Mitochondrial cytochrome P450 1B1 is involved in pregnenolone synthesis in human brain cells.

Neurosteroids, which are steroids synthesized by the nervous system, can exert neuromodulatory and neuroprotective effects via genomic and nongenomic pathways. The neurosteroid and major steroid precursor pregnenolone has therapeutical potential in various diseases, such as psychiatric and pain disorders, and may play important roles in myelination, neuroinflammation, neurotransmission, and neuroplasticity. Although pregnenolone is synthesized by CYP11A1 in peripheral steroidogenic organs, our recent study showed that pregnenolone must be synthesized by another mitochondrial cytochrome P450 (CYP450) enzyme other than CYP11A1 in human glial cells. Therefore, we sought to identify the CYP450 responsible for pregnenolone production in the human brain. Upon screening for CYP450s expressed in the human brain that have mitochondrial localization, we identified three enzyme candidates: CYP27A1, CYP1A1, and CYP1B1. We found that inhibition of CYP27A1 through inhibitors and siRNA knockdown did not negatively affect pregnenolone synthesis in human glial cells. Meanwhile, treatment of human glial cells with CYP1A1/CYP1B1 inhibitors significantly reduced pregnenolone production in the presence of 22(R)-hydroxycholesterol. We performed siRNA knockdown of CYP1A1 or CYP1B1 in human glial cells and found that only CYP1B1 knockdown significantly decreased pregnenolone production. Furthermore, overexpression of mitochondria-targeted CYP1B1 significantly increased pregnenolone production under basal conditions and in the presence of hydroxycholesterols and low-density lipoprotein. Inhibition of CYP1A1 and/or CYP1B1 via inhibitors or siRNA knockdown did not significantly reduce pregnenolone synthesis in human adrenal cortical cells, implying that CYP1B1 is not a major pregnenolone-producing enzyme in the periphery. These data suggest that mitochondrial CYP1B1 is involved in pregnenolone synthesis in human glial cells.

The neurosteroid pregnenolone is synthesized by a mitochondrial P450 enzyme other than CYP11A1 in human glial cells.

Neurosteroids, modulators of neuronal and glial cell functions, are synthesized in the nervous system from cholesterol. In peripheral steroidogenic tissues, cholesterol is converted to the major steroid precursor pregnenolone by the CYP11A1 enzyme. Although pregnenolone is one of the most abundant neurosteroids in the brain, expression of CYP11A1 is difficult to detect. We found that human glial cells produced pregnenolone, detectable by mass spectrometry and ELISA, despite the absence of observable immunoreactive CYP11A1 protein. Unlike testicular and adrenal cortical cells, pregnenolone production in glial cells was not inhibited by CYP11A1 inhibitors DL-aminoglutethimide and ketoconazole. Furthermore, addition of hydroxycholesterols increased pregnenolone synthesis, suggesting desmolase activity that was not blocked by DL-aminoglutethimide or ketoconazole. We explored three different possibilities for an alternative pathway for glial cell pregnenolone synthesis: (1) regulation by reactive oxygen species, (2) metabolism via a different CYP11A1 isoform, and (3) metabolism via another CYP450 enzyme. First, we found oxidants and antioxidants had no significant effects on pregnenolone synthesis, suggesting it is not regulated by reactive oxygen species. Second, overexpression of CYP11A1 isoform b did not alter synthesis, indicating use of another CYP11A1 isoform is unlikely. Finally, we show nitric oxide and iron chelators deferoxamine and deferiprone significantly inhibited pregnenolone production, indicating involvement of another CYP450 enzyme. Ultimately, knockdown of endoplasmic reticulum cofactor NADPH-cytochrome P450 reductase had no effect, while knockdown of mitochondrial CYP450 cofactor ferredoxin reductase inhibited pregnenolone production. These data suggest that pregnenolone is synthesized by a mitochondrial cytochrome P450 enzyme other than CYP11A1 in human glial cells.

Impairment of renal steroidogenesis at the onset of diabetes.

Accumulating evidence indicates the association between changes in circulating sex steroid hormone levels and the development of diabetic nephropathy. However, the renal synthesis of steroid hormones during diabetes is essentially unknown. Male Wistar rats were injected with streptozotocin (STZ) or vehicle. After one week, no changes in functional or structural parameters related to kidney damage were observed in STZ group; however, a higher renal expression of proinflammatory cytokines and HSP70 was found. Expression of Steroidogenic Acute Regulatory protein (StAR) and P450scc (CYP11A1) was decreased in STZ kidneys. Incubation of isolated mitochondria with 22R-hydroxycholesterol revealed a marked inhibition in CYP11A1 function at the medullary level in STZ group. The inhibition of these first steps of renal steroidogenesis in early STZ-induced diabetes led to a decreased local synthesis of pregnenolone and progesterone. These findings stimulate investigation of the probable role of nephrosteroids in kidney damage associated with diabetes.

Clock-Controlled Mitochondrial Dynamics Correlates with Cyclic Pregnenolone Synthesis.

Neurosteroids are steroids synthetized in the nervous system, with the first step of steroidogenesis taking place within mitochondria with the synthesis of pregnenolone. They exert important brain-specific functions by playing a role in neurotransmission, learning and memory processes, and neuroprotection. Here, we show for the first time that mitochondrial neurosteroidogenesis follows a circadian rhythm and correlates with the rhythmic changes in mitochondrial morphology. We used synchronized human A172 glioma cells, which are steroidogenic cells with a functional core molecular clock, to show that pregnenolone levels and translocator protein (TSPO) are controlled by the clock, probably via circadian regulation of mitochondrial fusion/fission. Key findings were recapitulated in mouse brains. We also showed that genetic or pharmacological abrogation of fusion/fission activity, as well as disturbing the core molecular clock, abolished circadian rhythms of pregnenolone and TSPO. Our findings provide new insights into the crosstalk between mitochondrial function (here, neurosteroidogenesis) and circadian cycles.

Sex dimorphism in an animal model of multiple sclerosis: Focus on pregnenolone synthesis.

Neuroactive steroids, molecules produced from cholesterol in steroidogenic cells (i.e., peripheral glands and nervous system) are physiological modulators and protective agents of nervous function. A possible role for neuroactive steroids in the sex-dimorphic clinical manifestation, onset and progression of Multiple Sclerosis (MS) has been recently suggested. To explore this possibility, we assessed the synthesis of the first steroidogenic product (pregnenolone; PREG) in the spinal cord of experimental autoimmune encephalomyelitis rats, a MS model. Data obtained indicate that the synthesis of PREG in the spinal cord is altered by the pathology in a sex-dimorphic way and depending on the pathological progression. Indeed, in male spinal cord the synthesis was already decreased at the acute phase of the disease (i.e., 14 days post induction - dpi) and maintained low during the chronic phase (i.e., 45 dpi), while in females this effect was observed only at the chronic phase. Substrate availability had also a role in the sex-dimorphic kinetics. Indeed, at the chronic phase, male animals showed a reduction in the levels of free cholesterol coupled to alteration of cholesterol metabolism into oxysterols; these effects were not observed in female animals. These findings suggest that the comprehension of the neurosteroidogenic processes could be relevant to better understand the sexual dimorphism of MS and to possibly design sex-oriented therapeutic strategies based on neuroactive steroids.

Quantum-Mechanical/Molecular-Mechanical Studies of CYP11A1-Catalyzed Biosynthesis of Pregnenolone from Cholesterol Reveal a C-C Bond Cleavage Reaction That Occurs by a Compound I-Mediated Electron Transfer.

We explore here a long-standing mechanistic question by using quantum-mechanical/molecular-mechanical (QM/MM) methodology. The question concerns the mechanism of steroid hormone biosynthesis, whereby the P450 enzyme, CYP11A1, catalyzes the C20-C22 bond-cleavage in the 20,22-hydroxylated cholesterol, 20R,22R-DiOHCH, leading to pregnenolone, which is critical for the subsequent production of all steroid hormones. This is an unusual feat whereby the P450 enzyme breaks two O-H bonds and one C-C bond, while making two C═O bonds. How does the enzyme perform such a complex and highly energy-demanding reaction? Our computational results rule out the previously proposed Compound I (Cpd I) electrophilic attack mechanism via the formation of a peroxide intermediate as well as the H-abstraction-mediated C-C cleavage mechanism. Notably, oxygen-rebound cannot transpire, in spite of the fact that the classical active species, Cpd I, participates in the catalytic process. Our findings reveal a mechanism whereby C-C bond cleavage is mediated by an electron transfer from the C22-O--deprotonated substrate to Cpd I. As such, our QM/MM calculations demonstrate that Cpd I acts as an electron sink that facilitates the C-C bond cleavage.

Pregnenolone Overproduction in Yarrowia lipolytica by Integrative Components Pairing of the Cytochrome P450scc System.

Microbial production of steroid drugs exhibits great potentials in much greener and more sustainable manners, in which engineering multiple cytochrome P450s is the prerequisite requirement. The pairing of multicomponents of P450 systems is a tremendous challenge. Herein, biosynthesis of pregnenolone (a common precursor of steroid drugs) in Yarrowia lipolytica was taken as a typical instance to explore the engineering strategy of the cytochrome P450 side-chain cleavage enzyme (P450scc) system. The mature forms of the components belonging to P450scc system, CYP11A1, adrenodoxin (Adx), and adrenodoxin reductase (AdR), were coexpressed in a former constructed campesterol producing strain. To maximize pregnenolone production, an integrative components pairing strategy was proposed for pairing the component sources and balancing the expression levels of CYP11A1, Adx, and AdR. Led by the above approaches, a 2344-fold improvement of pregnenolone titer was achieved at the shake flask level. Consequently, a highest reported pregnenolone titer of 78.0 mg/L in microbes was obtained in a 5 L bioreactor. Our study not only highlights the integrative components pairing of cytochrome P450scc as a general strategy for engineering other cytochrome P450s, but also provides a feasible and efficient platform of Y. lipolytica for other steroids production.

COSY catalyses trans-cis isomerization and lactonization in the biosynthesis of coumarins.

Coumarins, also known as 1,2-benzopyrones, comprise a large class of secondary metabolites that are ubiquitously found throughout the plant kingdom. In many plant species, coumarins are particularly important for iron acquisition and plant defence. Here, we show that COUMARIN SYNTHASE (COSY) is a key enzyme in the biosynthesis of coumarins. Arabidopsis thaliana cosy mutants have strongly reduced levels of coumarin and accumulate o-hydroxyphenylpropanoids instead. Accordingly, cosy mutants have reduced iron content and show growth defects when grown under conditions in which there is a limited availability of iron. Recombinant COSY is able to produce umbelliferone, esculetin and scopoletin from their respective o-hydroxycinnamoyl-CoA thioesters by two reaction steps-a trans-cis isomerization followed by a lactonization. This conversion happens partially spontaneously and is catalysed by light, which explains why the need for an enzyme for this conversion has been overlooked. The combined results show that COSY has an essential function in the biosynthesis of coumarins in organs that are shielded from light, such as roots. These findings provide routes to improving coumarin production in crops or by microbial fermentation.

TSPO Ligands Boost Mitochondrial Function and Pregnenolone Synthesis.

Translocator protein 18 kDa (TSPO) is located in the mitochondrial outer membrane and plays an important role in steroidogenesis and cell survival. In the central nervous system (CNS), its expression is upregulated in neuropathologies such as Alzheimer's disease (AD). Previously, we demonstrated that two new TSPO ligands based on an imidazoquinazolinone termed 2a and 2b, stimulated pregnenolone synthesis and ATP production in vitro. In the present study, we compared their effects to those of TSPO ligands described in the literature (XBD173, SSR-180,575, and Ro5-4864) by profiling the mitochondrial bioenergetic phenotype before and after treatment and investigating the protective effects of these ligands after oxidative injury in a cellular model of AD overexpressing amyloid-ß (Aß). Of note, ATP levels increased with rising pregnenolone levels suggesting that the energetic performance of mitochondria is linked to an increased production of this neurosteroid via TSPO modulation. Our results further demonstrate that the TSPO ligands 2a and 2b exerted neuroprotective effects by improving mitochondrial respiration, reducing reactive oxygen species and thereby decreasing oxidative stress-induced cell death as well as lowering Aß levels. The compounds 2a and 2b show similar or even better functional effects than those obtained with the reference TSPO ligands XBD173 and SSR-180.575. These findings indicate that the new TSPO ligands modulate mitochondrial bioenergetic phenotype and protect against oxidative injury probably through the de novo synthesis of neurosteroids, suggesting that these compounds could be potential new therapeutic tools for the treatment of neurodegenerative disease.

Neurosteroids: non-genomic pathways in neuroplasticity and involvement in neurological diseases.

Neurosteroids are neuroactive brain-born steroids. They can act through non-genomic and/or through genomic pathways. Genomic pathways are largely described for steroid hormones: the binding to nuclear receptors leads to transcription regulation. Pregnenolone, Dehydroepiandrosterone, their respective sulfate esters and Allopregnanolone have no corresponding nuclear receptor identified so far whereas some of their non-genomic targets have been identified. Neuroplasticity is the capacity that neuronal networks have to change their structure and function in response to biological and/or environmental signals; it is regulated by several mechanisms, including those that involve neurosteroids. In this review, after a description of their biosynthesis, the effects of Pregnenolone, Dehydroepiandrosterone, their respective sulfate esters and Allopregnanolone on their targets will be exposed. We then shall highlight that neurosteroids, by acting on these targets, can regulate neurogenesis, structural and functional plasticity. Finally, we will discuss the therapeutic potential of neurosteroids in the pathophysiology of neurological diseases in which alterations of neuroplasticity are associated with changes in neurosteroid levels.

Functionalized poly(3-hydroxybutyric acid) bodies as new in vitro biocatalysts.

Cytochromes P450 play a key role in the drug and steroid metabolism in the human body. This leads to a high interest in this class of proteins. Mammalian cytochromes P450 are rather delicate. Due to their localization in the mitochondrial or microsomal membrane, they tend to aggregate during expression and purification and to convert to an inactive form so that they have to be purified and stored in complex buffers. The complex buffers and low storage temperatures, however, limit the feasibility of fast, automated screening of the corresponding cytochrome P450-effector interactions, which are necessary to study substrate-protein and inhibitor-protein interactions. Here, we present the production and isolation of functionalized poly(3-hydroxybutyrate) granules (PHB bodies) from Bacillus megaterium MS941 strain. In contrast to the expression in Escherichia coli, where mammalian cytochromes P450 are associated to the cell membrane, when CYP11A1 is heterologously expressed in Bacillus megaterium, it is located on the PHB bodies. The surface of these particles provides a matrix for immobilization and stabilization of the CYP11A1 during the storage of the protein and substrate conversion. It was demonstrated that the PHB polymer basis is inert concerning the performed conversion. Immobilization of the CYP11A1 onto the PHB bodies allows freeze-drying of the complex without significant decrease of the CYP11A1 activity. This is the first lyophilization of a mammalian cytochrome P450, which allows storage over more than 18days at 4°C instead of storage at -80°C. In addition, we were able to immobilize the cytochrome P450 on the PHB bodies in vitro. In this case the expression of the protein is separated from the production of the immobilization matrix, which widens the application of this method. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Equine fetal adrenal, gonadal and placental steroidogenesis.

Equine fetuses have substantial circulating pregnenolone concentrations and thus have been postulated to provide significant substrate for placental 5α-reduced pregnane production, but the fetal site of pregnenolone synthesis remains unclear. The current studies investigated steroid concentrations in blood, adrenal glands, gonads and placenta from fetuses (4, 6, 9 and 10 months of gestational age (GA)), as well as tissue steroidogenic enzyme transcript levels. Pregnenolone and dehydroepiandrosterone (DHEA) were the most abundant steroids in fetal blood, pregnenolone was consistently higher but decreased progressively with GA. Tissue steroid concentrations generally paralleled those in serum with time. Adrenal and gonadal tissue pregnenolone concentrations were similar and 100-fold higher than those in allantochorion. DHEA was far higher in gonads than adrenals and progesterone was higher in adrenals than gonads. Androstenedione decreased with GA in adrenals but not in gonads. Transcript analysis generally supported these data. CYP17A1 was higher in fetal gonads than adrenals or allantochorion, and HSD3B1 was higher in fetal adrenals and allantochorion than gonads. CYP11A1 transcript was also significantly higher in adrenals and gonads than allantochorion and CYP19 and SRD5A1 transcripts were higher in allantochorion than either fetal adrenals or gonads. Given these data, and their much greater size, the fetal gonads are the source of DHEA and likely contribute more than fetal adrenal glands to circulating fetal pregnenolone concentrations. Low CYP11A1 but high HSD3B1 and SRD5A1 transcript abundance in allantochorion, and low tissue pregnenolone, suggests that endogenous placental pregnenolone synthesis is low and likely contributes little to equine placental 5α-reduced pregnane secretion.

Effect of Mifepristone on Corticosteroid Production in Vitro by Adrenal Glands of Rats with Streptozotocin Diabetes.

Changes in pregnenolone and corticosterone production by the adrenal glands of normoglycemic rats receiving the course of intraperitoneal mifepristone or NaCl administration were co-directed, but differed in magnitude. In rats with hyperglycemia, corticosteroid production increased after NaCl administration over 5 days and returned to the initial values after 15-day administration. On the contrary, pregnenolone and corticosterone production was suppressed after 5-day course of mifepristone, but significantly increased after mifepristone administration for 15 days. Intraperitoneal mifepristone administration almost completely abolished the response of rat adrenal glands with normo- and hyperglycemia to ACTH in vitro; this effect did not depend on the duration of administration.

Short-term effects of diabetes on neurosteroidogenesis in the rat hippocampus.

Diabetes may induce neurophysiological and structural changes in the central nervous system (i.e., diabetic encephalopathy). We here explored whether the levels of neuroactive steroids (i.e., neuroprotective agents) in the hippocampus may be altered by short-term diabetes (i.e., one month). To this aim, by liquid chromatography-tandem mass spectrometry we observed that in the experimental model of the rat raised diabetic by streptozotocin injection, one month of pathology induced changes in the levels of several neuroactive steroids, such as pregnenolone, progesterone and its metabolites (i.e., tetrahydroprogesterone and isopregnanolone) and testosterone and its metabolites (i.e., dihydrotestosterone and 3α-diol). Interestingly these brain changes were not fully reflected by the plasma level changes, suggesting that early phase of diabetes directly affects steroidogenesis and/or steroid metabolism in the hippocampus. These concepts are also supported by the findings that crucial steps of steroidogenic machinery, such as the gene expression of steroidogenic acute regulatory protein (i.e., molecule involved in the translocation of cholesterol into mitochondria) and cytochrome P450 side chain cleavage (i.e., enzyme converting cholesterol into pregnenolone) and 5α-reductase (enzyme converting progesterone and testosterone into their metabolites) are also affected in the hippocampus. In addition, cholesterol homeostasis as well as the functionality of mitochondria, a key organelle in which the limiting step of neuroactive steroid synthesis takes place, are also affected. Data obtained indicate that short-term diabetes alters hippocampal steroidogenic machinery and that these changes are associated with impaired cholesterol homeostasis and mitochondrial dysfunction in the hippocampus, suggesting them as relevant factors for the development of diabetic encephalopathy.

TSPO ligand residence time: a new parameter to predict compound neurosteroidogenic efficacy.

The pharmacological activation of the cholesterol-binding Translocator Protein (TSPO) leads to an increase of endogenous steroids and neurosteroids determining benefic pleiotropic effects in several pathological conditions, including anxiety disorders. The relatively poor relationship between TSPO ligand binding affinities and steroidogenic efficacies prompted us to investigate the time (Residence Time, RT) that a number of compounds with phenylindolylglyoxylamide structure (PIGAs) spends in contact with the target. Here, given the poor availability of TSPO ligand kinetic parameters, a kinetic radioligand binding assay was set up and validated for RT determination using a theoretical mathematical model successfully applied to other ligand-target systems. TSPO ligand RT was quantified and the obtained results showed a positive correlation between the period for which a drug interacts with TSPO and the compound ability to stimulate steroidogenesis. Specifically, the TSPO ligand RT significantly fitted both with steroidogenic efficacy (Emax) and with area under the dose-response curve, a parameter combining drug potency and efficacy. A positive relation between RT and anxiolytic activity of three compounds was evidenced. In conclusion, RT could be a relevant parameter to predict the steroidogenic efficacy and the in vivo anxiolytic action of new TSPO ligands.

2ß- and 16ß-hydroxylase activity of CYP11A1 and direct stimulatory effect of estrogens on pregnenolone formation.

The biosynthesis of steroid hormones in vertebrates is initiated by the cytochrome P450 CYP11A1, which performs the side-chain cleavage of cholesterol thereby producing pregnenolone. In this study, we report a direct stimulatory effect of the estrogens estradiol and estrone onto the pregnenolone formation in a reconstituted in vitro system consisting of purified CYP11A1 and its natural redox partners. We demonstrated the formation of new products from 11-deoxycorticosterone (DOC), androstenedione, testosterone and dehydroepiandrosterone (DHEA) during the in vitro reaction catalyzed by CYP11A1. In addition, we also established an Escherichia coli-based whole-cell biocatalytic system consisting of CYP11A1 and its redox partners to obtain sufficient yields of products for NMR-characterization. Our results indicate that CYP11A1, in addition to the previously described 6ß-hydroxylase activity, possesses a 2ß-hydroxylase activity towards DOC and androstenedione as well as a 16ß-hydroxylase activity towards DHEA. We also showed that CYP11A1 is able to perform the 6ß-hydroxylation of testosterone, a reaction that has been predominantly attributed to CYP3A4. Our results are the first evidence that sex hormones positively regulate the overall production of steroid hormones suggesting the need to reassess the role of CYP11A1 in steroid hormone biosynthesis and its substrate-dependent mechanistic properties.

Production of corticosteroid hormones in vitro by adrenals in rats with streptozotocin-induced diabetes.

We studied baseline and ACTH-stimulated in vitro production of corticosteroids by rat adrenals. Production of the basic corticosteroids pregnenolone (early precursor in corticosteroid synthesis), progesterone (intermediate precursor in synthesis of gluco- and mineralocorticoid hormones), and corticosterone (major glucocorticoid hormone in rodents) in animals with streptozotocin-induced diabetes was enhanced by 1.8-2.0 times in comparison with the control animals. Addition of ACTH to the incubation medium stimulated pregnenolone production by the adrenals equally in the control and experimental (diabetic) groups, while the increase in corticosterone production was less pronounced in the experimental group. Stimulation of corticosterone production in response to ACTH after saturation of the incubation medium with pregnenolone was also less pronounced in diabetic rats.

How steroids steer T cells.

Recent papers from Mahata et al. and Bereshchenko et al. reveal how steroids steer immune responses by tipping T helper (Th) subset balances and activities. Pregnenolone produced by Th2 cells mediates immunosuppressive responses, and glucocorticoids stimulate regulatory T cell development via the induction of GILZ expression.

Single-cell RNA sequencing reveals T helper cells synthesizing steroids de novo to contribute to immune homeostasis.

T helper 2 (Th2) cells regulate helminth infections, allergic disorders, tumor immunity, and pregnancy by secreting various cytokines. It is likely that there are undiscovered Th2 signaling molecules. Although steroids are known to be immunoregulators, de novo steroid production from immune cells has not been previously characterized. Here, we demonstrate production of the steroid pregnenolone by Th2 cells in vitro and in vivo in a helminth infection model. Single-cell RNA sequencing and quantitative PCR analysis suggest that pregnenolone synthesis in Th2 cells is related to immunosuppression. In support of this, we show that pregnenolone inhibits Th cell proliferation and B cell immunoglobulin class switching. We also show that steroidogenic Th2 cells inhibit Th cell proliferation in a Cyp11a1 enzyme-dependent manner. We propose pregnenolone as a "lymphosteroid," a steroid produced by lymphocytes. We speculate that this de novo steroid production may be an intrinsic phenomenon of Th2-mediated immune responses to actively restore immune homeostasis.