Steroid production after in vitro transcription, translation, and mitochondrial processing of protein products of complementary deoxyribonucleic acid for steroidogenic acute regulatory protein.

Endocrinology ◽  
1995 ◽  
Vol 136 (11) ◽  
pp. 5165-5176 ◽  
Author(s):  
S R King ◽  
T Ronen-Fuhrmann ◽  
R Timberg ◽  
B J Clark ◽  
J Orly ◽  
...  
Keyword(s):  
Deoxyribonucleic Acid ◽  
Regulatory Protein ◽  
In Vitro Transcription ◽  
Steroidogenic Acute Regulatory Protein ◽  
Steroid Production ◽  
Complementary Deoxyribonucleic Acid ◽  
Steroidogenic Acute Regulatory

scholarly journals The potential function of steroid sulphatase activity in steroid production and steroidogenic acute regulatory protein expression

2004 ◽  
Vol 380 (1) ◽  
pp. 153-160 ◽  
Author(s):  
Teruo SUGAWARA ◽  
Seiichiro FUJIMOTO
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Expression Vector ◽  
Regulatory Protein ◽  
Steroidogenic Acute Regulatory Protein ◽  
Steroid Production ◽  
Star Protein ◽  
Protein Levels ◽  
Steroid Sulphatase ◽  
Steroidogenic Acute Regulatory

The first step in the biosynthesis of steroid hormones is conversion of cholesterol into pregnenolone. StAR (steroidogenic acute regulatory) protein plays a crucial role in the intra-mitochondrial movement of cholesterol. STS (steroid sulphatase), which is present ubiquitously in mammalian tissues, including the placenta, adrenal gland, testis and ovary, desulphates a number of 3β-hydroxysteroid sulphates, including cholesterol sulphate. The present study was designed to examine the effect of STS on StAR protein synthesis and steroidogenesis in cells. Steroidogenic activities of COS-1 cells that had been co-transfected with a vector for the cholesterol P450scc (cytochrome P450 side-chain-cleavage enzyme) system, named F2, a StAR expression vector (pStAR), and an STS expression vector (pSTS) were assayed. Whole-cell extracts were subjected to SDS/PAGE and then to Western blot analysis. pSTS co-expressed in COS-1 cells with F2 and pStAR increased pregnenolone synthesis 2-fold compared with that of co-expression with F2 and pStAR. Western blot analysis using COS-1 cells that had been co-transfected with pSTS, F2 and pStAR revealed that StAR protein levels increased, whereas STS and P450scc protein levels did not change. The amount of StAR protein translation products increased when pSTS was added to an in vitro transcription–translation reaction mixture. Pulse–chase experiments demonstrated that the 37 kDa StAR pre-protein disappeared significantly (P<0.01) more slowly in COS-1 cells that had been transfected with pSTS than in COS-1 cells that had not been transfected with pSTS. The increase in StAR protein level is not a result of an increase in StAR gene expression, but is a result of both an increase in translation and a longer half-life of the 37 kDa pre-StAR protein. In conclusion, STS increases StAR protein expression level and stimulates steroid production.

scholarly journals Role of Intramitochondrial Arachidonic Acid and Acyl-CoA Synthetase 4 in Angiotensin II-Regulated Aldosterone Synthesis in NCI-H295R Adrenocortical Cell Line

Endocrinology ◽  
2012 ◽  
Vol 153 (7) ◽  
pp. 3284-3294 ◽  
Author(s):  
Pablo G. Mele ◽  
Alejandra Duarte ◽  
Cristina Paz ◽  
Alessandro Capponi ◽  
Ernesto J. Podestá
Keyword(s):  
Arachidonic Acid ◽  
Angiotensin Ii ◽  
Regulatory Protein ◽  
Steroidogenic Acute Regulatory Protein ◽  
Ang Ii ◽  
Steroid Production ◽  
Glomerulosa Cells ◽  
Steroidogenic Acute Regulatory ◽  
Export System

Although the role of arachidonic acid (AA) in angiotensin II (ANG II)- and potassium-stimulated steroid production in zona glomerulosa cells is well documented, the mechanism responsible for AA release is not fully described. In this study we evaluated the mechanism involved in the release of intramitochondrial AA and its role in the regulation of aldosterone synthesis by ANG II in glomerulosa cells. We show that ANG II and potassium induce the expression of acyl-coenzyme A (CoA) thioesterase 2 and acyl-CoA synthetase 4, two enzymes involved in intramitochondrial AA generation/export system well characterized in other steroidogenic systems. We demonstrate that mitochondrial ATP is required for AA generation/export system, steroid production, and steroidogenic acute regulatory protein induction. We also demonstrate the role of protein tyrosine phosphatases regulating acyl-CoA synthetase 4 and steroidogenic acute regulatory protein induction, and hence ANG II-stimulated aldosterone synthesis.

scholarly journals Peripheral-Type Benzodiazepine Receptor-Mediated Action of Steroidogenic Acute Regulatory Protein on Cholesterol Entry into Leydig Cell Mitochondria

2005 ◽  
Vol 19 (2) ◽  
pp. 540-554 ◽  
Author(s):  
Thierry Hauet ◽  
Zhi-Xing Yao ◽  
Himangshu S. Bose ◽  
Christopher T. Wall ◽  
Zeqiu Han ◽  
...  
Keyword(s):  
Regulatory Protein ◽  
Benzodiazepine Receptor ◽  
Steroidogenic Acute Regulatory Protein ◽  
Rate Determining Step ◽  
Human Choriogonadotropin ◽  
Leydig Tumor Cells ◽  
Peripheral Type ◽  
Steroidogenic Acute Regulatory ◽  
And Function

Abstract 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.

scholarly journals Steroidogenic acute regulatory protein and luteinizing hormone are required for normal ovarian steroidogenesis and oocyte maturation in zebrafish†

2019 ◽  
Vol 101 (4) ◽  
pp. 760-770 ◽  
Author(s):  
Guohui Shang ◽  
Xuyan Peng ◽  
Cheng Ji ◽  
Gang Zhai ◽  
Yonglin Ruan ◽  
...  
Keyword(s):  
Luteinizing Hormone ◽  
Oocyte Maturation ◽  
Mitochondrial Membrane ◽  
Regulatory Protein ◽  
Steroidogenic Acute Regulatory Protein ◽  
Transcription Activator ◽  
Inner Mitochondrial Membrane ◽  
Steroidogenic Acute Regulatory ◽  
Functional Deficiency

Abstract In recent studies, luteinizing hormone (LH) was reported to play important roles in oocyte maturation. However, the mechanism by which LH signaling, especially regarding the steroidogenesis process, affects oocyte maturation has not been clarified. In this study, zebrafish models with a functional deficiency in luteinizing hormone beta (Lhb) or steroidogenic acute regulatory protein (Star), an enzyme that promotes the transport of cholesterol into the inner mitochondrial membrane for maturation-induced hormone (MIH) production, were generated using transcription activator-like effector nucleases (TALENs). Similar phenotypes of the maturation-arrested oocytes in both female mutants have been observed. The levels of MIH in the oocytes of the female mutants were clearly decreased in both the lhb and star knockout zebrafish. The expression of star was dramatically down-regulated in the lhb mutant follicles and was clearly promoted by forskolin and hCG in vitro. Furthermore, treatment with the MIH precursors, pregnenolone or progesterone, as well as with MIH itself rescued the maturation-arrested oocyte phenotypes in both lhb and star mutants. The plasma levels of other steroids, including testosterone, estradiol, and cortisol, were not affected in the lhb mutants, while the levels of gonad hormones testosterone and estradiol were significantly increased in the star mutants. The cortisol levels were decreased in the star mutants. Collectively, our results confirm that LH plays important roles in the initiation of MIH synthesis from cholesterol and maintains oocyte maturation in zebrafish, as well as provide evidence that Star might act downstream of LH signaling in steroidogenesis.

scholarly journals The Contribution of Serine 194 Phosphorylation to Steroidogenic Acute Regulatory Protein Function

2014 ◽  
Vol 28 (7) ◽  
pp. 1088-1096 ◽  
Author(s):  
Goro Sasaki ◽  
Mohamad Zubair ◽  
Tomohiro Ishii ◽  
Toshikatsu Mitsui ◽  
Tomonobu Hasegawa ◽  
...  
Keyword(s):  
Adrenal Cortex ◽  
Protein Function ◽  
Regulatory Protein ◽  
Initial Step ◽  
Artificial Chromosome ◽  
Steroidogenic Acute Regulatory Protein ◽  
Inner Mitochondrial Membrane ◽  
Steroidogenic Acute Regulatory

The steroidogenic acute regulatory protein (StAR) facilitates the delivery of cholesterol to the inner mitochondrial membrane, where the cholesterol side-chain cleavage enzyme catalyzes the initial step of steroid hormone biosynthesis. StAR was initially identified in adrenocortical cells as a phosphoprotein, the expression and phosphorylation of which were stimulated by corticotropin. A number of in vitro studies have implicated cAMP-dependent phosphorylation at serine 194 (S194, S195 in human StAR) as an important residue for StAR activity. To explore the importance of S194 phosphorylation in StAR function in vivo, we developed a transgenic model using a bacterial artificial chromosome expressing either wild-type (WT) StAR or StAR mutation S194A to rescue StAR knockout (KO) mice. Despite StAR protein expression comparable to or higher than amounts seen with control animals or rescue with WT StAR, S194A StAR did not rescue the neonatal lethality and only partially rescued the sex reversal in male mice observed uniformly in StAR KO mice. Like the StAR KO mice, the adrenal cortex and testicular Leydig cells contained abundant lipid deposits when stained with oil red O. Adrenal StAR from S194A rescue animals lacks an acidic species, which appears upon corticotropin stimulation in animals rescued with WT StAR, consistent with defective StAR phosphorylation. These findings demonstrate that S194 is an essential residue for normal StAR function in the adrenal cortex and testes of mice.

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