Role of steroid sulfates in the regulation of steroid hormone biosynthesis
Zusammenfassung der Projektergebnisse
Steroid biosynthesis is realized by a cascade of reactions based on various protein-protein interactions taking place in steroidogenic organs such as adrenals, gonads and brain. Six cytochrome P450- dependent steroid hydroxylases are involved in the biosynthesis of glucocorticoids, mineralocorticoids and sexual hormones: CYP11A1, CYP11B1, CYP11B2, CYP17, CYP19, and CYP21. Three of these cytochromes P450 (CYP17, CYP19, CYP21) are located in the endoplasmic reticulum (microsomal P450s) and three (CYP11A1, CYP11B1, CYP11B2) are located in mitochondria. The hydroxylation reaction needs electrons to proceed. In case of microsomal P450s the electrons are being transferred from NADPH via an FAD- und FMN-containing reductase (CPR), while in case of mitochondrial P450s the electrons are provided from NADPH via an FAD-containing ferredoxin reductase, adrenodoxin reductase (AdR), and a ferredoxin (iron-sulphur protein), adrenodoxin (Adx). The effects of sulfated steroids on the biosynthesis of steroid hormones, especially the function of a mitochondrial (CYP11A1) and microsomal (CYP17 and CYP21) cytochromes P450 were studied in detail. First, it was investigated whether these compounds exert an effect on the kinetics of these steroidogenic enzymes. For this, these proteins and their redox partners were expressed in E. coli, purified, and studied in reconstituted in vitro systems. Potential binding of the compounds as substrates or inhibitors were investigated using the typical spectral changes (type I or type II) of cytochromes P450 upon substrate and inhibitor binding. Among the sulfated steroids tested only pregnenolone sulfate (PregS) induced a type I shift with CYP17. It was shown that PregS is converted into 17OH-PregS and the kinetics of this reaction was recorded for the first time. Interestingly, using 17OH-PregS as substrate, CYP17 was not able to perform its lyase reaction, even in the presence of cytochrome b5. These experimental results were further reproduced in a human cell culture system indicating a physiological function of sulfated steroids on steroid hormone biosynthesis. Furthermore, a potential influence of sulfated steroids was tested on the activity of CYP11, CYP17 and CYP21. It was demonstrated that the side-chain cleavage activity of CYP11 is increased in the presence of DHEAS using cholesterol as substrate. This effect was due to an increased substrate affinity as well as a tighter interaction with the electron transfer partner, Adx. However, the activities of CYP17 and CYP21 were not affected in the presence of sulfated steroids. Taken together, we were able to demonstrate that sulfated steroids have a direct effect on the activity of steroidogenic cytochromes P450 and that some of these sulfated steroids also serve as substrates. These findings demonstrate that sulfated steroids are significant and promising compounds in order to understand the regulation of the steroid hormone biosynthesis in more detail.
Projektbezogene Publikationen (Auswahl)
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(2014) A steroidogenic pathway for sulfonated steroids: the metabolism of pregnenolone sulfate. J Steroid Biochem Mol Biol 144: 324-333
Neunzig J, Sánchez-Guijo A, Mosa A, Hartmann MF, Geyer J, Wudy SA, Bernhardt R
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(2014) Dehydroepiandrosterone Sulfate (DHEAS) stimulates the first step in the biosynthesis of steroid hormones. PloS one 9(2): e89727
Neunzig J and Bernhardt R
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(2017) Effect of sulfonated steroids on steroidogenic cytochrome P450- dependent steroid hydroxylases. J Steroid Biochem Mol Biol
Neunzig J and Bernhardt R
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(2017) The steroid metabolite 16(ß)-OH-androstenedione generated by CYP21A2 serves as a substrate for CYP19A1. J Steroid Biochem Mol Biol 167:182-191
Neunzig J, Milhim M, Schiffer L, Kathri Y, Zapp J, Sánchez-Guijo A, Hartmann MF, Wudy SA, Bernhardt R
