9.A.24 The Mitochondrial Cholesterol/Porphyrin Uptake Translocator Protein (TSPO) Family
The central channel Tom40 of the preprotein translocase of the outer membrane (TOM) 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 transmembrane segments (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 reveal that PBR import (1) depends on the import receptor Tom70 but requires neither the Tom20 and Tom22 import receptors nor the import channel Tom40, (2) shares the post-Tom70 pathway with the 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. Thus, an unidentified pathway for the membrane integration of MOM proteins with multiple TMSs was identified (Otera et al., 2007).
Translocator protein (TSPO; PBR) has been reported to be an 18 kDa high affinity cholesterol, porphyrin uptake and drug-binding protein found primarily in the outer mitochondrial membrane. 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).
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).
The peripheral benzodiazepine receptor (PBR), which can also bind isoquinoline carboxamides (Riond et al. 1991) and integrates into the mitochondrial outer membrane (MOM) through five hydrophobic transmembrane segments (TMSs). Also called "translocator protein", TSPO). It is a mitochondrial cholesterol and porphyrin uptake transporter (Jaremko et al. 2014; Taylor et al. 2014) but is also part of the mitochondrial permeability transition pore (MPTP) which includes cyclophilin D, VDAC (TC#1.B.8) and the adenine nucleotide translocator (Austin et al. 2013). The 3-d structure has been determined at 2.4 Å resolution bound to its high affinity ligand, PK11195 which causes the otherwise loose 5 helix bundle to form a tight bundle with a hydrophobic pocket for PK11195 (Jaremko et al. 2014).
PBR of Homo sapiens (Q6ICF9)
Uncharacterized protein of 171 aas and 5 TMSs
UP of Loa loa (Eye worm) (Filaria loa)
TspO protein of 141 aas and 4-5 TMSs
TspO of Phaeocystis globosa virus
TspO-like; MBR-like protein of 163 aas
TspO-like protein of Galdieria sulfuraria
TspO/MBR family member of 151 aas and 5 TMSs. The crystal structure has been determined at 1.7 Å resolution (Guo et al. 2015). The protein was solved in complex with the benzodiazepine-like inhibitor, PK11195. TspO-mediated protoporphyrin IX (PpIX) reactions were also described, including catalytic degradation to a previously undescribed heme derivative. Structure-inspired mutations allowed the investigation of the reaction mechanisms, showing that TSPOs from Xenopus and man have similar PpIX-directed activities. Although TSPOs have been regarded as transporters, the catalytic activity in PpIX degradation suggests physiological importance for TSPOs in protection against oxidative stress (Guo et al. 2015).
TspO of Bacillus cereus
Uncharacterized protein of 134 aas and 4 TMSs (Hug et al. 2016).
UP of Candidatus Peribacter riflensis
Uncharacterized protein of 161 aas and 4 TMSs.
UP of Luteimonas mephitis
Tryptophan-rich sensory protein. TSPO, of 167 aas and 5 TMSs
TSPO of Halococcus sediminicola
The outer membrane tryptophan-rich sensory protein (TspO) of the TSPO/MBR family of 159 aas and 5 TMSs (Yeliseev et al. 1997; Yeliseev and Kaplan 1999). The 10 Å cryo electron microscopy structure is known (Korkhov et al. 2010) as are 1.8, 2.4 and 2.5 Å structures solved by x-ray crystallography (Li et al. 2015). Crystals obtained in the lipidic cubic phase reveal the binding site of an endogenous porphyrin ligand. The three crystal structures reveal a dimer, providing insights into the controversial physiological role of TSPO and how a mutation in the human homologue causes psychiatric disorders and reduced pregnenolone production (Li et al. 2015).
TspO of Rhodobacter spheroides
The Endoplasmic reticulum/Golgi TSPO protein is mainly detected in dry seeds, but can be induced in vegetative tissues by osmotic or salt stress or abscisic acid (ABA) treatment (Guillaumot et al. 2009).
TSPO of Arabidopsis thaliana
Tryptophan-rich sensory protein homologue, TspO, of 158 aas and 5 TMSs
TspO of Nostoc sp.
TspO/MBR family protein of 186 aas and 5 TMSs
TspO/MBR family protein of Acanthamoeba castellanii
TspO/MBR family protein of 186 aas and 5 TMSs
TspO of Niastella koreensis
TspO of 159 aas
TspO of Lactococcus lactis
Peripheral-type benzodiazepine receptor of 188 aas
Peripheral-type benzodiazepine receptor of Zea mays
TspO homologue of 193 aas
TspO of Oryza sativa
TspO homologue of 171 aas
TspO homologue of Maricaulis maris
CrtK protein of 166 aas and 5 TMSs.
CrtK of Oceanicaulis sp.
Uncharacterized protein of 177 aas
UP of Mycobacterium vanbaalenii
Tryptophan-rich sensory proteinof 160 aas and 5 TM
TpsO of Nocardia soli
Uncharacterized protein, WcoO of 272 aas and 8 TMSs.
WcoO of Clavibacter michiganensis
Uncharacterized protein of 155 aas and 5 TMSs.
UP of Methanosarcina mazei
Uncharacterized protein of 258 aas
UP of Bacillus selenitireducens
Uncharacterized protein of 290 aas and 8 TMSs
UP of Coriobacterium glomerans
Uncharacterized protein of 281 aas and 8 TMSs.
UP of Phytophthora infestans (Potato late blight fungus)
Uncharacterized protein of 264 aas and 8 TMSs.
UP of Rhizophagus irregularis (Arbuscular mycorrhizal fungus) (Glomus intraradices)
Uncharacterized protein of 350 aas and 8 TMSs.
UP of Guillardia theta
Uncharacterized protein of 277 aas and 7 TMSs
UP of Microbacterium yannicii
Uncharacterized protein of 236 aas and 8 TMSs.
UP of Paracoccus zeaxanthinifaciens
Uncharacterized protein of 250 aas and 8 TMSs.
UP of Henriciella aquimarina