Cholesterol transport in steroid biosynthesis: Role of protein–protein interactions and implications in disease states

The transfer of cholesterol from the outer to the inner mitochondrial membrane is the rate-limiting step in hormone-induced steroid formation. To ensure that this step is achieved efficiently, free cholesterol must accumulate in excess at the outer mitochondrial membrane and then be transferred to the inner membrane. This is accomplished through a series of steps that involve various intracellular organelles, including lysosomes and lipid droplets, and proteins such as the translocator protein (18 kDa, TSPO) and steroidogenic acute regulatory (StAR) proteins. TSPO, previously known as the peripheral-type benzodiazepine receptor, is a high-affinity drug- and cholesterol-binding mitochondrial protein. StAR is a hormone-induced mitochondria-targeted protein that has been shown to initiate cholesterol transfer into mitochondria. Through the assistance of proteins such as the cAMP-dependent protein kinase regulatory subunit Iα (PKA-RIα) and the PKA-RIα- and TSPO-associated acyl-coenzyme A binding domain containing 3 (ACBD3) protein, PAP7, cholesterol is transferred to and docked at the outer mitochondrial membrane. The TSPO-dependent import of StAR into mitochondria, and the association of TSPO with the outer/inner mitochondrial membrane contact sites, drives the intramitochondrial cholesterol transfer and subsequent steroid formation. The focus of this review is on (i) the intracellular pathways and protein–protein interactions involved in cholesterol transport and steroid biosynthesis and (ii) the roles and interactions of these proteins in endocrine pathologies and neurological diseases where steroid synthesis plays a critical role.     

PAP7, a PBR/PKA-RIα-associated protein: a new element in the relay of the hormonal induction of steroidogenesis

The precise mechanism by which the hormone-induced minimal cAMP levels act at the mitochondria to activate cholesterol transport and steroid synthesis is unknown. We propose that this mechanism involves a macromolecular signaling complex where a newly identified peripheral-type benzodiazepine receptor (PBR)-associated protein (PAP7) binds the regulatory subunit RIα of the cAMP-dependent protein kinase A (PKA), thus allowing for local efficient catalytic activation and phosphorylation of the substrate steroidogenesis acute regulatory protein (StAR), leading to cholesterol transfer from the low affinity StAR to the high affinity PBR cholesterol binding protein. The mouse and human PAP7 proteins were cloned, their genomic organization and chromosomal localization characterized, their tissue distribution evaluated and subcellular localization defined. PAP7 is highly expressed in steroidogenic tissues, where it follows the pattern of PKA-RIα expression and data from a human adrenal disease suggest that it participates in PKA-RIα-mediated tumorigenesis and hormone-independent hypercortisolism. PAP7 is localized in the Golgi and mitochondria and inhibition of PAP7 expression results in reduced hormone-induced cholesterol transport into mitochondria and decreased steroid formation. Taken together, these data suggest that PAP7 functions as an A-kinase anchoring protein (AKAP) critical in the cAMP-dependent steroid formation.     

PAP7, a PBR/PKA-RIalpha-associated protein: a new element in the relay of the hormonal induction of steroidogenesis

The precise mechanism by which the hormone-induced minimal cAMP levels act at the mitochondria to activate cholesterol transport and steroid synthesis is unknown. We propose that this mechanism involves a macromolecular signaling complex where a newly identified peripheral-type benzodiazepine receptor (PBR)-associated protein (PAP7) binds the regulatory subunit RIalpha of the cAMP-dependent protein kinase A (PKA), thus allowing for local efficient catalytic activation and phosphorylation of the substrate steroidogenesis acute regulatory protein (StAR), leading to cholesterol transfer from the low affinity StAR to the high affinity PBR cholesterol binding protein. The mouse and human PAP7 proteins were cloned, their genomic organization and chromosomal localization characterized, their tissue distribution evaluated and subcellular localization defined. PAP7 is highly expressed in steroidogenic tissues, where it follows the pattern of PKA-RIalpha expression and data from a human adrenal disease suggest that it participates in PKA-RIalpha-mediated tumorigenesis and hormone-independent hypercortisolism. PAP7 is localized in the Golgi and mitochondria and inhibition of PAP7 expression results in reduced hormone-induced cholesterol transport into mitochondria and decreased steroid formation. Taken together, these data suggest that PAP7 functions as an A-kinase anchoring protein (AKAP) critical in the cAMP-dependent steroid formation.     

A mitochondrial kinase complex is essential to mediate an ERK1/2-dependent phosphorylation of a key regulatory protein in steroid biosynthesis

ERK1/2 is known to be involved in hormone-stimulated steroid synthesis, but its exact roles and the underlying mechanisms remain elusive. Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not. We demonstrate herein by Western blot analysis and confocal studies that temporal mitochondrial ERK1/2 activation is obligatory for PKA-mediated steroidogenesis in the Leydig-transformed MA-10 cell line. PKA activity leads to the phosphorylation of a constitutive mitochondrial MEK1/2 pool with a lower effect in cytosolic MEKs, while EGF allows predominant cytosolic MEK activation and nuclear pERK1/2 localization. These results would explain why PKA favors a more durable ERK1/2 activation in mitochondria than does EGF. By means of ex vivo experiments, we showed that mitochondrial maximal steroidogenesis occurred as a result of the mutual action of steroidogenic acute regulatory (StAR) protein -a key regulatory component in steroid biosynthesis-, active ERK1/2 and PKA. Our results indicate that there is an interaction between mitochondrial StAR and ERK1/2, involving a D domain with sequential basic-hydrophobic motifs similar to ERK substrates. As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser(232). Directed mutagenesis of Ser(232) to a non-phosphorylable amino acid such as Ala (StAR S232A) inhibited in vitro StAR phosphorylation by active ERK1/2. Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production. In summary, here we show that StAR is a novel substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric protein kinase complex that regulates cholesterol transport. The role of MAPKs in mitochondrial function is underlined.     

Peripheral-type benzodiazepine receptor-mediated action of steroidogenic acute regulatory protein on cholesterol entry into leydig cell mitochondria

Hormone-induced steroid biosynthesis begins with the transfer of cholesterol from intracellular stores into mitochondria. Steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptor (PBR) have been implicated in this rate-determining step of steroidogenesis. MA-10 mouse Leydig tumor cells were treated with and without oligodeoxynucleotides (ODNs) antisense to PBR and StAR followed by treatment with saturating concentrations of human choriogonadotropin. Treatment with ODNs antisense but not missense for both proteins inhibited the respective protein expression and the ability of the cells to synthesize steroids in response to human choriogonadotropin. Treatment of the cells with either ODNs antisense to PBR or a transducible peptide antagonist to PBR resulted in inhibition of the accumulation of the mature mitochondrial 30-kDa StAR protein, suggesting that the presence of PBR is required for StAR import into mitochondria. Addition of in vitro transcribed/translated 37-kDa StAR or a fusion protein of Tom20 (translocase of outer membrane) and StAR (Tom/StAR) to mitochondria isolated from control cells increased pregnenolone formation. Mitochondria isolated from cells treated with ODNs antisense, but not missense, to PBR failed to form pregnenolone and respond to either StAR or Tom/StAR proteins. Reincorporation of in vitro transcribed/translated PBR, but not PBR missing the cholesterol-binding domain, into MA-10 mitochondria rescued the ability of the mitochondria to form steroids and the ability of the mitochondria to respond to StAR and Tom/StAR proteins. These data suggest that both StAR and PBR proteins are indispensable elements of the steroidogenic machinery and function in a coordinated manner to transfer cholesterol into mitochondria.     

Novel androstenetriol interacts with the mitochondrial translocator protein and controls steroidogenesis

Steroid hormones are metabolically derived from multiple enzymatic transformations of cholesterol. The controlling step in steroid hormone biogenesis is the delivery of cholesterol from intracellular stores to the cytochrome P450 enzyme CYP11A1 in the mitochondrial matrix. The 18-kDa translocator protein (TSPO) plays an integral part in this mitochondrial cholesterol transport. Consistent with its role in intracellular cholesterol movement, TSPO possesses a cholesterol recognition/interaction amino acid consensus (CRAC) motif that has been demonstrated to bind cholesterol. To further investigate the TSPO CRAC motif, we performed molecular modeling studies and identified a novel ligand, 3,17,19-androsten-5-triol (19-Atriol) that inhibits cholesterol binding at the CRAC motif. 19-Atriol could bind a synthetic CRAC peptide and rapidly inhibited hormonally induced steroidogenesis in MA-10 mouse Leydig tumor cells and constitutive steroidogenesis in R2C rat Leydig tumor cells at low micromolar concentrations. Inhibition at these concentrations was not due to toxicity or inhibition of the CYP11A1 enzyme and was reversed upon removal of the compound. In addition, 19-Atriol was an even more potent inhibitor of PK 11195-stimulated steroidogenesis, with activity in the high nanomolar range. This was accomplished without affecting PK 11195 binding or basal steroidogenesis. Finally, 19-Atriol inhibited mitochondrial import and processing of the steroidogenic acute regulatory protein without any effect on TSPO protein levels. In conclusion, we have identified a novel androstenetriol that can interact with the CRAC domain of TSPO, can control hormonal and constitutive steroidogenesis, and may prove to be a useful tool in the therapeutic control of diseases of excessive steroid formation.     

Hormone-induced 14-3-3γ adaptor protein regulates steroidogenic acute regulatory protein activity and steroid biosynthesis in MA-10 Leydig cells

Cholesterol is the sole precursor of steroid hormones in the body. The import of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroid biosynthesis, relies on the formation of a protein complex that assembles at the outer mitochondrial membrane called the transduceosome. The transduceosome contains several mitochondrial and cytosolic components, including the steroidogenic acute regulatory protein (STAR). Human chorionic gonadotropin (hCG) induces de novo synthesis of STAR, a process shown to parallel maximal steroid production. In the hCG-dependent steroidogenic MA-10 mouse Leydig cell line, the 14-3-3γ protein was identified in native mitochondrial complexes by mass spectrometry and immunoblotting, and its levels increased in response to hCG treatment. The 14-3-3 proteins bind and regulate the activity of many proteins, acting via target protein activation, modification and localization. In MA-10 cells, cAMP induces 14-3-3γ expression parallel to STAR expression. Silencing of 14-3-3γ expression potentiates hormone-induced steroidogenesis. Binding motifs of 14-3-3γ were identified in components of the transduceosome, including STAR. Immunoprecipitation studies demonstrate a hormone-dependent interaction between 14-3-3γ and STAR that coincides with reduced 14-3-3γ homodimerization. The binding site of 14-3-3γ on STAR was identified to be Ser-194 in the STAR-related sterol binding lipid transfer (START) domain, the site phosphorylated in response to hCG. Taken together, these results demonstrate that 14-3-3γ negatively regulates steroidogenesis by binding to Ser-194 of STAR, thus keeping STAR in an unfolded state, unable to induce maximal steroidogenesis. Over time 14-3-3γ homodimerizes and dissociates from STAR, allowing this protein to induce maximal mitochondrial steroid formation.     

Evolutionary Origin of the Mitochondrial Cholesterol Transport Machinery Reveals a Universal Mechanism of Steroid Hormone Biosynthesis in Animals

Steroidogenesis begins with the transport of cholesterol from intracellular stores into mitochondria via a series of protein-protein interactions involving cytosolic and mitochondrial proteins located at both the outer and inner mitochondrial membranes. In adrenal glands and gonads, this process is accelerated by hormones, leading to the production of high levels of steroids that control tissue development and function. A hormone-induced multiprotein complex, the transduceosome, was recently identified, and is composed of cytosolic and outer mitochondrial membrane proteins that control the rate of cholesterol entry into the outer mitochondrial membrane. More recent studies unveiled the steroidogenic metabolon, a bioactive, multimeric protein complex that spans the outer-inner mitochondrial membranes and is responsible for hormone-induced import, segregation, targeting, and metabolism of cholesterol by cytochrome P450 family 11 subfamily A polypeptide 1 (CYP11A1) in the inner mitochondrial membrane. The availability of genome information allowed us to systematically explore the evolutionary origin of the proteins involved in the mitochondrial cholesterol transport machinery (transduceosome, steroidogenic metabolon, and signaling proteins), trace the original archetype, and predict their biological functions by molecular phylogenetic and functional divergence analyses, protein homology modeling and molecular docking. Although most members of these complexes have a history of gene duplication and functional divergence during evolution, phylogenomic analysis revealed that all vertebrates have the same functional complex members, suggesting a common mechanism in the first step of steroidogenesis. An archetype of the complex was found in invertebrates. The data presented herein suggest that the cholesterol transport machinery is responsible for steroidogenesis among all vertebrates and is evolutionarily conserved throughout the entire animal kingdom.     

Differential regulation of steroid hormone biosynthesis in R2C and MA-10 Leydig tumor cells: role of SR-B1-mediated selective cholesteryl ester transport

The rat R2C Leydig tumor cell line is constitutively steroidogenic in nature, while the mouse MA-10 Leydig tumor cell line synthesizes large amounts of steroids only in response to hormonal stimulation. Earlier studies showed abundant cAMP-independent steroid production and constitutive expression of steroidogenic acute regulatory (StAR) protein in R2C cells. The objective of the current study was to identify possible genetic alterations in the R2C cell line responsible for rendering it a constitutively steroidogenic cell line, especially those that might have altered its cholesterol homeostatic mechanisms. Measurement of the levels of cholesterol esters and free cholesterol, precursors for steroidogenesis, indicated that R2C mitochondria were fourfold enriched in free cholesterol content compared with MA-10 mitochondria. In addition to the previously demonstrated increased expression of StAR protein, we show that R2C cells possess marginally enhanced protein kinase A activity, exhibit higher capacity to take up extracellular cholesterol esters, and express much higher levels of scavenger receptor-type B class 1 (SR-B1) and hormone sensitive lipase (HSL). These observations suggest that the high level of steroid biosynthesis in R2C cells is a result of the constitutive expression of the components involved in the uptake of cholesterol esters (SR-B1), their conversion to free cholesterol (HSL), and its mobilization to the inner mitochondrial membrane (StAR).     

Peripheral-type benzodiazepine receptor (PBR) aggregation and absence of steroidogenic acute regulatory protein (StAR)/PBR association in the mitochondrial membrane as determined by bioluminescence resonance energy transfer (BRET)

The steroidogenic acute regulatory protein (StAR) is responsible for acute control of cholesterol transport across the mitochondrial membrane, however the mechanism of StAR-associated cholesterol transport is unknown and may involve the peripheral-type benzodiazepine receptor (PBR)/endozepine system. Several molecules of PBR may associate to form a channel through which cholesterol passes to the inner mitochondrial membrane, and endozepine is the natural ligand for PBR. Bioluminescence resonance energy transfer (BRET) was used to test StAR/PBR/endozepine interactions, PBR aggregation, and the effect of second messengers on interactions. There was no evidence of StAR/PBR, StAR/endozepine, or PBR/endozepine interactions. The StAR and PBR fusion proteins were trafficking to the mitochondria as expected, but the endozepine fusion protein was not localized to the mitochondria indicating that it was not biologically active. Data were obtained indicating that PBR forms aggregates in the mitochondrial membrane. Energy transfer between PBR fusion proteins was dose and time dependent, but there was no effect induced by PK11195 ligand binding or pharmacologic activation of PKA or PKC second messenger pathways. It appears that PBR aggregates in the mitochondrial membrane, however there was no evidence that PBR aggregation is regulated in the acute control of steroidogenesis, or that PBR and StAR interact.     

Steroidogenic acute regulatory protein binds cholesterol and modulates mitochondrial membrane sterol domain dynamics

The steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step of steroidogenesis, delivery of cholesterol to the inner mitochondrial membrane. However, the mechanism whereby cholesterol translocation is accomplished has not been resolved. Recombinant StAR proteins lacking the first N-terminal 62 amino acids comprising the mitochondrial-targeting sequence were used to determine if StAR binds cholesterol and alters mitochondrial membrane cholesterol domains to enhance sterol transfer. First, a fluorescent NBD-cholesterol binding assay revealed 2 sterol binding sites (K(d) values near 32 nm), whereas the inactive A218V N-62 StAR mutant had only a single binding site with 8-fold lower affinity. Second, NBD-cholesterol spectral shifts and fluorescence resonance energy transfer from StAR Trp residues to NBD-cholesterol showed (i) close molecular interaction between these molecules (R(2/3) = 33 A) and (ii) sensitized NBD-cholesterol emission from only one of the two sterol binding sites. Third, circular dichroism showed that cholesterol binding induced a change in StAR secondary structure. Fourth, a fluorescent sterol transfer assay that did not require separation of donor and acceptor mitochondrial membranes demonstrated that StAR enhanced mitochondrial sterol transfer as much as 100-fold and induced/increased the formation of rapidly transferable cholesterol domains in isolated mitochondrial membranes. StAR was 67-fold more effective in transferring cholesterol from mitochondria of steroidogenic MA-10 cells than from human fibroblast mitochondria. In contrast, sterol carrier protein-2 (SCP-2) was only 2.2-fold more effective in mediating sterol transfer from steroidogenic cell mitochondria. Taken together these data showed that StAR is a cholesterol-binding protein, preferentially enhances sterol transfer from steroidogenic cell mitochondria, and interacts with mitochondrial membranes to alter their sterol domain structure and dynamics.     

Steroidogenic acute regulatory protein (StAR), a novel mitochondrial cholesterol transporter

Cholesterol is a vital component of cellular membranes, and is the substrate for biosynthesis of steroids, oxysterols and bile acids. The mechanisms directing the intracellular trafficking of this nearly insoluble molecule have received increased attention through the discovery of the steroidogenic acute regulatory protein (StAR) and similar proteins containing StAR-related lipid transfer (START) domains. StAR can transfer cholesterol between synthetic liposomes in vitro, an activity which appears to correspond to the trans-cytoplasmic transport of cholesterol to mitochondria. However, trans-cytoplasmic cholesterol transport in vivo appears to involve the recently-described protein StarD4, which is expressed in most cells. Steroidogenic cells must also move large amounts of cholesterol from the outer mitochondrial membrane to the first steroidogenic enzyme, which lies on the matrix side of the inner membrane; this action requires StAR. Congenital lipoid adrenal hyperplasia, a rare and severe disorder of human steroidogenesis, results from mutations in StAR, providing a StAR knockout of nature that has provided key insights into its activity. Cell biology experiments show that StAR moves large amounts of cholesterol from the outer to inner mitochondrial membrane, but acts exclusively on the outer membrane. Biophysical data show that only the carboxyl-terminal alpha-helix of StAR interacts with the outer membrane. Spectroscopic data and molecular dynamics simulations show that StAR's interactions with protonated phospholipid head groups on the outer mitochondrial membrane induce a conformational change (molten globule transition) needed for StAR's activity. StAR appears to act in concert with the peripheral benzodiazepine receptor, but the precise itinerary of a cholesterol molecule entering the mitochondrion remains unclear.     

Interaction of hormone-sensitive lipase with steroidogenic acute regulatory protein: facilitation of cholesterol transfer in adrenal

Hormone-sensitive lipase (HSL) is responsible for the neutral cholesteryl ester hydrolase activity in steroidogenic tissues. Through its action, HSL is involved in regulating intracellular cholesterol metabolism and making unesterified cholesterol available for steroid hormone production. Steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane and is a critical regulatory step in steroidogenesis. In the current studies we demonstrate a direct interaction of HSL with StAR using in vitro glutathione S-transferase pull-down experiments. The 37-kDa StAR is coimmunoprecipitated with HSL from adrenals of animals treated with ACTH. Deletional mutations show that HSL interacts with the N-terminal as well as a central region of StAR. Coexpression of HSL and StAR in Chinese hamster ovary cells results in higher cholesteryl ester hydrolytic activity of HSL. Transient overexpression of HSL in Y1 adrenocortical cells increases mitochondrial cholesterol content under conditions in which StAR is induced. It is proposed that the interaction of HSL with StAR in cytosol increases the hydrolytic activity of HSL and that together HSL and StAR facilitate cholesterol movement from lipid droplets to mitochondria for steroidogenesis.     

Effects of disruption of the mitochondrial electrochemical gradient on steroidogenesis and the Steroidogenic Acute Regulatory (StAR) proteinProceedings of Xth International Congress on Hormonal Steroids, Quebec, Canada, 17–21 June 1998.

The steroidogenic acute regulatory (StAR) protein, which mediates cholesterol delivery to the inner mitochondrial membrane and the P450scc enzyme, has been shown to require a mitochondrial electrochemical gradient for its activity in vitro. To characterize the role of this gradient in cholesterol transfer, investigations were conducted in whole cells, utilizing the protonophore carbonyl cyanide m-chlorophenylhydrazone (m-CCCP) and the potassium ionophore valinomycin. These reagents, respectively, dissipate the mitochondrial electrochemical gradient and inner mitochondrial membrane potential. Both MA-10 Leydig tumor cell steroidogenesis and mitochondrial import of StAR were inhibited by m-CCCP or valinomycin at concentrations which had only minimal effects on P450scc activity. m-CCCP also inhibited import and processing of both StAR and the truncated StAR mutants, N-19 and C-28, in transfected COS-1 cells. Steroidogenesis induced by StAR and N-47, an active N-terminally truncated StAR mutant, was reduced in transfected COS-1 cells when treated with m-CCCP. This study shows that StAR action requires a membrane potential, which may reflect a functional requirement for import of StAR into the mitochondria, or more likely, an unidentified factor which is sensitive to ionophore treatment. Furthermore, the ability of N-47 to stimulate steroidogenesis in nonsteroidogenic HepG2 liver tumor cells, suggests that the mechanism by which StAR acts may be common to many cell types.     

The regulatory mechanism of Tremella mesenterica on steroidogenesis in MA-10 mouse Leydig tumor cells

Tremella mesenterica (TM), a yellow jelly mushroom, has been traditionally used as tonic food to improve body condition in Chinese society for a long time. We have previously demonstrated that TM reduced in vitro hCG-treated steroidogenesis in MA-10 mouse Leydig tumor cells without any toxicity effect. In the present study, the mechanism how TM suppressed hCG-treated steroidogenesis in MA-10 cells was investigated. MA-10 cells were treated with vehicle, human chorionic gonadotropin (hCG, 50 ng/ml), or different reagents with or without TM to clarify the effects. TM significantly suppressed progesterone production with the presences of forskolin (10 and 100 microM) or dbcAMP (0.5 and 1mM), respectively, in MA-10 cells (p<0.05), which indicated that TM suppressed steroidogenesis after PKA activation along the signal pathway. Beyond our expectation, TM induced the expression of steroidogenic acute regulatory (StAR) protein with or without hCG treatments. However, TM profoundly decreased P450 side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzyme activities without any influences on the expression of both enzymes. These inhibitions on steroidogenic enzyme activities might counteract the stimulation of StAR protein expression. In conclusion, results suggest that TM suppressed hCG-treated steroidogenesis in MA-10 cells by inhibiting PKA signal pathway and steroidogenic enzyme activities.