Characterization of CYP82 genes involved in the biosynthesis of structurally diverse benzylisoquinoline alkaloids in Corydalis yanhusuo.

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.

Levosimendan Relaxes Thoracic Aortic Smooth Muscle in Mice by Inhibiting PKC and Activating Inwardly Rectifying Potassium Channels.

ABSTRACT: Studies have examined the therapeutic effect of levosimendan on cardiovascular diseases such as heart failure, perioperative cardiac surgery, and septic shock, but the specific mechanism in mice remains largely unknown. This study aimed to investigate the relaxation mechanism of levosimendan in the thoracic aorta smooth muscle of mice. Levosimendan-induced relaxation of isolated thoracic aortic rings that were precontracted with norepinephrine or KCl was recorded in an endothelium-independent manner. Vasodilatation by levosimendan was not associated with the production of the endothelial relaxation factors nitric oxide and prostaglandins. The voltage-dependent K + channel (K V ) blocker (4-aminopyridine) and selective K Ca blocker (tetraethylammonium) had no effect on thoracic aortas treated with levosimendan, indicating that K V and K Ca channels may not be involved in the levosimendan-induced relaxation mechanism. Although the inwardly rectifying K + channel (K ir ) blocker (barium chloride) and the K ATP channel blocker (glibenclamide) significantly inhibited levosimendan-induced vasodilation in the isolated thoracic aorta, barium chloride had a much stronger inhibitory effect on levosimendan-induced vasodilation than glibenclamide, suggesting that levosimendan-induced vasodilation may be mediated by K ir channels. The vasodilation effect and expression of K ir 2.1 induced by levosimendan were further enhanced by the PKC inhibitor staurosporine. Extracellular calcium influx was inhibited by levosimendan without affecting intracellular Ca 2+ levels in the isolated thoracic aorta. These results suggest that K ir channels play a more important role than K ATP channels in regulating vascular tone in larger arteries and that the activity of the K ir channel is enhanced by the PKC pathway.

LZP is required for hepatic triacylglycerol transportation through maintaining apolipoprotein B stability.

The conserved zona pellucida (ZP) domain is found in hundreds of extracellular proteins that are expressed in various organs and play a variety of roles as structural components, receptors and tumor suppressors. A liver-specific zona pellucida domain-containing protein (LZP), also named OIT3, has been shown to be mainly expressed in human and mouse hepatocytes; however, the physiological function of LZP in the liver remains unclear. Here, we show that Lzp deletion inhibited very low-density lipoprotein (VLDL) secretion, leading to hepatic TG accumulation and lower serum TG levels in mice. The apolipoprotein B (apoB) levels were significantly decreased in the liver, serum, and VLDL particles of LZP-deficient mice. In the presence of LZP, which is localized to the endoplasmic reticulum (ER) and Golgi apparatus, the ER-associated degradation (ERAD) of apoB was attenuated; in contrast, in the absence of LZP, apoB was ubiquitinated by AMFR, a known E3 ubiquitin ligase specific for apoB, and was subsequently degraded, leading to lower hepatic apoB levels and inhibited VLDL secretion. Interestingly, hepatic LZP levels were elevated in mice challenged with a high-fat diet and humans with simple hepatic steatosis, suggesting that LZP contributes to the physiological regulation of hepatic TG homeostasis. In general, our data establish an essential role for LZP in hepatic TG transportation and VLDL secretion by preventing the AMFR-mediated ubiquitination and degradation of apoB and therefore provide insight into the molecular function of LZP in hepatic lipid metabolism.

Identification of the cytochrome P450s responsible for the biosynthesis of two types of aporphine alkaloids and their de novo biosynthesis in yeast.

Aporphine alkaloids have diverse pharmacological activities; however, our understanding of their biosynthesis is relatively limited. Previous studies have classified aporphine alkaloids into two categories based on the configuration and number of substituents of the D-ring and have proposed preliminary biosynthetic pathways for each category. In this study, we identified two specific cytochrome P450 enzymes (CYP80G6 and CYP80Q5) with distinct activities toward (S)-configured and (R)-configured substrates from the herbaceous perennial vine Stephania tetrandra, shedding light on the biosynthetic mechanisms and stereochemical features of these two aporphine alkaloid categories. Additionally, we characterized two CYP719C enzymes (CYP719C3 and CYP719C4) that catalyzed the formation of the methylenedioxy bridge, an essential pharmacophoric group, on the A- and D-rings, respectively, of aporphine alkaloids. Leveraging the functional characterization of these crucial cytochrome P450 enzymes, we reconstructed the biosynthetic pathways for the two types of aporphine alkaloids in budding yeast (Saccharomyces cerevisiae) for the de novo production of compounds such as (R)-glaziovine, (S)-glaziovine, and magnoflorine. This study provides key insight into the biosynthesis of aporphine alkaloids and lays a foundation for producing these valuable compounds through synthetic biology.

Phylogenetic analysis and functional characterization of norcoclaurine synthase involved in benzylisoquinoline alkaloids biosynthesis in Stephania tetrandra.

Benzylisoquinoline alkaloids (BIAs) are a class of secondary metabolites that possess diverse pharmaceutical properties and are exclusively accumulated in specific plant genera. The Pictet-Spengler condensation, catalyzed by norcoclaurine synthase (NCS), represents a key enzymatic reaction in the biosynthetic pathway of BIAs. While NCS genes have been identified in several plant families such as Papaveraceae, Berberidaceae, and Ranunculaceae, no NCS genes have been reported in Menispermaceae, which is another genus known to accumulate BIAs. Here, NCSs were isolated and functionally characterized from the Menispermaceae family plant Stephania tetrandra. In vitro enzyme assay identified two functional StNCSs which could catalyze the formation of (S)-norcoclaurine. These functionally characterized genes were then integrated into engineered yeast to enable the production of norcoclaurine. Phylogenetic analysis of the NCS enzymes revealed that the StNCSs predominantly clustered into two clades. The functional StNCSs clustered with known NCSs, highlighting the presence of a specific NCS catalytic domain. This study not only provides additional genetic components for the synthetic biology-based production of BIAs in yeast but also contributes to the understanding of the phylogenetic relationships and structure-function relationship of NCS genes involved in the origin and production of BIAs.

Structure-function analysis of CYP719As involved in methylenedioxy bridge-formation in the biosynthesis of benzylisoquinoline alkaloids and its de novo production.

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.

Colorectal cancer risk susceptibility loci in a Swedish population.

To search for colorectal cancer (CRC) risk loci, Swedish samples were used for a genome-wide haplotype analysis. A logistic regression model was employed in 2663 CRC cases and 1642 controls in the discovery analysis. Three analyses were done, on all, familial-, and nonfamilial CRC samples and only results with odds ratio (OR) > 1 were analyzed. single nucleotide polymorphism (SNP) analysis did not generate any statistically significant results. Haplotype analysis suggested novel loci, on chromosome 2q36.1 (OR = 1.71, p value = 5.6924 × 10-8 ) in all CRC samples, chromosome 1q43 (OR = 4.04 p value = 3.24 × 10-8 ) in familial CRC samples, and two hits in nonfamilial CRC samples, chromosomes 2q36.1 (OR = 1.71 p value = 5.69 × 10-8 ) and 3p24.3 (OR = 1.62 p value = 6.21 × 10-9 ). Moreover, one locus on chromosome 20q13.33 was suggested in analyses of all samples, and five more novel loci were suggested on chromosomes 10q25.3, 15q,22.31, 17p11.2, 1p34.2, and 3q24. The haplotypes from the analysis of all samples were replicated in a second study of CRC cases and controls from the same part of Sweden. In summary, using haplotype analysis in Swedish CRC samples, the best hits were novel loci and the locus on chromosomes 2q36.1 and 20q13.33 suggested in the analysis of all samples were confirmed in a second cohort. The ORs were often higher than ORs from published genome-wide association study (GWAS). The study suggested it was possible that a risk locus could involve more than one gene, and that haplotypes could give information on the gene or genes possibly involved in the risk at specific locus.

Application of qPCR assays based on haloacids transporter gene dehp2 for discrimination of Burkholderia and Paraburkholderia.

BACKGROUND: A major facilitator superfamily transporter Dehp2 was recently shown to be playing an important role in transport and biodegradation of haloacids in Paraburkholderia caribensis MBA4, and Dehp2 is phylogenetically conserved in Burkholderia sensu lato. RESULTS: We designed both Burkholderia sensu stricto-specific and Paraburkholderia-specific qPCR assays based on dehp2 and 16S rRNA, and validated the qPCR assays in 12 bacterial strains. The qPCR assays could detect single species of Burkholderia sensu stricto or Paraburkholderia with high sensitivity and discriminate them in mixtures with high specificity over a wide dynamic range of relative concentrations. At relatively lower cost compared with sequencing-based approach, the qPCR assays will facilitate discrimination of Burkholderia sensu stricto and Paraburkholderia in a large number of samples. CONCLUSIONS: For the first time, we report the utilization of a haloacids transporter gene for discriminative purpose in Burkholderia sensu lato. This enables not only quick decision on proper handling of putative pathogenic samples in Burkholderia sensu stricto group but also future exploitation of relevant species in Paraburkholderia group for haloacids biodegradation purposes.

RNA interference in the oleaginous yeast Rhodosporidium toruloides.

The red yeast Rhodosporidium toruloides is an excellent microbial host for production of carotenoids, neutral lipids and valuable enzymes. In recent years, genetic tools for gene expression and gene disruption have been developed for this red yeast. However, methods remain limited in terms of fine-tuning gene expression. In this study, we first demonstrated successful implementation of RNA interference (RNAi) in R. toruloides NP11, which was applied to down-regulate the expression of autophagy related gene 8 (ATG8), and fatty acid synthase genes (FAS1 and FAS2), respectively. Compared with the control strain, RNAi-engineered strains showed a silencing efficiency ranging from 11% to 92%. The RNAi approach described here ensures selective inhibition of the target gene expression, and should expand our capacity in the genetic manipulation of R. toruloides for both fundamental research and advanced cell factory development.

Cellular prion protein is involved in decidualization of mouse uterus.

Prion protein (PrP) is encoded by a single copy gene Prnp in many cell and tissue types. PrP is very famous for its infectious conformers (PrPSC) resulting in transmissible spongiform encephalopathies. At present, physiological functions of its cellular isoform (PrPC) remain ambiguous. Although PrPC expression has been found in uterus, whether it functions in maternal-fetal dialogue during early pregnant is unknown. In this study, we examined PrPC mRNA and protein in the uterus of peri-implantation mice, and found that they were expressed with a spatiotemporal dynamic pattern. Interestingly, PrPC was significantly increased in the decidual zones around the implanting embryos at the implantation window stage. To further demonstrate that PrPC is involved in the decidualization of mouse uterus during embryo implantation, we constructed the artificial decidualization models and the delayed implantation models. Once the pseudopregnant mice were artificially induced to decidualization, the PrPC expression then increased significantly in the decidua zone. And also, if the delayed implantation embryos were allowed to implant, PrPC protein was also simultaneously improved in stromal cells surrounding the implanting embryos. Moreover, PrPC expression can be inhibited by progesterone but upregulated by estrogen in mouse uterus. These results suggest that PrPC may play an important role in embryo implantation and decidualization.