Structure of the human steroidogenic acute regulatory (StAR) protein gene: StAR stimulates mitochondrial cholesterol 27-hydroxylase (original) (raw)
Related papers
Journal of Biological Chemistry, 2001
The metabolism of cholesterol by cytochrome P450 side chain cleavage enzyme is hormonally regulated in steroidogenic tissues via intramitochondrial cholesterol transport. The mediating steroidogenic acute regulatory protein (StAR) is synthesized as a 37-kDa (p37) precursor that is phosphorylated by protein kinase A and cleaved within the mitochondria to generate 30-kDa forms (p30, pp30). The effectiveness of modified recombinant StAR forms in COS-1 cells without mitochondrial import has led to a prevailing view that cholesterol transport is mediated by p37 StAR via activity on the outer mitochondrial membrane. The present study of the activation of cholesterol metabolism by bromo-cAMP in adrenal cells in relation to 35 S-StAR turnover indicates that targeting of pp30 to the inner membrane provides the dominant cholesterol transport mechanism. We show that 1) only newly synthesized StAR is functional, 2) phosphorylation and processing of p37 to pp30 occurs rapidly and stoichiometrically, 3) both steps are necessary for optimum transport, and 4) newly synthesized pp30 exhibits very high activity (400 molecules of cholesterol/StAR/min). Segregation of cAMP activation and synthesis of StAR from cholesterol metabolism showed that very low levels of newly synthesized StAR (1 fmol/min/10 6 cells) sustained activated cholesterol metabolism (0.4 pmol/min/10 6 cells, t1 ⁄2 ؍ 70 min) long after complete removal of p37 (t1 ⁄2 ؍ 5 min). This activity was highly sensitive to inhibition of processing by CCCP only until sufficient pp30 was formed. Maximum activation preceded bromo-cAMP-induced StAR expression, indicating other limiting steps in cholesterol metabolism.
Role of the steroidogenic acute regulatory protein (StAR) in steroidogenesis
Biochemical Pharmacology, 1996
The rate-limiting, hormone-regulated, enzymatic step in steroidogenesis is the conversion of cholesterol to pregnenolone by the cholesterol side-chain cleavage enzyme system (CSCC), which is located on the matrix side of the inner mitochondrial membrane. However, it has long been observed that hydrophilic cholesterol-like substrates capable of traversing the mitochondrial membranes are cleaved to pregnenolone by the CSCC in the absence of any hormone stimulation. Therefore, the true regulated step in the acute response of steroidogenic cells to hormone stimulation is the delivery of cholesterol to the inner mitochondrial membrane and the CSCC. It has been known for greater than three decades that transfer of cholesterol requires de novo protein synthesis; however, prior to this time the regulatory protein(s) had yet to be identified conclusively. It is the purpose of this commentary to briefly review a number of the candidates that have been proposed as the acute regulatory protein. As such, we have summarized the available information that describes the roles of transcription, translation, and phosphorylation in this regulation, and have also reviewed the supporting cases that have been made for several of the proteins put forth as the acute regulator. We close with a comprehensive description of the Steroidogenic Acute Regulatory protein (StAR) that we and others have identified and characterized as a family of proteins that are synthesized and imported into the mitochondria in response to hormone stimulation, and for which strong evidence exists indicating that it is the long sought acute regulatory protein.
Current Knowledge on the Acute Regulation of Steroidogenesis
Biology of reproduction, 2018
How rapid induction of steroid hormone biosynthesis occurs in response to trophic hormone stimulation of steroidogenic cells has been a subject of intensive investigation for approximately six decades. A key observation made very early was that acute regulation of steroid biosynthesis required swift and timely synthesis of a new protein whose role appeared to be involved in the delivery of the substrate for all steroid hormones, cholesterol, from the outer to the inner mitochondrial membrane where the process of steroidogenesis begins. It was quickly learned that this transfer of cholesterol to the inner mitochondrial membrane was the regulated and rate limiting step in steroidogenesis. Following this observation, the quest for this putative regulator protein(s) began in earnest in the late 1950s. This review provides a history of this quest, the candidate proteins that arose over the years, and facts surrounding their rise or decline. Only two have persisted-Translocator Protein (T...
The Journal of Steroid Biochemistry and Molecular Biology, 1999
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 eects 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 re¯ect a functional requirement for import of StAR into the mitochondria, or more likely, an unidenti®ed 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.
Endocrinology, 1998
It has been proposed that the steroidogenic acute regulatory (StAR) protein controls hormone-stimulated steroid production by mediating cholesterol transfer to the mitochondrial inner membrane. This study was conducted to determine the effect of wild-type StAR and several modified forms of StAR on intramitochondrial cholesterol transfer. Forty-seven N-terminal or 28 C-terminal amino acids of the StAR protein were removed, and COS-1 cells were transfected with pCMV vector only, wild-type StAR, N-47, or the C-28 constructs. Lysates from the transfected COS-1 cells were then incubated with mitochondria from MA-10 mouse Leydig tumor cells that were preloaded with [ 3 H]cholesterol. After incubation, mitochondria were collected and fractionated on sucrose gradients into outer membranes, inner membranes, and membrane contact sites, and [ 3 H]cholesterol content was determined in each membrane fraction. Incubation of MA-10 mitochondria with wild-type StAR containing cell lysate resulted in a significant 34.9% increase in [ 3 H]cholesterol content in contact sites and a significant 32.8% increase in inner mitochondrial membranes. Incubations with cell lysate containing N-47 StAR protein also resulted in a 16.4% increase in [ 3 H]cholesterol in contact sites and a significant 26.1% increase in the inner membrane fraction. In contrast, incubation with the C-28 StAR protein had no effect on cholesterol transfer. The cholesterol-transferring activity of the N-47 truncation, in contrast to that of the C-28 mutant, was corroborated when COS-1 cells were cotransfected with F2 vector (containing cytochrome P450 side-chain cleavage enzyme, ferridoxin, and ferridoxin reductase) and either pCMV empty vector or the complementary DNAs of wild-type StAR, N-47 StAR, or C-28 StAR. Pregnenolone production was significantly increased in both wild-type and N-47transfected cells, whereas that in C-28-transfected cells was similar to the control value. Finally, immunolocalization studies with confocal image and electron microscopy were performed to determine the cellular location of StAR and its truncated forms in transfected COS-1 cells. The results showed that wild-type and most of the C-28 StAR protein were imported into the mitochondria, whereas most of N-47 protein remained in the cytosol. These studies demonstrate a direct effect of StAR protein on cholesterol transfer to the inner mitochondrial membrane, that StAR need not enter the mitochondria to produce this transfer, and the importance of the C-terminus of StAR in this process.
StAR—A tissue specific acute mediator of steroidogenesis
Trends in Endocrinology & Metabolism, 1996
The rate-limiting and acutely regulated step in steroid hormone biosynthesis is the translocation of cholesterol, the precursor of all steroid hormones, from the mitochondrial outer membrane to the inner membrane, where it is converted to pregnenolone by the cytochrome P450 cholesterol side-chain cleavage enzyme (P45Oscc). This step has long been known to be dependent upon the de novo synthesis of a labile protein factol; which is required for the intramitochondrial translocation of cholesterol, Recently, the Steroidogenic Acute Regulatory (StAR) protein has been shown to have an indispensable role in acute steroid production and is proposed to be this labile protein factor. Given the fundamental importance of StAR as a key regulator of steroid hormone biosynthesis, the next frontier for researchers is elucidating the molecular mechanisms that control StAR expression and function.
Cholesterol Binding Does Not Predict Activity of the Steroidogenic Acute Regulatory Protein, StAR
Journal of Biological Chemistry, 2007
Steroidogenic acute regulatory protein (StAR) stimulates adrenal and gonadal steroidogenesis by increasing the influx of cholesterol into mitochondria, where it is converted to pregnenolone to initiate steroidogenesis. StAR acts on the outer mitochondrial membrane where each molecule stimulates the mitochondrial import of several hundred molecules of cholesterol, but the precise mechanism of the action of StAR remains uncertain. StAR has a sterol-binding pocket that can accommodate one molecule of cholesterol. Direct assays show that StAR can bind cholesterol with stoichiometry approaching 1:1, and several disease-causing mutants with decreased or absent activity have correspondingly decreased cholesterol binding. We show that the StAR mutant R182L, which causes severe disease and is devoid of measurable activity in transfected cells or with isolated steroidogenic mitochondria, nevertheless, can bind as much [ 14 C]-or NBD-cholesterol as wild-type StAR under equilibrium conditions and can transfer cholesterol between liposomes in vitro. Similarly, the artificial mutant S195A had 46.5% of the activity of wild-type StAR but bound cholesterol indistinguishably from wild-type. Competition assays showed that the rate of binding (t1 ⁄ 2on) for R182L was only 36% of the wild-type and the rate of dissociation (t1 ⁄ 2off) was 57% of wild-type, whereas the t1 ⁄ 2on and t1 ⁄ 2off for S195A and S195D were essentially the same for wild-type. These data indicate that cholesterol binding and transfer activities are distinct from its activity to induce steroidogenesis. StAR appears to act by other mechanisms in addition to cholesterol binding.
Endocrinology, 1998
It has been proposed that the steroidogenic acute regulatory (StAR) protein controls hormone-stimulated steroid production by mediating cholesterol transfer to the mitochondrial inner membrane. This study was conducted to determine the effect of wild-type StAR and several modified forms of StAR on intramitochondrial cholesterol transfer. Forty-seven N-terminal or 28 C-terminal amino acids of the StAR protein were removed, and COS-1 cells were transfected with pCMV vector only, wild-type StAR, N-47, or the C-28 constructs. Lysates from the transfected COS-1 cells were then incubated with mitochon- dria from MA-10 mouse Leydig tumor cells that were preloaded with (3H)cholesterol. After incubation, mitochondria were collected and fractionated on sucrose gradients into outer membranes, inner mem- branes, and membrane contact sites, and (3H)cholesterol content was determined in each membrane fraction. Incubation of MA-10 mito- chondria with wild-type StAR containing cell lysate resulted ...
Protein-Protein Interactions Mediate Mitochondrial Cholesterol Transport and Steroid Biosynthesis
Journal of Biological Chemistry, 2006
Transport of cholesterol into the mitochondria is the ratedetermining, hormone-sensitive step in steroid biosynthesis. Here we report that the mechanism underlying mitochondrial cholesterol transport involves the formation of a macromolecular signaling complex composed of the outer mitochondrial membrane translocator protein (TSPO), previously known as peripheral-type benzodiazepine receptor; the TSPO-associated protein PAP7, which binds and brings to mitochondria the regulatory subunit RI␣ of the cAMP-dependent protein kinase (PKARI␣); and the hormone-induced PKA substrate, steroidogenic acute regulatory protein (StAR). Hormone treatment of MA-10 Leydig cells induced the co-localization of TSPO, PAP7, PKARI␣, and StAR in mitochondria, visualized by confocal microscopy, and the formation in living cells of a high molecular weight multimeric complex identified using photoactivable amino acids. The hormone-induced recruitment of exogenous TSPO in this complex was found to parallel the increased presence of 7-azi-5␣-cholestan-3-ol in the samples. Co-expression of Tspo, Pap7, PkarI␣, and Star genes resulted in the stimulation of steroid formation in both steroidogenic MA-10 and non-steroidogenic COS-F2-130 cells that were engineered to metabolize cholesterol. Disruption of these protein-protein interactions and specifically the PKARI␣-PAP7 and PAP7-TSPO interactions, using PAP7 mutants where the N0 area homologous to dual A-kinase-anchoring protein-1 or the acyl-CoA signature motif were deleted or using the peptide Ht31 known to disrupt the anchoring of PKA, inhibited both basal and hormone-induced steroidogenesis. These results suggest that the initiation of cAMP-induced protein-protein interactions results in the formation of a multivalent scaffold in the outer mitochondrial membrane that mediates the effect of hormones on mitochondrial cholesterol transport and steroidogenesis. . The abbreviations used are: R, regulatory; AKAP, A-kinase-anchoring protein; D-AKAP, dual AKAP; hCG, human chorionic gonadotropin; PAP7, peripheral-type benzodiazepine receptor and PKARI␣-associated protein 7; PKA, cAMP-dependent protein kinase, protein kinase A; PKARI␣, regulatory subunit I␣; photocholesterol, 7-azi-5␣-cholestan-3-ol; StAR, steroidogenic acute regulatory protein; TSPO, translocator protein (18 kDa); GFP, green fluorescent protein; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; PBS, phosphate-buffered saline; BFA, brefeldin A; RIA, radioimmunoassay; ANT, adenine nucleotide transporter; VDAC, voltagedependent anion channel.