Corticosteroid Biosynthesis Revisited: No Direct Hydroxylation of Pregnenolone by Steroid 21-Hydroxylase.

Cytochrome P450s (CYPs) are an essential family of enzymes in the human body. They play a crucial role in metabolism, especially in human steroid biosynthesis. Reactions catalyzed by these enzymes are highly stereo- and regio-specific. Lack or severe malfunctions of CYPs can cause severe diseases and even shorten life. Hence, investigations on metabolic reactions and structural requirements of substrates are crucial to gain further knowledge on the relevance of different enzymes in the human body functions and the origin of diseases. One key enzyme in the biosynthesis of gluco- and mineralocorticoids is CYP21A2, also known as steroid 21-hydroxylase. To investigate the steric and regional requirements of substrates for this enzyme, we performed whole-cell biotransformation assays using a strain of fission yeast Schizosaccharomyces pombe recombinantly expressing CYP21A2. The progestogens progesterone, pregnenolone, and their 17α-hydroxy-derivatives were used as substrates. After incubation, samples were analyzed using gas chromatography coupled to mass spectrometry. For progesterone and 17α-hydroxyprogesterone, their corresponding 21-hydroxylated metabolites 11-deoxycorticosterone and 11-deoxycortisol were detected, while after incubation of pregnenolone and 17α-hydroxypregnenolone, no hydroxylated product was observed. Findings were confirmed with authentic reference material. Molecular docking experiments agree with these results and suggest that interaction between the 3-oxo group and arginine-234 of the enzyme is a strict requirement. The presented results demonstrate once more that the presence of an oxo-group in position 3 of the steroid is indispensable, while a 3-hydroxy group prevents hydroxylation in position C-21 by CYP21A2. This knowledge may be transferred to other CYP21A2 substrates and hence help to gain essential insights into steroid metabolism

Pentacyclic triterpenes from Terminalia arjuna show multiple benefits on aged and dry skin

BACKGROUND Pentacyclic triterpenoids improve epidermal barrier function and induce collagen production. Here, their effects on cutaneous aging by means of objective instrumental measurements were elucidated. METHODS Reconstituted human epidermis, cultivated keratinocytes and fibroblasts were incubated with Terminalia arjuna triterpenes (T. arjuna bark extract), and mRNA and protein expression of various genes was determined using microarray analysis, qRT-PCR and ELISA techniques. Clinical efficacy of T. arjuna bark extract versus vehicle control cream was elucidated in 30 patients and transepidermal water loss (TEWL), skin hydration and elasticity were measured. Another 30 female patients in their postmenopausal phase were treated with a similar regime, and skin sebum content, cutaneous blood microcirculation and skin density/echogenicity were assessed. RESULTS Incubation with T. arjuna triterpenes increased FGF-2, TSP-1, TGF-\textgreekb and CTGF expression, and VEGF secretion in vitro. Elevated lactate dehydrogenase release upon sodium dodecyl sulphate challenge was reversed by the application of T. arjuna bark extract. T. arjuna bark extract decreased TEWL, improved skin moisturization, reduced scaliness and led to significantly improved skin elasticity. Also, increases in blood microflow and skin sebum content as well as improved skin thickness/echogenicity were noted on postmenopausal skin, resulting in visible reduction of sagging skin on the jowls as demonstrated by digital photography. CONCLUSION T. arjuna bark extract appears as an innovative active ingredient that exerts versatile antiaging properties in vitro and in vivo

Th17 micro-milieu regulates NLRP1-dependent caspase-5 activity in skin autoinflammation.

IL-1β is a potent player in cutaneous inflammation and central for the development of a Th17 micro-milieu in autoinflammatory diseases including psoriasis. Its production is controlled at the transcriptional level and by subsequent posttranslational processing via inflammatory caspases. In this study, we detected inflammatory caspase-5 active in epidermal keratinocytes and in psoriatic skin lesions. Further, interferon-γ and interleukin-17A synergistically induced caspase-5 expression in cultured keratinocytes, which was dependent on the antimicrobial peptide psoriasin (S100A7). However, diseases-relevant triggers for caspase-5 activity and IL-1β production remain unknown. Recently, extranuclear DNA has been identified as danger-signals abundant in the psoriatic epidermis. Here, we could demonstrate that cytosolic double-stranded (ds) DNA transfected into keratinocytes triggered the activation of caspase-5 and the release of IL-1β. Further, interleukin-17A promoted caspase-5 function via facilitation of the NLRP1-inflammasome. Anti-inflammatory vitamin D interfered with the IL-1β release and suppressed caspase-5 in keratinocytes and in psoriatic skin lesions. Our data link the disease-intrinsic danger signals psoriasin (S100A7) and dsDNA for NLPR1-dependent caspase-5 activity in psoriasis providing potential therapeutic targets in Th17-mediated skin autoinflammation

Title page Glucuronide production by whole-cell biotransformation using genetically engineered fission yeast S. pombe

Abstract Drug metabolites generated by UDP glycosyltransferases (UGTs) are needed for drug development and toxicity studies, especially in the context of safety testing of metabolites during drug development. Since chemical metabolite synthesis can be arduous, various biological approaches have been developed; however, no whole-cell biotransformation with recombinant microbes that express human UGTs was yet achieved. In this study we expressed human UDP glucose-6-dehydrogenase (UGDH) together with several human or rat UGT isoforms in the fission yeast Schizosaccharomyces pombe and generated strains that catalyze the whole-cell glucuronidation of standard substrates. Moreover, we established two methods to obtain stable isotope-labeled glucuronide metabolites: The first uses a labeled aglycon, while the second employs 13 C 6 -glucose as a metabolic precursor of isotope-labeled UDPglucuronic acid (UDP-GA) and yields a sixfold labeled glucuronide. The system described here should lead to a significant facilitation in the production of both labeled and unlabeled drug glucuronides for industry and academia. DMD 30965

Biosynthesis of Salbutamol-4′-O-sulfate as Reference for Identification of Intake Routes and Enantiopure Salbutamol Administration by Achiral UHPLC-MS/MS

The aim of the study was a comprehensive and quantitative determination of salbutamol and its sulfoconjugated major metabolite in urine samples using achiral ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Therefore, salbutamol-4′-O-sulfate was biosynthesized as a reference using genetically modified fission yeast cells, and the product was subsequently characterized by NMR and HRMS. In competitive sports, salbutamol is classified as a prohibited drug; however, inhalation at therapeutic doses is permitted with a maximum allowance of 600 µg/8 h. In contrast, the enantiopure levosalbutamol is prohibited under any condition. For analytical discrimination, the amount of salbutamol and its main metabolite excreted in the urine was studied. As proof of concept, a longitudinal study in one healthy volunteer was performed in order to investigate excreted amounts and to study potential discrimination using achiral chromatography. Discrimination of administration of racemic salbutamol or the enantiopure levosalbutamol was not achieved by solely analyzing salbutamol as the parent compound. However, a distinction was possible by evaluation of the proportion of salbutamol-4′-O-sulfate in relation to salbutamol. Therefore, reference material of metabolites is of great importance in doping control, especially for threshold substances

Ferredoxin 1b (Fdx1b) Is the essential mitochondrial redox partner for cortisol biosynthesis in zebrafish

Mitochondrial cytochrome P450 (CYP) enzymes rely on electron transfer from the redox partner ferredoxin 1 (FDX1) for catalytic activity. Key steps in steroidogenesis require mitochondrial CYP enzymes and FDX1. Over 30 ferredoxin mutations have been explored in vitro; however, no spontaneously occurring mutations have been identified in humans leaving the impact of FDX1 on steroidogenesis in the whole organism largely unknown. Zebrafish are an important model to study human steroidogenesis, because they have similar steroid products and endocrine tissues. This study aimed to characterize the influence of ferredoxin on steroidogenic capacity in vivo by using zebrafish. Zebrafish have duplicate ferredoxin paralogs: fdx1 and fdx1b. Although fdx1 was observed throughout development and in most tissues, fdx1b was expressed after development of the zebrafish interrenal gland (counterpart to the mammalian adrenal gland). Additionally, fdx1b was restricted to adult steroidogenic tissues, such as the interrenal, gonads, and brain, suggesting that fdx1b was interacting with steroidogenic CYP enzymes. By using transcription activator-like effector nucleases, we generated fdx1b mutant zebrafish lines. Larvae with genetic disruption of fdx1b were morphologically inconspicuous. However, steroid hormone analysis by liquid chromatography tandem mass spectrometry revealed fdx1b mutants failed to synthesize glucocorticoids. Additionally, these mutants had an up-regulation of the hypothalamus-pituitary-interrenal axis and showed altered dark-light adaptation, suggesting impaired cortisol signaling. Antisense morpholino knockdown confirmed Fdx1b is required for de novo cortisol biosynthesis. In summary, by using zebrafish, we generated a ferredoxin knockout model system, which demonstrates for the first time the impact of mitochondrial redox regulation on glucocorticoid biosynthesis in vivo

Bystander signaling via oxidative metabolism

Humaira Aziz Sawal,1 Kashif Asghar,2 Matthias Bureik,3 Nasir Jalal4 1Healthcare Biotechnology Department, Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Islamabad, 2Basic Sciences Research, Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore, Pakistan; 3Health Science Platform, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, China; 4Health Science Platform, Department of Molecular and Cellular Pharmacology, Tianjin University, Tianjin, China Abstract: The radiation-induced bystander effect (RIBE) is the initiation of biological end points in cells (bystander cells) that are not directly traversed by an incident-radiation track, but are in close proximity to cells that are receiving the radiation. RIBE has been indicted of causing DNA damage via oxidative stress, besides causing direct damage, inducing tumorigenesis, producing micronuclei, and causing apoptosis. RIBE is regulated by signaling proteins that are either endogenous or secreted by cells as a means of communication between cells, and can activate intracellular or intercellular oxidative metabolism that can further trigger signaling pathways of inflammation. Bystander signals can pass through gap junctions in attached cell lines, while the suspended cell lines transmit these signals via hormones and soluble proteins. This review provides the background information on how reactive oxygen species (ROS) act as bystander signals. Although ROS have a very short half-life and have a nanometer-scale sphere of influence, the wide variety of ROS produced via various sources can exert a cumulative effect, not only in forming DNA adducts but also setting up signaling pathways of inflammation, apoptosis, cell-cycle arrest, aging, and even tumorigenesis. This review outlines the sources of the bystander effect linked to ROS in a cell, and provides methods of investigation for researchers who would like to pursue this field of science. Keywords: radiation-induced bystander effect (RIBE), reactive oxygen species (ROS), oxidative stress, bystander signaling, tumorigenesis&nbsp

Challenges of steroid biotransformation with human Cytochrome P450 monooxygenase CYP21 using recombinant Schizosaccharomyces pombe

Since cytochrome P450 monooxygenases enable the regio- and stereo-selective hydroxylation of C-H bonds, they are of outstanding interest for the synthesis of pharmaceuticals and fine chemicals. Nevertheless, for industrial applications of such enzymes, e.g., steroid hydroxylation, several challenges like cofactor and oxygen supply, limited stability and activity, or low substrate solubility have to be overcome. To identify the limiting factors in a P450 catalyzed whole cell biotransformation, 21-hydroxylation of 17-alpha-hydroxyprogesterone in Schizosaccharomyces pombe expressing human CYP21 was chosen as model reaction. We report here that resting cells of this recombinant yeast strain can be used for efficient biotransformation. In the present study, we analyzed the intracellular redox cofactor pool of S. pombe by LC-MS/MS measurements and report the first quantification of the intracellular cofactor pool during P450 hydroxylation. Thereby a limitation caused by the redox cofactor could be excluded for resting cells. In contrary, low substrate solubility and its transport into the cell affect activity. Screening for an appropriate cosolvent identified methanol as the most promising candidate, since it showed the lowest inactivation effect on the biocatalyst. Through permeabilization of the membrane with the detergent tween 80 steroid hydroxylation activity increases, leading to a productivity of 540 microM d(-1) in a final batch experiment under optimized reaction conditions