These patients define a new disorder, nonclassic lipoid congenital adrenal hyperplasia, and represent a new cause of nonautoimmune Addison disease (primary adrenal failure).

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 [ 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.In the adrenal and gonad, the steroidogenic acute regulatory protein (StAR) 2 facilitates the flow of cholesterol from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM) where it is converted to pregnenolone by the cholesterol side chain cleavage enzyme, P450scc, thus initiating the synthesis of steroid hormones (1, 2). Loss of StAR activity ablates adrenal and gonadal steroidogenesis, causing lifethreatening congenital lipoid adrenal hyperplasia (3). Fulllength StAR is expressed as a 37-kDa, 285-residue phosphoprotein having a mitochondrial leader sequence that is cleaved to a 30-kDa intramitochondrial form (4 -7). Nevertheless, StAR acts on the OMM, and is inactive inside the mitochondrion: deletion of 62 NH 2 -terminal residues yields a protein (N-62 StAR) that remains in the cytoplasm, yet retains full biologic activity (8), and affixing StAR to the OMM yields constituitive activity, whereas localizing it to the intramembranous space or the matrix side of the IMM ablates activity (9). Only the COOH-terminal ␣-helix (C-helix) of StAR interacts with the OMM (10). Biophysical data show that StAR undergoes an acid-induced structural change to a molten globule state (11), and the association of StAR with the OMM is stronger under acidic conditions (10,(12)(13)(14)(15).The structure of StAR has not been determined experimentally, but the structures of three clos...

The steroidogenic acute regulatory protein (StAR) belongs to a family of 15 StAR-related lipid transfer (START) domain proteins termed StARD1-StARD15. StAR (StARD1) induces adrenal and gonadal steroidogenesis by moving cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane by an unclear process that involves conformational changes that have been characterized as a molten globule transition. We expressed, purified, and assessed the activity and cholesterol-binding behavior of StARD1 and StARD3-D7, showing that StARD6 had activity equal to StARD1, whereas StARD4, D5, and D7 had little or no activity with adrenal mitochondria in vitro. Partial proteolysis examined by mass spectrometry suggests that StARD6 has a protease-sensitive C-terminus, similar to but smaller than that of StARD1. Experiments using urea denaturation, stopped-flow kinetics and measurements of mitochondrial membrane association suggests that StARD1 and StARD6 both unfold and refold slowly with similar kinetic patterns. Isothermal titration calorimetry suggests that StARD6 interacts with mitochondrial membranes as well as or better than StARD1. Computational modeling of StARD6 suggests that it has a similar fold to StARD1, with a hydrophobic sterol-binding pocket and a unique C-terminal extension. StARD6, which is expressed only in male germ-line cells, thus exhibits biological and biophysical properties that imply a role in steroidogenesis.

The steroidogenic acute regulatory protein (StAR) simulates steroid biosynthesis by increasing the flow of cholesterol from the outer mitochondrial membrane (OMM) to the inner membrane. StAR acts exclusively on the OMM, and only StAR's carboxyl-terminal ␣-helix (C-helix) interacts with membranes. Biophysical studies have shown that StAR becomes a molten globule at acidic pH, but a physiologic role for this structural transition has been controversial. Molecular modeling shows that the C-helix, which forms the floor of the sterol-binding pocket, is stabilized by hydrogen bonding to adjacent loops. Molecular dynamics simulations show that protonation of the C-helix and adjacent loops facilitates opening and closing the sterol-binding pocket. Two disulfide mutants, S100C/S261C (SS) and D106C/A268C (DA), designed to limit the mobility of the C-helix but not disrupt overall conformation, were prepared in bacteria, and their correct folding and positioning of the disulfide bonds was confirmed. The SS mutant lost half, and the DA mutant lost all cholesterol binding capacity and steroidogenic activity with isolated mitochondria in vitro, but full binding and activity was restored to each mutant by disrupting the disulfide bonds with dithiothreitol. These data strongly support the model that StAR activity requires a pH-dependent molten globule transition on the OMM.Molten globules are compact, partially unfolded proteins that retain their secondary structure but have lost some tertiary structure (1); such partially unfolded structures are typically inactive but may be intermediates in membrane insertion (2). Steroidogenic acute regulatory protein (StAR), 3 which is essential for normal adrenal and gonadal steroidogenesis, facilitates the flow of cholesterol from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM), where cholesterol is converted to pregnenolone by the cholesterol side chain cleavage enzyme, P450scc (3, 4). Mutation of human StAR causes potentially lethal congenital lipoid adrenal hyperplasia (5, 6); all missense mutations that cause this disease are found in the carboxyl-terminal 50% of the protein, indicating that these sequences are required for StAR activity (7-9). StAR is synthesized as a 37-kDa phosphoprotein with an amino-terminal mitochondrial leader sequence that is cleaved during mitochondrial entry (3, 10, 11).The mechanism by which StAR moves cholesterol from the OMM to IMM remains unclear. The x-ray crystal structures of two closely related proteins, N-216 MLN64 (12) and StarD4 (13), reveal a ␤-barrel structure with a hydrophobic sterol-binding pocket (SBP) that will accommodate one cholesterol molecule, although the apparent access channel to the SBP is too small to accommodate a cholesterol molecule. Models of StAR show the same fold (12,14,15), suggesting action as a transport protein. Recent data suggest that members of the StarD4 family of related proteins, which lack mitochondrial targeting sequences, serve as cytosolic transporters for insoluble lipid molecu...