MLN64 contains a domain with homology to the steroidogenic acute regulatory protein (StAR) that stimulates steroidogenesis

MLN64 is a protein that is highly expressed in certain breast carcinomas. The C terminus of MLN64 shares significant homology with the steroidogenic acute regulatory protein (StAR), which plays a key role in steroid hormone biosynthesis by enhancing the intramitochondrial translocation of cholesterol to the cholesterol side-chain cleavage enzyme. We tested the ability of MLN64 to stimulate steroidogenesis by using COS-1 cells cotransfected with plasmids expressing the human cholesterol side-chain cleavage enzyme system and wild-type and mutant MLN64 proteins. Wild-type MLN64 increased pregnenolone secretion in this system 2-fold. The steroidogenic activity of MLN64 was found to reside in the C terminus of the protein, because constructs from which the C-terminal StAR homology domain was deleted had no steroidogenic activity. In contrast, removal of N-terminal sequences increased MLN64’s steroidogenesis-enhancing activity. MLN64 mRNA was found in many human tissues, including the placenta and brain, which synthesize steroid hormones but do not express StAR. Western blot analysis revealed the presence of lower molecular weight immunoreactive MLN64 species that contain the C-terminal sequences in human tissues. Homologs of both MLN64 and StAR were identified in Caenorhabditis elegans, indicating that the two proteins are ancient. Mutations that inactivate StAR were correlated with amino acid residues that are identical or similar among StAR and MLN64, indicating that conserved motifs are important for steroidogenic activity. We conclude that MLN64 stimulates steroidogenesis by virtue of its homology to StAR.     

Molten globule structure and steroidogenic activity of N-218 MLN64 in human placental mitochondria

Progesterone synthesis by the human placenta requires the conversion of mitochondrial cholesterol to pregnenolone by cytochrome P450scc. Most steroidogenic tissues use the steroidogenic acute regulatory protein (StAR) to deliver cholesterol to the inner mitochondrial membrane where P450scc is located, but StAR is not expressed in the human placenta. However, the human placenta does express MLN64, which has a C-terminal domain homologous to StAR that can also transport cholesterol. We investigated the ability of bacterially expressed N-218 MLN64 and N-62 StAR to transport cholesterol between artificial membranes and to its inner membrane site of use in placental mitochondria. Urea denaturation experiments show that N-218 MLN64 undergoes a pH-dependent and denaturant-dependent structural transition to a molten globule state, as reported previously for N-62 StAR. N-218 MLN64 stimulated cholesterol transfer between artificial phospholipid vesicles with an initial rate of 6.5 mol/min.mol N-218 MLN64. Both N-218 MLN64 and N-62 StAR stimulated cholesterol transfer to the inner mitochondrial membrane, as evidenced by a 6-fold stimulation of pregnenolone synthesis with saturating transporter. This stimulation was seen only after the endogenous cholesterol in the steroidogenic pool of the isolated mitochondria was first depleted. No stimulation was observed by N-218 MLN64 or N-62 StAR when 20alpha-hydroxycholesterol was added as substrate for P450scc, confirming that these proteins stimulate P450scc activity by enhancing cholesterol transport. MLN64 levels in placental JEG-3 cells were unresponsive to stimulation by 8-bromo-cAMP over 24 h. These data show that human N-218 MLN64 and N-62 StAR have similar biophysical and functional properties and are able to stimulate steroidogenesis in a human placental system, which normally lacks StAR. The results reveal that with saturating MLN64, steroidogenesis by placental mitochondria proceeds at near-maximal rate.     

Folding, activity and import of steroidogenic acute regulatory protein into mitochondria changed by nicotine exposure

Nicotine, a pharmacologically active constituent of tobacco smoke, decreases sex steroid production and impairs reproductive function. The rate-limiting step in steroid hormone biosynthesis is the transport of substrate cholesterol from the outer to inner mitochondrial membrane by the steroidogenic acute regulatory protein (StAR). StAR is a 37 kDa cytoplasmic phosphoprotein processed as a 32 kDa intermediate to a mature 30 kDa inactive mitochondrial protein. StAR's cholesterol transport capacity is proportional to its residency time at the outer mitochondrial membrane (OMM). Nonsteroidogenic COS-1 cells transfected with StAR/F2, steroidogenic MA-10 cells induced with cAMP or transfected with StAR or the isolated steroidogenic mitochondria preincubated with nicotine reduced StAR expression, import and activity. Mitochondria isolated from steroidogenic tissues or cells, pretreated with nicotine, also reduced the association of StAR with the OMM, but had no effect on the import of signal sequence substituted SCC/N-62StAR. The fluorescence emission maximum of StAR was unchanged with nicotine, but StAR's free energy of unfolding and the surface area (m) increased in the presence of nicotine. Nicotine also blocked StAR from proteolysis with trypsin, suggesting that nicotine partially stabilised protein conformation by insertion into the molten globule conformation of StAR.     

Nigericin inhibits accumulation of the steroidogenic acute regulatory protein but not steroidogenesis

The steroidogenic acute regulatory (StAR) protein mediates the delivery of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol side chain cleavage complex converts it to pregnenolone. While the mechanism by which this mitochondrial protein acts is poorly understood, one component of the mitochondrial electrochemical gradient, the electrochemical potential (DeltaPsi), appears to be essential. In this study, the importance of the other component, the proton gradient (DeltapH), was examined. Disruption of DeltapH with the electroneutral K(+)/H(+) exchanger, nigericin, had no effect on steroidogenesis in MA-10 mouse Leydig tumor cells at concentrations which significantly reduced StAR protein levels. These data indicate for the first time in true steroidogenic cells, that StAR can act prior to being fully imported into the mitochondria and are consistent with observations made in COS-1 cells using mutant forms of StAR. These results support the hypothesis that a DeltaPsi-dependent factor is required for StAR activity and demonstrate that nigericin is the first compound described, capable of inhibiting StAR accumulation without affecting steroidogenesis.     

Creation and activity of COS-1 cells stably expressing the F2 fusion of the human cholesterol side-chain cleavage enzyme system

A fusion construct for the human cholesterol side-chain cleavage enzyme system termed F2 (H(2)N-P450scc-adrenodoxin reductase-adrenodoxin-COOH), was stably expressed in nonsteroidogenic COS-1 cells. Multiple clones were obtained and analyzed, identifying the clone COS-F2-130 as the most active in converting 22R-hydroxycholesterol (22R-OH-C) to pregnenolone. The F2 fusion construct was properly transcribed and translated in COS-F2-130 cells, indicating that these cells did not proteolytically cleave the F2 protein. Steroid analyses show that the COS-F2-130 cells do not convert appreciable quantities of pregnenolone to other steroids. Isolated COS-F2-130 mitochondria showed enhanced steroidogenesis when incubated with biosynthetic N-62 StAR protein in vitro. The cells were easily transfectable with StAR expression vectors, showing that COS-F2-130 cells exhibited both StAR-independent and StAR-dependent activity. Transient expression of either full-length or N-62 StAR stimulated steroidogenesis to approximately 45% of the maximal steroidogenic capacity, as indicated by incubation with 22R-OH-C. Single, double, and triple transfections of individual vectors expressing P450scc, adrenodoxin reductase, and adrenodoxin demonstrated that the P450 moiety of the F2 fusion protein could only receive electrons from the covalently linked adrenodoxin moiety, but that free adrenodoxin reductase could foster activity of the fusion enzyme. COS-F2-130 cells provide a useful system for studying steroidogenesis, as these are the only cells described to date that convert cholesterol to pregnenolone but lack downstream enzymes that catalyze other steroidogenic reactions.     

The active form of the steroidogenic acute regulatory protein, StAR, appears to be a molten globule

The steroidogenic acute regulatory protein (StAR) increases the movement of cholesterol from the outer to the inner membrane of adrenal and gonadal mitochondria, thus providing the substrate for steroid hormone biosynthesis. Deletion of 62 amino-terminal aa produces a cytoplasmic form of StAR (N-62 StAR) that lacks the mitochondrial leader sequence but retains full activity and appears to act at the outer mitochondrial membrane. At neutral pH the native state of bacterially expressed N-62 StAR protein displays cooperative unfolding under the influence of urea with DeltaGH2O = -4.1 kcal/mol, and it remains correctly folded down to pH 4. Limited proteolysis at different pHs shows that the biologically essential C-terminal region is accessible to solvent, and that the N-terminal domain is compact at pH 8 and partially unfolds below pH 4. Secondary structural analysis of CD curves suggests that the unfolding may coincide with an increase in alpha-helical character at pH 3.5. Fluorescence spectroscopy at pH 3-8 and at 0-6 M urea is consistent with two distinct domains, a compact N-terminal domain containing tryptophans 96 and 147 and a more solvent-accessible C-terminal domain containing tryptophans 241 and 250. These observations suggest that StAR forms a molten globule structure at pH 3.5-4.0. As the mitochondrial proton pump results in an electrochemical gradient, and as StAR must unfold during mitochondrial entry, StAR probably undergoes a similar conformational shift to an extended structure while interacting with the mitochondrial outer membrane, allowing this apparent molten globule form to act as an on/off switch for cholesterol entry into the mitochondria.     

Evidence that StAR and MLN64 act on the outer mitochondrial membrane as molten globules

StAR increases the flow of cholesterol from the outer to inner mitochondrial membrane (OMM to IMM), but its mechanism of action remains unclear. MLN64 is a 445 amino acid protein of unknown function that has four N-terminal transmembrane domains and whose C-terminal domain from 218-445 is 37% identical to StAR. N-62 StAR is as active as wild-type StAR, and N-234 MLN64 has 1/3 to 1/2 of StAR's activity. N-62 StAR lacks a mitochondrial leader and is confined to the cytoplasm, indicating that it acts on the OMM. Bacterially expressed N-62 StAR and N-218 MLN64 are active with isolated MA-10 cell mitochondria, indicating the proteins were properly folded. Far-UV CD spectroscopy, unfolding in urea, and fluorescence spectroscopy indicate that StAR undergoes a pH-dependent transition to a molten globule (retained secondary structure, partially lost tertiary structure) and stabilizes in mildly acid conditions. Far-UV CD data indicate that MLN64 undergoes a much less pronounced transition. Western blotting shows that normal human placenta has abundant N-terminally-cleaved 30 kDa MLN64. Partial proteolysis followed by mass spectrometry shows that the C-termini of StAR and MLN64 are sensitive to proteolysis, indicating looser folding. Our model of StAR action is that the protease-resistant domain unfolds slowly during normal mitochondrial entry, keeping StAR in contact with the OMM longer, increasing activity. The transition to the molten globule may be related to interaction with the OMM. These data are consistent with the recent crystallographic structure of N-216 MLN64 in which MLN64 binds cholesterol one molecule at a time, but are not consistent with the suggestion that StAR/MLN64 must reside in the intramembraneous space to transfer cholesterol form the OMM to the IMM.     

Replacement of alanine with asparagic acid at position 203 in human steroidogenic acute regulatory protein impairs the ability to enhance steroidogenesis in vitro

Steroidogenic acute regulatory protein (StAR) is a 30-kDa phosphorylated protein that rapidly appears in mitochondria of steroidogenic cells following tropic stimulation, and is required in the acute regulation of steroidogenesis. It was reported that mutations in the STAR gene encoding StAR cause congenital lipoid adrenal hyperplasia (CLAH), an autosomal recessive disorder characterized by impaired synthesis of all adrenal and gonadal steroid hormones. We previously reported a D203A polymorphism in the STAR gene in Japanese patients with CLAH as well as in normal Japanese subjects. In the present study, we analyzed the ability of the A203 StAR and D203 StAR to stimulate steroidogenesis using the in vitro functional expression system. The A203 StAR caused a twelve-fold increase in pregnenolone secretion over COS-1 cells transfected with an NH2-cholesterol side-chain cleavage enzyme (P450scc)-adrenodoxin reductase-adrenodoxin-COOH fusion protein expressing plasmid (F2) and an empty vector, whereas the D203 StAR increased pregnenolone production no more than threefold. Western blot analysis detected mainly two species of StAR consisting of the 37-kDa precursor and the 30-kDa mature form. Together, these results indicate that the alanine at position 203 in human StAR is functionally important and that the D203 StAR is extremely unlikely to be a polymorphism.     

Involvement of the C-terminal residues of the 20,000-dalton light chain of myosin on the regulation of smooth muscle actomyosin.

The segment of smooth muscle regulatory light chain essential for the phosphorylation dependent activation of actomyosin motor activity and the binding of myosin heavy chain was identified. The C-terminal domain of the 20-kDa light chain, which is less conserved than the rest of the polypeptide among various muscle types, was mutated by either deletion or substitution of amino acid residues and the mutant light chains were then incorporated into myosin by subunit exchange. Deletion of Lys149-Ala166 markedly reduced the affinity of the light chain for the heavy chain, whereas the C-terminal five residues, Lys167-Asp171, did not contribute to the binding affinity. Deletion of Lys149-Phe158 abolished the phosphorylation-dependent activation of actomyosin ATPase activity as well as superprecipitation activity. These results suggest that the C-terminal domain of the regulatory light chain is critical for transmitting the change in the conformation of the regulatory light chain induced by phosphorylation at Ser19 to the heavy chain.     

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.     

Reactive oxygen disrupts mitochondria in MA-10 tumor Leydig cells and inhibits steroidogenic acute regulatory (StAR) protein and steroidogenesis

Reactive oxygen species (ROS) are involved in a variety of pathophysiological conditions of the testis, and oxidative stress is known to inhibit ovarian and testicular steroidogenesis. The site of ROS-mediated inhibition of steroidogenesis in the corpus luteum and MA-10 tumor Leydig cells was shown to be the hormone-sensitive mitochondrial cholesterol transfer step. The purpose of this study was to examine the effects of ROS on steroidogenic acute regulatory (StAR) protein in MA-10 cells and determine the extent to which MA-10 cell mitochondria are sensitive to oxidative stress. cAMP-stimulated progesterone production was inhibited in a dose-dependent manner in MA-10 cells exposed to H(2)O(2). StAR protein, but not mRNA levels, was decreased in parallel to changes in progesterone production. Even at the highest concentrations of H(2)O(2) tested, there was no effect on P450 side-chain cleavage enzyme protein levels. Oxidative stress from exposure to exogenous xanthine oxidase and xanthine resulted in the inhibition of both progesterone production and StAR protein expression. The mature 30- and 32-kDa intramitochondrial forms of StAR were decreased relative to the 37-kDa extramitochondrial precursor form of StAR, indicating that the ROS-mediated inhibition of StAR protein was due, in part, to the inhibition of mitochondrial import and processing. Vital staining with the fluorescent dye tetramethylrhodamine ethyl ester was used to visualize changes in the mitochondrial electrochemical gradient-dependent membrane potential (Deltapsim). ROS caused a significant dissipation of Deltapsi(m) and time-dependent loss of tetramethylrhodamine ethyl ester fluorescence. The inhibitory effects of H(2)O(2) were transient. There was no evidence for ROS-induced cell death, and following H(2)O(2) removal in the presence of continuous treatment with 8-bromo-cAMP, StAR protein levels and progesterone production were restored. In addition, there was no loss of cell viability following treatment with H(2)O(2) or xanthine/xanthine oxidase as determined by trypan blue exclusion. H(2)O(2) did not cause a significant decrease in total cellular ATP levels. These data indicate that oxidative stress-mediated perturbation of the mitochondria and dissipation of Deltapsi(m) results in the inhibition of StAR protein expression and its import, processing, and cholesterol transfer activity. These findings confirm earlier studies demonstrating the requirement for maintenance of an intact Deltapsi(m) for StAR protein function in cholesterol transport. The significant reduction in the 32- to 30-kDa mature forms of StAR, cessation of cholesterol transport, and loss of Deltapsi(m) are consistent with mitochondrial perturbation because of oxidative stress. This mechanism likely contributes to a host of pathophysiological events evident in testicular disorders such as infection, reperfusion injury, aging, cryptorchidism, and varicocele.     

The steroidogenic acute regulatory protein,StAR, works only at the outer mitochondrial membrane

The steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol into mitochondria to initiate steroidogenesis, but its site of action on the mitochondria has been uncertain. One model states that StAR has a fairly rigid structure and functions in the intramembranous space (IMS) where it transports cholesterol from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM); another model states that StAR works solely on or in the OMM and undergoes a partially open molten globule conformation while picking up and discharging cholesterol. We designed, built and tested a series of StAR fusion proteins designed to immobilize StAR on the OMM, the IMS, or the matrix side of the IMM. Only the constructs at the OMM were active, either in vivo or in vitro. As these data indicated that StAR acts at or in the OMM we hypothesized that StAR' s activity would be proportional to the amount of time it spends on the OMM. To test this hypothesis, we built a series of StAR proteins with altered mitochondrial leaders designed to speed or slow StAR's mitochondrial entry. Cell transfections showed that the constructs that slowed entry had more activity and those designed to speed entry had less activity. Analysis of import kinetics in vitro confirmed that these constructs accelerated import inversely proportional to their activity. These data show that StAR works only on the OMM, providing an unusual example of a protein that exerts its biological activity in a cellular location it occupies only transiently, rather than in the location (the matrix) to which it is targeted.     

Transmembrane domain length of viral K+ channels is a signal for mitochondria targeting

K⁺ channels operate in the plasma membrane and in membranes of organelles including mitochondria. The mechanisms and topogenic information for their differential synthesis and targeting is unknown. This article describes 2 similar viral K⁺ channels that are differentially sorted; one protein (Kesv) is imported by the Tom complex into the mitochondria, the other (Kcv) to the plasma membrane. By creating chimeras we discovered that mitochondrial sorting of Kesv depends on a hierarchical combination of N- and C-terminal signals. Crucial is the length of the second transmembrane domain; extending its C terminus by ≥2 hydrophobic amino acids redirects Kesv from the mitochondrial to the plasma membrane. Activity of Kesv in the plasma membrane is detected electrically or by yeast rescue assays only after this shift in sorting. Hence only minor structural alterations in a transmembrane domain are sufficient to switch sorting of a K⁺ channel between the plasma membrane and mitochondria.     

Toward the NMR structure of StAR

The steroidogenic acute regulatory (StAR) protein plays a crucial role in steroidogenesis, as it accelerates the transport of cholesterol to the inner mitochondrial membrane where the cytochrome P450scc enzyme is located. Mutations in the StAR gene can lead to lipoid congenital adrenal hyperplasia (LCAH), a disease that is fatal if not treated with hormone replacement therapy. Solving the structure of StAR is an important aspect of understanding LCAH. Point mutations or truncations in the StAR gene produce a partial to non-functional protein that hinders the StAR-induced delivery of cholesterol to the mitochondria during an acute hormonal stimulation of steroidogenic cells. So far, homology modeling, structure-based thermodynamics and biophysical studies have allowed us to propose the existence of an open state of StAR where the C-terminal α-helix 4 undergoes partial unfolding. This may act as a gating mechanism to the cholesterol binding site. Once bound, cholesterol leads to the stabilization and the refolding of α-helix 4, and eventually to the interaction with an import complex at the surface of the mitochondria. Though the current homology models have proven useful in understanding StAR function, only the full determination of the 3D structure of the apo- and holo-states will further validate this two-state model. In this context, we have used solution-state nuclear magnetic resonance (NMR) and obtained high-resolution ¹H–¹⁵N-HSQC spectra of StAR in its apo- and holo-states at physiological pH. Both spectra displayed well-dispersed resonances. However, key differences are observed on the spectra which indicate that both states have stable but slightly different tertiary structures. In conjunction with the binding/activity assays and biophysical methods, this original NMR data constitutes another structural step into the validation of the two-state model and the three-dimensional structure of StAR.     

In vitro assembly of apophytochrome and apophytochrome deletion mutants expressed in yeast with phycocyanobilin.

Recombinant pea type I phytochrome apoprotein expressed in yeast is shown to assemble in vitro with phycocyanobilin to produce a photoreversible phytochrome-like adduct. As an initial investigation of the amino acid sequence requirements for chromophore incorporation, three phyA gene product deletion mutants were produced in yeast. Truncation of the N-terminal tail to residue 46 demonstrates that this region is not critical to bilin attachment, but a deletion mutant lacking 222 amino acids from the N terminus failed to yield holophytochrome in vitro, under the same conditions. A mutant comprising a deletion of the C terminus to residue 548 showed bilin incorporation and red/far-red photoreversibility, indicating that bilin-apophytochrome assembly still occurred even when the entire C-terminal domain was truncated.