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The FEBS Journal Nov 2021Mitochondrial cytochromes P450 presumably originated from a common microsomal P450 ancestor. However, it is still unknown how ancient mitochondrial P450s were able to...
Mitochondrial cytochromes P450 presumably originated from a common microsomal P450 ancestor. However, it is still unknown how ancient mitochondrial P450s were able to retain their oxygenase function following relocation to the mitochondrial matrix and later emerged as enzymes specialized for steroid hormone biosynthesis in vertebrates. Here, we used the approach of ancestral sequence reconstruction (ASR) to resurrect ancient CYP11A1 enzymes and characterize their unique biochemical properties. Two ancestral CYP11A1 variants, CYP11A_Mammal_N101 and CYP11A_N1, as well as an extant bovine form were recombinantly expressed and purified to homogeneity. All enzymes showed characteristic P450 spectral properties and were able to convert cholesterol as well as other sterol substrates to pregnenolone, yet with different specificities. The vertebrate CYP11A_N1 ancestor preferred the cholesterol precursor, desmosterol, as substrate suggesting a convergent evolution of early cholesterol metabolism and CYP11A1 enzymes. Both ancestors were able to withstand increased levels of hydrogen peroxide but only the ancestor CYP11A_N1 showed increased thermostability (˜ 25 °C increase in T ) compared with the extant CYP11A1. The extraordinary robustness of ancient mitochondrial P450s, as demonstrated for CYP11A_N1, may have allowed them to stay active when presented with poorly compatible electron transfer proteins and resulting harmful ROS in the new environment of the mitochondrial matrix. To the best of our knowledge, this work represents the first study that describes the resurrection of ancient mitochondrial P450 enzymes. The results will help to understand and gain fundamental functional insights into the evolutionary origins of steroid hormone biosynthesis in animals.
Topics: Cholesterol Side-Chain Cleavage Enzyme; Humans; Phylogeny
PubMed: 34092040
DOI: 10.1111/febs.16054 -
Neurochemistry International Feb 2023Pregnenolone is a precursor of various steroid hormones involved in osteoblast proliferation, microtubules polymerization and cell survival protection. Previous reports...
BACKGROUND
Pregnenolone is a precursor of various steroid hormones involved in osteoblast proliferation, microtubules polymerization and cell survival protection. Previous reports focused on the effects of pregnenolone metabolites on stem cell proliferation and differentiation; however, the effects of pregnenolone itself has not been well explored. The present study aimed to investigate the role of pregnenolone on NSC proliferation and to determine the doses required for NSC differentiation as well as the various genes involved in its mechanism of action.
METHODS
NSCs were isolated from the embryonic cortex of E14 mice, incubated for 5 days, and then treated with pregnenolone doses of 2, 5, 10, 15 and 20 μM for another 5 days. The number of neurospheres and neurosphere derived cells were then counted. Flow cytometry was used to evaluate the differentiation of NSCs into oligodendrocytes, astrocytes, and neurons. The expression level of Notch1, Pax6 and Sox10 genes were also measured by Real Time PCR after 5 days of treatment.
RESULTS
Our data suggest that treatment with 10 μM pregnenolone is optimal for NSC proliferation. In fact, this concentration caused the highest increase in the number of neurospheres and neurosphere derived cells, compared to the control group. In addition, treatment with low doses of pregnenolone (5 and 10 μM) caused a significant increase in NSC differentiation towards immature (Olig2) and mature (MBP) oligodendrocyte cell populations, compared to controls. However, NSC differentiation into neurons (beta III tubulin cells) increased in all treatment groups, with the highest and most significant increase obtained at 15 μM concentration. It is worth noting that pregnenolone at the highest concentration of 15 μM decreased the number of astrocytes (GFAP+). Furthermore, there was an increase of Sox10 expression with low pregnenolone doses, leading to oligodendrogenesis, whereas Notch1 and Pax6 gene expression increased in pregnenolone groups with more neurogenesis.
CONCLUSION
Pregnenolone regulates NSCs proliferation in vitro. Treatment with low doses of pregnenolone caused an increase in the differentiation of NSCs into mature oligodendrocytes while higher doses increased the differentiation of NSCs into neurons. Oligodendrogenesis was accompanied by Sox10 while neurogenesis occurred together with Notch1 and Pax6 expression.
Topics: Animals; Mice; Cell Differentiation; Cell Proliferation; Cells, Cultured; Neural Stem Cells; Neurogenesis; Neurons; PAX6 Transcription Factor; SOXE Transcription Factors; Tubulin; Pregnenolone; Receptor, Notch1
PubMed: 36657722
DOI: 10.1016/j.neuint.2023.105489 -
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 -
Human Genomics Feb 2020First discovered in a light-sensitive retinal mutant of Drosophila, the transient receptor potential (TRP) superfamily of non-selective cation channels serve as... (Review)
Review
First discovered in a light-sensitive retinal mutant of Drosophila, the transient receptor potential (TRP) superfamily of non-selective cation channels serve as polymodal cellular sensors that participate in diverse physiological processes across the animal kingdom including the perception of light, temperature, pressure, and pain. TRPM3 belongs to the melastatin sub-family of TRP channels and has been shown to function as a spontaneous calcium channel, with permeability to other cations influenced by alternative splicing and/or non-canonical channel activity. Activators of TRPM3 channels include the neurosteroid pregnenolone sulfate, calmodulin, phosphoinositides, and heat, whereas inhibitors include certain drugs, plant-derived metabolites, and G-protein subunits. Activation of TRPM3 channels at the cell membrane elicits a signal transduction cascade of mitogen-activated kinases and stimulus response transcription factors. The mammalian TRPM3 gene hosts a non-coding microRNA gene specifying miR-204 that serves as both a tumor suppressor and a negative regulator of post-transcriptional gene expression during eye development in vertebrates. Ocular co-expression of TRPM3 and miR-204 is upregulated by the paired box 6 transcription factor (PAX6) and mutations in all three corresponding genes underlie inherited forms of eye disease in humans including early-onset cataract, retinal dystrophy, and coloboma. This review outlines the genomic and functional complexity of the TRPM3_miR-204 locus in mammalian eye development and disease.
Topics: Eye Diseases; Gene Expression Regulation; Genetic Loci; Humans; MicroRNAs; TRPM Cation Channels
PubMed: 32070426
DOI: 10.1186/s40246-020-00258-4 -
Applied Microbiology and Biotechnology Jan 2022In this paper, we studied the transformation of two 3β-hydroxy-5-ene-steroids-pregnenolone and dehydroepiandrosterone (DHEA) by Backusella lamprospora VKM F- 944. The...
In this paper, we studied the transformation of two 3β-hydroxy-5-ene-steroids-pregnenolone and dehydroepiandrosterone (DHEA) by Backusella lamprospora VKM F- 944. The soil-dwelling zygomycete wild-type strain has been earlier selected during the screening and previously unexplored for this purpose. The fungus fully converted pregnenolone to form a mixture of axial 7α-hydroxy-pregnenolone and 7α,11α-dihydroxy-pregnenolone, while no metabolites with β-orientation of the hydroxyl group were detected. The pathway to 7α,11α-diOH-pregnenolone seems to include 7α-hydroxylation of 11α-hydroxylated derivative. The only product from DHEA was identified as 7α-hydroxy-DHEA. The structures of steroid metabolites were confirmed by HPLC, mass-spectrometry (MS), and H and C NMR analyses. Under the optimized conditions, the yield of 7α-OH-DHEA reached 94% (w/w) or over 14 g/L in absolute terms, even at high concentration of the substrate (DHEA) (15 g/L). To our knowledge, it is the highest yield of the value-added 7α-OH-DHEA reported so far. The results contribute to the knowledge of the diversity of the wild-type fungal strains capable of effective steroid hydroxylation. They could be applied for the production of allylic steroid 7α-alcohols that are widely used in medicine. KEY POINTS: • Zygomycete Backusella lamprospora actively hydroxylates 3β-hydroxy-5-en-steroids. • Axial 7α-hydroxylation is the preferable reaction by the strain towards pregnenolone and DHEA. • The strain selectively produces 7α-OH-DHEA even at high substrate concentrations (up to 15 g/L).
Topics: Dehydroepiandrosterone; Hydroxylation; Mucorales; Pregnenolone
PubMed: 34939135
DOI: 10.1007/s00253-021-11737-6 -
Toxicology Sep 2019Human placental 3β-hydroxysteroid dehydrogenase/steroid Δ5, 4-isomerase 1 (HSD3B1), a high-affinity type I enzyme, uses pregnenolone to make progesterone, which is... (Review)
Review
Human placental 3β-hydroxysteroid dehydrogenase/steroid Δ5, 4-isomerase 1 (HSD3B1), a high-affinity type I enzyme, uses pregnenolone to make progesterone, which is critical for maintenance of pregnancy. HSD3B1 is located in the mitochondrion and the smooth endoplasmic reticulum of placental cells and is encoded by HSD3B1 gene. HSD3B1 contains GATA and TEF-5 regulatory elements. Many endocrine disruptors, including phthalates, methoxychlor and its metabolite, organotins, and gossypol directly inhibit placental HSD3B1 thus blocking progesterone production. In this review, we discuss the placental HSD3B1, its gene regulation, biochemistry, subcellular location, and inhibitors from the environment.
Topics: Environmental Pollutants; Female; Gene Expression Regulation; Humans; Multienzyme Complexes; Placenta; Pregnancy; Progesterone Reductase; Steroid Isomerases
PubMed: 31351905
DOI: 10.1016/j.tox.2019.152253 -
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 -
The Journal of Steroid Biochemistry and... Nov 2023Starting with pregnenolone, a 20-carbonyl group was converted into an amino group through a series of chemical reactions. This amino group was further converted into...
Starting with pregnenolone, a 20-carbonyl group was converted into an amino group through a series of chemical reactions. This amino group was further converted into selenocyanoalkylamide, leading to the synthesis of six pregnenolone selenocyanoalkylamide derivatives. These compounds were then screened for antitumor activity in vitro, yielding promising results. Compounds 4b-4f show higher inhibitory activity than the positive control abiraterone and 2-methoxyestradiol, with IC values lower than 10 μmol/L against breast, ovarian, and cervical cancer cell lines that closely related to human hormone expression levels. The Annexin V assay of compound 4f revealed that compounds inhibited tumor cell proliferation primarily through the induction of programmed apoptosis. The zebrafish test results indicated that compound 4d had significant inhibitory activity against MCF-7 cell xenografts in vivo. Moreover, the antibacterial test indicated that compounds 4a and 4d-4e had better inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) than the positive controls vancomycin and ampicillin. These results suggest that these compounds may hold promise as novel antitumor agents or antimicrobial agents for further study.
Topics: Animals; Humans; Vancomycin; Methicillin-Resistant Staphylococcus aureus; Pregnenolone; Zebrafish; Anti-Bacterial Agents; Antineoplastic Agents
PubMed: 37652364
DOI: 10.1016/j.jsbmb.2023.106388 -
Journal of Biomedical Science Aug 2022CYP11A1 is a protein located in the inner membrane of mitochondria catalyzing the first step of steroid synthesis. As a marker gene for steroid-producing cells, the...
BACKGROUND
CYP11A1 is a protein located in the inner membrane of mitochondria catalyzing the first step of steroid synthesis. As a marker gene for steroid-producing cells, the abundance of CYP11A1 characterizes the extent of steroidogenic cell differentiation. Besides, the mitochondria of fully differentiated steroidogenic cells are specialized with tubulovesicular cristae. The participation of CYP11A1 in the change of mitochondrial structure and the differentiation of steroid-producing cells, however, has not been investigated.
METHODS
We engineered nonsteroidogenic monkey kidney COS1 cells to express CYP11A1 upon doxycycline induction and examined the mitochondrial structure of these cells. We also mapped the CYP11A1 domains that confer structural changes of mitochondria. We searched for CYP11A1-interacting proteins and investigated the role of this interacting protein in shaping mitochondrial structure. Finally, we examined the effect of CYP11A1 overexpression on the amount of mitochondrial contact site and cristae organizing system.
RESULTS
We found that CYP11A1 overexpression led to the formation of tubulovesicular cristae in mitochondria. We also identified the A'-helix located at amino acid #57-68 to be sufficient for membrane insertion and crista remodeling. We identified heat shock protein 60 (Hsp60) as the CYP11A1-interacting protein and showed that Hsp60 is required for CYP11A1 accumulation and crista remodeling. Finally, we found that the small MIC10 subcomplex of the mitochondrial contact site and cristae organizing system was reduced when CYP11A1 was overexpressed.
CONCLUSIONS
CYP11A1 participates in the formation of tubulovesicular cristae in the mitochondria of steroidogenic cells. Its A'-helix is sufficient for the formation of tubulovesicular cristae and for protein integration into the membrane. CYP11A1 interacts with Hsp60, which is required for CYP11A1 accumulation. The accumulation of CYP11A1 leads to the reduction of MIC10 complex and changes mitochondrial structure.
Topics: Cholesterol Side-Chain Cleavage Enzyme; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Steroids
PubMed: 35978408
DOI: 10.1186/s12929-022-00846-7 -
Toxicological Sciences : An Official... Mar 2022Perfluorooctanoic acid (PFOA) is a synthetic fluorosurfactant used in the manufacturing of fluorotelomers. Although PFOA is no longer produced in the United States, it...
Perfluorooctanoic acid (PFOA) is a synthetic fluorosurfactant used in the manufacturing of fluorotelomers. Although PFOA is no longer produced in the United States, it is environmentally persistent and found in imported food packaging, cookware, and textiles. Previous studies have identified developmental toxicity of PFOA, but little is known about the effects of PFOA on the adult ovary. Thus, this study examined the effects of PFOA on hormone levels, ovarian steroidogenic gene expression, and folliculogenesis in mice in vitro and in vivo. For the in vitro studies, antral follicles from adult female mice were cultured with vehicle control or 1, 10, or 100 μg/ml PFOA for 96 h. For the in vivo studies, adult CD-1 female mice were orally dosed with vehicle control or 1, 5, 10, or 20 mg/kg/day PFOA for 10 days. Gene expression of steroidogenic enzymes, levels of sex steroid hormones, and follicle counts were analyzed. In vitro, PFOA (100 μg/ml) significantly decreased follicle growth, estradiol and estrone levels, and gene expression of StaR, Cyp11a1, and Hsd3b1 compared with controls. In vivo, exposure to PFOA significantly decreased progesterone and pregnenolone levels (5 mg/kg), increased testosterone levels (1 mg/kg), and increased gene expression of Cyp19a1 (1 mg/kg) compared with controls. Exposure to PFOA also significantly altered follicle counts by decreasing primordial follicles and increasing preantral and antral follicles (5 and 10 mg/kg) compared with controls. Collectively, these data show that PFOA disrupts adult ovarian function in a nonmonotonic matter and may pose a risk for premature ovarian failure.
Topics: Animals; Caprylates; Estradiol; Female; Fluorocarbons; Mice; Ovarian Follicle; Ovary
PubMed: 35104888
DOI: 10.1093/toxsci/kfac005