Steroid hormones are critical for organismal development and health. The rate-limiting step in steroidogenesis is the transport of cholesterol from the outer mitochondrial membrane (OMM) to the cytochrome P450 enzyme CYP11A1 in the inner mitochondrial membrane (IMM). Cholesterol transfer occurs through a complex termed the "transduceosome," in which cytosolic steroidogenic acute regulatory protein interacts with OMM proteins translocator protein and voltage-dependent anion channel (VDAC) to assist with the transfer of cholesterol to OMM. It has been proposed that cholesterol transfer from OMM to IMM occurs at specialized contact sites bridging the two membranes composed of VDAC and IMM adenine nucleotide translocase (ANT). Blue native PAGE of Leydig cell mitochondria identified two protein complexes that were able to bind cholesterol at 66- and 800-kDa. Immunoblot and mass spectrometry analyses revealed that the 800-kDa complex contained the OMM translocator protein (18-kDa) and VDAC along with IMM CYP11A1, ATPase family AAA domain-containing protein 3A (ATAD3A), and optic atrophy type 1 proteins, but not ANT. Knockdown of ATAD3A, but not ANT or optic atrophy type 1, in Leydig cells resulted in a significant decrease in hormone-induced, but not 22R-hydroxycholesterol-supported, steroid production. Using a 22-phenoxazonoxy-5-cholene-3-beta-ol CYP11A1-specific probe, we further demonstrated that the 800-kDa complex offers the microenvironment needed for CYP11A1 activity. Addition of steroidogenic acute regulatory protein to the complex mobilized the cholesterol bound at the 800-kDa complex, leading to increased steroid formation. These results identify a bioactive, multimeric protein complex spanning the OMM and IMM unit that is responsible for the hormone-induced import, segregation, targeting, and metabolism of cholesterol.
Translocator protein (TSPO) is a key member of the mitochondrial cholesterol transport complex in steroidogenic tissues. To assess the function of TSPO, we generated two lines of Cre-mediated Tspo conditional knockout (cKO) mice. First, gonadal somatic celltargeting Amhr2-Cre mice were crossed with Tspo-floxed mice to obtain F1 Tspo Amhr2 cKO mice (Tspo fl/fl ;Amhr2-Cre /+). The unexpected Mendelian ratio of 4.4% cKO mice was confirmed by genotyping of 12.5-day-postcoitum (dpc) embryos. As Amhr2-Cre is expressed in gonads at 12.5 dpc, these findings suggest preimplantation selection of embryos. Analysis of expression databases revealed elevated levels of Amhr2 in two-and eight-cell zygotes, suggesting ectopic Tspo silencing before the morula stage and demonstrating elevated embryonic lethality and involvement of TSPO in embryonic development. To circumvent this issue, steroidogenic cell-targeting Nr5a1-Cre mice were crossed with Tspofloxed mice. The resulting Tspo fl/fl ;Nr5a1-Cre /+ mice were born at a normal Mendelian ratio. Nr5a1-driven Tspo cKO mice exhibited highly reduced Tspo levels in adrenal cortex and gonads. Treatment of mice with human chorionic gonadotropin (hCG) resulted in increased circulating testosterone levels despite extensive lipid droplet depletion. In contrast, Nr5a1-driven Tspo cKO mice lost their ability to form corticosterone in response to adrenocorticotropic hormone (ACTH). Important for ACTH-dependent steroidogenesis, Mc2r, Stard1, and Cypa11a1 levels were unaffected, whereas Scarb1 levels were increased and accumulation of lipid droplets was observed, indicative of a blockade of cholesterol utilization for steroidogenesis. TSPO expression in the adrenal medulla and increased epinephrine production were also observed. In conclusion, TSPO was found necessary for preimplantation embryo development and ACTH-stimulated steroid biosynthesis.translocator protein | anti-Mullerian hormone receptor type II | nuclear receptor subfamily 5 group A member 1 | knockout mice | steroidogenesis T he 18-kDa translocator protein (TSPO) is an outer mitochondrial membrane (OMM) protein, originally named the peripheral benzodiazepine receptor (PBR) due to its discovery as a high-affinity binding site for the benzodiazepine diazepam outside of the central nervous system (1). Pharmacological, biochemical, and structural research has revealed TSPO's ability to bind numerous classes of drugs (1-4). Additional investigations also demonstrated that TSPO is a high-affinity cholesterol-binding protein. Cholesterol binding is localized to the C-terminal portion of its fifth transmembrane helix at a conserved cholesterol recognition/association amino acid consensus motif (1, 5). This finding was further supported by recent structural data (6). A search for additional physiological ligands of TSPO revealed that both the tetrapyrole protoporphyrin IX, a heme precursor, and the polypeptide endozepine, a GABA A modulator, bind TSPO, suggesting a role for TSPO in numerous physiological processes (1, 2, 5). Interestingly...
Steroidogenesis begins with cholesterol transfer into mitochondria through the transduceosome, a complex composed of cytosolic proteins that include steroidogenesis acute regulatory protein (STAR), 14-3-3 adaptor proteins, and the outer mitochondrial membrane proteins Translocator Protein (TSPO) and Voltage-Dependent Anion Channel (VDAC). TSPO is a drug- and cholesterol- binding protein found at particularly high levels in steroid synthesizing cells. Its aberrant expression has been linked to cancer, neurodegeneration, neuropsychiatric disorders and primary hypogonadism. Brain steroids serve as local regulators of neural development and excitability. Reduced levels of these steroids have been linked to depression, anxiety and neurodegeneration. Reduced serum testosterone is common among subfertile young men and aging men, and is associated with depression, metabolic syndrome and reduced sexual function. Although testosterone-replacement therapy is available, there are undesired side-effects. TSPO drug ligands have been proposed as therapeutic agents to regulate steroid levels in the brain and testis.
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