RESUMO
Discovery of the Asgard superphylum of archaea provides new evidence supporting the two-domain model of life: eukaryotes originated from an Asgard-related archaeon that engulfed a bacterial endosymbiont. However, how eukaryotes acquired bacterial-like membrane lipids with a sn-glycerol-3-phosphate (G3P) backbone instead of the archaeal-like sn-glycerol-1-phosphate (G1P) backbone remains unknown. In this study, we reconstituted archaeal lipid production in Saccharomyces cerevisiae by expressing unsaturated archaeol-synthesizing enzymes. Using Golden Gate cloning for pathway assembly, modular gene replacement was performed, revealing the potential biosynthesis of both G1P- and G3P-based unsaturated archaeol by uncultured Asgard archaea. Unexpectedly, hybrid neutral lipids containing both archaeal isoprenoids and eukaryotic fatty acids were observed in recombinant S. cerevisiae. The ability of yeast and archaeal diacylglycerol acyltransferases to synthesize such hybrid lipids was demonstrated.
Assuntos
Archaea , Saccharomyces cerevisiae , Archaea/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Glicerol/metabolismo , Lipídeos de Membrana/metabolismo , Bactérias/metabolismo , Fosfatos/metabolismoRESUMO
Lanthipeptides are a major class of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by thioether cross-links called lanthionine (Lan) and methyllanthionine (MeLan). Previously, we developed a method to produce mature lanthipeptides in recombinant Escherichia coli, but manual steps hinder large-scale analogue screening. Here we devised an automated workflow for creating and screening variant libraries of haloduracin, a two-component class II lanthipeptide. An integrated work cell of a synthetic biology foundry was programmed to robotically execute DNA library construction, host transformation, peptide production, mass spectrometry analysis, and activity screening by agar diffusion assay. For recombinantly produced Halα peptides, the sequence-activity relationship of 380 single-residue variants and >1300 triple-residue combinatorial variants were rapidly analyzed in microplates within weeks. The peptide expression levels in E. coli were also visualized via robotic creation and analysis of GFP-lanthipeptide fusions for select peptide mutants. Following shake-flask fermentation and purification, one Halα mutant was confirmed with enhanced specific antimicrobial activity relative to the wild-type peptide. Overall, this approach may be generally applicable for the high-throughput characterization and engineering of RiPP natural products.
Assuntos
Bacteriocinas , Procedimentos Cirúrgicos Robóticos , Escherichia coli/genética , Escherichia coli/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Processamento de Proteína Pós-Traducional , Peptídeos/metabolismoRESUMO
d-alanine (d-Ala) and several other d-amino acids (d-AAs) act as hormones and neuromodulators in nervous and endocrine systems. Unlike the endogenously synthesized d-serine in animals, d-Ala may be from exogenous sources, e.g., diet and intestinal microorganisms. However, it is unclear if the capability to produce d-Ala and other d-AAs varies among different microbial strains in the gut. We isolated individual microorganisms of rat gut microbiota and profiled their d-AA production in vitro, focusing on d-Ala. Serial dilutions of intestinal contents from adult male rats were plated on agar to obtain clonal cultures. Using MALDI-TOF MS for rapid strain typing, we identified 38 unique isolates, grouped into 11 species based on 16S rRNA gene sequences. We then used two-tier screening to profile bacterial d-AA production, combining a d-amino acid oxidase-based enzymatic assay for rapid assessment of non-acidic d-AA amount and chiral LC-MS/MS to quantify individual d-AAs, revealing 19 out of the 38 isolated strains as d-AA producers. LC-MS/MS analysis of the eight top d-AA producers showed high levels of d-Ala in all strains tested, with substantial inter- and intra-species variations. Though results from the enzymatic assay and LC-MS/MS analysis aligned well, LC-MS/MS further revealed the existence of d-glutamate and d-aspartate, which are poor substrates for this enzymatic assay. We observed large inter- and intra-species variation of d-AA production profiles from rat gut microbiome species, demonstrating the importance of chemical profiling of gut microbiota in addition to sequencing, furthering the idea that microbial metabolites modulate host physiology.
Assuntos
Microbioma Gastrointestinal , Alanina , Aminoácidos/metabolismo , Animais , Cromatografia Líquida , Microbioma Gastrointestinal/fisiologia , Masculino , RNA Ribossômico 16S/genética , Ratos , Espectrometria de Massas em TandemRESUMO
Lanthipeptides are a group of ribosomally synthesized and post-translationally modified peptides with diverse structural features and bioactivities. Gut-microbiota-derived lanthipeptides play important roles in gut homeostasis of the host. Herein, we report the discovery and biosynthesis of class III lantibiotics named amylopeptins, which are derived from the gut microbiota of Sprague-Dawley rats and display a narrow antimicrobial spectrum. In contrast to known class III lanthipeptides, the biosynthesis of amylopeptins employs AmyP, which belongs to a subgroup of S8 family serine proteases, to remove the leader of corresponding precursor peptides in a site-specific manner during the last step of their maturation. Overall, this study shows for the first time that S8 family proteases participate in the biosynthesis of class III lanthipeptides.
Assuntos
Bacillus amyloliquefaciens/genética , Bacteriocinas/biossíntese , Peptídeo Hidrolases/metabolismo , Animais , Bacillus amyloliquefaciens/metabolismo , Bacteriocinas/química , Bacteriocinas/genética , Microbioma Gastrointestinal , Peptídeo Hidrolases/genética , Ratos , Ratos Sprague-DawleyRESUMO
Pseudomonas aeruginosa displays an impressive metabolic versatility, which ensures its survival in diverse environments. Reported herein is the identification of rare azetidine-containing alkaloids from P. aeruginosa PAO1, termed azetidomonamides, which are derived from a conserved, quorum-sensing regulated nonribosomal peptide synthetase (NRPS) pathway. Biosynthesis of the azetidine motif has been elucidated by gene inactivation, feeding experiments, and biochemical characterization in vitro, which involves a new S-adenosylmethionine-dependent enzyme to produce azetidine 2-carboxylic acid as an unusual building block of NRPS. The mutants of P. aeruginosa unable to produce azetidomonamides had an advantage in growth at high cell density inâ vitro and displayed rapid virulence in Galleria mellonella model, inferring functional roles of azetidomonamides in the host adaptation. This work opens the avenue to study the biological functions of azetidomonamides and related compounds in pathogenic and environmental bacteria.
Assuntos
Alcaloides/metabolismo , Azetidinas/metabolismo , Proteínas de Bactérias/metabolismo , Peptídeo Sintases/metabolismo , Pseudomonas aeruginosa/fisiologia , Alcaloides/química , Azetidinas/química , Vias Biossintéticas , Humanos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismo , Percepção de QuorumRESUMO
Bone homeostasis is maintained by formation and destruction of bone, which are two processes tightly coupled and controlled. Targeting both stimulation on bone formation and suppression on bone resorption becomes a promising strategy for treating osteoporosis. In this study, we examined the effect of wedelolactone, a natural product from Ecliptae herba, on osteoblastogenesis as well as osteoclastogenesis. In mouse bone marrow mesenchymal stem cells (BMSC), wedelolactone stimulated osteoblast differentiation and bone mineralization. At the molecular level, wedelolactone directly inhibited GSK3ß activity and enhanced the phosphorylation of GSK3ß, thereafter stimulated the nuclear translocation of ß-catenin and runx2. The expression of osteoblastogenesis-related marker gene including osteorix, osteocalcin and runx2 increased. At the same concentration range, wedelolactone inhibited RANKL-induced preosteoclastic RAW264.7 actin-ring formation and bone resorption pits. Further, wedelolactone blocked NF-kB/p65 phosphorylation and abrogated the NFATc1 nuclear translocation. As a result, osteoclastogenesis-related marker gene expression decreased, including c-src, c-fos, and cathepsin K. In ovariectomized mice, administration of wedelolactone prevented ovariectomy-induced bone loss by enhancing osteoblast activity and inhibiting osteoclast activity. Together, these data demonstrated that wedelolactone facilitated osteoblastogenesis through Wnt/GSK3ß/ß-catenin signaling pathway and suppressed RANKL-induced osteoclastogenesis through NF-κB/c-fos/NFATc1 pathway. These results suggested that wedelolacone could be a novel dual functional therapeutic agent for osteoporosis.
Assuntos
Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Cumarínicos/farmacologia , Células-Tronco Mesenquimais/metabolismo , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Células da Medula Óssea/citologia , Clotrimazol/análogos & derivados , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos BALB C , Osteoblastos/citologia , Células RAW 264.7 , beta Catenina/metabolismoRESUMO
The essential attributes of a solvent system for separation polar compounds on CCC are polarity, selectively and performance. Here, hydrophilic organic/salt-containing aqueous two-phase system (HO/S TPS) was evaluated as an alternative solvent system for CCC separation of polar compounds. Polarity measurements based on Rohrschneider-Snyder parameter was developed as quantitative assessing the polarity of HO/S TPS and comparing with an organic/aqueous system. All investigated 1-butanol/ethanol/saturated ammonium sulfate solution/water (BEAsWat) and 1-butanol/ethanol/saturated dipotassium hydrogen phosphate solution/water (BEDhpWat) systems with polarity values of organic phase from 4.5 to 6.8, were more polar than chloroform/methanol/water (1/1/1). The considerable water content of BEAsWat and BEDhpWat (0/1/1/1/) was 45.4 and 42.6% (w%) of hydrophilic organic phase, and 66.4 and 51.2% (w%) of salt-containing aqueous phase, respectively, closed to conventional aqueous two-phase system. Therefore, the polarity of HO/S TPS is in the middle of organic/aqueous and aqueous two-phase system. The LogKC values of twenty four polar compounds as model mixture confirmed that the polarities of HO/S TPSs were matched to that of the polar compounds and shown to be a very selective technique capable of separating positional isomers. Moreover, BEAsWat and BEDhpWat systems can be easily retained in CCC column with suitable elution mode. The hydrodynamic behavior reversion of HO/S TPS on hydrodynamic CCC was observed and was tentatively explained based on the density difference. Finally, caffeoylquinic acid isomers and dihydroxybenzoic acid isomers were successfully separated with HO/S TPS on CCC, respectively. Those results demonstrate that HO/S TPS on CCC is a performant and stable way to separate polar compounds from natural products.
Assuntos
1-Butanol/química , Sulfato de Amônio/química , Clorofórmio/química , Distribuição Contracorrente/métodos , Etanol/química , Metanol/química , Fosfatos/química , Compostos de Potássio/química , Cromatografia Líquida de Alta Pressão/métodos , Interações Hidrofóbicas e Hidrofílicas , Extratos Vegetais/química , Solventes , Água/químicaRESUMO
Twelve new ent-abietane diterpenoids, chlorabietins A-L (), were isolated from the roots of Chloranthus oldhamii. Their structures and absolute configurations were determined by extensive spectroscopic analyses, X-ray diffraction, and experimental/calculated electronic circular dichroism (ECD) spectroscopy. Among the new isolates, chlorabietins D () and E () are the first two naturally occurring 8-spiro-fused 9,10-seco-ent-abietanes containing an unexpected cis-fused A/B ring system. Chlorabietin F () is a rare chinane-type diterpenoid featuring a hitherto unknown C-ring cleavage between C-13 and C-14, which might be derived from a common precursor of the above spiro-diterpenoid epimers and , and their biosynthetic relationships are briefly discussed. Meanwhile, chlorabietin I () is the first representative of the abietane-type diterpenoids possessing a tetrahydrofurano function bridging C-6 and C-19. Chlorabietins B (), C (), F (), and G () showed anti-neuroinflammatory effects by inhibiting the nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine BV-2 microglial cells, with IC50 values ranging from 16.4 to 33.8 µM.
Assuntos
Abietanos/química , Abietanos/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Magnoliopsida/química , Microglia/efeitos dos fármacos , Animais , Linhagem Celular , Camundongos , Microglia/metabolismo , Modelos Moleculares , Conformação Molecular , Óxido Nítrico/biossíntese , Raízes de Plantas/químicaRESUMO
Three unprecedented phloroglucinol-diterpene adducts, chlorabietols A-C (1-3), were isolated from the roots of the rare Chloranthaceae plant Chloranthus oldhamii. They represent a new class of compounds, featuring an abietane-type diterpenoid coupled with different alkenyl phloroglucinol units by forming a 2,3-dihydrofuran ring. Their structures were elucidated by detailed spectroscopic analysis, molecular modeling studies, and electronic circular dichroism calculations. Compounds 1-3 showed inhibitory activity against protein tyrosine phosphatase 1B (PTP1B) with IC50 values of 12.6, 5.3, and 4.9 µM, respectively.
Assuntos
Diterpenos/química , Furanos/química , Floroglucinol/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/farmacologia , Diterpenos/isolamento & purificação , Concentração Inibidora 50 , Estrutura Molecular , Floroglucinol/isolamento & purificação , Raízes de Plantas , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/químicaRESUMO
Bioassay-guided phytochemical studies on Stellera chamaejasme led to the isolation of two new biflavones, chamaejasmenin E (1) and chamaejasmin D (2), together with ten known compounds. The structures of new compounds were elucidated by extensive spectroscopic analyses and their absolute configurations on 2, 3, 2â³ and 3â³ were confirmed by TDDFT quantum chemical calculated ECD spectra combined with experimental ECD spectra. All isolated biflavones were evaluated for their cytotoxic activities against Bel-7402 and A549 tumor cell lines, and sikokianin D (3) was found to possess the most potential cytotoxic activities against both the two cell lines with IC50 values of 1.29 ± 0.21 and 0.75 ± 0.25 µM, respectively. Moreover, some structure-function relationships of these bioflavones for cytotoxic activities were explored and summarized.
Assuntos
Flavonas/química , Thymelaeaceae/química , Biflavonoides/química , Biflavonoides/isolamento & purificação , Linhagem Celular Tumoral , Flavonas/isolamento & purificação , Humanos , Estrutura Molecular , Raízes de Plantas/química , Relação Estrutura-AtividadeRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The dried stems of Clematis armandii (Caulis clematidis armandii), named "Chuan-Mu-Tong" in Chinese Pharmacopoeia, have been traditionally used as an herbal remedy mainly for inflammation-associated diseases. The Aim of the study is to identify the potential anti-neuroinflammatory components from Clematis armandii. MATERIALS AND METHODS: The ethanol extract of "Chuan-Mu-Tong" was suspended in H2O and exhaustively extracted with CH2Cl2. The CH2Cl2 fraction was successively subjected to column chromatography (CC) over silica gel, Sephadex LH-20, and semi-preparative HPLC. The structures of the isolated compounds were identified by spectroscopic methods and by comparison with those reported in the literature. Their anti-neuroinflammatory activities were evaluated by inhibitory effects on pro-inflammatory mediators [e.g. nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α)] in lipopolysaccharide (LPS)-activated BV-2 cells. RESULTS: One new and sixteen known lignans were isolated and characterized. The absolute configuration of the new lignan, (7R,8S)-9-acetyl-dehydrodiconiferyl alcohol (1), was elucidated by a combination of 1D/2D NMR techniques and the Electronic Circular Dichroism (ECD) spectroscopy based on the empirical helicity rules. The anti-neuroinflammatory bioassay showed that compounds 1, (7R,8S)-dehydrodiconiferyl alcohol (2), erythro-guaiacylglycerol-ß-coniferyl ether (5), and threo-guaiacylglycerol-ß-coniferyl ether (6) displayed significant inhibitory effects on NO production. Among them, neolignans 1 and 2 exhibited more potent activities than the positive control (N(G)-monomethyl-L-arginine, L-NMMA), with an IC50 value of 9.3 and 3.9 µM, respectively. Moreover, both 1 and 2 were also found to concentration-dependently suppress the TNF-α release in LPS-stimulated BV-2 cells. CONCLUSION: The results revealed that lignans are the major components of "Chuan-Mu-Tong", and their anti-neuroinflammatory activities strongly support the traditional application of this herb medicine on inflammation. Moreover, the dihydrobenzo[b]furan neolignans 1 and 2 as well as Caulis clematidis armandii could be further exploited as new therapeutic agents to treat inflammation-mediated neurodegenerative and aging-associated diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Clematis , Lignanas/farmacologia , Fármacos Neuroprotetores/farmacologia , Animais , Anti-Inflamatórios/isolamento & purificação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Clematis/química , Lignanas/isolamento & purificação , Lipopolissacarídeos , Camundongos , Fármacos Neuroprotetores/isolamento & purificação , Óxido Nítrico/metabolismo , Extratos Vegetais/química , Caules de Planta/química , Fator de Necrose Tumoral alfa/metabolismoRESUMO
One (formosumone A, 1) new and fifteen (2-16) known phenolic compounds were isolated from the leaves of Cratoxylum formosum ssp. pruniflorumm, a substitute for the popular bitter nail tea ("Kuding Tea") generally used in Southeast Asia. Their structures were determined by extensive spectroscopic analysis and by comparison with literature data. Compound 1 possesses a rare scaffold of a flavanone coupled with a phloroglucinol moiety, representing the first example of such a scaffold from the Clusiaceae family. Among the isolates, toxyloxanthone B (11) and vismione D (12) were found to show remarkable anti-neuroinflammatory effects by inhibiting nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated murine BV-2 microglial cells. Additionally, toxyloxanthone B (11) exhibited significant neuroprotective effect against ß-amyloid(25-35) (Aß(25-35))-induced cell viability decrease in SH-SY5Y neuroblastoma cells.
Assuntos
Anti-Inflamatórios/química , Clusiaceae/química , Flavonoides/química , Fármacos Neuroprotetores/química , Fenóis/química , Extratos Vegetais/química , Animais , Antraquinonas/química , Antraquinonas/isolamento & purificação , Antraquinonas/farmacologia , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Lipopolissacarídeos/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Estrutura Molecular , Fármacos Neuroprotetores/isolamento & purificação , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico/metabolismo , Fenóis/isolamento & purificação , Fenóis/farmacologia , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Folhas de Planta/química , Xantonas/química , Xantonas/isolamento & purificação , Xantonas/farmacologiaRESUMO
The crosslinked polystyrene nanocapsules with controllable crosslinking degree have been prepared by the ultraviolet (UV)-induced photocrosslinking of the polystyrene grafted silica nanoparticles (SN-PS), which was obtained by the surface-initiated atom transfer radical polymerisation of styrene from the modified silica nanoparticle templates, after the silica templates were etched with hydrofluoric acid. The effect of the UV-irradiating time on the inner diameter of the nanocapsules, and the degree of crosslinking and the thickness of the shells was investigated. The dynamic light scattering results showed that the degree of crosslinking of the obtained nanocapsules increased with the prolongation of the UV-irradiation time, therefore the inner diameter of the nanocapsules increased. However, the percentage of grafting of the crosslinked polymer shells decreased with increasing the UV-irradiation time because of the photodecomposition of the polystyrene grafted during the UV-irradiated crosslinking process, according to the thermogravimetric analysis.
Assuntos
Nanocápsulas/química , Nanotecnologia/métodos , Poliestirenos/química , Luz , Tamanho da Partícula , Polimerização , Espalhamento de Radiação , Dióxido de Silício , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície , TermogravimetriaRESUMO
Tetracyclic triterpenoids (named as altissimanins A-E, 1-5) and a terpenylated coumarin (denominated as altissimacoumarin G, 6), along with fifteen known compounds (7-21) were isolated from the bark of Ailanthus altissima. Structures of compounds 1-6 were established by spectroscopic methods and chemical transformations. Altissimanin A (1) is a tirucallane-type triterpenoid bearing an uncommon oxetane ring in the side-chain, while altissimanins D (4) and E (5) are two unprecedented dimers each consisting of one tirucallane-type and one dammarane-type triterpenoid moiety. All the isolates were evaluated for their cytotoxic effects against a small panel of human cancer cell lines.