title: Chaperones rejuvenate folding and activity of 3-β hydroxysteroid dehydrogenase 2.
abstract: The steroidogenic enzyme 3-β hydroxysteroid dehydrogenase 2 (3βHSD2) mediates the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione through both its dehydrogenase and isomerase activities, making it necessary for the protein to undergo a reversible conformational change. We hypothesized that chaperones assist 3βHSD2 in switching between the conformations to initiate, enhance, and maintain activity. In the presence of the chaperone lauryl maltoside (LM), 3βHSD2 immediately converted pregnenolone to progesterone, with a 6.4-fold increase in synthesis. Using far-UV circular dichroism (CD), we found that addition of LM increased 3βHSD2's α-helical content, which over time reverted to control levels, suggesting the formation of a stable but reversible conformation possibly due to hydrophobic interactions of the protein with LM micelles. We also found that LM increased fluorescence resonance energy transfer (FRET) about 11-fold between 3βHSD2 and fluorescing ANS molecules. This observation supports the idea that detergent(s) act as chaperones to assist 3βHSD2 in forming stable complexes, which in turn promotes proper folding. Mass spectrometric fingerprinting illustrated that LM incubation resulted in an ordered fragmentation of molecular mass from 39 to 13 kDa, as compared to limited or no proteolysis in the absence of LM. In addition, space-filling modeling demonstrated that 3βHSD2 association with detergents likely exposed the hydrophobic region, leading to its proteolysis. We conclude that detergents help 3βHSD2 to refold in order to rejuvenate, contributing to the ability of cells to rapidly produce steroids when needed.
authors.affiliation: Department of Biochemistry, Mercer University School of Medicine, Savannah, GA 31404, USA.
authors.forename: Maheshinie
authors.initials: M
authors.lastname: Rajapaksha
authors.forename: Manoj
authors.initials: M
authors.lastname: Prasad
authors.forename: James L
authors.initials: JL
authors.lastname: Thomas
authors.forename: Randy M
authors.initials: RM
authors.lastname: Whittal
authors.forename: Himangshu S
authors.initials: HS
authors.lastname: Bose
mesh_terms: D015096:3-Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002784:Cholesterol / Q000378:metabolism; D002942:Circular Dichroism; D003902:Detergents / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005960:Glucosides / Q000378:metabolism / Q000494:pharmacology; D057927:Hydrophobic and Hydrophilic Interactions; D007985:Leydig Cells / Q000187:drug effects / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism* / Q000494:pharmacology; D008970:Molecular Weight; D011284:Pregnenolone / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding
chemical_list: D003902:Detergents; D005960:Glucosides; D018832:Molecular Chaperones; D011374:Progesterone; C040358:dodecyl maltoside; D011284:Pregnenolone; D002784:Cholesterol; D015096:3-Hydroxysteroid Dehydrogenases