ABSTRACT
Prolyl hydroxylase domain (PHD) enzymes change HIF activity according to oxygen signal; whether it is regulated by other physiological conditions remains largely unknown. Here, we report that PHD3 is induced by fasting and regulates hepatic gluconeogenesis through interaction and hydroxylation of CRTC2. Pro129 and Pro615 hydroxylation of CRTC2 following PHD3 activation is necessary for its association with cAMP-response element binding protein (CREB) and nuclear translocation, and enhanced binding to promoters of gluconeogenic genes by fasting or forskolin. CRTC2 hydroxylation-stimulated gluconeogenic gene expression is independent of SIK-mediated phosphorylation of CRTC2. Liver-specific knockout of PHD3 (PHD3 LKO) or prolyl hydroxylase-deficient knockin mice (PHD3 KI) show attenuated fasting gluconeogenic genes, glycemia, and hepatic capacity to produce glucose during fasting or fed with high-fat, high-sucrose diet. Importantly, Pro615 hydroxylation of CRTC2 by PHD3 is increased in livers of fasted mice, diet-induced insulin resistance or genetically obese ob/ob mice, and humans with diabetes. These findings increase our understanding of molecular mechanisms linking protein hydroxylation to gluconeogenesis and may offer therapeutic potential for treating excessive gluconeogenesis, hyperglycemia, and type 2 diabetes.
Subject(s)
Diabetes Mellitus, Type 2 , Glucose , Humans , Mice , Animals , Glucose/metabolism , Proline/metabolism , Hydroxylation , Diabetes Mellitus, Type 2/metabolism , Liver/metabolism , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Gluconeogenesis/physiology , Prolyl Hydroxylases/metabolism , Hepatocytes/metabolism , Mice, Inbred C57BLABSTRACT
Epithelial-mesenchymal transition (EMT) has been implicated in various aspects of tumor development, including tumor invasion and metastasis, cancer stemness, and therapy resistance. Diverse stroma cell types along with biochemical and biophysical factors in the tumor microenvironment impinge on the EMT program to impact tumor progression. Here we provide an in-depth review of various tumor microenvironmental signals that regulate EMT in cancer. We discuss the molecular mechanisms underlying the role of EMT in therapy resistance and highlight new therapeutic approaches targeting the tumor microenvironment to impact EMT and tumor progression.
Subject(s)
Neoplasms , Tumor Microenvironment , Humans , Tumor Microenvironment/genetics , Epithelial-Mesenchymal Transition/genetics , Neoplasms/etiology , Neoplasms/geneticsABSTRACT
Benzylisoquinoline alkaloids (BIAs) represent a significant class of secondary metabolites with crucial roles in plant physiology and substantial potential for clinical applications. CYP82 genes are involved in the formation and modification of various BIA skeletons, contributing to the structural diversity of compounds. In this study, Corydalis yanhusuo, a traditional Chinese medicine rich in BIAs, was investigated to identify the catalytic function of CYP82s during BIA formation. Specifically, 20 CyCYP82-encoding genes were cloned, and their functions were identified in vitro. Ten of these CyCYP82s were observed to catalyze hydroxylation, leading to the formation of protopine and benzophenanthridine scaffolds. Furthermore, the correlation between BIA accumulation and the expression of CyCYP82s in different tissues of C. yanhusuo was assessed their. The identification and characterization of CyCYP82s provide novel genetic elements that can advance the synthetic biology of BIA compounds such as protopine and benzophenanthridine, and offer insights into the biosynthesis of BIAs with diverse structures in C. yanhusuo.
Subject(s)
Alkaloids , Benzylisoquinolines , Corydalis , Benzophenanthridines , Corydalis/genetics , Corydalis/chemistry , Corydalis/metabolism , Alkaloids/metabolism , Plant Extracts/chemistryABSTRACT
BACKGROUND AND AIMS: NASH has emerged as a leading cause of chronic liver disease. However, the mechanisms that govern NASH fibrosis remain largely unknown. CREBZF is a CREB/ATF bZIP transcription factor that causes hepatic steatosis and metabolic defects in obesity. APPROACH AND RESULTS: Here, we show that CREBZF is a key mechanism of liver fibrosis checkpoint that promotes hepatocyte injury and exacerbates diet-induced NASH in mice. CREBZF deficiency attenuated liver injury, fibrosis, and inflammation in diet-induced mouse models of NASH. CREBZF increases HSC activation and fibrosis in a hepatocyte-autonomous manner by stimulating an extracellular matrix protein osteopontin, a key regulator of fibrosis. The inhibition of miR-6964-3p mediates CREBZF-induced production and secretion of osteopontin in hepatocytes. Adeno-associated virus -mediated rescue of osteopontin restored HSC activation, liver fibrosis, and NASH progression in CREBZF-deficient mice. Importantly, expression levels of CREBZF are increased in livers of diet-induced NASH mouse models and humans with NASH. CONCLUSIONS: Osteopontin signaling by CREBZF represents a previously unrecognized intrahepatic mechanism that triggers liver fibrosis and contributes to the severity of NASH.
Subject(s)
Non-alcoholic Fatty Liver Disease , Osteopontin , Animals , Humans , Mice , Basic-Leucine Zipper Transcription Factors/metabolism , Disease Models, Animal , Fatty Liver/genetics , Fatty Liver/metabolism , Fibrosis , Hepatocytes/metabolism , Hepatocytes/pathology , Liver/metabolism , Liver/pathology , Liver Cirrhosis/pathology , Mice, Inbred C57BL , Non-alcoholic Fatty Liver Disease/genetics , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/pathology , Osteopontin/genetics , Osteopontin/metabolismABSTRACT
We present a systematic theoretical study on the angular distribution and linear polarization of x-ray line emissions of neon-like ions following the electron-impact excitation from the ground state to the excited levels [(2p5)1/23d3/2]J=1, [(2p5)3/23d5/2]J=1, [(2p5)3/23d3/2]J=1, and [(2p5)1/23s]J=1. The cross sections are calculated by using the flexible atomic code under configuration-interaction plus many-body perturbation theory method. The angular distribution and linear polarization are obtained based on density matrix theory. Emphasis has been placed on the effect of the configuration mixing on the angular distribution and polarization. It has been proved that the strong mixing of configuration [(2p5)3/23d3/2]J=1 with configuration [(2p5)1/23s]J=1 can result in the abrupt change of Z-dependence of angular distribution and polarization. It indicates that angular distribution and polarization can be expected to serve as a tool for investigation of configuration mixing effect.
ABSTRACT
BACKGROUND: Mycobacterium tuberculosis(MTB) most often infects the lungs and results in pulmonary tuberculosis(TB). MTB-specific memory T cells are able to respond quickly against antigens and help reduce the burden of pulmonary bacteria. The characteristics, function and chemotaxis axis of memory T cells in the lung remain unclear. The current study aimed to clarify the classification, function and recruitment of local antigen-specific memory T cells in the lung and the periphery blood of patients with pulmonary TB. METHODS: A total of 85 patients with active pulmonary TB were included in the study. Bronchoalveolar lavage fluid (BALF) and Peripheral blood were collected for further detection. The cell-surface markers and intracellular staining of memory T cell subtypes were measured by flow cytometry. The level of CXCL9, CXCL10 and CXCL11 in Bronchoalveolar lavage fluid cells and peripheral blood mononuclear cells (PBMC) were measured by Real-time PCR. RESULTS: The ratio of effective Memory T cells (TEM) were the highest in BALF of patients with pulmonary TB. In patients, CXCR3 and its ligands was increased in memory T cells of BALF compared with PBMC. IFN-γ+TNF-α+ effective Memory T cells and central memory T cells from BALF were increased after antigen stimulation. CXCR3 was higher in IFN-γ+ compared with IFN-γ- in CD4+ TCM and TEM from BALF of patients. Compared with PBMC, the PD-1 levels of terminal effector memory RA+(TEMRA) and TEM cells in CD4+ memory T cells of BALF were significantly increased. In addition, PD-1 was increased in IFN-γ+ compared with IFN-γ- in CD4+TEM from BALF of patients. There was no difference in Treg ratio between PBMC and BALF of TB patients. CONCLUSIONS: The CXCL9/CXCL11-CXCR3 axis may participate in the chemotaxis of memory T cells from the peripheral to lung. CD4+TEM and TEMRA in BALF may have exhausted, especially the cytokine producing TEM. Our study clarified the characteristics of antigen-specific memory T cells in local lung and may have impact on strategies of therapy and vaccine.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Pulmonary , Humans , Memory T Cells , Leukocytes, Mononuclear , Programmed Cell Death 1 Receptor , Bronchoalveolar Lavage , Bronchoalveolar Lavage Fluid , CD4-Positive T-LymphocytesABSTRACT
Benzylisoquinoline alkaloids (BIAs) are a type of secondary metabolite with clinical application value. (S)-stylopine is a special BIA which contains methylenedioxy bridge structures. CYP719As could catalyze the methylenedioxy bridge-formation on the A or D rings of protoberberine alkaloids, while displaying significant substrate regiospecificity. To explore the substrate preference of CYP719As, we cloned and identified five CyCYP719A candidates from Corydalis yanhusuo. Two CyCYP719As (CyCYP719A39 and CyCYP719A42) with high catalytic efficiency for the methylenedioxy bridge-formation on the D or A rings were characterized, respectively. The residues (Leu 294 for CyCYP719A42 and Asp 289 for CyCYP719A39) were identified as the key to controlling the regioselectivity of CYP719As affecting the methylenedioxy bridge-formation on the A or D rings by homology modeling and mutation analysis. Furthermore, for de novo production of BIAs, CyCYP719A39, CyCYP719A42, and their mutants were introduced into the (S)-scoulerine-producing yeast to produce 32 mg/L (S)-stylopine. These results lay a foundation for understanding the structure-function relationship of CYP719A-mediated methylenedioxy bridge-formation and provide yeast strains for the BIAs production by synthetic biology.
Subject(s)
Alkaloids , Benzylisoquinolines , Benzylisoquinolines/metabolism , Saccharomyces cerevisiae/metabolism , Alkaloids/metabolismABSTRACT
The resonance 3C ([(2p5)1/23d3/2]J=1 â [2p6]J=0) to intercombination 3D ([(2p5)3/23d5/2]J=1 â [2p6]J=0) line intensity ratio of neonlike ions has been studied. The measured line intensity ratio for neonlike Xe44+ ions shows an apparent change, which is reproduced by the calculations using the relativistic configuration interaction plus many-body perturbation theory. It is clearly elucidated that the change in the 3C/3D line intensity ratio is caused by strong configuration mixing between the upper levels of the 3D and 3F ([(2p5)1/23s]J=1 â [2p6]J=0) lines. The present measurement allows us to discuss the 3C/3D line intensity ratio for the highest-Z ions hitherto, which suggests that the experiment-theory discrepancy in the 3C/3D line intensity ratio of neonlike ions diminishes with increasing atomic number Z and further trends to vanish at higher-Z ions. Furthermore, the present study provides benefits to better understand configuration mixing effect in the radiative opacity of hot plasmas.
ABSTRACT
Paeoniflorin, a representative pinane monoterpene glycoside, is the main active component and quality index of Paeoniae Radix Alba and Paeoniae Radix Rubra.The possible biosynthesis of paeoniflorin is as follows: GPP is derived from mevalonate(MVA) and/or 2-C-methyl-D-erythritol 4-phosphate(MEP) pathway(s) followed by the catalysis with terpene synthase, cytochrome P450(CYP450), UDP-glucuronosyltransferase(UGT), and acyltransferase(AT), respectively.This study aims to explore the genes rela-ted to the biosynthesis of paeoniflorin.To be specific, the cDNA libraries for flowers, leaves, and roots of Paeonia lactiflora were established and sequenced.A total of 30 609 open reading frames(ORFs) were yielded.Through functional annotation and expression analysis of all CYP450 genes in the transcriptome, 11 CYP450 genes belonging to CYP71 A and CYP71 D subfamilies and showing expression trend consistent with monoterpene synthase PlPIN that may be involved in paeoniflorin biosynthesis were screened out.Subsequently, 7 UGT genes and 9 AT genes demonstrating the expression trend consistent with PlPIN which were possibly involved in paeoniflorin biosynthesis were further screened by functional annotation analysis, full-length sequence analysis, expression analysis, and phylogeny analysis.This study provided a systematic screening method with smaller number of candidate genes, thus reducing the workload of functional gene verification.The result laid a foundation for analyzing the biosynthesis pathway of paeoniflorin and the formation mechanism.
Subject(s)
Paeonia , Bridged-Ring Compounds , Gene Expression Profiling , Glucosides/genetics , Glucosides/metabolism , Monoterpenes/metabolism , Paeonia/geneticsABSTRACT
Diterpenoids, including more than 18,000 compounds, represent an important class of metabolites that encompass both phytohormones and some industrially relevant compounds. These molecules with complex, diverse structures and physiological activities, have high value in the pharmaceutical industry. Most medicinal diterpenoids are extracted from plants. Major advances in understanding the biosynthetic pathways of these active compounds are providing unprecedented opportunities for the industrial production of diterpenoids by metabolic engineering and synthetic biology. Here, we summarize recent developments in the field of diterpenoid biosynthesis from medicinal herbs. An overview of the pathways and known biosynthetic enzymes is presented. In particular, we look at the main findings from the past decade and review recent progress in the biosynthesis of different groups of ringed compounds. We also discuss diterpenoid production using synthetic biology and metabolic engineering strategies, and draw on new technologies and discoveries to bring together many components into a useful framework for diterpenoid production.
Subject(s)
Diterpenes , Plants, Medicinal , Biosynthetic Pathways , Diterpenes/chemistry , Diterpenes/metabolism , Humans , Synthetic BiologyABSTRACT
BACKGROUND AND AIMS: STAT3, a member of the signal transducer and activator of transcription (STAT) family, is strongly associated with liver injury, inflammation, regeneration, and hepatocellular carcinoma development. However, the signals that regulate STAT3 activity are not completely understood. APPROACH AND RESULTS: Here we characterize CREB/ATF bZIP transcription factor CREBZF as a critical regulator of STAT3 in the hepatocyte to repress liver regeneration. We show that CREBZF deficiency stimulates the expression of the cyclin gene family and enhances liver regeneration after partial hepatectomy. Flow cytometry analysis reveals that CREBZF regulates cell cycle progression during liver regeneration in a hepatocyte-autonomous manner. Similar results were observed in another model of liver regeneration induced by intraperitoneal injection of carbon tetrachloride (CCl4 ). Mechanistically, CREBZF potently associates with the linker domain of STAT3 and represses its dimerization and transcriptional activity in vivo and in vitro. Importantly, hepatectomy-induced hyperactivation of cyclin D1 and liver regeneration in CREBZF liver-specific knockout mice was reversed by selective STAT3 inhibitor cucurbitacin I. In contrast, adeno-associated virus-mediated overexpression of CREBZF in the liver inhibits the expression of the cyclin gene family and attenuates liver regeneration in CCl4 -treated mice. CONCLUSIONS: These results characterize CREBZF as a coregulator of STAT3 to inhibit regenerative capacity, which may represent an essential cellular signal to maintain liver mass homeostasis. Therapeutic approaches to inhibit CREBZF may benefit the compromised liver during liver transplantation.
Subject(s)
Basic-Leucine Zipper Transcription Factors/metabolism , Gene Expression Regulation , Liver Regeneration/genetics , Liver/physiology , STAT3 Transcription Factor/genetics , Animals , Basic-Leucine Zipper Transcription Factors/genetics , Carbon Tetrachloride/toxicity , Cell Cycle/genetics , Gene Deletion , Hepatocytes/drug effects , Hepatocytes/physiology , Liver/drug effects , Liver/injuries , Metabolic Networks and Pathways , Mice , Mice, KnockoutABSTRACT
The extracellular polysaccharide (EPS) matrix embedding microbial cells and soil particles plays an important role in the development of biological soil crusts (BSCs), which is widely recognized as beneficial to soil fertility in dryland worldwide. This study examined the EPS-producing bacterial strains YL24-1 and YL24-3 isolated from sandy soil in the Mu Us Desert in Yulin, Shaanxi province, China. The strains YL24-1 and YL24-3 were able to efficiently produce EPS; the levels of EPS were determined to be 257.22 µg/mL and 83.41 µg/mL in cultures grown for 72 h and were identified as Sinorhizobium meliloti and Pedobacter sp., respectively. When the strain YL24-3 was compared to Pedobacter yulinensis YL28-9T using 16S rRNA gene sequencing, the resemblance was 98.6% and the strain was classified as Pedobacter sp. using physiological and biochemical analysis. Furthermore, strain YL24-3 was also identified as a subspecies of Pedobacter yulinensis YL28-9T on the basis of DNA-DNA hybridization and polar lipid analysis compared with YL28-9T. On the basis of the EPS-related genes of relevant strains in the GenBank, several EPS-related genes were cloned and sequenced in the strain YL24-1, including those potentially involved in EPS synthesis, assembly, transport, and secretion. Given the differences of the strains in EPS production, it is possible that the differences in gene sequences result in variations in the enzyme/protein activities for EPS biosynthesis, assembly, transport, and secretion. The results provide preliminary evidence of various contributions of bacterial strains to the formation of EPS matrix in the Mu Us Desert.
Subject(s)
Extracellular Polymeric Substance Matrix/chemistry , Pedobacter/isolation & purification , Pedobacter/physiology , Sinorhizobium meliloti/isolation & purification , Sinorhizobium meliloti/physiology , Bacterial Typing Techniques , China , DNA, Bacterial/genetics , Desert Climate , Extracellular Polymeric Substance Matrix/genetics , Extracellular Polymeric Substance Matrix/metabolism , Extracellular Space/chemistry , Fatty Acids/analysis , Metals, Heavy/pharmacology , Nucleic Acid Hybridization , Pedobacter/cytology , Pedobacter/drug effects , Phylogeny , RNA, Ribosomal, 16S/genetics , Sinorhizobium meliloti/cytology , Sinorhizobium meliloti/drug effects , Soil MicrobiologyABSTRACT
Surface plasmon resonance (SPR) with two-dimensional (2D) materials is proposed to enhance the sensitivity of sensors. A novel Goos-Hänchen (GH) shift sensing scheme based on blue phosphorene (BlueP)/transition metal dichalogenides (TMDCs) and graphene structure is proposed. The significantly enhanced GH shift is obtained by optimizing the layers of BlueP/TMDCs and graphene. The maximum GH shift of the hybrid structure of Ag-Indium tin oxide (ITO)-BlueP/WS2-graphene is -2361λ with BlueP/WS2 four layers and a graphene monolayer. Furthermore, the GH shift can be positive or negative depending on the layer number of BlueP/TMDCs and graphene. For sensing performance, the highest sensitivity of 2.767 × 107λ/RIU is realized, which is 5152.7 times higher than the traditional Ag-SPR structure, 2470.5 times of Ag-ITO, 2159.2 times of Ag-ITO-BlueP/WS2, and 688.9 times of Ag-ITO-graphene. Therefore, such configuration with GH shift can be used in various chemical, biomedical and optical sensing fields.
ABSTRACT
Lotus (Nelumbo nucifera Gaertn.), one of the earliest plants in angiosperms, is a perennial aquatic herb widely distributed throughout Eastern Asia. Quercetin and its glycosides are the most abundant phenolic compounds in lotus with multiple pharmacological activities. Although many flavonoid O-glycosyltransferases involved in the biosynthesis of quercetin glycosides have been identified from terrestrial plants, no glycosyltransferase has been identified in aquatic plants. In this study, a new glycosyltransferase (NpUGT6) was identified from the embryo of Nelumbo nucifera (Nelumbinis Plumula). Function characterization demonstrated that NpUGT6 exhibited a robust capability to regio- and stereo-specific O-glycosylation at the 3-hydroxy group of quercetin scaffolds with UDP-glucose. Moreover, the O-glycosylation catalyzed by NpUGT6 was reversible. NpUGT6 is the first identified flavonoid O-glycosyltransferase from aquatic plants. Its sequence will provide useful guidance for the discovery of additional flavonoid glycosyltransferses in Nymphaeaceae and other aquatic plants.
Subject(s)
Flavonoids/chemistry , Glycosyltransferases/chemistry , Nelumbo/enzymology , Plant Proteins/chemistry , Catalysis , Cloning, Molecular , Escherichia coli/metabolism , Glycosides/chemistry , Glycosylation , Hydrogen-Ion Concentration , Kinetics , Phylogeny , Quercetin/analogs & derivatives , Quercetin/chemistry , Recombinant Proteins/chemistry , Seeds/enzymology , Substrate Specificity , Uridine Diphosphate Glucose/chemistryABSTRACT
The RIG-I-like RNA helicase (RLR)-mediated interferon (IFN) response plays a pivotal role in the hepatic antiviral immunity. The hepatitis A virus (HAV) and the hepatitis C virus (HCV) counter this response by encoding a viral protease that cleaves the mitochondria antiviral signaling protein (MAVS), a common signaling adaptor for RLRs. However, a third hepatotropic RNA virus, the hepatitis E virus (HEV), does not appear to encode a functional protease yet persists in infected cells. We investigated HEV-induced IFN responses in human hepatoma cells and primary human hepatocytes. HEV infection resulted in persistent virus replication despite poor spread. This was companied by a type III IFN response that upregulated multiple IFN-stimulated genes (ISGs), but type I IFNs were barely detected. Blocking type III IFN production or signaling resulted in reduced ISG expression and enhanced HEV replication. Unlike HAV and HCV, HEV did not cleave MAVS; MAVS protein size, mitochondrial localization, and function remained unaltered in HEV-replicating cells. Depletion of MAVS or MDA5, and to a less extent RIG-I, also diminished IFN production and increased HEV replication. Furthermore, persistent activation of the JAK/STAT signaling rendered infected cells refractory to exogenous IFN treatment, and depletion of MAVS or the receptor for type III IFNs restored the IFN responsiveness. Collectively, these results indicate that unlike other hepatotropic RNA viruses, HEV does not target MAVS and its persistence is associated with continuous production of type III IFNs.
Subject(s)
Hepatitis E virus/physiology , Hepatitis E/immunology , Interferons/immunology , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/immunology , Hepatitis E/genetics , Hepatitis E/virology , Hepatitis E virus/genetics , Hepatitis E virus/immunology , Hepatocytes/immunology , Hepatocytes/virology , Humans , Interferons/genetics , Virus ReplicationABSTRACT
Insulin is critical for the regulation of de novo fatty acid synthesis, which converts glucose to lipid in the liver. However, how insulin signals are transduced into the cell and then regulate lipogenesis remains to be fully understood. Here, we identified CREB/ATF bZIP transcription factor (CREBZF) of the activating transcription factor/cAMP response element-binding protein (ATF/CREB) gene family as a key regulator for lipogenesis through insulin-Akt signaling. Insulin-induced gene 2a (Insig-2a) decreases during refeeding, allowing sterol regulatory element binding protein 1c to be processed to promote lipogenesis; but the mechanism of reduction is unknown. We show that Insig-2a inhibition is mediated by insulin-induced CREBZF. CREBZF directly inhibits transcription of Insig-2a through association with activating transcription factor 4. Liver-specific knockout of CREBZF causes an induction of Insig-2a and Insig-1 and resulted in repressed lipogenic program in the liver of mice during refeeding or upon treatment with streptozotocin and insulin. Moreover, hepatic CREBZF deficiency attenuates hepatic steatosis in high-fat, high-sucrose diet-fed mice. Importantly, expression levels of CREBZF are increased in livers of diet-induced insulin resistance or genetically obese ob/ob mice and humans with hepatic steatosis, which may underscore the potential role of CREBZF in the development of sustained lipogenesis in the liver under selective insulin resistance conditions. CONCLUSION: These findings uncover an unexpected mechanism that couples changes in extracellular hormonal signals to hepatic lipid homeostasis; disrupting CREBZF function may have the therapeutic potential for treating fatty liver disease and insulin resistance. (Hepatology 2018).
Subject(s)
Basic-Leucine Zipper Transcription Factors/genetics , Fatty Liver/pathology , Gene Expression Regulation , Insulin Resistance/genetics , Lipogenesis/genetics , Analysis of Variance , Animals , Biopsy, Needle , Diet, High-Fat , Disease Models, Animal , Fatty Liver/genetics , Hepatocytes/metabolism , Hepatocytes/pathology , Humans , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Random Allocation , Signal TransductionABSTRACT
Smoking is considered to be the main source of indoor pollution, and it has been identified as an important environmental factor contributing to asthma onset. We know that T helper 2 (Th2) response plays a crucial role in the process of asthma disease. We have investigated the reaction of cigarette smoke extract (CSE) on Th polarization which is controlled by dendritic cells (DCs). Stimulated by CSE, immature DCs from murine bone marrow showed upregulated levels of TIM4. Cocultured with CD4+ T cells, stimulated DCs increased the ratio of IL-4+ versus IFN-γ+ of CD4+ T cells. This suggests a differentiation towards Th2 response. Moreover, antibodies against TIM4 reversed the upexpression of the IL-4+/IFN-γ+ ratio provoked by CSE, indicating that the Th2 polarization which was induced by CSE is via TIM4 mechanisms. CSE could activate mitogen-activated protein kinase pathways like ERK and p38. Upregulation of TIM4 expression by CSE stimulation was found to be inhibited by an ERK inhibitor but not p38 and JNK. In conclusion, DC-induced Th2 polarization is a hallmark of CSE allergy, and this aspect can be explained by CSE-induced TIM4 expression.
Subject(s)
Dendritic Cells/physiology , Extracellular Signal-Regulated MAP Kinases/physiology , MAP Kinase Signaling System/physiology , Membrane Proteins/physiology , Nicotiana/adverse effects , Smoke/adverse effects , Th2 Cells/immunology , Animals , Cell Polarity , Cells, Cultured , Interferon-gamma/analysis , Interleukin-4/analysis , Membrane Proteins/antagonists & inhibitors , MiceABSTRACT
Herein, the catalytic promiscuity of TcCGT1, a new C-glycosyltransferase (CGT) from the medicinal plant Trollius chinensis is explored. TcCGT1 could efficiently and regio-specifically catalyze the 8-C-glycosylation of 36 flavones and other flavonoids and could also catalyze the O-glycosylation of diverse phenolics. The crystal structure of TcCGT1 in complex with uridine diphosphate was determined at 1.85â Å resolution. Molecular docking revealed a new model for the catalytic mechanism of TcCGT1, which is initiated by the spontaneous deprotonation of the substrate. The spacious binding pocket explains the substrate promiscuity, and the binding pose of the substrate determines C- or O-glycosylation activity. Site-directed mutagenesis at two residues (I94E and G284K) switched C- to O-glycosylation. TcCGT1 is the first plant CGT with a crystal structure and the first flavone 8-C-glycosyltransferase described. This provides a basis for designing efficient glycosylation biocatalysts.
Subject(s)
Cloning, Molecular , Glycosyltransferases/metabolism , Plant Proteins/chemistry , Ranunculaceae/enzymology , Catalytic Domain , Glycosyltransferases/genetics , Models, Molecular , Plant Proteins/metabolism , Protein ConformationABSTRACT
Prenylated pterocarpans are valuable natural products that play significant roles in plant defence and possess diverse biological activities. However, structural diversity of prenylated pterocarpans is still limited. Prenyltransferases (PTs) could catalyze the transfer of prenyl moieties to acceptor molecules and increase the structural diversity and biological activity of natural products. Up to date, only two pterocarpan PTs have been identified from plants. In this study, a new pterocarpan prenyltransferase gene, designated as PcM4DT, was identified from Psoralea corylifolia. The deduced polypeptide is predicted to be a membrane-bound protein with eight transmembrane regions. Functional characterization of recombinant PcM4DT demonstrated this enzyme could catalyze C-4 prenylation of pterocarpans, and exhibited strict substrate specificity to maackiain and 3-hydroxy-9-methoxy-pterocarpan. It also showed a strict donor specificity to DMAPP. Furthermore, removal of the putative transit peptide of PcM4DT obviously increased the catalytic activity (up to 90%). PcM4DT represents the first PT identified from the Psoralea genus.
Subject(s)
Cell Membrane/metabolism , Dimethylallyltranstransferase/metabolism , Prenylation , Psoralea/enzymology , Pterocarpans/chemistry , Pterocarpans/metabolism , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , HL-60 Cells , Humans , Kinetics , Psoralea/cytology , Pterocarpans/pharmacology , Stereoisomerism , Substrate SpecificityABSTRACT
Glycosylation is an effective approach to improve the druggability of natural products by increasing their water solubility. In this work, we report the glycosylation of oleanane-type triterpenoids by a recombinant microbial glycosyltransferase YjiC1. A preliminary screening test indicated YjiC1 exhibited robust capabilities for O-glycosylation of triterpenoids, based on LC/MS analysis. Among the products, two new compounds (2a and 3a), together with a known one (1a), were isolated and characterized. These products exhibited improved water solubility, and 3a showed moderate anti-HIV activities at 100 µM. This reaction provides a facile and efficient approach to synthesize the glucosides of triterpenoids.