StAR enhances transcription of genes encoding the mitochondrial proteases involved in its own degradation.

Steroidogenic acute regulatory protein (StAR) is essential for steroid hormone synthesis in the adrenal cortex and the gonads. StAR activity facilitates the supply of cholesterol substrate into the inner mitochondrial membranes where conversion of the sterol to a steroid is catalyzed. Mitochondrial import terminates the cholesterol mobilization activity of StAR and leads to mounting accumulation of StAR in the mitochondrial matrix. Our studies suggest that to prevent mitochondrial impairment, StAR proteolysis is executed by at least 2 mitochondrial proteases, ie, the matrix LON protease and the inner membrane complexes of the metalloproteases AFG3L2 and AFG3L2:SPG7/paraplegin. Gonadotropin administration to prepubertal rats stimulated ovarian follicular development associated with increased expression of the mitochondrial protein quality control system. In addition, enrichment of LON and AFG3L2 is evident in StAR-expressing ovarian cells examined by confocal microscopy. Furthermore, reporter studies of the protease promoters examined in the heterologous cell model suggest that StAR expression stimulates up to a 3.5-fold increase in the protease gene transcription. Such effects are StAR-specific, are independent of StAR activity, and failed to occur upon expression of StAR mutants that do not enter the matrix. Taken together, the results of this study suggest the presence of a novel regulatory loop, whereby acute accumulation of an apparent nuisance protein in the matrix provokes a mitochondria to nucleus signaling that, in turn, activates selected transcription of genes encoding the enrichment of mitochondrial proteases relevant for enhanced clearance of StAR.

Expression and roles of steroidogenic acute regulatory (StAR) protein in 'non-classical', extra-adrenal and extra-gonadal cells and tissues.

The activity of the steroidogenic acute regulatory (StAR) protein is indispensable and rate limiting for high output synthesis of steroid hormones in the adrenal cortex and the gonads, known as the 'classical' steroidogenic organs (StAR is not expressed in the human placenta). In addition, studies of recent years have shown that StAR is also expressed in many tissues that produce steroid hormones for local use, potentially conferring some functional advantage by acting via intracrine, autocrine or paracrine fashion. Others hypothesized that StAR might also function in non-steroidogenic roles in specific tissues. This review highlights the evidence for the presence of StAR in 17 extra-adrenal and extra-gonadal organs, cell types and malignancies. Provided is the physiological context and the rationale for searching for the presence of StAR in such cells. Since in many of the tissues the overall level of StAR is relatively low, we also reviewed the methods used for StAR detection. The gathered information suggests that a comprehensive understanding of StAR activity in 'non-classical' tissues will require the use of experimental approaches that are able to analyze StAR presence at single-cell resolution.

Transcription of steroidogenic acute regulatory protein in the rodent ovary and placenta: alternative modes of cyclic adenosine 3', 5'-monophosphate dependent and independent regulation.

Steroid hormone synthesis is a vital function of the adrenal cortex, serves a critical role in gonadal function, and maintains pregnancy if normally executed in the placenta. The substrate for the synthesis of all steroid hormones is cholesterol, and its conversion to the first steroid, pregnenolone, by the cholesterol side-chain cleavage cytochrome P450 (CYP11A1) enzyme complex takes place in the inner mitochondrial membranes. Steroidogenic acute regulatory protein (STAR) facilitates the rate-limiting transfer of cholesterol from the outer mitochondrial membrane to CYP11A1 located in the inner organelle membranes. The current study explored the mechanisms controlling transcription of the Star gene in primary cell cultures of mouse placental trophoblast giant cells and rat ovarian granulosa cells examined throughout the course of their functional differentiation. Our findings show that the cis-elements required for Star transcription in the rodent placenta and the ovary are centered in a relatively small proximal region of the promoter. In placental trophoblast giant cells, cAMP is required for activation of the Star promoter, and the cis-elements mediating a maximal response were defined as cAMP response element 2 and GATA. EMSA studies show that placental cAMP-responsive element binding protein (CREB)-1 and activating transcription factor-2 (ATF2) bind to a -81/-78 sequence, whereas GATA-2 binds to a -66/-61 sequence. In comparison, patterns of Star regulation in the ovary suggested tissue-specific and developmental controlled modes of Star transcription. During the follicular phase, FSH/cAMP induced CREB-1 dependent activity, whereas upon luteinization STAR expression becomes cAMP and CREB independent, a functional shift conferred by FOS-related antigen-2 displacement of CREB-1 binding, and the appearance of a new requirement for CCAAT enhancer-binding protein beta and steroidogenic factor 1 that bind to upstream elements (-117/-95). These findings suggest that during evolution, the promoters of the Star gene acquired nonconsensus sequence elements enabling expression of a single gene in different organs, or allowing dynamic temporal changes corresponding to progressing phases of differentiation in a given cell type.

Turnover of mitochondrial steroidogenic acute regulatory (StAR) protein by Lon protease: the unexpected effect of proteasome inhibitors.

Steroidogenic acute regulatory protein (StAR) is a vital mitochondrial protein promoting transfer of cholesterol into steroid making mitochondria in specialized cells of the adrenal cortex and gonads. Our previous work has demonstrated that StAR is rapidly degraded upon import into the mitochondrial matrix. To identify the protease(s) responsible for this rapid turnover, murine StAR was expressed in wild-type Escherichia coli or in mutant strains lacking one of the four ATP-dependent proteolytic systems, three of which are conserved in mammalian mitochondria-ClpP, FtsH, and Lon. StAR was rapidly degraded in wild-type bacteria and stabilized only in lon (-)mutants; in such cells, StAR turnover was fully restored upon coexpression of human mitochondrial Lon. In mammalian cells, the rate of StAR turnover was proportional to the cell content of Lon protease after expression of a Lon-targeted small interfering RNA, or overexpression of the protein. In vitro assays using purified proteins showed that Lon-mediated degradation of StAR was ATP-dependent and blocked by the proteasome inhibitors MG132 (IC(50) = 20 microm) and clasto-lactacystin beta-lactone (cLbetaL, IC(50) = 3 microm); by contrast, epoxomicin, representing a different class of proteasome inhibitors, had no effect. Such inhibition is consistent with results in cultured rat ovarian granulosa cells demonstrating that degradation of StAR in the mitochondrial matrix is blocked by MG132 and cLbetaL but not by epoxomicin. Both inhibitors also blocked Lon-mediated cleavage of the model substrate fluorescein isothiocyanate-casein. Taken together, our former studies and the present results suggest that Lon is the primary ATP-dependent protease responsible for StAR turnover in mitochondria of steroidogenic cells.

Transcriptional regulation of the cholesterol side chain cleavage cytochrome P450 gene (CYP11A1) revisited: binding of GATA, cyclic adenosine 3',5'-monophosphate response element-binding protein and activating protein (AP)-1 proteins to a distal novel cluster of cis-regulatory elements potentiates AP-2 and steroidogenic factor-1-dependent gene expression in the rodent placenta and ovary.

The first and key enzyme controlling the synthesis of steroid hormones is cholesterol side chain cleavage cytochrome P450 (P450scc, CYP11A1). This study sought to elucidate overlooked modes of regulation of P450scc transcription in the rodent placenta and ovary. Transcription of P450scc requires two clusters of cis-regulatory elements: a proximal element (-40) known to bind either activating protein 2 (AP-2) in the placenta, or steroidogenic factor 1 in the ovary, and a distal region of the promoter (-475/-447) necessary for potentiation of the AP-2/steroidogenic factor 1-dependent activity up to 7-fold. In primary cultures of mouse trophoblast giant cells and rat ovarian granulosa cells, binding of trans-factors to the distal regulatory sequences generated transcriptional activity in a tissue-specific pattern: in the placenta, cAMP response element (CRE)-binding protein 1 (CREB-1) and GATA-2 binding generates promoter activity in a cAMP-independent manner, whereas in ovarian cells, CREB-1 and GATA-4 are required for FSH responsiveness. However, as ovarian follicles advance toward ovulation, elevated Fra-2 expression replaces CREB-1 function by binding the same CRE(1/2) motif. Our findings suggest that upon onset of follicular recruitment, CREB-1 mediates FSH/cAMP signaling, which switches to cAMP-independent expression of P450scc in luteinizing granulosa cells expressing Fra-2. In the placenta, the indispensable role of CREB-1 was demonstrated by use of dominant-negative CREB-1 mutant, but neither cAMP nor Ser133 phosphorylation of CREB-1 is required for P450scc transcription. These observations suggest that placental regulation of P450scc expression is subjected to alternative signaling pathway(s) yet to be found.

A trimerizing GxxxG motif is uniquely inserted in the severe acute respiratory syndrome (SARS) coronavirus spike protein transmembrane domain.

In an attempt to understand what distinguishes severe acute respiratory syndrome (SARS) coronavirus (SCoV) from other members of the coronaviridae, we searched for elements that are unique to its proteins and not present in any other family member. We identified an insertion of two glycine residues, forming the GxxxG motif, in the SCoV spike protein transmembrane domain (TMD), which is not found in any other coronavirus. This surprising finding raises an "oligomerization riddle": the GxxxG motif is a known dimerization signal, while the SCoV spike protein is known to be trimeric. Using an in vivo assay, we found that the SCoV spike protein TMD is oligomeric and that this oligomerization is driven by the GxxxG motif. We also found that the GxxxG motif contributes toward the trimerization of the entire spike protein; in that, mutations in the GxxxG motif decrease trimerization of the full-length protein expressed in mammalian cells. Using molecular modeling, we show that the SCoV spike protein TMD adopts a distinct and unique structure as opposed to all other coronaviruses. In this unique structure, the glycine residues of the GxxxG motif are facing each other, enhancing helix-helix interactions by allowing for the close positioning of the helices. This unique orientation of the glycine residues also stabilizes the trimeric bundle during multi-nanosecond molecular dynamics simulation in a hydrated lipid bilayer. To the best of our knowledge, this is the first demonstration that the GxxxG motif can potentiate other oligomeric forms beside a dimer. Finally, according to recent studies, the stabilization of the trimeric bundle is linked to a higher fusion activity of the spike protein, and the possible influence of the GxxxG motif on this feature is discussed.

Analysis of trophoblast giant cell steroidogenesis in primary cultures.

Placental progesterone synthesis in humans prevents abortion of the fetus by maintaining uterine quiescence and low myometrial excitability. In rodents, a transient steroidogenic output is observed in the trophoblast giant cells during mid-pregnancy. Although the exact role of this locally produced progesterone is not clear, rodent trophoblast giant cells are an important cell model for studying the regulation of placental steroidogenesis. This chapter describes the methods we developed to analyze the regulation of genes involved in progesterone biosynthesis in miniature cultures of primary trophoblast cells from rodents. These genes include cholesterol side chain cleavage cytochrome P450 (P450scc) and its accessory proteins, steroidogenic acute regulatory protein (StAR) and 3beta-hydroxysteroid dehydrogenase/isomerase (3betaHSD). To obtain giant cells, uterine implantation sites are sliced in half, and the trophoblast giant cell layers are separated from the surrounding decidua by scraping. Cells can subsequently be separated by gentle enzymatic digestion with trypsin, or collagenase, and plated for further study in vitro. This chapter provides instructions, insights, and comments instrumental for performing in situ visualization of giant cell mRNA and proteins, analyzing enzyme activities, and conducting promoter analyses with a limited number of cells.

Cleavage site selection within a folded substrate by the ATP-dependent lon protease.

Mechanistic studies of ATP-dependent proteolysis demonstrate that substrate unfolding is a prerequisite for processive peptide bond hydrolysis. We show that mitochondrial Lon also degrades folded proteins and initiates substrate cleavage non-processively. Two mitochondrial substrates with known or homology-derived three-dimensional structures were used: the mitochondrial processing peptidase alpha-subunit (MPPalpha) and the steroidogenic acute regulatory protein (StAR). Peptides generated during a time course of Lon-mediated proteolysis were identified and mapped within the primary, secondary, and tertiary structure of the substrate. Initiating cleavages occurred preferentially between hydrophobic amino acids located within highly charged environments at the surface of the folded protein. Subsequent cleavages proceeded sequentially along the primary polypeptide sequence. We propose that Lon recognizes specific surface determinants or folds, initiates proteolysis at solvent-accessible sites, and generates unfolded polypeptides that are then processively degraded.

Transcription of cholesterol side-chain cleavage cytochrome P450 in the placenta: activating protein-2 assumes the role of steroidogenic factor-1 by binding to an overlapping promoter element.

Progesterone is essential to the sustenance of pregnancy in humans and other mammals. From the second trimester on, the human placenta is the sole origin of de novo synthesized steroid hormones. In mice, placentation at midgestation is accompanied by a temporal rise of steroid hormone synthesis commencing in the giant cells of the mouse trophoblast. In doing so, the giant trophoblasts, as any other steroidogenic cell, express high levels of the key steroidogenic enzyme, cholesterol side-chain cleavage cytochrome P450 (P450scc). Because steroidogenic factor 1 (SF-1), the transcription factor required for expression of P450scc in the adrenals and the gonads, is not expressed in the placenta, we hypothesized that placenta-specific nuclear factor(s) (PNF) assumes the role of SF-1 by binding to the same promoter region that harbors the SF-1 recognition site in the P450scc gene. To address this possibility, we used SCC1, a well conserved proximal region in the P450scc genes (-60/-32 in the rat gene) to purify PNF from human term placenta. Sequencing of the purified PNF revealed that it is the alpha isoform of the human activating protein-2 (AP-2alpha). Specific antibodies tested in EMSA confirmed that AP-2alpha is the predominant isoform that binds SCC1 in the human placenta, whereas AP-2gamma is the only mouse placental protein that binds this oligonucleotide. Functional studies showed that coexpression of the rat P450scc promoter (-378/+8 CAT) and AP-2 isoforms (alpha or gamma) in human embryonic kidney 293 cells results in a marked activation of chloramphenicol acetyltransferase (CAT) transcription that is dependent on an intact AP-2 motif, GCCTTGAGC. This motif conforms with consensus sequences previously determined for binding of the AP-2 alpha and gamma isoforms. Mutations of the AP-2 element ablated binding of AP-2 to SCC1, as well as severely diminished the promoter activity in primary mouse giant trophoblasts and human choriocarcinoma JAR cells. Collectively, these studies suggest that expression of placental P450scc is governed by AP-2 factors that bind to a cis-element that largely overlaps the sequence required for recognition of SF-1 in other steroidogenic tissues.

The murine 3beta-hydroxysteroid dehydrogenase (3beta-HSD) gene family: a postulated role for 3beta-HSD VI during early pregnancy.

The enzyme 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) is essential for the biosynthesis of all active steroid hormones. The 3beta-HSD enzyme consists in multiple isoforms, each the product of a distinct gene. In the mouse, six tissue-specific isoforms have been identified. These isoforms are expressed in a tissue- and temporal specific manner. Mouse 3beta-HSD VI is the only isoform expressed in decidua and giant trophoblast cells during the first half of mouse pregnancy. The tissue- and temporal-specific expression of 3beta-HSD VI during mouse pregnancy, as determined by in situ hybridization and immunohistochemistry, shows that 3beta-HSD is expressed exclusively in the antimesometrial decidua on E6.5 and E7.5. By E9.5, expression of 3beta-HSD is observed in giant trophoblast cells with a marked increase in expression by E10.5. No expression of 3beta-HSD is seen in decidua after E7.5 and no expression of 3beta-HSD is seen in the embryo at any of the times investigated. Giant trophoblast cells in culture from E9.5 and E10.5 synthesize progesterone with cells from E10.5 producing about 3.5-fold more progesterone during the first 24 h in culture. Western blot analysis of 3beta-HSD VI protein demonstrates that the amount of 3beta-HSD VI protein correlates with the amount of progesterone biosynthesis in giant trophoblast cells from E9.5 and E10.5. We propose that progesterone produced during the first half of mouse pregnancy in decidua and giant trophoblast cells acts as an immunosuppressant at the fetal maternal interface to prevent rejection of the fetus.

The life cycle of the steroidogenic acute regulatory (StAR) protein: from transcription through proteolysis.

The Steroidogenic Acute Regulatory (StAR) protein is a mitochondrial protein required for the transport of cholesterol substrate to the P450scc enzyme located in the inner mitochondrial membranes of steroid producing cells. This study suggests that the acute regulation of the rodent StAR gene in the ovary is mediated by two factors, C/EBPbeta and GATA-4. Once translated, the StAR precursor protein is either imported into the mitochondria, or it is rapidly degraded in the cytosol. We predicted that in order to perpetuate StAR activity cycles, imported StAR should turn over rapidly to avoid a potentially harmful accumulation of the protein in sub-mitochondrial compartments. Pulse-chase experiments in metabolically labeled cells showed that: (a) the turnover rate of mature mitochondrial StAR protein (30 kDa) is much faster (t(1/2) = 4-5 h) than that of other mitochondrial proteins; (b) dissipation of the inner membrane potential (-delta psi) by carbonyl cyanide m-chlorophenylhydrazone (mCCCP) accelerates the mitochondrial degradation of StAR; (c) unexpectedly, the mitochondrial degradation of StAR is inhibited by MG132 and lactacystin, but not by epoxomicin. Furthermore, StAR degradation becomes inhibitor-resistant two hours after import. Therefore, these studies suggest a bi-phasic route of StAR turnover in the mitochondria. Shortly after import, StAR is degraded by inhibitor-sensitive protease(s) (phase I), whereas at later times, StAR turnover proceeds to completion through an MG132-resistant proteolytic activity (phase II). Collectively, this study defines StAR as a unique protein that can authentically be used to probe multiple proteolytic activities in mammalian mitochondria.