Acute Stimulation by Peptide Hormones of Mitochondrial Steroid Hormone Synthesis: Production of a Similiar Protein in Three Steroidogenic Tissues

Pon, Liza A.; Hartigan, Jennifer A.; Epstein, Linda F.; Orme-Johnson, N R

doi:10.1007/978-1-4684-8640-7_50

Cited by 13 publications

(21 citation statements)

References 40 publications

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“…5C). These observations are in agreement with earlier reports from the laboratories of Orme-Johnson and Stocco (2,(12)(13)(14). In contrast to wild-type StAR, the C-His-tag StAR was not proteolytically processed in the COS-1 cells and the C-His-tag StAR that was associated with the organelles was digested by proteinase K (Fig.…”

Section: His-tag Star Proteinssupporting

confidence: 94%

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The Mechanism of Action of Steroidogenic Acute Regulatory Protein (StAR)

Arakane

Kallen

Watari

et al. 1998

Journal of Biological Chemistry

178

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Steroidogenic acute regulatory protein (StAR) plays an essential role in steroidogenesis, facilitating delivery of cholesterol to cytochrome P450 scc on the inner mitochondrial membrane. StAR is synthesized in the cytoplasm and is subsequently imported by mitochondria and processed to a mature form by cleavage of the NH 2 -terminal mitochondrial targeting sequence. To explore the mechanism of StAR action, we produced 6-histidinetagged N-62 StAR (His-tag StAR) constructs lacking the NH 2 -terminal 62 amino acids that encode the mitochondrial targeting sequence and examined their steroidogenic activity in intact cells and on isolated mitochondria. His-tag StAR proteins stimulated pregnenolone synthesis to the same extent as wild-type StAR when expressed in COS-1 cells transfected with the cholesterol side-chain cleavage system. His-tag StAR was diffusely distributed in the cytoplasm of transfected COS-1 cells whereas wild-type StAR was localized to mitochondria. There was no evidence at the light or electron microscope levels for selective localization of His-tag StAR protein to mitochondrial membranes. In vitro import assays demonstrated that wild-type StAR preprotein was imported and processed to mature protein that was protected from subsequent trypsin treatment. In contrast, His-tag StAR was not imported and protein associated with mitochondria was sensitive to trypsin. Using metabolically labeled COS-1 cells transfected with wild-type or His-tag StAR constructs, we confirmed that wild-type StAR preprotein was imported and processed by mitochondria, whereas His-tag StAR remained largely cytosolic and unprocessed. To determine whether cytosolic factors are required for StAR action, we developed an assay system using washed mitochondria isolated from bovine corpora lutea and purified recombinant His-tag StAR proteins expressed in Escherichia coli. Recombinant His-tag StAR stimulated pregnenolone production in a dose-and time-dependent manner, functioning at nanomolar concentrations. A point mutant of StAR (A218V) that causes lipoid congenital adrenal hyperplasia was incorporated into the His-tag protein. This mutant was steroidogenically inactive in COS-1 cells and on isolated mitochondria. Our observations conclusively document that StAR acts on the outside of mitochondria, independent of mitochondrial import, and in the absence of cytosol. The ability to produce bioactive recombinant StAR protein paves the way for refined structural studies of StAR and StAR mutants.

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Section: His-tag Star Proteinssupporting

confidence: 94%

“…Cyclic AMP influences StAR gene expression, like many proteins important for steroidogenesis (8 -10), and enhances StAR activity by triggering posttranslational modifications (11)(12)(13).…”

Section: Steroidogenic Acute Regulatory Protein (Star)mentioning

confidence: 99%

The Mechanism of Action of Steroidogenic Acute Regulatory Protein (StAR)

Arakane

Kallen

Watari

et al. 1998

Journal of Biological Chemistry

178

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“…How free cholesterol in the outer mitochondrial membrane is transferred to the P450-cholesterol side chain cleavage (P450-SCC) complex on the inner mitochondrial membrane is unclear, and the existence of a hormonally regulated, labile protein which facilitates this transfer had been predicted for many years (1, 3). The recently discovered steroidogenic acute regulatory protein (StAR) is a 30-kDa mitochondrial protein which has many of the characteristics of this regulator (3,4). StAR has been demonstrated to promote movement of cholesterol from the outer to the inner mitochondrial membrane where P450-SCC catalyzes the first, and rate-limiting, enzymatic step in steroid synthesis (5, 6).…”

mentioning

confidence: 99%

“…In adrenocortical cells, adrenocorticotropin (ACTH) 1 stimulates hydrolysis of cholesteryl esters in lipid droplets, and transport of free cholesterol to mitochondria for use in steroid synthesis (1,2). How free cholesterol in the outer mitochondrial membrane is transferred to the P450-cholesterol side chain cleavage (P450-SCC) complex on the inner mitochondrial membrane is unclear, and the existence of a hormonally regulated, labile protein which facilitates this transfer had been predicted for many years (1,3). The recently discovered steroidogenic acute regulatory protein (StAR) is a 30-kDa mitochondrial protein which has many of the characteristics of this regulator (3,4).…”

mentioning

confidence: 99%

Lipoproteins Regulate Expression of the Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenocortical Cells

Reyland¹,

Evans²,

White³

2000

Journal of Biological Chemistry

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The production of hormones in steroidogenic tissues is dependent upon the availability of free cholesterol at the inner mitochondrial membrane, the site of the cholesterol side chain cleavage. In adrenocortical cells, adrenocorticotropin (ACTH) 1 stimulates hydrolysis of cholesteryl esters in lipid droplets, and transport of free cholesterol to mitochondria for use in steroid synthesis (1, 2). How free cholesterol in the outer mitochondrial membrane is transferred to the P450-cholesterol side chain cleavage (P450-SCC) complex on the inner mitochondrial membrane is unclear, and the existence of a hormonally regulated, labile protein which facilitates this transfer had been predicted for many years (1, 3). The recently discovered steroidogenic acute regulatory protein (StAR) is a 30-kDa mitochondrial protein which has many of the characteristics of this regulator (3,4). StAR has been demonstrated to promote movement of cholesterol from the outer to the inner mitochondrial membrane where P450-SCC catalyzes the first, and rate-limiting, enzymatic step in steroid synthesis (5, 6). In addition, expression of StAR promotes steroidogenesis, and directly facilitates the transfer of cholesterol and other sterols in reconstituted mitochondria (7-9). Perhaps the most convincing evidence that StAR is critical for this process in vivo is the observation by Miller and others (10, 11) that mutations in the StAR gene cause congenital lipoid adrenal hypoplasia. In this autosomal recessive disorder, gonadal and adrenal steroid production are blocked at the level of cholesterol side chain cleavage, resulting in a massive accumulation of cholesterol in the adrenal glands (10). Likewise, mice in which the StAR gene is disrupted have a severe reduction in corticosterone synthesis and accumulate massive lipid deposits in their adrenal cortex (12).Expression of StAR is acutely regulated in response to hormonal stimulation by a process which does not require de novo protein synthesis (5). Phosphorylation of StAR has also been demonstrated to regulate its steroidogenic activity (13). cAMPdependent protein kinase (PKA), as well as the transcription factors steroidogenic factor-1 (SF-1) and C/EBP␤, are required for basal transcription of StAR (14 -19). Other reports demonstrate that StAR transcription can be regulated by oxysterols (20) and agents known to increase cellular calcium can also regulate StAR transcription (21). Since the net result of ACTH stimulation is an increase in free cholesterol at the outer mitochondrial membrane, the current studies were undertaken to ask if the expression of StAR is regulated directly by lipoproteins, the major source of cholesterol for steroid synthesis in adrenocortical cells. Our studies demonstrate that lipoproteins regulate StAR expression by a mechanism that is dependent on cAMP-dependent protein kinase, and the transcription factor SF-1. The regulation of StAR expression by cholesterol may represent a positive feedback circuit designed to maintain maximal output of steroid hormone. * This work ...

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“…Several cycloheximide-sensitive proteins that deliver cholesterol to the inner mitochondrial membrane and effect rapid change in the rate of adrenal steroidogenesis have been described (2,(8)(9)(10). Steroidogenic activator peptides isolated from rat adrenal and rat Leydig cell tumor stimulate in vitro pregnenolone synthesis 1.6-to 17-fold, with half-maximal stimulation in the range of 10-100 nM (8,9).…”

mentioning

confidence: 99%

Identification of des-(Gly-Ile)-endozepine as an effector of corticotropin-dependent adrenal steroidogenesis: stimulation of cholesterol delivery is mediated by the peripheral benzodiazepine receptor.

Besman

Yanagibashi

Lee

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

201

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Delivery ofcholesterol to inner mitochondrial membranes is rate-limiting for steroidogenesis in the zona fasciculata of adrenal cortex. A protein that stimulates this process was isolated to homogeneity from bovine adrenal tissue. This protein's primary structure has been determined in its entirety by a combination of automated Edman microsequencing, fast-atom bombardment mass spectrometry (FAB-MS). The sequence was identical to that previously reported for bovine brain endozepine, except that it lacks the last two residues, -Gly-Ile, at the C terminus. To our knowledge, isolation of an endozepine-related protein from a tissue other than brain has not been reported previously. Endozepine competes with benzodiazepines for saturable binding sites in synaptosomes and in mitochondria of specific peripheral tissues. Previous reports have localized the adrenal benzodiazepine receptor to the outer mitochondrial membrane. In this report, we show that the prototypic benzoiazepine, diazepam, effects a stimulation of adrenal mitochondrial cholesterol delivery similar to that observed for endozepine. The effective diazepam concentration was consistent with that previously shown to displace a high-affinity ligand of the mitochondrial benzodiazepine receptor. The action of diazepam in adrenal mitochondria suggests that the mediation of corticotropininduced steroidogenesis may be the physiological function of the peripheral-type benzodiazepine receptor. These studies provide new insights into the previously unknown function of peripheral benzodiazepine receptors and should allow new investigations into the stimulation of steroidogenesis by endozepines and benzodiazepines in the brain and in certain peripheral tissues.

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Acute Stimulation by Peptide Hormones of Mitochondrial Steroid Hormone Synthesis: Production of a Similiar Protein in Three Steroidogenic Tissues

Cited by 13 publications

References 40 publications

The Mechanism of Action of Steroidogenic Acute Regulatory Protein (StAR)

The Mechanism of Action of Steroidogenic Acute Regulatory Protein (StAR)

Lipoproteins Regulate Expression of the Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenocortical Cells

Identification of des-(Gly-Ile)-endozepine as an effector of corticotropin-dependent adrenal steroidogenesis: stimulation of cholesterol delivery is mediated by the peripheral benzodiazepine receptor.

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