Showing papers by "Himangshu S. Bose published in 2011"

Lipid-mediated unfolding of 3β-hydroxysteroid dehydrogenase 2 is essential for steroidogenic activity.

Abstract: For inner mitochondrial membrane (IMM) proteins that do not undergo N-terminal cleavage, the activity may occur in the absence of a receptor present in the mitochondrial membrane. One such protein is human 3β-hydroxysteroid dehydrogenase 2 (3βHSD2), the IMM resident protein responsible for catalyzing two key steps in steroid metabolism: the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione. Conversion requires that 3βHSD2 serve as both a dehydrogenase and an isomerase. The dual functionality of 3βHSD2 results from a conformational change, but the trigger for this change remains unknown. Using fluorescence resonance energy transfer, we found that 3βHSD2 interacted strongly with a mixture of dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidylcholine (DPPC). 3βHSD2 became less stable when incubated with the individual lipids, as indicated by the decrease in thermal denaturation (T(m)) from 42 to 37 °C. DPPG, alone or in combination with DPPC, led to a decrease in α-helical content without an effect on the β-sheet conformation. With the exception of the 20 N-terminal amino acids, mixed vesicles protected 3βHSD2 from trypsin digestion. However, protein incubated with DPPC was only partially protected. The lipid-mediated unfolding completely supports the model in which a cavity forms between the α-helix and β-sheet. As 3βHSD2 lacks a receptor, opening the conformation may activate the protein.

Decreased Cytochrome c Oxidase IV Expression Reduces Steroidogenesis

Abstract: Steroidogenic acute regulatory protein facilitates the translocation of cholesterol to the inner mitochondrial membrane, thereby initiating steroidogenesis. At the inner mitochondrial membrane, cytochrome P450 side-chain cleavage enzyme converts cholesterol to pregnenolone, an oxidative process requiring electrons from NADPH. Pregnenolone then serves as the substrate for the formation of progesterone or dehydroepiandrosterone by downstream enzymes. Studies have shown that cigarette smoke (CS) influences steroid hormone levels. To better understand the underlying mechanisms, we used a mouse model to study the effects of chronic CS exposure on steroidogenesis. Through radioimmunoassay and metabolic conversion assays, we found that CS reduced progesterone and dehydroepiandrosterone without affecting cytochrome P450 side-chain cleavage enzyme or 3β-hydroxysteroid dehydrogenase 2 expression. However, CS did reduce expression of cytochrome c oxidase IV (COX IV), a component of the mitochondrial complex that serves as the last enzyme in the electron transport chain. Small interfering RNA-mediated COX IV knockdown indeed decreased progesterone synthesis in steroidogenic cells. In summary, COX IV likely plays a role in steroidogenesis, and passive smoking may negatively affect steroidogenesis by disrupting the electron transport chain.

Inner Mitochondrial Translocase Tim50 Interacts with 3-Hydroxysteroid Dehydrogenase Type 2 to Regulate

Abstract: In the adrenals, testes, and ovaries, 3-hydroxysteroid dehydrogenase type 2 (3HSD2) catalyzes the conversion of pregnenolone to progesterone and dehydroepiandrostenedione to androstenedione. Alterations in this pathway can have deleterious effects, including sexual development impairment, spontaneous abortion, and breast cancer. 3HSD2, synthesized in the cytosol, is imported into the inner mitochondrial membrane (IMM) by translocases. Steroidogenesis requires that 3HSD2 acts as both a dehydrogenase and isomerase. To achieve this dual functionality, 3HSD2 must undergo a conformational change; however, what triggers that change remains unknown. We propose that 3HSD2 associates with IMM or outer mitochondrial membrane translocases facing the intermembrane space (IMS) and that this interaction promotes the conformational change needed for full activity. Fractionation assays demonstrate that 3HSD2 associated with the IMM but did not integrate into the membrane. Through mass spectrometry and Western blotting of mitochondrial complexes and density gradient ultracentrifugation, we show that that 3HSD2 formed a transient association with the translocases Tim50 and Tom22 and with Tim23. This association occurred primarily through the interaction of Tim50 with the N terminus of 3HSD2 and contributed to enzymatic activity. Tim50 knockdown inhibited catalysis of dehydroepiandrostenedione to androstenedione and pregnenolone to progesterone. Although Tim50 knockdown decreased 3HSD2 expression, restoration of expression via proteasome and protease inhibition did not rescue activity. In addition, protein fingerprinting and CD spectroscopy reveal the flexibility of 3HSD2, a necessary characteristic for forming multiple associations. In summary, Tim50 regulates 3HSD2 expression and activity, representing a new role for translocases in steroidogenesis.