Mahuya Bose

Steroidogenic Activity of StAR Requires Contact with Mitochondrial VDAC1 and Phosphate Carrier Protein

The steroidogenic acute regulatory protein (StAR) is required for adrenal and gonadal steroidogenesis and for male sexual differentiation. StAR acts on the outer mitochondrial membrane (OMM) to facilitate movement of cholesterol from the OMM to the inner mitochondrial membrane to be converted to pregnenolone, the precursor of all steroid hormones. The mechanisms of the action of StAR remain unclear; the peripheral benzodiazepine receptor, an OMM protein, appears to be involved, but the identity of OMM proteins that interact with StAR remain unknown. Here we demonstrate that phosphorylated StAR interacts with voltage-dependent anion channel 1 (VDAC1) on the OMM, which then facilitates processing of the 37-kDa phospho-StAR to the 32-kDa intermediate. In the absence of VDAC1, phospho-StAR is degraded by cysteine proteases prior to mitochondrial import. Phosphorylation of StAR by protein kinase A requires phosphate carrier protein on the OMM, which appears to interact with StAR before it interacts with VDAC1. VDAC1 and phosphate carrier protein are the first OMM proteins shown to contact StAR.

Characterization of a Recombinant Adeno-Associated Virus Type 2 Reference Standard Material

A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18 x 10¹¹ particles/ml; 95% confidence interval [CI], 7.89 x 10¹¹ to 1.05 x 10¹² particles/ml), vector genomes ({X}, 3.28 x 10¹⁰ vector genomes/ml; 95% CI, 2.70 x 10¹⁰ to 4.75 x 10¹⁰ vector genomes/ml), transducing units ({X}, 5.09 x 10⁸ transducing units/ml; 95% CI, 2.00 x 10⁸ to 9.60 x 10⁸ transducing units/ml), and infectious units ({X}, 4.37 x 10⁹ TCID₅₀ IU/ml; 95% CI, 2.06 x 10⁹ to 9.26 x 10⁹ TCID₅₀ IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed.

StAR-like Activity and Molten Globule Behavior of StARD6, A Male Germ-Line Protein

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.

Mitochondria-associated Endoplasmic Reticulum Membrane (MAM) Regulates Steroidogenic Activity via Steroidogenic Acute Regulatory Protein (StAR)-Voltage-dependent Anion Channel 2 (VDAC2) Interaction

Background: Steroidogenic acute regulatory protein (StAR) fosters cholesterol into the adrenal and gonadal mitochondria to initiate steroidogenesis. Results: Voltage-dependent anion channel 2 (VDAC2) knockdown ablated pregnenolone synthesis and StAR processing into the mitochondria. Conclusion: Interaction between StAR and VDAC2 is critical for steroidogenesis. Significance: VDAC2 is a crucial regulator for initiating steroidogenesis. Steroid hormones are essential for carbohydrate metabolism, stress management, and reproduction and are synthesized from cholesterol in mitochondria of adrenal glands and gonads/ovaries. In acute stress or hormonal stimulation, steroidogenic acute regulatory protein (StAR) transports substrate cholesterol into the mitochondria for steroidogenesis by an unknown mechanism. Here, we report for the first time that StAR interacts with voltage-dependent anion channel 2 (VDAC2) at the mitochondria-associated endoplasmic reticulum membrane (MAM) prior to its translocation to the mitochondrial matrix. In the MAM, StAR interacts with mitochondrial proteins Tom22 and VDAC2. However, Tom22 knockdown by siRNA had no effect on pregnenolone synthesis. In the absence of VDAC2, StAR was expressed but not processed into the mitochondria as a mature 30-kDa protein. VDAC2 interacted with StAR via its C-terminal 20 amino acids and N-terminal amino acids 221–229, regulating the mitochondrial processing of StAR into the mature protein. In the absence of VDAC2, StAR could not enter the mitochondria or interact with MAM-associated proteins, and therefore steroidogenesis was inhibited. Furthermore, the N terminus was not essential for StAR activity, and the N-terminal deletion mutant continued to interact with VDAC2. The endoplasmic reticulum-targeting prolactin signal sequence did not affect StAR association with the MAM and thus its mitochondrial targeting. Therefore, VDAC2 controls StAR processing and activity, and MAM is thus a central location for initiating mitochondrial steroidogenesis.

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. StARs 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 StARs 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.

Cholesterol-Mediated Conformational Changes in the Steroidogenic Acute Regulatory Protein Are Essential for Steroidogenesis

Although the mechanism by which the steroidogenic acute regulatory protein (StAR) promotes steroidogenesis has been studied extensively, it remains incompletely characterized. Because structural analysis has revealed a hydrophobic sterol-binding pocket (SBP) within StAR, this study sought to examine the regulatory role of cholesterol concentrations on protein folding and mitochondrial import. Stopped-flow analyses revealed that at low concentrations, cholesterol promotes StAR folding. With increasing cholesterol concentrations, an intermediate state is reached followed by StAR unfolding. With 5 μg/mL cholesterol, the apparent binding was 0.011 s(-1), and the unfolding time (t1/2) was 63 s. The apparent binding increased from 0.036 to 0.049 s(-1) when the cholesterol concentration was increased from 50 μg/mL to 100 μg/mL while t1/2 decreased from 19 to 14 s. These cholesterol-induced conformational changes were not mediated by chemical chaperones. Protein fingerprinting analysis of StAR in the absence and presence of cholesterol by mass spectrometry revealed that the cholesterol binding region, comprising amino acids 132-188, is protected from proteolysis. In the absence of cholesterol, a longer region of amino acids from position 62 to 188 was protected, which is suggestive of organization into smaller, tightly folded regions with cholesterol. In addition, rapid cholesterol metabolism was required for the import of StAR into the mitochondria, suggesting that the mitochondria have a limited capacity for import and processing of steroidogenic proteins, which is dependent on cholesterol storage. Thus, cholesterol regulates StAR conformation, activating it to an intermediate flexible state for mitochondrial import and its enhanced cholesterol transfer capacity.

Identification of unknown protein complex members by radiolocalization and analysis of low‐abundance complexes resolved using native polyacrylamide gel electrophoresis

Identification of unknown binding partners of a protein of interest can be a difficult process. Current strategies to determine protein binding partners result in a high amount of false‐positives, requiring use of several different methods to confirm the accuracy of the apparent association. We have developed and utilized a method that is reliable and easily substantiated. Complexes are isolated from cell extract after exposure to the radiolabeled protein of interest, followed by resolution on a native polyacrylamide gel. Native conformations are preserved, allowing the complex members to maintain associations. By radiolabeling the protein of interest, the complex can be easily identified at detection levels below the threshold of Serva Blue, Coomassie, and silver stains. The visualized radioactive band is analyzed by MS to identify binding partners, which can be subsequently verified by antibody shift and immunoprecipitation of the complex. By using this method we have successfully identified binding partners of two proteins that reside in different locations of a cellular organelle.

Steroidogenic Acute Regulatory Protein Has a More Open Conformation Than the Independently Folded Smaller Subdomains

The acute steroidogenic response, which produces steroids in response to stress, requires the steroidogenic acute regulatory protein (StAR). StAR, a mitochondrial matrix protein, acts on the outer mitochondrial membrane (OMM) to facilitate the movement of cholesterol from the outer to inner mitochondrial membrane via an unknown mechanism. The N-terminal sequence was reported to be nonessential for activity. We show that alteration of the StAR amino-terminal sequence does not change the thermodynamic stability of StAR but offers protection from proteolytic degradation. A longer association between StAR and the OMM strengthens the interaction with cholesterol. Far-UV CD spectra showed that the smaller fragments of StAR domains were less alpha-helical compared to N-62 StAR but were structured as determined by limited proteolysis followed by mass spectrometry. The START domain consisting of amino acids 63-193 also exhibited protease protection for amino acids 84-193. The Stern-Volmer quenching constant (K(SV)) of the N-62 StAR protein is 12.1 x 10(5) M(-1), with all other START fragments having significantly smaller K(SV) values ranging from 6 to 10 x 10(5) M(-1), showing that N-62 StAR has a more open conformation. Only N-62 StAR protein is stabilized with cholesterol having an increased DeltaH value of -5.6 +/- 0.3 kcal/mol at 37 degrees C. These findings demonstrate a mechanism in which StAR is stabilized at the OMM by cholesterol to initiate its massive import into mitochondria.

Hydrophobic core of the steroidogenic acute regulatory protein for cholesterol transport.

The steroidogenic acute regulatory protein (StAR), the first family member of START (StAR-related lipid transport) proteins, plays an essential role by facilitating the movement of cholesterol from the outer to inner mitochondrial membrane. Wild-type and mutant StAR binds cholesterol with similar intensity, but only wild-type StAR can transport it to mitochondria. Here, we report that the hydrophobic core is crucial for biological activity of proteins with START domains. Wild-type StAR increased steroidogenic activity by 7-9-fold compared to mutant R182L StAR, but both of them showed similar near-UV CD spectra. The fluorescence maximum of wild-type StAR is red shifted in comparison to mutant StAR under identical urea concentration. TFE increased the alpha-helical contribution of wild-type StAR more than the mutant protein. Acrylamide quenching for the wild-type protein (K(SV) = 12.0 +/- 0.2-11.2 +/- 0.5 M(-1)) exceeded that of the mutant protein (K(SV) = 4 +/- 0.2 M(-1)). Consistent with these findings, the hydrophobic probe ANS bound wild-type StAR (K(app) = 8.1 x 10(5) M(-1)) to a greater degree than mutant StAR (K(app) = 3.75 x 10(5) M(-1)). Partial proteolysis examined by mass spectrometry suggests that only wild-type StAR has a protease-sensitive C-terminus, but not the mutant. Stopped-flow CD revealed that the time of unfolding of mutant StAR was 0.017 s. In contrast, the wild-type StAR protein is unfolded in 16.3 s. In summary, these results demonstrate that wild-type StAR adopts a very flexible form due to the accommodation of more water molecules, while mutant StAR is generated by an alternate folding pathway making it inactive.