Effect of vitamin A deprivation on the cholesterol side-chain cleavage enzyme activity of testes and ovaries of rats.
Ontology highlight
ABSTRACT:
SUBMITTER: Jayaram M
PROVIDER: S-EPMC1165941 | biostudies-other | 1973 Sep
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Mitochondrial cytochrome P450 11A1 (CYP11A1 or P450 11A1) is the only known enzyme that cleaves the side chain of cholesterol, yielding pregnenolone, the precursor of all steroid hormones. Pregnenolone is formed via three sequential monooxygenation reactions that involve the progressive production of 22R-hydroxycholesterol (22HC) and 20α,22R-dihydroxycholesterol, followed by the cleavage of the C20-C22 bond. Herein, we present the 2.5-Å crystal structure of CYP11A1 in complex with the first reaction intermediate, 22HC. The active site cavity in CYP11A1 represents a long curved tube that extends from the protein surface to the heme group, the site of catalysis. 22HC occupies two-thirds of the cavity with the 22R-hydroxyl group nearest the heme, 2.56 Å from the iron. The space at the entrance to the active site is not taken up by 22HC but filled with ordered water molecules. The network formed by these water molecules allows the "soft" recognition of the 22HC 3β-hydroxyl. Such a mode of 22HC binding suggests shuttling of the sterol intermediates between the active site entrance and the heme group during the three-step reaction. Translational freedom of 22HC and torsional motion of its aliphatic tail are supported by solution studies. The CYP11A1-22HC co-complex also provides insight into the structural basis of the strict substrate specificity and high catalytic efficiency of the enzyme and highlights conserved structural motifs involved in redox partner interactions by mitochondrial P450s.
| S-EPMC3037674 | biostudies-literature
Project description:ContextThe cholesterol side-chain cleavage enzyme (P450scc), encoded by the CYP11A1 gene, converts cholesterol to pregnenolone to initiate steroidogenesis. Genetic defects in P450scc cause a rare autosomal recessive disorder that is clinically indistinguishable from congenital lipoid adrenal hyperplasia (lipoid CAH). Nonclassic lipoid CAH is a recently recognized disorder caused by mutations in the steroidogenic acute regulatory protein (StAR) that retain partial function.ObjectiveWe describe two siblings with hormonal findings suggesting nonclassic lipoid CAH, who had a P450scc mutation that retains partial function.Patients and methodsA 46,XY male presented with underdeveloped genitalia and partial adrenal insufficiency; his 46,XX sister presented with adrenal insufficiency. Hormonal studies suggested nonclassic lipoid CAH. Sequencing of the StAR gene was normal, but compound heterozygous mutations were found in the CYP11A1 gene. Mutations were recreated in the F2 plasmid expressing a fusion protein of the cholesterol side-chain cleavage system. P450scc activity was measured as Vmax/Km for pregnenolone production in transfected COS-1 cells.ResultsThe patients were compound heterozygous for the previously described frameshift mutation 835delA and the novel missense mutation A269V. When expressed in the P450scc moiety of F2, the A269V mutant retained 11% activity of the wild-type F2 protein.ConclusionsThere is a broad clinical spectrum of P450scc deficiency. Partial loss-of-function CYP11A1 mutation can present with a hormonal phenotype indistinguishable from nonclassic lipoid CAH.
| S-EPMC3047228 | biostudies-literature
Project description:The cholesterol side-chain cleavage enzyme P450scc, encoded by CYP11A1, converts cholesterol to pregnenolone to initiate steroidogenesis. P450scc deficiency can disrupt adrenal and gonadal steroidogenesis, resembling congenital lipoid adrenal hyperplasia clinically and hormonally; only 12 such patients have been reported previously.We sought to expand clinical and genetic experience with P450scc deficiency.We sequenced candidate genes in 7 children with adrenal insufficiency who lacked disordered sexual development. P450scc missense mutations were recreated in the F2 vector, which expresses the fusion protein P450scc-Ferredoxin Reductase-Ferredoxin. COS-1 cells were transfected, production of pregnenolone was assayed, and apparent kinetic parameters were calculated. Previously described P450scc mutants were assayed in parallel.Four of five Bedouin children in one kindred were compound heterozygotes for mutations c.694C>T (Arg232Stop) and c.644T>C (Phe215Ser). Single-nucleotide polymorphism analysis confirmed segregation of these mutations. The fifth kindred member and another Bedouin patient presented in infancy and were homozygous for Arg232Stop. A patient from Fiji presenting in infancy was homozygous for c.358T>C (Arg120Stop). All mutations are novel. As assayed in the F2 fusion protein, P450scc Phe215Ser retained 2.5% of wild-type activity; previously described mutants Leu141Trp and Ala269Val had 2.6% and 12% of wild-type activity, respectively, and Val415Glu and c.835delA lacked detectable activity.Although P450scc is required to produce placental progesterone required to maintain pregnancy, severe mutations in P450scc are compatible with term gestation; milder P450scc mutations may present later without disordered sexual development. Enlarged adrenals usually distinguish steroidogenic acute regulatory protein deficiency from P450scc deficiency, but only DNA sequencing is definitive.
| S-EPMC3565115 | biostudies-literature
Project description:ContextMitochondrial cytochrome P450scc converts cholesterol to pregnenolone in all steroidogenic tissues. Although progesterone production from the fetally-derived placenta is necessary to maintain pregnancy to term, four patients with mutations in the gene encoding P450scc (CYP11A1), have been described, one in a 46,XX female and three in underandrogenized 46,XY individuals, all with primary adrenal failure.ObjectiveOur aim was to determine whether P450scc mutations might be found in other children and to explore genotype/phenotype correlations.Methods and patientsWe performed mutational analysis of CYP11A1 in individuals with 46,XY disorders of sex development and primary adrenal failure, followed by functional studies of P450scc activity and of P450scc RNA splicing.ResultsAmong nine 46,XY infants with adrenal failure and disordered sexual differentiation, two infants had compound heterozygous mutations in CYP11A1. One patient harbored the novel P450scc missense mutations L141W and V415E, which retained 38 and 0% activity, respectively. The other carried a CYP11A1 frameshift mutation c835delA (0% activity) and a splice site mutation [IVS3+(2-3)insT] that prevented correct splicing of P450scc mRNA.ConclusionsP450scc deficiency is a recently recognized disorder that may be more frequent than originally thought. The phenotypic spectrum ranges from severe loss-of-function mutations associated with prematurity, complete underandrogenization, and severe, early-onset adrenal failure, to partial deficiencies found in children born at term with clitoromegaly and later-onset adrenal failure. In contradistinction to congenital lipoid adrenal hyperplasia caused by steroidogenic acute regulatory protein mutations, adrenal hyperplasia has not been reported in any of the six patients with P450scc deficiency.
| S-EPMC2266942 | biostudies-literature
Project description:Bufo bufo gargarizans Cantor are precious medicinal animals in traditional Chinese medicine (TCM). Bufadienolides as the major pharmacological components are generated from the venomous glands of B. bufo gargarizans. Bufadienolides are one type of cardiac aglycone with a six-member lactone ring and have properties of antitumor, cardiotonic, tonsillitis, and anti-inflammatory. The biosynthesis of bufadienolides is complex and unclear. This study explored the transcriptome of three different tissues (skin glands, venom glands, and muscles) of B. bufo gargarizans by high-throughput sequencing. According to the gene tissue-specific expression profile, 389 candidate genes were predicted possibly participating in the bufadienolides biosynthesis pathway. Then, BbgCYP11A1 was identified as a cholesterol side chain cleaving the enzyme in engineering yeast producing cholesterol. Furthermore, the catalytic activity of BbgCYP11A1 was studied with various redox partners. Interestingly, a plant NADPH-cytochrome P450 reductase (CPR) from Anemarrhena asphodeloides showed notably higher production than BbgAdx-2A-BbgAdR from B. bufo gargarizans. These results will provide certainly molecular research to reveal the bufadienolides biosynthesis pathway in B. bufo gargarizans.
| S-EPMC9037069 | biostudies-literature
Project description:BACKGROUND:CYP11A1, a gene belonging to the family 11 of cytochrome P450, encodes a crucial steroidogenic enzyme that catalyzes the initial step in the production of all classes of steroids. Many studies show that CYP11A1 plays a role in ovary function. However, the role of CYP11A1 in goose reproductive cycle remains largely unknown. RESULTS:In this study, full-length CYP11A1 cDNA of Zhedong goose was obtained using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA consisted of a 96-base pair (bp) 5'untranslated region (UTR), a 179-bp 3'UTR and a 1509-bp open reading frame. The open reading frame encodes a putative 503 amino acid protein that shares high homology with CYP11A1 of other birds. The amino acid sequence possesses conserved domains of the P450 superfamily, which include the steroid-binding domain and the heme-binding region. Real-time quantitative polymerase chain reaction (qPCR) analysis revealed CYP11A1 mRNA was expressed ubiquitously in every Zhedong goose tissue analyzed, including the heart, liver, glandular stomach, lung, spleen, kidney, intestinum tenue, intestinum crassum, cerebrum, cerebellum, muscle, oviduct, pituitary, hypothalamus and ovary.. The relatively low levels of CYP11A1 mRNA were detected in pituitary, ovary and oviduct tissues at ovulation when compared with levels at oviposition. Interestingly, higher expression was observed in ovary and oviduct tissues during brooding. Lastly, higher mRNA expression of Yangzhou geese was detected during the ovulation period than that of Zhedong geese. CONCLUSIONS:Our findings reveal the sequence characterization and expression patterns of the CYP11A1 gene during the goose reproductive cycle, which may provides correlative evidence that CYP11A1 expression is important in reproduction activity.
| S-EPMC4688999 | biostudies-literature
Project description:Mycophenolic acid (MPA, 1) and its derivatives are first-line immunosuppressants used in organ transplantation and for treating autoimmune diseases. Despite chemical synthetic achievements, the biosynthetic formation of a seven-carbon carboxylic acid pharmacophore side chain of 1, especially the processes involving the cleavage of the prenyl side chain between DHMP (4) and DMMPA (5), remains unknown. In this work, we identified a membrane-bound prenyltransferase, PgMpaA, that transfers FPP to 4 to yield FDHMP (6). Compound 6 undergoes the first cleavage step via a new globin-like enzyme PgMpaB to form a cryptic intermediate 12. Heterologous expression of PgMpa genes in Aspergillus nidulans demonstrates that the second cleavage step (from 12 to 5) of 1 is a PgMpa cluster-independent process in vivo. Our results, especially the discovery of the broad tolerance of substrates recognized by PgMpaB, set up a strategy for the formation of "pseudo-isopentenyl" natural products using fungal globin-like enzymes.
| S-EPMC6900556 | biostudies-literature
Project description:The enzyme cytochrome P450 11A1 cleaves the C20-C22 carbon-carbon bond of cholesterol to form pregnenolone, the first 21-carbon precursor of all steroid hormones. Various reaction mechanisms are possible for the carbon-carbon bond cleavage step of P450 11A1, and most current proposals involve the oxoferryl active species, Compound I (FeO(3+)). Compound I can either (i) abstract an O-H hydrogen atom or (ii) be attacked by a nucleophilic hydroxy group of its substrate, 20R,22R-dihydroxycholesterol. The mechanism of this carbon-carbon bond cleavage step was tested using (18)O-labeled molecular oxygen and purified P450 11A1. P450 11A1 was incubated with 20R,22R-dihydroxycholesterol in the presence of molecular oxygen ((18)O2), and coupled assays were used to trap the labile (18)O atoms in the enzymatic products (i.e., isocaproaldehyde and pregnenolone). The resulting products were derivatized and the (18)O content was analyzed by high-resolution mass spectrometry. P450 11A1 showed no incorporation of an (18)O atom into either of its carbon-carbon bond cleavage products, pregnenolone and isocaproaldehyde . The positive control experiments established retention of the carbonyl oxygens in the enzymatic products during the trapping and derivatization processes. These results reveal a mechanism involving an electrophilic Compound I species that reacts with nucleophilic hydroxy groups in the 20R,22R-dihydroxycholesterol intermediate of the P450 11A1 reaction to produce the key steroid pregnenolone.
| S-EPMC5289895 | biostudies-literature
Project description:Canine hypoadrenocorticism likely arises from immune-mediated destruction of adrenocortical tissue, leading to glucocorticoid and mineralocorticoid deficiency. In humans with autoimmune Addison's disease (AAD) or autoimmune polyendocrine syndrome (APS), circulating autoantibodies have been demonstrated against enzymes associated with adrenal steroid synthesis. The current study investigates autoantibodies against steroid synthesis enzymes in dogs with spontaneous hypoadrenocorticism. Coding regions of canine CYP21A2 (21-hydroxylase; 21-OH), CYP17A1 (17-hydroxylase; 17-OH), CYP11A1 (P450 side-chain cleavage enzyme; P450scc) and HSD3B2 (3β hydroxysteroid dehydrogenase; 3βHSD) were amplified, cloned and expressed as 35S-methionine radiolabelled recombinant protein. In a pilot study, serum samples from 20 dogs with hypoadrenocorticism and four unaffected control dogs were screened by radio-immunoprecipitation assay. There was no evidence of reactivity against 21-OH, 17-OH or 3βHSD, but five dogs with hypoadrenocorticism showed immunoreactivity to P450scc compared with controls. Serum samples were subsequently obtained from 213 dogs diagnosed with hypoadrenocorticism and 110 dogs from a hospital control population. Thirty control dogs were randomly selected to establish a threshold for antibody positivity (mean + 3 × standard deviation). Dogs with hypoadrenocorticism were more likely to be P450scc autoantibody positive than hospital controls (24% vs. 1.2%, respectively; p = 0.0016). Sex was significantly associated with the presence of P450scc autoantibodies in the case population, with 30% of females testing positive compared with 17% of males (p = 0.037). Significant associations with breed (p = 0.015) and DLA-type (DQA1*006:01 allele; p = 0.017) were also found. This cross-sectional study indicates that P450scc autoantibodies are present in a proportion of dogs affected with hypoadrenocorticism.
| S-EPMC4664467 | biostudies-literature
Project description:The first step in the synthesis of steroids is catalysed by cytochrome P-450ssc (cholesterol-side-chain cleavage enzyme). We have investigated the synthesis of this enzyme in three cultured cell lines at the protein and hormone secretion levels. Hormone levels were measured by an enzyme immunoassay using a monoclonal antibody against progesterone. The protein level was detected using polyclonal antibodies directed against a P-450scc fusion protein overproduced in Escherichia coli. Utilizing a bacteriophage T7 promoter expression system, a large amount of human P-450scc fusion protein was produced and easily purified. P-450scc was synthesized in the mouse adrenal tumour cell line Y1 and human choriocarcinoma cell line JEG-3, but not in monkey kidney cell line COS-1. The production of P-450scc in Y1 and JEG-3 cells was stimulated by 8-bromo cyclic AMP, the effect of which was not observed until 6 h after induction and was more pronounced at 24 h. Y1 and JEG-3 cells exhibited a difference in progesterone secretion after induction.
| S-EPMC1149983 | biostudies-other