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Frontiers in Cell and Developmental... 2023The sperm-specific channel CatSper (cation channel of sperm) controls the intracellular Ca concentration ([Ca]) and plays an essential role in sperm function. It is...
The sperm-specific channel CatSper (cation channel of sperm) controls the intracellular Ca concentration ([Ca]) and plays an essential role in sperm function. It is mainly activated by the steroid progesterone (P4) but is also promiscuously activated by a wide range of synthetic and physiological compounds. These compounds include diverse steroids whose action on the channel is so far still controversial. To investigate the effect of these compounds on CatSper and sperm function, we developed a high-throughput screening (HTS) assay to measure changes in [Ca] in human sperm and screened 1,280 approved and off-patent drugs including 90 steroids from the Prestwick chemical library. More than half of the steroids tested (53%) induced an increase in [Ca] and reduced the P4-induced Ca influx in human sperm in a dose-dependent manner. Ten of the most potent steroids (activating and P4-inhibiting) were selected for a detailed analysis of their action on CatSper and their ability to act on sperm acrosome reaction (AR) and penetration in viscous media. We found that these steroids show an inhibitory effect on P4 but not on prostaglandin E1-induced CatSper activation, suggesting that they compete for the same binding site as P4. Pregnenolone, dydrogesterone, epiandrosterone, nandrolone, and dehydroepiandrosterone acetate (DHEA) were found to activate CatSper at physiologically relevant concentrations within the nanomolar range. Like P4, most tested steroids did not significantly affect the AR while stanozolol and estropipate slightly increased sperm penetration into viscous medium. Furthermore, using a hybrid approach integrating pharmacophore analysis and statistical modelling, we were able to screen for steroids that can activate the channel and define the physicochemical and structural properties required for a steroid to exhibit agonist activity against CatSper. Overall, our results indicate that not only physiological but also synthetic steroids can modulate the activity of CatSper with varying potency and if bound to CatSper prior to P4, could impair the timely CatSper activation necessary for proper fertilization to occur.
PubMed: 37547474
DOI: 10.3389/fcell.2023.1221578 -
CNS Neuroscience & Therapeutics Nov 2023As a devastating neurological disease, spinal cord injury (SCI) results in severe tissue loss and neurological dysfunction. Pregnane X receptor (PXR) is a...
INTRODUCTION
As a devastating neurological disease, spinal cord injury (SCI) results in severe tissue loss and neurological dysfunction. Pregnane X receptor (PXR) is a ligand-activated nuclear receptor with a major regulatory role in xenobiotic and endobiotic metabolism and recently has been implicated in the central nervous system. In the present study, we aimed to investigate the role and mechanism of PXR in SCI.
METHODS
The clip-compressive SCI model was performed in male wild-type C57BL/6 (PXR ) and PXR-knockout (PXR ) mice. The N2a H O -induced injury model mimicked the pathological process of SCI in vitro. Pregnenolone 16α-carbonitrile (PCN), a mouse-specific PXR agonist, was used to activate PXR in vivo and in vitro. The siRNA was applied to knock down the PXR expression in vitro. Transcriptome sequencing analysis was performed to discover the relevant mechanism, and the NRF2 inhibitor ML385 was used to validate the involvement of PXR in influencing the NRF2/HO-1 pathway in the SCI process.
RESULTS
The expression of PXR decreased after SCI and reached a minimum on the third day. In vivo, PXR knockout significantly improved the motor function of mice after SCI, meanwhile, inhibited apoptosis, inflammation, and oxidative stress induced by SCI. On the contrary, activation of PXR by PCN negatively influenced the recovery of SCI. Mechanistically, transcriptome sequencing analysis revealed that PXR activation downregulated the mRNA level of heme oxygenase-1 (HO-1) after SCI. We further verified that PXR deficiency activated the NRF2/HO-1 pathway and PXR activation inhibited this pathway in vitro.
CONCLUSION
PXR is involved in the recovery of motor function after SCI by regulating NRF2/HO-1 pathway.
Topics: Animals; Male; Mice; Heme Oxygenase-1; Mice, Inbred C57BL; NF-E2-Related Factor 2; Pregnane X Receptor; Spinal Cord Injuries
PubMed: 37269088
DOI: 10.1111/cns.14279 -
Biological Procedures Online Nov 2023Renal cancer therapies are challenging owing to the extensive spreading of this cancer to other organs and its ability to pose resistance to current medications....
BACKGROUND
Renal cancer therapies are challenging owing to the extensive spreading of this cancer to other organs and its ability to pose resistance to current medications. Therefore, drugs targeting novel targets are urgently required to overcome these challenges. The cholesterol side-chain cleavage enzyme (CYP11A1) is closely associated with steroidogenesis, and its downregulation is linked to adrenal dysfunction and several types of carcinoma. We previously found that overexpression of CYP11A1 inhibited epithelial-mesenchymal transition and induced G2/M arrest in the kidney cancer Caki-1 cell line. In this context, natural compounds that exhibit potent CYP11A1 stimulation activity can be promising therpaeutic agents for kidney cancer.
METHODS
We screened a panel of 1374 natural compounds in a wound-healing assay using CYP11A1-transfected Caki-1 cells. Of these, 167 promising biologically active compounds that inhibited cancer cell migration by more than 75% were selected, and their half-maximal inhibitory concentrations (IC) were determined. The IC of 159 compounds was determined and 38 compounds with IC values less than 50 µM were selected for further analysis. Steroid hormones (cholesterol and pregnenolone) levels in cells treated with the selected compounds were quantitated using LC-MS/MS to determine their effect on CYP11A1 activity. Western blotting for CYP11A1, autophagy signaling proteins, and ferroptosis regulators were performed to ivestigate the mechanisms underlying the action of the selected compounds.
RESULTS
We screened five promising natural lead compounds that inhibited cancer cell proliferation after three screening steps. The IC of these compounds was determined to be between 5.9 and 14.6 μM. These candidate compounds increased the expression of CYP11A1 and suppressed cholesterol levels while increasing pregnenolone levels, which is consistent with the activation of CYP11A1. Our results showed that CYP11A1 activation inhibited the migration of cancer cells, promoted ferroptosis, and triggered autophagy signaling.
CONCLUSIONS
This study indicates that the CYP11A1-overexpressing Caki-1 cell line is useful for screening drugs against kidney cancer. The two selected compounds could be utilized as lead compounds for anticancer drug discovery, and specifically for the development of antirenal cancer medication.
PubMed: 38036976
DOI: 10.1186/s12575-023-00225-y -
Nature Plants Oct 2023Cardenolides are specialized, steroidal metabolites produced in a wide array of plant families. Cardenolides play protective roles in plants, but these molecules,...
Cardenolides are specialized, steroidal metabolites produced in a wide array of plant families. Cardenolides play protective roles in plants, but these molecules, including digoxin from foxglove (Digitalis spp.), are better known for treatment of congenital heart failure, atrial arrhythmia, various cancers and other chronic diseases. However, it is still unknown how plants synthesize 'high-value', complex cardenolide structures from, presumably, a sterol precursor. Here we identify two cytochrome P450, family 87, subfamily A (CYP87A) enzymes that act on both cholesterol and phytosterols (campesterol and β-sitosterol) to form pregnenolone, the first committed step in cardenolide biosynthesis in the two phylogenetically distant plants Digitalis purpurea and Calotropis procera. Arabidopsis plants overexpressing these CYP87A enzymes ectopically accumulated pregnenolone, whereas silencing of CYP87A in D. purpurea leaves by RNA interference resulted in substantial reduction of pregnenolone and cardenolides. Our work uncovers the key entry point to the cardenolide pathway, and expands the toolbox for sustainable production of high-value plant steroids via synthetic biology.
Topics: Cardenolides; Plants; Digitalis; Pregnenolone
PubMed: 37723202
DOI: 10.1038/s41477-023-01515-9 -
RSC Advances May 2024The amyloid state, which is a specific conformation of proteins, offers valuable information about both functional protein structures and the pathological assemblies...
Pregnenolone derivatives for the treatment of Alzheimer's disease: synthesis, and inhibition of amyloid β peptide aggregation, acetylcholinesterase and carbonic anhydrase-II.
The amyloid state, which is a specific conformation of proteins, offers valuable information about both functional protein structures and the pathological assemblies associated with various diseases. One of the major hallmarks of Alzheimer's disease includes primarily the extracellular build-up of a peptide known as amyloid-β, which has a sequence consisting of 39 to 42 amino acid residues, and the formation of intracellular neurofibrillary tangles mostly consisting of hyperphosphorylated tau protein. Drugs that are expected to reduce Aβ production, prevent Aβ aggregation, and promote Aβ clearance are promising approaches for treating AD. Current work is focused on identifying the compounds that have balanced even mild biological activities against multiple targets instead of finding one-target compound with high potency. We synthesized pregnenolone derivatives and evaluated their potential against inhibition of eeAChE/eqBChE, hCA-II and self-mediated Aβ peptide aggregation. Our synthesized derivatives 23, and 25-27 exhibited concomitant inhibition of all the tested macromolecular targets. All the active compounds were found to be BBB penetrants in the PAMPA assay. Furthermore, these selected compounds were found to be non-neurotoxic in the MTT assay on neuroblastoma SH-SY5Y cells. Docking studies support dual binding site (PAS and CAS) inhibition of AChE which showed Aβ aggregation and AChE inhibition. Moreover, docking studies carried out on the 3D crystallographic structure of Aβ peptide (PDB ID = 1IYT) showed significant interactions with amino acid residues Asp 23 and Lys 28, and hydrophobic interactions with the Phe19, Phe20, and Ala 30 effectively impeding the formation of β-sheet structures.
PubMed: 38716099
DOI: 10.1039/d4ra01536c -
Environmental Health : a Global Access... Sep 2023Knowledge of whether prenatal exposure to ambient air pollution disrupts steroidogenesis is currently lacking. We investigated the association between prenatal ambient...
Knowledge of whether prenatal exposure to ambient air pollution disrupts steroidogenesis is currently lacking. We investigated the association between prenatal ambient air pollution and highly accurate measurements of cord blood steroid hormones from the androgenic pathway.This study included 397 newborns born between the years 2010 and 2015 from the ENVIRONAGE cohort in Belgium of whom six cord blood steroid levels were measured: 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, dehydroepiandrosterone, pregnenolone, androstenedione, and testosterone. Maternal ambient exposure to PM (particles with aerodynamic diameter ≤ 2.5 μm), NO and black carbon (BC) were estimated daily during the entire pregnancy using a high-resolution spatiotemporal model. The associations between the cord blood steroids and the air pollutants were tested and estimated by first fitting linear regression models and followed by fitting weekly prenatal exposures to distributed lag models (DLM). These analyses accounted for possible confounders, coexposures, and an interaction effect between sex and the exposure. We examined mixture effects and critical exposure windows of PM, NO and BC on cord blood steroids via the Bayesian kernel machine regression distributed lag model (BKMR-DLM).An interquartile range (IQR) increment of 7.96 µg/m in PM exposure during pregnancy trimester 3 was associated with an increase of 23.01% (99% confidence interval: 3.26-46.54%) in cord blood levels of 17α-hydroxypregnenolone, and an IQR increment of 0.58 µg/m³ in BC exposure during trimester 1 was associated with a decrease of 11.00% (99% CI: -19.86 to -0.012%) in cord blood levels of androstenedione. For these two models, the DLM statistics identified sensitive gestational time windows for cord blood steroids and ambient air pollution exposures, in particular for 17α-hydroxypregnenolone and PM exposure during trimester 3 (weeks 28-36) and for androsterone and BC exposure during early pregnancy (weeks 2-13) as well as during mid-pregnancy (weeks 18-26). We identified interaction effects between pollutants, which has been suggested especially for NO.Our results suggest that prenatal exposure to ambient air pollutants during pregnancy interferes with steroid levels in cord blood. Further studies should investigate potential early-life action mechanisms and possible later-in-life adverse effects of hormonal disturbances due to air pollution exposure.
Topics: Infant, Newborn; Female; Pregnancy; Humans; 17-alpha-Hydroxypregnenolone; Androstenedione; Bayes Theorem; Birth Cohort; Fetal Blood; Nitrogen Dioxide; Prenatal Exposure Delayed Effects; Air Pollution; Steroids; Air Pollutants; Particulate Matter
PubMed: 37674219
DOI: 10.1186/s12940-023-01010-w -
Frontiers in Immunology 2023Pregnenolone (P5) is synthesized as the first bioactive steroid in the mitochondria from cholesterol. Clusters of differentiation 4 (CD4+) and Clusters of...
Pregnenolone (P5) is synthesized as the first bioactive steroid in the mitochondria from cholesterol. Clusters of differentiation 4 (CD4+) and Clusters of differentiation 8 (CD8+) immune cells synthesize P5 ; P5, in turn, play important role in immune homeostasis and regulation. However, P5's biochemical mode of action in immune cells is still emerging. We envisage that revealing the complete spectrum of P5 target proteins in immune cells would have multifold applications, not only in basic understanding of steroids biochemistry in immune cells but also in developing new therapeutic applications. We employed a CLICK-enabled probe to capture P5-binding proteins in live T helper cell type 2 (Th2) cells. Subsequently, using high-throughput quantitative proteomics, we identified the P5 interactome in CD4+ Th2 cells. Our study revealed P5's mode of action in CD4+ immune cells. We identified novel proteins from mitochondrial and endoplasmic reticulum membranes to be the primary mediators of P5's biochemistry in CD4+ and to concur with our earlier finding in CD8+ immune cells. Applying advanced computational algorithms and molecular simulations, we were able to generate near-native maps of P5-protein key molecular interactions. We showed bonds and interactions between key amino acids and P5, which revealed the importance of ionic bond, hydrophobic interactions, and water channels. We point out that our results can lead to designing of novel molecular therapeutics strategies.
Topics: Pregnenolone; Th2 Cells; Molecular Dynamics Simulation; Steroids; Carrier Proteins
PubMed: 38022565
DOI: 10.3389/fimmu.2023.1229703 -
Cell Reports Mar 2024Osteoclasts play a central role in cancer-cell-induced osteolysis, but the molecular mechanisms of osteoclast activation during bone metastasis formation are...
Osteoclasts play a central role in cancer-cell-induced osteolysis, but the molecular mechanisms of osteoclast activation during bone metastasis formation are incompletely understood. By performing RNA sequencing on a mouse breast carcinoma cell line with higher bone-metastatic potential, here we identify the enzyme CYP11A1 strongly upregulated in osteotropic tumor cells. Genetic deletion of Cyp11a1 in tumor cells leads to a decreased number of bone metastases but does not alter primary tumor growth and lung metastasis formation in mice. The product of CYP11A1 activity, pregnenolone, increases the number and function of mouse and human osteoclasts in vitro but does not alter osteoclast-specific gene expression. Instead, tumor-derived pregnenolone strongly enhances the fusion of pre-osteoclasts via prolyl 4-hydroxylase subunit beta (P4HB), identified as a potential interaction partner of pregnenolone. Taken together, our results demonstrate that Cyp11a1-expressing tumor cells produce pregnenolone, which is capable of promoting bone metastasis formation and osteoclast development via P4HB.
Topics: Humans; Female; Osteogenesis; Cholesterol Side-Chain Cleavage Enzyme; Cell Line, Tumor; Bone Neoplasms; Osteoclasts; Pregnenolone; Breast Neoplasms; Cell Differentiation
PubMed: 38489269
DOI: 10.1016/j.celrep.2024.113936 -
Life (Basel, Switzerland) Apr 2024Pregnane neuroactive steroids, notably allopregnanolone and pregnenolone, exhibit efficacy in mitigating inflammatory signals triggered by toll-like receptor (TLR)... (Review)
Review
Pregnane neuroactive steroids, notably allopregnanolone and pregnenolone, exhibit efficacy in mitigating inflammatory signals triggered by toll-like receptor (TLR) activation, thus attenuating the production of inflammatory factors. Clinical studies highlight their therapeutic potential, particularly in conditions like postpartum depression (PPD), where the FDA-approved compound brexanolone, an intravenous formulation of allopregnanolone, effectively suppresses TLR-mediated inflammatory pathways, predicting symptom improvement. Additionally, pregnane neurosteroids exhibit trophic and anti-inflammatory properties, stimulating the production of vital trophic proteins and anti-inflammatory factors. Androstane neuroactive steroids, including estrogens and androgens, along with dehydroepiandrosterone (DHEA), display diverse effects on TLR expression and activation. Notably, androstenediol (ADIOL), an androstane neurosteroid, emerges as a potent anti-inflammatory agent, promising for therapeutic interventions. The dysregulation of immune responses via TLR signaling alongside reduced levels of endogenous neurosteroids significantly contributes to symptom severity across various neuropsychiatric disorders. Neuroactive steroids, such as allopregnanolone, demonstrate efficacy in alleviating symptoms of various neuropsychiatric disorders and modulating neuroimmune responses, offering potential intervention avenues. This review emphasizes the significant therapeutic potential of neuroactive steroids in modulating TLR signaling pathways, particularly in addressing inflammatory processes associated with neuropsychiatric disorders. It advances our understanding of the complex interplay between neuroactive steroids and immune responses, paving the way for personalized treatment strategies tailored to individual needs and providing insights for future research aimed at unraveling the intricacies of neuropsychiatric disorders.
PubMed: 38792602
DOI: 10.3390/life14050582 -
Deubiquitinase UCHL1 regulates estradiol synthesis by stabilizing voltage-dependent anion channel 2.The Journal of Biological Chemistry Nov 2023Lack of estradiol production by granulosa cells blocks follicle development, causes failure of estrous initiation, and results in an inability to ovulate. The...
Lack of estradiol production by granulosa cells blocks follicle development, causes failure of estrous initiation, and results in an inability to ovulate. The ubiquitin-proteasome system plays a critical role in maintaining protein homeostasis and stability of the estrous cycle, but knowledge of deubiquitination enzyme function in estradiol synthesis is limited. Here, we observe that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) is more significant in estrous sows and high litter-size sows than in nonestrous sows and low-yielding sows. Overexpression of UCHL1 promotes estradiol synthesis in granulosa cells, and interference with UCHL1 has the opposite effect. UCHL1 binds, deubiquitinates, and stabilizes voltage-dependent anion channel 2 (VDAC2), promoting the synthesis of the estradiol precursor pregnenolone. Cysteine 90 (C90) of UCHL1 is necessary for its deubiquitination activity, and Lys45 and Lys64 in VDAC2 are essential for its ubiquitination and degradation. In vivo, compared with WT and sh-NC-AAV groups, the estrus cycle of female mice is disturbed, estradiol level is decreased, and the number of antral follicles is decreased after the injection of sh-UCHL1-AAV into ovarian tissue. These findings suggest that UCHL1 promotes estradiol synthesis by stabilizing VDAC2 and identify UCHL1 as a candidate gene affecting reproductive performance.
Topics: Animals; Female; Mice; Estradiol; Granulosa Cells; Ovarian Follicle; Swine; Ubiquitin Thiolesterase; Voltage-Dependent Anion Channel 2; Sus scrofa
PubMed: 37797697
DOI: 10.1016/j.jbc.2023.105316