PI3K/AKT/mTOR and TLR4/MyD88/NF-κB Signaling Inhibitors Attenuate Pathological Mechanisms of Allergic Asthma.

Asthma is an inflammatory airway disease wherein bronchoconstriction, airway inflammation, and airway obstruction during asthma attacks are the main problems. It is recognized that imbalance of Th1/Th2 and Th17/Treg is a critical factor in asthma pathogenesis. Manipulation of these with signaling molecules such as mTOR, PI3K, Akt, and MyD88 can control asthma. Mouse model of allergic asthma was produced and treated with ketamine, metformin, metformin and ketamine, triciribine, LY294002, and torin2. MCh challenge test, BALf's Eos Count, the IL-4, 5, INF-γ, eicosanoid, total IgE levels were determined. The MUC5a, Foxp3, RORγt, PI3K, mTOR, Akt, PU.1, and MyD88 gene expressions and histopathology study were done. Asthma groups that were treated with all six components had reduced Penh value, total IgE, IL-4 and IL-5 levels, MUC5a, RORγt, MyD88 and mTOR expression, goblet cell hyperplasia, and mucus hyper-secretion. The eosinophil percentage and Cys-LT level were decreased by metformin and ketamine, triciribine, LY294002, and torin2. The level of IFN-γ was increased in triciribine, LY294002, and torin2. Metformin, metformin and ketamine, triciribine, LY294002, and torin2 reduced Akt and PI3K expression, peribronchial and perivascular inflammation, and increased expression of Foxp3. Torin2 had an effect on PU.1 expression. Inhibition of PI3K/AKT/mTOR and TLR4/MyD88/NF-κB signaling with targeted molecules can attenuate asthma pathology and play an important role in airways protection.

The chromosome-level reference genome assembly for Panax notoginseng and insights into ginsenoside biosynthesis.

Panax notoginseng, a perennial herb of the genus Panax in the family Araliaceae, has played an important role in clinical treatment in China for thousands of years because of its extensive pharmacological effects. Here, we report a high-quality reference genome of P. notoginseng, with a genome size up to 2.66 Gb and a contig N50 of 1.12 Mb, produced with third-generation PacBio sequencing technology. This is the first chromosome-level genome assembly for the genus Panax. Through genome evolution analysis, we explored phylogenetic and whole-genome duplication events and examined their impact on saponin biosynthesis. We performed a detailed transcriptional analysis of P. notoginseng and explored gene-level mechanisms that regulate the formation of characteristic tubercles. Next, we studied the biosynthesis and regulation of saponins at temporal and spatial levels. We combined multi-omics data to identify genes that encode key enzymes in the P. notoginseng terpenoid biosynthetic pathway. Finally, we identified five glycosyltransferase genes whose products catalyzed the formation of different ginsenosides in P. notoginseng. The genetic information obtained in this study provides a resource for further exploration of the growth characteristics, cultivation, breeding, and saponin biosynthesis of P. notoginseng.

Investigating the Catalytic Activity of Glycosyltransferase on Quercetin from Tripterygium wilfordii.

Flavonoid glycosides have shown many pharmacological activities in clinical studies. However, the main way to obtain flavonoid glycosides is to extract and separate them from plants, which wastes both time and resources. Here, we identified the O-glycosyltransferase (UGTs) TwUGT3 from Tripterygium wilfordii and analyzed its bioinformatics. First, the enzyme was found to utilize phloretin and uridine diphosphate glucose (UDPG) as substrates to produce an acid-tolerant glucoside. Then, it also can use quercetin and UDPG as substrates to produce the corresponding O-glucoside. In addition, we further explored the substrate specificity of TwUGT3, which suggested that it also accepts luteolin, pinocembrin, and genistein to produce the corresponding O-glucosides. Subsequently, the optimum pH, reaction time, reaction temperature, and enzymatic kinetic parameters of TwUGT3 were determined.

Isolation and characterization of a glycosyltransferase with specific catalytic activity towards flavonoids from Tripterygium wilfordii.

Flavonoids are important secondary metabolites that exist in many medicinal plants. Flavonoid glycosyltransferases can transfer sugar moieties to their parent rings, producing various flavonoid glycosides with significant pharmacological activities. Here, we report the molecular cloning of the O-glycosyltransferase TwUGT2 from Tripterygium wilfordii and its catalytic activity was explored by heterologous expression in E. coli. The results showed that TwUGT2 has specific glycosyltransferase activity towards C-3 and 7 hydroxyl groups of flavonoids, thereby converting quercetin and pinocembrin into isoquercitrin and pinocembrin 7-O-beta-D-glucoside, respectively. The identification of TwUGT2 will provide a useful molecular tool for synthetic biology and contribute to drug discovery.[Formula: see text].

A specific UDP-glucosyltransferase catalyzes the formation of triptophenolide glucoside from Tripterygium wilfordii Hook. f.

Tripterygium wilfordii Hook. f. is a perennial woody vine member of the Celastraceae family. As a traditional Chinese medicine, it contains complex chemical components and exerts various pharmacological activities. In the present study, we identified a glucosyltransferase, TwUGT1, that can catalyze the synthesis of an abietane-type diterpene glucoside, namely, triptophenolide14-O-beta-D-glucopyranoside, and investigated the pharmacological activity of triptophenolide glucoside in diverse cancer cells. Triptophenolide glucoside exhibited significant inhibitory effects on U87-MG, U251, C6, MCF-7, HeLa, K562, and RBL-2H3 cells as determined by pharmacological analysis. The triptophenolide glucoside content of T. wilfordii was analyzed using Agilent Technologies 6490 Triple Quad LC/MS. The glucosyltransferase TwUGT1 belongs to subfamily 88 and group E in family 1. Molecular docking and site-directed mutagenesis of TwUGT1 revealed that the His30, Asp132, Phe134, Thr154, Ala370, Leu376, Gly382, His387, Glu395 and Gln412 residues play crucial roles in the catalytic activity of triptophenolide 14-O-glucosyltransferase. In addition, TwUGT1 was also capable of glucosylating phenolic hydroxyl groups, such as those in liquiritigenin, pinocembrin, 4-methylumbelliferone, phloretin, and rhapontigenin.

Analysis of the role of geranylgeranyl diphosphate synthase 8 from Tripterygium wilfordii in diterpenoids biosynthesis.

Tripterygium wilfordii is known to contain various types of bioactive diterpenoids that exhibit many remarkable activities. Many studies have recently been targeted toward the elucidation of the diterpenoids biosynthetic pathways in attempts to obtain these compounds with a view to solving the dilemma of low yield in plants. However, the short-chain prenyltransferases (SC-PTSs) responsible for the formation of geranylgeranyl diphosphate (GGPP), a crucial precursor for synthesizing the skeleton structures of diterpenoids, have not been characterized in depth. Here, T. wilfordii transcriptome data were used to identify eight putative GGPPSs, including two small subunits of geranyl diphosphate synthase (GPPS.SSU). Of them, GGPPS1, GGPPS7, GGPPS8, GPPS.SSU II and GPPS.SSU were translocated mainly into chloroplasts, and GGPPS8 exhibited the optimal catalytic efficiency with respect to catalyzing the formation of GGPP. In addition, the expression pattern of GGPPS8 was similar to that of downstream terpene synthase genes that are directly correlated with triptolide production in roots, indicating that GGPPS8 was most likely to participate in triptolide biosynthesis in roots among the studied enzymes. GPPS.SSU was inactive alone but interacted with GGPPS1, GGPPS7 and GGPPS8 to change the product from GGPP to GPP. These findings implicate that these candidate genes can be regulated to shift the metabolic flux toward diterpenoid formation, increasing the yields of bioactive diterpenoids in plants.

Probing the function of protein farnesyltransferase in Tripterygium wilfordii.

KEY MESSAGE: We found two subunits FTase/GGTaseI-α and FTase-ß formed a heterodimer to transfer a farnesyl group from FPP to protein N-dansyl-GCVLS, confirming they are responsible for protein farnesylation in planta. Tripterygium wilfordii is a medicinal plant with a broad spectrum of anti-inflammatory, immunosuppressive and anti-cancer activities. Recently, a number of studies have focused on investigating the biosynthetic pathways of its bioactive compounds, whereas little attention has been paid to the enzymes which play important roles in regulating diverse developmental processes of T. wilfordii. In this study, we report for the first time the identification and characterization of two subunits of farnesyltransferase (FTase), farnesyltransferase/geranylgeranyltransferase I-α (TwFTase/GGTase I-α) and farnesyltransferase-ß (TwFTase-ß), in this important medicinal plant. Cell-free in vivo assays, yeast two-hybrid (Y2H) and pull-down assays showed that the two subunits interact with each other to form a heterodimer to perform the role of specifically transferring a farnesyl group from FPP to the CAAX-box protein N-dansyl-GCVLS. Furthermore, we discovered that the two subunits had the same cytoplasmic localization pattern and displayed the same tissue expression pattern. These results indicated that we identified a functional TwFTase enzyme which contains two functionally complementary subunits TwFTase/GGTase I-α and TwFTase-ß, which provides us promising genetic targets to construct transgenic plants or screen for more adaptable T. wilfordii mutants, which are able to survive in changing environments.

Glucosyltransferase Capable of Catalyzing the Last Step in Neoandrographolide Biosynthesis.

ApUGT, a diterpene glycosyltransferase from Andrographis paniculata, could transfer a glucose to the C-19 hydroxyl moiety of andrograpanin to form neoandrographolide. This glycosyltransferase has a broad substrate scope, and it can glycosylate 26 natural and unnatural compounds of different structural types. This study provides a basis for exploring the glycosylation mechanism of ent-labdane-type diterpenes and plays an important role in diversifying the structures used in drug discovery.

Overexpression and RNAi-mediated downregulation of TwIDI regulates triptolide and celastrol accumulation in Tripterygium wilfordii.

The aim of this study was to verify the effects of TwIDI (GenBank: KT279355.1) on triptolide and celastrol accumulation in the biosynthesis of terpenoids in Tripterygium wilfordii and the regulation of the expression of related genes in the triptolide and celastrol biosynthesis pathway. After bioinformatics analysis of TwIDI, we cloned the full-length CDS and a specific 398 bp fragment to construct overexpression and RNAi vectors, respectively. The specific amplification of hygromycin and kanamycin resistance gene fragments confirmed that the expression vectors had been successfully delivered into Tripterygium wilfordii suspension cells. qRT-PCR was used to detect the expression of TwIDI and related genes in the triptolide and celastrol biosynthesis pathway. The expression of TwIDI was increased to 157% of the control group (empty vector) in the overexpression group, and was reduced to 71% of the control group in the RNAi group. Notably, the expression of other genes in the triptolide and celastrol biosynthesis pathway also showed differences. For example, TwMCS was reduced to 62% of the control when TwIDI was overexpressed and increased to 188% in the RNAi group. The expression of TwDXS did not change significantly both during TwIDI overexpression and RNAi group. The accumulation of triptolide and celastrol in the suspension cells of Tripterygium wilfordii was detected by UPLC, revealing that the contents of triptolide and celastrol were increased 1.36- and 1.20-fold over the control group in the overexpression group, and decreased to 0.16 and 0.36 of the control group in the RNAi group. Based on these findings, the effect on the accumulation of active terpenoids in Tripterygium wilfordii and the feedback regulation of genes in the triptolide and celastrol biosynthesis pathway was verified through TwIDI overexpression and RNAi experiments.

Probing the Single Key Amino Acid Responsible for the Novel Catalytic Function of ent-Kaurene Oxidase Supported by NADPH-Cytochrome P450 Reductases in Tripterygium wilfordii.

Tripterygium wilfordii produces not only ent-kaurene, which is an intermediate of gibberellin (GA) biosynthesis in flowering plants, but also 16α-hydroxy-ent-kaurane, whose physiological role has not been characterized. The two compounds are biosynthesized from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) by diterpene synthases, which have been discovered and functionally characterized in T. wilfordii. Here, we described the functional characterization of four cytochrome P450 reductases (TwCPR) and one ent-kaurene oxidase (TwKO). Four TwCPRs were found to have relatively ubiquitous expression in T. wilfordii root, stem, leaf, and flower tissues. Co-expression of both a TwCPR and TwKO in yeast showed that TwCPR3 has a slightly better activity for providing the electrons required for these reactions, indicating that TwCPR3 is more suitable for use in the functional analysis of other cytochrome P450 monooxygenases. TwKO catalyzed the three-step oxidation of the C4α methyl of the tetracyclic diterpene intermediate ent-kaurene to form ent-kaurenoic acid as an early step in GA biosynthesis. Notably, TwKO could also convert 16α-hydroxy-ent-kaurane to 16α-hydroxy-ent-kaurenoic acid, indicating an important function of 16α-hydroxy-ent-kaurane in the anti-HIV principle tripterifordin biosynthetic pathway in planta. Homology modeling and molecular docking were used to investigate the unknown crucial active amino acid residue involved in the catalytic reaction of TwKO, and one key residue (Leu387) contributed to the formation of 16α-hydroxy-ent-kaurenoic acid, most likely by forming hydrogen bonds with the hydroxyl group (-OH) of 16α-hydroxy-ent-kaurane, which laid the basis for further investigation of the multifunctional nature of KO catalysis. Also, our findings paved the way for the complete biosynthesis of the anti-HIV principle tripterifordin.

Phylogeographic and phylogenetic analysis for Tripterygium species delimitation.

Tripterygium wilfordii (Celastraceae) is a traditional Chinese medicine; and the dried root and rhizome constitute the main officinal parts. Tripterygium wilfordii has been identified as a potential candidate for the treatment of systemic lupus erythematosus, rheumatoid arthritis, nephritis, asthma, leprosy, and cancer. The phylogenetic relationships within the Tripterygium genus are ambiguous; thus, our aim is to clarify the relationships within this genus using phylogeographic and phylogenetic analyses. Here, we first sequenced three plastid DNA regions (i.e., psbA-trnH, rpl32-trnL, and trnL-trnF) and found that Tripterygium hypoglaucum and T. wilfordii were clustered together based on the strength of the topology in the phylogenetic analysis: T. hypoglaucum is polyphyletic, and T. wilfordii is paraphyletic. A spatial analysis of molecular variance showed that the best group value is 4, and the groups were almost consistent with the topology of in the phylogenetic analysis. The Mantel analyses of Tripterygium using IBD web showed statistically significant relationships between genetic and geographical distance distributions (r = .3479, p < .0001). The molecular dating using Fossil calibration indicated that the divergence in Tripterygium was approximately 8.13 Ma. Furthermore, we also analyzed four DNA regions (i.e., ITS2, psbA-trnH, matK, and rbcL) that were obtained from the NCBI nucleotide database; these results showed that T. wilfordii and T. hypoglaucum clustered together, while Tripterygium regelii represented a separate cluster. Tripterygium hypoglaucum and T. wilfordii were never distinct lineages, and the species circumscriptions are artificial. We propose that T. wilfordii and T. hypoglaucum are conspecific, while T. regelii likely constitutes a separate species.