RESUMO
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.
Assuntos
Saponinas , Esqualeno/análogos & derivados , Triterpenos , Glicosídeos , Flavonoides , Saponinas/genética , Glicosiltransferases , Difosfato de UridinaRESUMO
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.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Animais , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Humanos , Simulação de Dinâmica Molecular , Estabilidade Proteica , Ratos , Receptores Adrenérgicos alfa 2/química , Receptores Adrenérgicos alfa 2/genética , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 1/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de Neurotensina/química , Receptores de Neurotensina/genética , Receptores de Neurotensina/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo , Especificidade por Substrato , PerusRESUMO
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.
Assuntos
Canais Iônicos/metabolismo , Lipídeos/química , Receptores Acoplados a Proteínas G/metabolismo , Animais , Sítios de Ligação , Colesterol/metabolismo , Simulação por Computador , Humanos , Canais Iônicos/química , Ligantes , Receptores Acoplados a Proteínas G/químicaRESUMO
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.
Assuntos
Ginsenosídeos/metabolismo , Glicosiltransferases/metabolismo , Ácido Oleanólico/análogos & derivados , Panax/metabolismo , Difosfato de Uridina/metabolismo , Glicosiltransferases/análise , Glicosiltransferases/genética , Ácido Oleanólico/metabolismo , Panax/enzimologiaRESUMO
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.
Assuntos
Erigeron , Genes myb , Proteínas de Plantas , Fatores de Transcrição , Erigeron/genética , Regulação da Expressão Gênica de Plantas , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Glicoesfingolipídeos , Receptores de Glucagon , Simulação de Dinâmica Molecular , Ligação Proteica , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Glucagon/metabolismoRESUMO
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.
Assuntos
Genes de Plantas/genética , Ginsenosídeos/biossíntese , Glucuronosiltransferase/genética , Ácido Oleanólico/análogos & derivados , Panax/genética , Proteínas de Plantas/genética , Cromatografia Líquida de Alta Pressão , Perfilação da Expressão Gênica , Glucuronatos/biossíntese , Espectrometria de Massas , Redes e Vias Metabólicas/genética , Ácido Oleanólico/biossíntese , Ácido Oleanólico/metabolismo , Panax/enzimologia , Panax/metabolismo , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes , Análise de Sequência de DNARESUMO
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.
Assuntos
Acriflavina/farmacologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Fatores de Transcrição/genética , Fatores de Virulência/genética , Xanthomonas campestris/efeitos dos fármacos , Xanthomonas campestris/patogenicidade , Análise Mutacional de DNA , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Doenças das Plantas/microbiologia , Sítio de Iniciação de Transcrição , Virulência , Xanthomonas campestris/genéticaRESUMO
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.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Porinas/química , Porinas/metabolismo , Providencia/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação , Biologia Computacional , Simulação por Computador , Ligação de Hidrogênio , Ativação do Canal Iônico , Modelos Biológicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Eletricidade EstáticaRESUMO
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.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Córtex Cerebral/efeitos dos fármacos , Receptores ErbB/efeitos dos fármacos , Glucosídeos/farmacologia , Monoterpenos/farmacologia , Degeneração Neural , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptor Cross-Talk/efeitos dos fármacos , Receptor A1 de Adenosina/efeitos dos fármacos , Animais , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Hipóxia Celular , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Citoproteção , Relação Dose-Resposta a Droga , Ativação Enzimática , Receptores ErbB/metabolismo , Idade Gestacional , Células HEK293 , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Fosforilação , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Receptor A1 de Adenosina/genética , Receptor A1 de Adenosina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Quinases da Família src/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Antagonistas do Receptor A2 de Adenosina/farmacologia , Agonistas de Dopamina/farmacologia , Desenho de Fármacos , Multimerização Proteica , Receptor A1 de Adenosina/metabolismo , Receptores de Dopamina D1/metabolismo , Antagonistas do Receptor A2 de Adenosina/síntese química , Antagonistas do Receptor A2 de Adenosina/química , Animais , Ligação Competitiva , Encéfalo/metabolismo , Agonistas de Dopamina/síntese química , Agonistas de Dopamina/química , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Ligantes , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Ratos , Ratos Wistar , Receptor A1 de Adenosina/química , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/química , Relação Estrutura-AtividadeRESUMO
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.
RESUMO
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.
Assuntos
Ginsenosídeos , Panax , Triterpenos , Panax/genética , Panax/metabolismo , Triterpenos/química , Triterpenos/metabolismo , Genômica , Plantas/metabolismoRESUMO
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.
Assuntos
Proteínas de Membrana , Simulação de Dinâmica Molecular , Proteínas de Membrana/química , Microscopia Crioeletrônica , Lipídeos de Membrana , Conformação ProteicaRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Virulência/metabolismo , Xanthomonas campestris/genética , Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Sequência de Bases , Brassica/microbiologia , Mapeamento Cromossômico , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Sítio de Iniciação de Transcrição , Virulência , Fatores de Virulência/genética , Xanthomonas campestris/metabolismo , Xanthomonas campestris/patogenicidadeRESUMO
Erigeron breviscapus is a Compositae plant, and its rich flavonoids have shown strong preventative and curative effects in the treatment of cardio- and cerebrovascular diseases. bHLH genes play a crucial role in plant growth and development. There are 116 EbbHLH genes in E. breviscapus, and each gene has been named based on its chromosome location. Our phylogenetic analysis divided these genes into 18 subfamilies. To further investigate its function, EbbHLH80 was isolated from E. breviscapus leaves. Next, transcriptomic and metabolomic analyses of tobacco leaves were performed. Among 421 differentially accumulated compounds, 98 flavonoids were identified. In addition, differentially expressed genes were identified using RNA-seq, and further analysis suggested that EbbHLH80-OE could not only regulate the expression of some structural genes in the flavonoid biosynthesis pathway to achieve flavonoid accumulation but also be involved in the regulation of a series of downstream pathways, such as stress response, ABA and ethylene signal transduction, to affect plant growth and development. The results of our analysis provide new insights into the function of EbbHLH80 and lay the foundation for future functional studies on E. breviscapus.
RESUMO
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.
Assuntos
Proteínas de Membrana , Simulação de Dinâmica Molecular , Sítios de Ligação , Ligantes , Bicamadas Lipídicas/química , Lipídeos , Proteínas de Membrana/químicaRESUMO
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.
RESUMO
G-protein-coupled receptors signal through cognate G proteins. Despite the widespread importance of these receptors, their regulatory mechanisms for G-protein selectivity are not fully understood. Here we present a native mass spectrometry-based approach to interrogate both biased signalling and allosteric modulation of the ß1-adrenergic receptor in response to various ligands. By simultaneously capturing the effects of ligand binding and receptor coupling to different G proteins, we probed the relative importance of specific interactions with the receptor through systematic changes in 14 ligands, including isoprenaline derivatives, full and partial agonists, and antagonists. We observed enhanced dynamics of the intracellular loop 3 in the presence of isoprenaline, which is capable of acting as a biased agonist. We also show here that endogenous zinc ions augment the binding in receptor-Gs complexes and propose a zinc ion-binding hotspot at the TM5/TM6 intracellular interface of the receptor-Gs complex. Further interrogation led us to propose a mechanism in which zinc ions facilitate a structural transition of the intermediate complex towards the stable state.