Functional characterization of genes related to triterpene and flavonoid biosynthesis in Cyclocarya paliurus.

MAIN CONCLUSION: The oxidosqualene cyclases (OSCs) generating triterpenoid skeletons in Cyclocarya paliurus were identified for the first time, and two uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyzing the glycosylation of flavonoids were characterized. Cyclocarya paliurus, a native rare dicotyledonous plant in China, contains an abundance of triterpenoid saponins and flavonoid glycosides that exhibit valuable pharmaceutical effects in preventing hypertension, hyperlipidemia, and diabetes. However, the molecular mechanism explaining the biosynthesis of triterpenoid saponin and flavonoid glycoside in C. paliurus remains unclear. In this study, the triterpene content in different tissues and the expression pattern of genes encoding the key enzymes associated with triterpenoid saponin and flavonoid glycoside biosynthesis were studied using transcriptome and metabolome analysis. The eight upstream oxidosqualene cyclases (OSCs) involved in triterpenoid saponin biosynthesis were functionally characterized, among them CpalOSC6 catalyzed 2,3;22,23-dioxidosqualene to form 3-epicabraleadiol; CpalOSC8 cyclized 2,3-oxidosqualene to generate dammarenediol-II; CpalOSC2 and CpalOSC3 produced ß-amyrin and CpalOSC4 produced cycloartenol, while CpalOSC2-CpalOSC5, CpalOSC7, and CpalOSC8 all produced lanosterol. However, no catalytic product was detected for CpalOSC1. Moreover, two downstream flavonoid uridine diphosphate (UDP)-glycosyltransferases (UGTs) (CpalUGT015 and CpalUGT100) that catalyze the last step of flavonoid glycoside biosynthesis were functionally elucidated. These results uncovered the key genes involved in the biosynthesis of triterpenoid saponins and flavonoid glycosides in C. paliurus that could be applied to produce flavonoid glycosides and key triterpenoid saponins in the future via a synthetic strategy.

PtdIns(4,5)P2 stabilizes active states of GPCRs and enhances selectivity of G-protein coupling.

G-protein-coupled receptors (GPCRs) are involved in many physiological processes and are therefore key drug targets1. Although detailed structural information is available for GPCRs, the effects of lipids on the receptors, and on downstream coupling of GPCRs to G proteins are largely unknown. Here we use native mass spectrometry to identify endogenous lipids bound to three class A GPCRs. We observed preferential binding of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) over related lipids and confirm that the intracellular surface of the receptors contain hotspots for PtdIns(4,5)P2 binding. Endogenous lipids were also observed bound directly to the trimeric Gαsßγ protein complex of the adenosine A2A receptor (A2AR) in the gas phase. Using engineered Gα subunits (mini-Gαs, mini-Gαi and mini-Gα12)2, we demonstrate that the complex of mini-Gαs with the ß1 adrenergic receptor (ß1AR) is stabilized by the binding of two PtdIns(4,5)P2 molecules. By contrast, PtdIns(4,5)P2 does not stabilize coupling between ß1AR and other Gα subunits (mini-Gαi or mini-Gα12) or a high-affinity nanobody. Other endogenous lipids that bind to these receptors have no effect on coupling, highlighting the specificity of PtdIns(4,5)P2. Calculations of potential of mean force and increased GTP turnover by the activated neurotensin receptor when coupled to trimeric Gαißγ complex in the presence of PtdIns(4,5)P2 provide further evidence for a specific effect of PtdIns(4,5)P2 on coupling. We identify key residues on cognate Gα subunits through which PtdIns(4,5)P2 forms bridging interactions with basic residues on class A GPCRs. These modulating effects of lipids on receptors suggest consequences for understanding function, G-protein selectivity and drug targeting of class A GPCRs.

Lipid-Dependent Regulation of Ion Channels and G Protein-Coupled Receptors: Insights from Structures and Simulations.

Ion channels and G protein-coupled receptors (GPCRs) are regulated by lipids in their membrane environment. Structural studies combined with biophysical and molecular simulation investigations reveal interaction sites for specific lipids on membrane protein structures. For K channels, PIP2 plays a key role in regulating Kv and Kir channels. Likewise, several recent cryo-EM structures of TRP channels have revealed bound lipids, including PIP2 and cholesterol. Among the pentameric ligand-gated ion channel family, structural and biophysical studies suggest the M4 TM helix may act as a lipid sensor, e.g., forming part of the binding sites for neurosteroids on the GABAA receptor. Structures of GPCRs have revealed multiple cholesterol sites, which may modulate both receptor dynamics and receptor oligomerization. PIP2 also interacts with GPCRs and may modulate their interactions with G proteins. Overall, it is evident that multiple lipid binding sites exist on channels and receptors that modulate their function allosterically and are potential druggable sites.

Identification of two UDP-glycosyltransferases involved in the main oleanane-type ginsenosides in Panax japonicus var. major.

MAIN CONCLUSION: Two UDP-glycosyltransferases from Panax japonicus var. major were identified, and the biosynthetic pathways of three oleanane-type ginsenosides (chikusetsusaponin IVa, ginsenoside Ro, zingibroside R1) were elucidated. Chikusetsusaponin IVa and ginsenoside Ro are primary active components formed by stepwise glycosylation of oleanolic acid in five medicinal plants of the genus Panax. However, the key UDP-glycosyltransferases (UGTs) in the biosynthetic pathway of chikusetsusaponin IVa and ginsenoside Ro are still unclear. In this study, two UGTs (PjmUGT1 and PjmUGT2) from Panax japonicus var. major involved in the biosynthesis of chikusetsusaponin IVa and ginsenoside Ro were identified based on bioinformatics analysis, heterologous expression and enzyme assays. The results show that PjmUGT1 can transfer a glucose moiety to the C-28 carboxyl groups of oleanolic acid 3-O-ß-D-glucuronide and zingibroside R1 to form chikusetsusaponin IVa and ginsenoside Ro, respectively. Meanwhile, PjmUGT2 can transfer a glucose moiety to oleanolic acid 3-O-ß-D-glucuronide and chikusetsusaponin IVa to form zingibroside R1 and ginsenoside Ro. This work uncovered the biosynthetic mechanism of chikusetsusaponin IVa and ginsenoside Ro, providing the rational production of valuable saponins through synthetic biology strategy.

[Identification and expression profiling of R2R3-MYB transcription factors in Erigeron breviscapus].

R2 R3-MYB transcription factors are ubiquitous in plants, playing a role in the regulation of plant growth, development, and secondary metabolism. In this paper, the R2 R3-MYB transcription factors were identified by bioinformatics analysis of the genomic data of Erigeron breviscapus, and their gene sequences, structures, physical and chemical properties were analyzed. The functions of R2 R3-MYB transcription factors were predicted by cluster analysis. Meanwhile, the expression patterns of R2 R3-MYB transcription factors in response to hormone treatments were analyzed. A total of 108 R2 R3-MYB transcription factors, named EbMYB1-EbMYB108, were identified from the genome of E. breviscapus. Most of the R2 R3-MYB genes carried 2-4 exons. The phylogenetic tree of MYBs in E. breviscapus and Arabidopsis thaliala was constructed, which classified 234 MYBs into 30 subfamilies. The MYBs in the five MYB subfamilies of A.thaliala were clustered into independent clades, and those in E. breviscapus were clustered into four clades. The transcriptome data showed that MYB genes were differentially expressed in different tissues of E. breviscapus and in response to the treatments with exogenous hormones such as ABA, SA, and GA for different time. The transcription of 13 R2 R3-MYB genes did not change significantly, and the expression patterns of some genes were up-regulated or down-regulated with the extension of hormone treatment time. This study provides a theoretical basis for revealing the mechanisms of R2 R3-MYB transcription factors in regulating the growth and development, stress(hormone) response, and active ingredient accumulation in E. breviscapus.

The Glycosphingolipid GM3 Modulates Conformational Dynamics of the Glucagon Receptor.

The extracellular domain (ECD) of class B1 G-protein-coupled receptors (GPCRs) plays a central role in signal transduction and is uniquely positioned to sense both the extracellular and membrane environments. Although recent studies suggest a role for membrane lipids in the modulation of class A and class F GPCR signaling properties, little is known about the effect of lipids on class B1 receptors. In this study, we employed multiscale molecular dynamics simulations to access the dynamics of the glucagon receptor (GCGR) ECD in the presence of native-like membrane bilayers. Simulations showed that the ECD could move about a hinge region formed by residues Q122-E126 to adopt both closed and open conformations relative to the transmembrane domain. ECD movements were modulated by binding of the glycosphingolipid GM3. These large-scale fluctuations in ECD conformation may affect the ligand binding and receptor activation properties. We also identify a unique phosphatidylinositol (4,5)-bisphosphate (PIP2) interaction profile near intracellular loop (ICL) 2/TM3 at the G-protein-coupling interface, suggesting a mechanism of engaging G-proteins that may have a distinct dependence on PIP2 compared with class A GPCRs. Given the structural conservation of class B1 GPCRs, the modulatory effects of GM3 and PIP2 on GCGR may be conserved across these receptors, offering new insights into potential therapeutic targeting.

Transcriptome analysis of Panax zingiberensis identifies genes encoding oleanolic acid glucuronosyltransferase involved in the biosynthesis of oleanane-type ginsenosides.

MAIN CONCLUSION: Oleanolic acid glucuronosyltransferase (OAGT) genes synthesizing the direct precursor of oleanane-type ginsenosides were discovered. The four recombinant proteins of OAGT were able to transfer glucuronic acid at C-3 of oleanolic acid that yields oleanolic acid 3-O-ß-glucuronide. Ginsenosides are the primary active components in the genus Panax, and great efforts have been made to elucidate the mechanisms underlying dammarane-type ginsenoside biosynthesis. However, there is limited information on oleanane-type ginsenosides. Here, high-performance liquid chromatography analysis demonstrated that oleanane-type ginsenosides (particularly ginsenoside Ro and chikusetsusaponin IV and IVa) are the abundant ginsenosides in Panax zingiberensis, an extremely endangered Panax species in southwest China. These ginsenosides are derived from oleanolic acid 3-O-ß-glucuronide, which may be formed from oleanolic acid catalyzed by an unknown oleanolic acid glucuronosyltransferase (OAGT). Transcriptomic analysis of leaves, stems, main roots, and fibrous roots of P. zingiberensis was performed, and a total of 46,098 unigenes were obtained, including all the identified homologous genes involved in ginsenoside biosynthesis. The most upstream genes were highly expressed in the leaves, and the UDP-glucosyltransferase genes were highly expressed in the roots. This finding indicated that the precursors of ginsenosides are mainly synthesized in the leaves and transported to different parts for the formation of particular ginsenosides. For the first time, enzyme activity assay characterized four genes (three from P. zingiberensis and one from P. japonicus var. major, another Panax species with oleanane-type ginsenosides) encoding OAGT, which particularly transfer glucuronic acid at C-3 of oleanolic acid to form oleanolic acid 3-O-ß-glucuronide. Taken together, our study provides valuable genetic information for P. zingiberensis and the genes responsible for synthesizing the direct precursor of oleanane-type ginsenosides.

XCC2366, A Gene Encoding A Putative TetR Family Transcriptional Regulator, is Required for Acriflavin Resistance and Virulence of Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris (Xcc) is the phytopathogen that causes black rot disease in cruciferous plants. The XCC2366 gene product is annotated as a protein belonging to the TetR family of transcriptional regulators. In this study, we evaluated the function and expression of the XCC2366 gene. Mutational analysis demonstrated that XCC2366 is involved in the resistance to acriflavin and is necessary for virulence in Xcc. In addition, the XCC2366 transcription initiation site was mapped at nucleotide A, 63 nucleotide upstream of the XCC2366 translation start codon. Furthermore, transcriptional analysis revealed that the expression of XCC2366 is induced in the presence of acriflavin. Reporter assay also showed that XCC2366 regulates its own expression under acriflavin-supplemented condition. To the best of our knowledge, acriflavin resistance-related gene in the crucifer pathogen Xcc was characterized for the first time.

Understanding Voltage Gating of Providencia stuartii Porins at Atomic Level.

Bacterial porins are water-filled ß-barrel channels that allow translocation of solutes across the outer membrane. They feature a constriction zone, contributed by the plunging of extracellular loop 3 (L3) into the channel lumen. Porins are generally in the open state, but undergo gating in response to external voltages. To date the underlying mechanism is unclear. Here we report results from molecular dynamics simulations on the two porins of Providenica stuartii, Omp-Pst1 and Omp-Pst2, which display distinct voltage sensitivities. Voltage gating was observed in Omp-Pst2, where the binding of cations in-between L3 and the barrel wall results in exposing a conserved aromatic residue in the channel lumen, thereby halting ion permeation. Comparison of Omp-Pst1 and Omp-Pst2 structures and trajectories suggests that their sensitivity to voltage is encoded in the hydrogen-bonding network anchoring L3 onto the barrel wall, as we observed that it is the strength of this network that governs the probability of cations binding behind L3. That Omp-Pst2 gating is observed only when ions flow against the electrostatic potential gradient of the channel furthermore suggests a possible role for this porin in the regulation of charge distribution across the outer membrane and bacterial homeostasis.

Paeoniflorin ameliorates ischemic neuronal damage in vitro via adenosine A1 receptor-mediated transactivation of epidermal growth factor receptor.

AIM: Paeoniflorin from Chinese herb Paeoniae Radix has been shown to ameliorate middle cerebral artery occlusion-induced ischemia in rats. The aim of this study was to investigate the mechanisms underlying the neuroprotective action of PF in cultured rat cortical neurons. METHODS: Primary cultured cortical neurons of rats were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R) insult. Cell survival was determined using MTT assay. HEK293 cells stably transfected with A1R (HEK293/A1R) were used for detailed analysis. Phosphorylation of the signaling proteins was evaluated by Western blot or immunoprecipitation. Receptor interactions were identified using co-immunoprecipitation and immunofluorescence staining. RESULTS: Paeoniflorin (10 nmol/L to 1 µmol/L) increased the survival of neurons subjected to OGD/R. Furthermore, paeoniflorin increased the phosphorylation of Akt and ERK1/2 in these neurons. These effects were blocked by PI3K inhibitor wortmannin or MEK inhibitor U0126. Paeoniflorin also increased the phosphorylation of Akt and ERK1/2 in HEK293/A1R cells. Both A1R antagonist DPCPX and EGFR inhibitor AG1478 not only blocked paeoniflorin-induced phosphorylation of ERK1/2 and Akt in HEK293/A1R cells, but also paeoniflorin-increased survival of neurons subjected to OGD/R. In addition, paeoniflorin increased the phosphorylation of Src kinase and activation of MMP-2 in HEK293/A1R cells. Both Src inhibitor PP2 and MMP-2/MMP-9 inhibitor BiPs not only blocked paeoniflorin-induced phosphorylation of ERK1/2 (and Akt) in HEK293/A1R cells, but also paeoniflorin-increased survival of neurons subjected to OGD/R. CONCLUSION: Paeoniflorin promotes the survival of cultured cortical neurons by increasing Akt and ERK1/2 phosphorylation via A1R-mediated transactivation of EGFR.

Genome-wide profiling of WRKY genes involved in flavonoid biosynthesis in Erigeron breviscapus.

The transcription factors of WRKY genes play essential roles in plant growth, stress responses, and metabolite biosynthesis. Erigeron breviscapus, a traditional Chinese herb, is abundant in flavonoids and has been used for centuries to treat cardiovascular and cerebrovascular diseases. However, the WRKY transcription factors that regulate flavonoid biosynthesis in E. breviscapus remain unknown. In this study, a total of 75 EbWRKY transcription factors were predicted through comprehensive genome-wide characterization of E. breviscapus and the chromosomal localization of each EbWRKY gene was investigated. RNA sequencing revealed transient responses of 74 predicted EbWRKY genes to exogenous abscisic acid (ABA), salicylic acid (SA), and gibberellin 3 (GA3) after 4 h of treatment. In contrast, the expression of key structural genes involved in flavonoid biosynthesis increased after 4 h in GA3 treatment. However, the content of flavonoid metabolites in leaves significantly increased at 12 h. The qRT-PCR results showed that the expression patterns of EbWRKY11, EbWRKY30, EbWRKY31, EbWRKY36, and EbWRKY44 transcription factors exhibited a high degree of similarity to the 11 structural genes involved in flavonoid biosynthesis. Protein-DNA interactions were performed between the key genes involved in scutellarin biosynthesis and candidate WRKYs. The result showed that F7GAT interacts with EbWRKY11, EbWRKY36, and EbWRKY44, while EbF6H has a self-activation function. This study provides comprehensive information on the regulatory control network of flavonoid accumulation mechanisms, offering valuable insights for breeding E. breviscapus varieties with enhanced scutellarin content.

Design, synthesis and biological evaluation of bivalent ligands against A(1)-D(1) receptor heteromers.

AIM: To design and synthesize bivalent ligands for adenosine A1-dopamine D1 receptor heteromers (A1-D1R), and evaluate their pharmacological activities. METHODS: Bivalent ligands and their corresponding A1R monovalent ligands were designed and synthesized. The affinities of the bivalent ligands for A1R and D1R in rat brain membrane preparation were examined using radiolabeled binding assays. To demonstrate the formation of A1-D1R, fluorescence resonance energy transfer (FRET) was conducted in HEK293 cells transfected with D1-CFP and A1-YFP. Molecular modeling was used to analyze the possible mode of protein-protein and protein-ligand interactions. RESULTS: Two bivalent ligands for A1R and D1R (20a, 20b), as well as the corresponding A1R monovalent ligands (21a, 21b) were synthesized. In radiolabeled binding assays, the bivalent ligands showed affinities for A1R 10-100 times higher than those of the corresponding monovalent ligands. In FRET experiments, the bivalent ligands significantly increased the heterodimerization of A1R and D1R compared with the corresponding monovalent ligands. A heterodimer model with the interface of helixes 3, 4, 5 of A1R and helixes 1, 6, 7 from D1R was established with molecular modeling. The distance between the two ligand binding sites in the heterodimer model was approximately 48.4 Å, which was shorter than the length of the bivalent ligands. CONCLUSION: This study demonstrates the existence of A1-D1R in situ and a simultaneous interaction of bivalent ligands with both the receptors.

Comparative genomics reveals the diversification of triterpenoid biosynthesis and origin of ocotillol-type triterpenes in Panax.

Gene duplication is assumed to be the major force driving the evolution of metabolite biosynthesis in plants. Freed from functional burdens, duplicated genes can mutate toward novelties until fixed due to selective fitness. However, the extent to which this mechanism has driven the diversification of metabolite biosynthesis remains to be tested. Here we performed comparative genomics analysis and functional characterization to evaluate the impact of gene duplication on the evolution of triterpenoid biosynthesis using Panax species as models. We found that whole-genome duplications (WGDs) occurred independently in Araliaceae and Apiaceae lineages. Comparative genomics revealed the evolutionary trajectories of triterpenoid biosynthesis in plants, which was mainly promoted by WGDs and tandem duplication. Lanosterol synthase (LAS) was likely derived from a tandem duplicate of cycloartenol synthase that predated the emergence of Nymphaeales. Under episodic diversifying selection, the LAS gene duplicates produced by γ whole-genome triplication have given rise to triterpene biosynthesis in core eudicots through neofunctionalization. Moreover, functional characterization revealed that oxidosqualene cyclases (OSCs) responsible for synthesizing dammarane-type triterpenes in Panax species were also capable of producing ocotillol-type triterpenes. Genomic and biochemical evidence suggested that Panax genes encoding the above OSCs originated from the specialization of one OSC gene duplicate produced from a recent WGD shared by Araliaceae (Pg-ß). Our results reveal the crucial role of gene duplication in diversification of triterpenoid biosynthesis in plants and provide insight into the origin of ocotillol-type triterpenes in Panax species.

LipIDens: simulation assisted interpretation of lipid densities in cryo-EM structures of membrane proteins.

Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures. The pipeline integrates the implementation and analysis of multi-scale MD simulations for identification, ranking and refinement of lipid binding poses which superpose onto cryo-EM map densities. Thus, LipIDens enables direct integration of experimental and computational structural approaches to facilitate the interpretation of lipid-like cryo-EM densities and to reveal the molecular identities of protein-lipid interactions within a bilayer environment. We demonstrate this by application of our open-source LipIDens code to ten diverse membrane protein structures which exhibit lipid-like densities.

BMF-AS1/BMF Promotes Diabetic Vascular Calcification and Aging both In Vitro and In Vivo.

Vascular calcification and aging often increase morbidity and mortality in patients with diabetes mellitus (DM); however, the underlying mechanisms are still unknown. In the present study, we found that Bcl-2 modifying factor (BMF) and BMF antisense RNA 1 (BMF-AS1) were significantly increased in high glucose-induced calcified and senescent vascular smooth muscle cells (VSMCs) as well as artery tissues from diabetic mice. Inhibition of BMF-AS1 and BMF reduced the calcification and senescence of VSMCs, whereas overexpression of BMF-AS1 and BMF generates the opposite results. Mechanistic analysis showed that BMF-AS1 interacted with BMF directly and up-regulated BMF at both mRNA and protein levels, but BMF did not affect the expression of BMF-AS1. Moreover, knocking down BMF-AS1 and BMF suppressed the calcification and senescence of VSMCs, and BMF knockout (BMF-/-) diabetic mice presented less vascular calcification and aging compared with wild type diabetic mice. In addition, higher coronary artery calcification scores (CACs) and increased plasma BMF concentration were found in patients with DM, and there was a positive correlation between CACs and plasma BMF concentration. Thus, BMF-AS1/BMF plays a key role in promoting high glucose-induced vascular calcification and aging both in vitro and in vivo. BMF-AS1 and BMF represent potential therapeutic targets in diabetic vascular calcification and aging.

Transcriptional analysis and functional characterization of XCC1294 gene encoding a GGDEF domain protein in Xanthomonas campestris pv. campestris.

The nucleotide cyclic di-GMP is a second messenger in bacteria that regulates a range of cellular functions including the virulence of pathogens. GGDEF is a protein domain involved in the synthesis of cyclic di-GMP. The genome of the crucifer pathogen Xanthomonas campestris pv. campestris (Xcc) encodes 21 proteins with a GGDEF domain. Clp, a homolog of the model transcription factor Crp of Escherichia coli, is a global regulator in Xcc. The aim of this study is to identify genes encoding GGDEF domain proteins whose expression is regulated by Clp. Results of reporter assay and RT-PCR analysis suggested that Clp regulates the expression of a set of genes encoding proteins harboring GGDEF domain. The transcription initiation site of XCC1294, one of the Clp regulated gene encoding a GGDEF domain protein, was mapped. Promoter analysis and gel retardation assay indicated that the transcription of XCC1294 is positively and directly regulated by Clp. Furthermore, transcription of XCC1294 was subject to catabolite repression and affected by several stress conditions. We also showed that mutation of XCC1294 results in enhanced surface attachment. In addition, transcription of three putative adhesin genes (xadA, fhaC, and yapH) was increased in the XCC1294 mutant. Taken together, the data presented here indicate that Clp positively regulates expression of XCC1294, and that XCC1294 serves a regulator of bacterial attachment and regulates different adhesin genes expression.

Hepatocyte cholesterol content modulates glucagon receptor signalling.

OBJECTIVE: To determine whether glucagon receptor (GCGR) actions are modulated by cellular cholesterol levels. METHODS: We determined the effects of experimental cholesterol depletion and loading on glucagon-mediated cAMP production, ligand internalisation and glucose production in human hepatoma cells, mouse and human hepatocytes. GCGR interactions with lipid bilayers were explored using coarse-grained molecular dynamic simulations. Glucagon responsiveness was measured in mice fed a high cholesterol diet with or without simvastatin to modulate hepatocyte cholesterol content. RESULTS: GCGR cAMP signalling was reduced by higher cholesterol levels across different cellular models. Ex vivo glucagon-induced glucose output from mouse hepatocytes was enhanced by simvastatin treatment. Mice fed a high cholesterol diet had increased hepatic cholesterol and a blunted hyperglycaemic response to glucagon, both of which were partially reversed by simvastatin. Simulations identified likely membrane-exposed cholesterol binding sites on the GCGR, including a site where cholesterol is a putative negative allosteric modulator. CONCLUSIONS: Our results indicate that cellular cholesterol content influences glucagon sensitivity and indicate a potential molecular basis for this phenomenon. This could be relevant to the pathogenesis of non-alcoholic fatty liver disease, which is associated with both hepatic cholesterol accumulation and glucagon resistance.

PyLipID: A Python Package for Analysis of Protein-Lipid Interactions from Molecular Dynamics Simulations.

Lipids play important modulatory and structural roles for membrane proteins. Molecular dynamics simulations are frequently used to provide insights into the nature of these protein-lipid interactions. Systematic comparative analysis requires tools that provide algorithms for objective assessment of such interactions. We introduce PyLipID, a Python package for the identification and characterization of specific lipid interactions and binding sites on membrane proteins from molecular dynamics simulations. PyLipID uses a community analysis approach for binding site detection, calculating lipid residence times for both the individual protein residues and the detected binding sites. To assist structural analysis, PyLipID produces representative bound lipid poses from simulation data, using a density-based scoring function. To estimate residue contacts robustly, PyLipID uses a dual-cutoff scheme to differentiate between lipid conformational rearrangements while bound from full dissociation events. In addition to the characterization of protein-lipid interactions, PyLipID is applicable to analysis of the interactions of membrane proteins with other ligands. By combining automated analysis, efficient algorithms, and open-source distribution, PyLipID facilitates the systematic analysis of lipid interactions from large simulation data sets of multiple species of membrane proteins.

Integrated Metabolomic and Transcriptomic Analysis and Identification of Dammarenediol-II Synthase Involved in Saponin Biosynthesis in Gynostemma longipes.

Gynostemma longipes contains an abundance of dammarane-type ginsenosides and gypenosides that exhibit extensive pharmacological activities. Increasing attention has been paid to the elucidation of cytochrome P450 monooxygenases (CYPs) and UDP-dependent glycosyltransferases (UGTs) that participate downstream of ginsenoside biosynthesis in the Panax genus. However, information on oxidosqualene cyclases (OSCs), the upstream genes responsible for the biosynthesis of different skeletons of ginsenoside and gypenosides, is rarely reported. Here, an integrative study of the metabolome and the transcriptome in the leaf, stolon, and rattan was conducted and the function of GlOSC1 was demonstrated. In total, 46 triterpenes were detected and found to be highly abundant in the stolon, whereas gene expression analysis indicated that the upstream OSC genes responsible for saponin skeleton biosynthesis were highly expressed in the leaf. These findings indicated that the saponin skeletons were mainly biosynthesized in the leaf by OSCs, and subsequently transferred to the stolon via CYPs and UGTs biosynthesis to form various ginsenoside and gypenosides. Additionally, a new dammarane-II synthase (DDS), GlOSC1, was identified by bioinformatics analysis, yeast expression assay, and enzyme assays. The results of the liquid chromatography-mass spectrometry (LC-MS) analysis proved that GlOSC1 could catalyze 2,3-oxidosqualene to form dammarenediol-II via cyclization. This work uncovered the biosynthetic mechanism of dammarenediol-II, an important starting substrate for ginsenoside and gypenosides biosynthesis, and may achieve the increased yield of valuable ginsenosides and gypenosides produced under excess substrate in a yeast cell factory through synthetic biology strategy.

EbMYBP1, a R2R3-MYB transcription factor, promotes flavonoid biosynthesis in Erigeron breviscapus.

Erigeron breviscapus, a traditional Chinese medicinal plant, is enriched in flavonoids that are beneficial to human health. While we know that R2R3-MYB transcription factors (TFs) are crucial to flavonoid pathway, the transcriptional regulation of flavonoid biosynthesis in E. breviscapus has not been fully elucidated. Here, EbMYBP1, a R2R3-MYB transcription factor, was uncovered as a regulator involved in the regulation of flavonoid accumulation. Transcriptome and metabolome analysis revealed that a large group of genes related to flavonoid biosynthesis were significantly changed, accompanied by significantly increased concentrations of the flavonoid in EbMYBP1-OE transgenic tobacco compared with the wild-type (WT). In vitro and in vivo investigations showed that EbMYBP1 participated in flavonoid biosynthesis, acting as a nucleus-localized transcriptional activator and activating the transcription of flavonoid-associated genes like FLS, F3H, CHS, and CHI by directly binding to their promoters. Collectively, these new findings are advancing our understanding of the transcriptional regulation that modulates the flavonoid biosynthesis.