In Vivo and in Vitro Peripheral-Type Benzodiazepine Receptor Polymerization:  Functional Significance in Drug Ligand and Cholesterol Binding

Abstract

Peripheral-type benzodiazepine receptor (PBR) is an 18 kDa high-affinity drug ligand and cholesterol binding protein involved in various cell functions. Antisera for distinct PBR areas identified immunoreactive proteins of 18, 40, and 56 kDa and occasionally 72, 90, and 110 kDa in testicular Leydig and breast cancer cells. These sizes may correspond to PBR polymers and correlated to the levels of reactive oxygen species. Treatment of Leydig cells with human chorionic gonadotropin rapidly induced free radical, PBR polymer, and steroid formation. UV photoirradiation generates ROS species, which increased the size of intramembraneous particles of recombinant PBR reconstituted into proteoliposomes consistent with polymer formation, determined both by SDS-PAGE and by freeze-fracture electron microscopy. Spectroscopic analysis revealed the formation of dityrosines as the covalent cross-linker between PBR monomers. Moreover, photoirradiation increased PK 11195 drug ligand binding and reduced cholesterol binding capacity of proteoliposomes. Further addition of PK 11195 drug ligand to polymers increased the rate of cholesterol binding. These data indicate that reactive oxygen species induce in vivo and in vitro the formation of covalent PBR polymers. We propose that the PBR polymer might be the functional unit responsible for ligand-activated cholesterol binding and that PBR polymerization is a dynamic process modulating the function of this receptor in cholesterol transport and other cell-specific PBR-mediated functions.

The 18 kDa Translocator Protein as a Potential Participant in Atherosclerosis

“…Recently, we have established that the TSPO appears to be an active participant in the generation of ROS at mitochondrial levels and maintenance of the mitochondrial membrane potential, in relation to apoptosis (Kugler et al, 2008;Zeno et al, 2009). In turn, ROS levels also affect TSPO function (Delavoie et al, 2003). As a result of this, we suggest that the TSPO may be involved in oxidative stress related to cardiovascular disorders.…”

“…The suggestion of TSPO involvement in steroidogenesis modulated by TSPO ligands was further supported by TSPO antisense knockdowns in MA-10 Leydig cells, which reduced steroid production (Hauet et al, 2005). It has been proposed that TSPO polymerization modulates the function of this receptor in cholesterol transport, since polymer formation induced by ROS increased both TSPO ligand binding and cholesterol-binding capacities (Delavoie et al, 2003). The monomer binds cholesterol with high affinity but not so with TSPO ligands.…”

“…Recently, TSPO has been found to occur not only in the 18 kDa form, but also as 36-, 54-, and 72 kDa TSPO polymers (Delavoie et al, 2003). This topic has arisen from the electron microscopic observation that the 18 kDa TSPO protein was organized in clusters of 2-7 molecules on Leydig cell mitochondrial membranes (Papadopoulos et al, 1997).…”

“…In particular, activation of the TSPO may lead to a decrease of the mitochondrial membrane potential, mitochondrial dysfunction, and subsequent release of mitochondrial cytochrome c, followed by the activation of a caspase cascade leading to apoptosis (Levin et al, 2005). Interestingly, as ROS generation accompanies in cardiovascular diseases, it has also been shown that oxidative stress modulates TSPO structure and function (Delavoie et al, 2003). Vice versa, TSPO appears to be an essential participant in ROS generation at mitochondrial levels induced by various agents Zeno et al, 2009;Choi et al, 2011).…”

“…TSPO ligands are reported to induce TSPO-mediated translocation of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane (Papadopoulos et al, 1997). Moreover, PK 11195 and Ro5-4864 (the classical specific TSPO ligands) increased cholesterol transport into the mitochondria and subsequent steroid synthesis in gonadal, adrenal, brain, and liver cells (Delavoie et al, 2003). Ro5-4864 directly stimulated the release of corticotrophin releasing hormone (CRH) in rats, whereas PK 11195 directly stimulated the secretion of ACTH.…”