Mammalian cytochrome P450scc enzyme system catalyzes the initial step in steroid hormone biosynthesis—cholesterol hydroxylation followed by cleavage of the side-chain to yield pregnenolone. This system consists of th...Mammalian cytochrome P450scc enzyme system catalyzes the initial step in steroid hormone biosynthesis—cholesterol hydroxylation followed by cleavage of the side-chain to yield pregnenolone. This system consists of three components—the cytochrome P450scc (CYP11A1), a flavoprotein (NADPH-adrenodoxin reductase, AdR) and an iron-sulfur protein (adrenodoxin, Adx). In this work, the three-component electron transport chain (AdR/Adx/CYP11A1) from bovine adrenal cortex has been implemented in Escherichia coli by co-expression of the corresponding coding sequences from a tricistronic plasmid. The cDNAs of AdR, Adx and CYP11A1 are situated in a single transcription unit and separated by ribosome binding sequences. The recombinant strain created was capable of synthesizing functional proteins identical to the bovine CYP11A1, AdR and Adx on molecular weights and immuno-specificity. The experiments in vivo showed pregnenolone production from cholesterol by the transformed bacteria. Maximal productivity of 0.42 ± 0.015 mg/l pregnenolone for 24 h has been reached for the induced cells in the presence of cholesterol solubilizing agent—methyl-β-cyclodextrin. Thus, a stable transgenic E. coli strain with the functional reconstructed bovine cholesterol side-chain cleavage system has been firstly generated in this work. The findings are of importance for studies of mammalian steroidogenic system features, and may open some perspectives for further generation of novel microbial biocatalysts.展开更多
Background The clinical manifestations of nonclassical 11beta-hydroxylase deficiency are very similar to those of nonclassical 21-hydroxylase deficiency.For this study,we investigated the relationship between the clin...Background The clinical manifestations of nonclassical 11beta-hydroxylase deficiency are very similar to those of nonclassical 21-hydroxylase deficiency.For this study,we investigated the relationship between the clinical and molecular features of congenital adrenal hyperplasia caused by 11beta-hydroxylase deficiency and reviewed the related literature,which are expected to provide assistance for the clinical diagnosis and analysis of congenital adrenal hyperplasia.Methods Clinical data for 10 Chinese patients diagnosed with congenital adrenal hyperplasia in our hospital from 2018 to 2022 were retrospectively analyzed.We examined the effects of gene mutations on protease activity and constructed threedimensional structure prediction models of proteins.Results We describe 10 patients with 11beta-hydroxylase gene mutations(n=5,46,XY;n=5,46,XX),with 10 novel mutations were reported.Female patients received treatment at an early stage,with an average age of 2.08±1.66 years,whereas male patients received treatment significantly later,at an average age of 9.77±3.62 years.The most common CYP11B1 pathogenic variant in the Chinese population was found to be c.1360C>T.All mutations lead to spatial conformational changes that affect protein stability.Conclusions Our study found that there was no significant correlation between each specific mutation and the severity of clinical manifestations.Different patients with the same gene pathogenic variant may have mild or severe clinical manifestations.The correlation between genotype and phenotype needs further study.Three-dimensional protein simulations may provide additional support for the physiopathological mechanism of genetic mutations.展开更多
过量激素刺激骨髓间质干细胞(bone marrow stromal cells,BMSCs)使得成骨能力减弱、成脂能力增强是导致股骨头坏死(osteonecrosis of the femoral head,ONFH)的主要原因,调控机制尚未阐明。最新的研究发现激素引起骨组织中11β-HSD1活...过量激素刺激骨髓间质干细胞(bone marrow stromal cells,BMSCs)使得成骨能力减弱、成脂能力增强是导致股骨头坏死(osteonecrosis of the femoral head,ONFH)的主要原因,调控机制尚未阐明。最新的研究发现激素引起骨组织中11β-HSD1活性增强,导致骨局部内源性激素积聚可抑制骨形成。抑制11β-HSD1活性可促进成骨细胞分化,抑制脂肪分化。故推测内源性激素在ONFH的发病过程中起到重要作用,在激素与BMSCs活性变化之间可能存在11β-HSD1调控的内源性激素代谢途径参与ONFH的发病过程。因此,深入研究11β-HSD1介导的内源性激素代谢途径在ONFH发病中的作用,对深入了解ONFH的发病机制具有重要意义。展开更多
Steroid saponins are secondary metabolites with multiple medicinal values that are found in large quantities in natural medicines,especially Vernonia amygdalina,a famous nature medicine for the treatment of tonsilliti...Steroid saponins are secondary metabolites with multiple medicinal values that are found in large quantities in natural medicines,especially Vernonia amygdalina,a famous nature medicine for the treatment of tonsillitis,diabetes,pneumonia.The current study was designed to combine molecular networking(MN)with diagnostic ions for rapid identification ofΔ^(7,9(11))stigmastane-type saponins which were theα-glucosidase inhibitory active substances in V.amygdalina.First,theα-glucosidase inhibitory activities of fiveΔ^(7,9(11))stigmastane-type steroid saponins that were previously isolated were screened,which indicated that theΔ^(7,9(11))stigmastane-type steroid saponin was one of the active constituents responsible for ameliorating diabetes.Furthermore,a strategy was proposed to identify stigmastane-type steroid saponins and verify the plausibility of derived fragmentation pathways by applying MN,MolNetEnhancer and unsupervised substructure annotation(MS2LDA).Based on this strategy,other sevenΔ^(7,9(11))stigmastane-type steroid saponins were identified from this plant.Our research provide scientific evidence for the antidiabetic potential of the steroid saponin-rich extract of V.amygdalina leaf.展开更多
文摘Mammalian cytochrome P450scc enzyme system catalyzes the initial step in steroid hormone biosynthesis—cholesterol hydroxylation followed by cleavage of the side-chain to yield pregnenolone. This system consists of three components—the cytochrome P450scc (CYP11A1), a flavoprotein (NADPH-adrenodoxin reductase, AdR) and an iron-sulfur protein (adrenodoxin, Adx). In this work, the three-component electron transport chain (AdR/Adx/CYP11A1) from bovine adrenal cortex has been implemented in Escherichia coli by co-expression of the corresponding coding sequences from a tricistronic plasmid. The cDNAs of AdR, Adx and CYP11A1 are situated in a single transcription unit and separated by ribosome binding sequences. The recombinant strain created was capable of synthesizing functional proteins identical to the bovine CYP11A1, AdR and Adx on molecular weights and immuno-specificity. The experiments in vivo showed pregnenolone production from cholesterol by the transformed bacteria. Maximal productivity of 0.42 ± 0.015 mg/l pregnenolone for 24 h has been reached for the induced cells in the presence of cholesterol solubilizing agent—methyl-β-cyclodextrin. Thus, a stable transgenic E. coli strain with the functional reconstructed bovine cholesterol side-chain cleavage system has been firstly generated in this work. The findings are of importance for studies of mammalian steroidogenic system features, and may open some perspectives for further generation of novel microbial biocatalysts.
文摘Background The clinical manifestations of nonclassical 11beta-hydroxylase deficiency are very similar to those of nonclassical 21-hydroxylase deficiency.For this study,we investigated the relationship between the clinical and molecular features of congenital adrenal hyperplasia caused by 11beta-hydroxylase deficiency and reviewed the related literature,which are expected to provide assistance for the clinical diagnosis and analysis of congenital adrenal hyperplasia.Methods Clinical data for 10 Chinese patients diagnosed with congenital adrenal hyperplasia in our hospital from 2018 to 2022 were retrospectively analyzed.We examined the effects of gene mutations on protease activity and constructed threedimensional structure prediction models of proteins.Results We describe 10 patients with 11beta-hydroxylase gene mutations(n=5,46,XY;n=5,46,XX),with 10 novel mutations were reported.Female patients received treatment at an early stage,with an average age of 2.08±1.66 years,whereas male patients received treatment significantly later,at an average age of 9.77±3.62 years.The most common CYP11B1 pathogenic variant in the Chinese population was found to be c.1360C>T.All mutations lead to spatial conformational changes that affect protein stability.Conclusions Our study found that there was no significant correlation between each specific mutation and the severity of clinical manifestations.Different patients with the same gene pathogenic variant may have mild or severe clinical manifestations.The correlation between genotype and phenotype needs further study.Three-dimensional protein simulations may provide additional support for the physiopathological mechanism of genetic mutations.
文摘过量激素刺激骨髓间质干细胞(bone marrow stromal cells,BMSCs)使得成骨能力减弱、成脂能力增强是导致股骨头坏死(osteonecrosis of the femoral head,ONFH)的主要原因,调控机制尚未阐明。最新的研究发现激素引起骨组织中11β-HSD1活性增强,导致骨局部内源性激素积聚可抑制骨形成。抑制11β-HSD1活性可促进成骨细胞分化,抑制脂肪分化。故推测内源性激素在ONFH的发病过程中起到重要作用,在激素与BMSCs活性变化之间可能存在11β-HSD1调控的内源性激素代谢途径参与ONFH的发病过程。因此,深入研究11β-HSD1介导的内源性激素代谢途径在ONFH发病中的作用,对深入了解ONFH的发病机制具有重要意义。
基金supported by the National Natural Science Foundation of China(No.81573550)。
文摘Steroid saponins are secondary metabolites with multiple medicinal values that are found in large quantities in natural medicines,especially Vernonia amygdalina,a famous nature medicine for the treatment of tonsillitis,diabetes,pneumonia.The current study was designed to combine molecular networking(MN)with diagnostic ions for rapid identification ofΔ^(7,9(11))stigmastane-type saponins which were theα-glucosidase inhibitory active substances in V.amygdalina.First,theα-glucosidase inhibitory activities of fiveΔ^(7,9(11))stigmastane-type steroid saponins that were previously isolated were screened,which indicated that theΔ^(7,9(11))stigmastane-type steroid saponin was one of the active constituents responsible for ameliorating diabetes.Furthermore,a strategy was proposed to identify stigmastane-type steroid saponins and verify the plausibility of derived fragmentation pathways by applying MN,MolNetEnhancer and unsupervised substructure annotation(MS2LDA).Based on this strategy,other sevenΔ^(7,9(11))stigmastane-type steroid saponins were identified from this plant.Our research provide scientific evidence for the antidiabetic potential of the steroid saponin-rich extract of V.amygdalina leaf.
