Pregnenolone

About: Pregnenolone is a research topic. Over the lifetime, 3539 publications have been published within this topic receiving 126444 citations. The topic is also known as: (3b)-3-hydroxy-Pregn-5-en-20-one & 3-Hydroxypregn-5-en-20-one.

Differential Inhibition of 17α-Hydroxylase and 17,20-Lyase Activities by Three Novel Missense CYP17 Mutations Identified in Patients with P450c17 Deficiency

Abstract: The microsomal enzyme cytochrome P450c17 is an important regulator of steroidogenesis. The enzyme has two functions: 17alpha-hydroxylase and 17,20-lyase activities. These functions determine the ability of adrenal glands and gonads to synthesize 17alpha-hydroxylated glucocorticoids (17alpha-hydroxylase activity) and/or sex steroids (17,20-lyase activity). Both enzyme functions depend on correct steroid binding, but it was recently shown that isolated lyase deficiency can also be caused by mutations located in the redox partner interaction domain. In this article we present the clinical history and molecular analysis of two patients with combined 17alpha-hydroxylase/17,20-lyase deficiency and four patients with isolated 17,20-lyase deficiency. In these six patients, four missense CYP17 mutations were identified. Two mutations were located in the steroid-binding domain (F114V and D116V), and the other two mutations were found in the redox partner interaction domain (R347C and R347H). We investigated the activity of these mutated proteins by transfection experiments in COS-1 cells using pregnenolone, progesterone, or their hydroxylated products as a substrate and measuring 17alpha-hydroxylase- and 17,20-lyase-dependent metabolites in the medium. The mutations in the steroid-binding domain (F114V and D116V) of P450c17 caused combined, complete (F114V), or partial (D116V) 17alpha-hydroxylase and 17,20-lyase deficiencies, whereas mutations in the redox partner interaction domain (R347C and R347H) displayed less severe 17alpha-hydroxylase deficiency, but complete 17,20-lyase deficiency. These findings are consistent with the clinical data and support the observation that the redox partner interaction domain is essential for normal 17,20-lyase function of P450c17.

The mitochondrial environment is required for activity of the cholesterol side-chain cleavage enzyme, cytochrome P450scc

Abstract: Steroidogenesis is initiated by the conversion of cholesterol to pregnenolone by mitochondrial cytochrome P450scc [cholesterol, reduced-adrenal-ferredoxin:oxygen oxidoreductase (side-chain-cleaving); EC 1.14.15.6]. Several subsequent steroidal conversions occur in the endoplasmic reticulum (ER), but the last step in the production of glucocorticoids and mineralocorticoids again occurs in the mitochondria. Although cellular compartmentalization of steroidogenic enzymes appears to be a feature of all steroidogenic pathways, some reports indicate that cholesterol can be converted to pregnenolone outside the mitochondria. To investigate whether P450scc can function outside the mitochondria, we constructed vectors producing P450scc and various fusion enzymes of P450scc with electron-transport proteins and directed their expression to either the ER or the mitochondria. Whether targeted to mitochondria or to the ER, plasmid vectors encoding P450scc and fusion proteins of P450scc with either mitochondrial or microsomal electron-transport proteins produced immunodetectable protein. When expressed in mitochondria, all of these constructions converted 22-hydroxycholesterol to pregnenolone, but when expressed in the ER none of them produced pregnenolone. These results show that P450scc can function only in the mitochondria. Furthermore, it appears to be the mitochondrial environment that is required, rather than the specific mitochondrial electron-transport intermediates.

Steroid synthesis-dependent, oxygen-mediated damage of mitochondrial and microsomal cytochrome P-450 enzymes in rat Leydig cell cultures.

Abstract: Treatment of primary cultures of rat Leydig cells with 1 mM 8-bromo-cAMP for 2 days at ambient oxygen tension (19%) caused a 59% decrease in mitochondrial cholesterol sidechain cleavage (P-450scc) activity. This decrease was completely prevented when the oxygen tension was reduced to 1% O2 or when steroid synthesis was inhibited by aminoglutethimide. When the endogenous concentration of pregnenolone was increased by inhibiting its further metabolism, P-450scc activity was reduced by 80% in unstimulated cultures and was completely eliminated in cAMP-treated cultures. These losses were prevented when cells were maintained at 1% O2. The amount of immunoreactive P-450scc was also decreased by treatments that reduced P-450scc activity. Stimulation with cAMP also lowered microsomal C17_20 lyase activity by an oxygen-mediated, steroid synthesis-dependent mechanism. Treatment of cultures with testosterone caused a similar oxygen tension-sensitive decrease in C17_20 lyase activity. These results suggest that the e...