown to associate with cytosolic and mitochondrial proteins as part of a large multiprotein complex involved in mitochondrial cholesterol transport, the rate-limiting step in steroidogenesis. It has been concluded that TSPO is a unique mitochondrial pharmacological target for diseases that involve increased mitochondrial activity, including steroidogenesis, but the specific function is not clear (Papadopoulos et al. 2017). However, TSPO has been shown to interact with other cellular proteins: VDAC, 30 kDa adenine nucleotide translocase (ANT), cyclophilin D, hexokinase, creatinine kinase, diazepam binding inhibitor (DBI), phosphate carrier and Bcl-2 family proteins (Kołodziejczyk 2015).

TSPO functions in cholesterol import, mitochondrial metabolism, apoptosis, cell proliferation, Ca²⁺ signaling, oxidative stress, and inflammation. TSPO forms a complex with VDAC, a protein that mediates the flux of ions, including Ca²⁺, nucleotides, and metabolites across the OMM, and it controls metabolism and apoptosis while interacting with many proteins. Both TSPO and VDAC are over-expressed in brains from Alzheimer's disease patients. TSPO-interacting ligands have been considered as a potential basis for drug development (Shoshan-Barmatz et al. 2019). The Translocator Protein of 18 kDa (TSPO) has an alternative binding site for the benzodiazepine diazepam. It is an evolutionary well-conserved and tryptophan-rich 169-amino acids protein with five alpha helical transmembrane domains stretching the outer mitochondrial membrane, with the carboxyl-terminus in the cytosol and a short amino-terminus in the intermembrane space of mitochondrion. Together with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT), it forms the mitochondrial permeability transition pore (MPTP). TSPO expression is ubiquitary, with higher levels in steroid producing tissues; in the central nervous system, it is mainly expressed in glial cells and neurons. TSPO is implicated in a variety of fundamental cellular processes including steroidogenesis, heme biosynthesis, mitochondrial respiration, mitochondrial membrane potential, cell proliferation and differentiation, cell life/death balance, and oxidative stress. Altered TSPO expression has been found in some pathological conditions. In particular, high TSPO expression levels have been documented in cancer, neuroinflammation, and brain injury. Conversely, low TSPO expression levels have been evidenced in anxiety disorders. Therefore, TSPO is not only an interesting drug target for therapeutic purpose (anticonvulsant, anxiolytic, etc.), but also a valid diagnostic marker of related-diseases detectable by fluorescent or radiolabeled ligands. Barresi et al. 2020 have presented an update of previous reviews dealing with the medicinal chemistry of TSPO and highlighted the most outstanding advances in the development of TSPO ligands as potential therapeutic or diagnostic tools. Compounds enhancing GABAergic neurotransmission such as neurosteroids and TSPO ligands, which also may exert anti-inflammatory properties in concert with immunomodulators such as C1q may open new avenues for the treatment of psychiatric disorders (Rupprecht et al. 2021). Together with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT), it forms the mitochondrial permeability transition pore (MPTP) (Rupprecht et al. 2021).

TSPO is phylogenetically widespread from archaea and bacteria to insects, vertebrates, plants, and fungi. TSPO's primary amino acid sequence is only modestly conserved between diverse species, although its five transmembrane helical structure appears mainly conserved (Hiser et al. 2021). Its cellular location and orientation in membranes have been reported to vary between species and tissues, with implications for potential diverse binding partners and function. Most TSPO functions relate to stress-induced changes in metabolism; it could be a receptor, a sensor, a transporter, or a translocator or more than one of these. TSPO may act indirectly by association with various protein binding partners or with endogenous or exogenous ligands. Hiser et al. 2021 review proteins that have commonly been invoked as TSPO binding partners. Possibly TSPO was originally a bacterial receptor/stress sensor associated with porphyrin binding as its most ancestral function and that it later developed additional stress-related roles in eukaryotes as its ability to bind new partners evolved. Mitochondrial interaction between TSPO and STAR promotes cholesterol and deleterious sterol mitochondrial accumulation during myocardial ischemia-reperfusion (Bréhat et al. 2024). This interaction regulates mitochondrial homeostasis and plays a key role during mitochondrial injury.

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