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1.
Microb Pathog ; 173(Pt A): 105837, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36257566

RESUMO

Gamma-proteobacteria is a class of gram-negative opportunistic pathogens existing in the intestinal flora, often leading to diarrhea and intestinal infectious diseases, and plays an important role in maintaining intestinal homeostasis. Type III secretion system (T3SS), an important virulence system, is closely related to the adhesion and invasion and pathogenicity to host cells. Therefore, anti-virulence agents targeting T3SS are important strategies for controlling pathogenic infections. In this study, the anti-Salmonella T3SS active compounds neochebulagic acid (1), ellagic acid (2) and urolithin M5 (3) were isolated from seed extract of Terminalia citrina by activity-guided isolation method. Based on the fact that urolithins are the main and stable intestinal microbiota metabolites of hydrolysable tannins, we found that the metabolite urolithin B repressed translation and secretion of SipC through the Hha-H-NS-HilD-HilC-RtsA-HilA regulatory pathway. The results provide evidence for Terminalia seeds and ellagitannin-rich berries and nuts in regulating intestinal homeostasis and treating bacterial infection.


Assuntos
Terminalia , Sistemas de Secreção Tipo III , Sistemas de Secreção Tipo III/metabolismo , Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium , Taninos Hidrolisáveis/farmacologia , Taninos Hidrolisáveis/metabolismo , Fatores de Transcrição/genética , Proteínas de Bactérias/genética
2.
Nat Chem Biol ; 14(9): 876-886, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30120361

RESUMO

Signals from 800 G-protein-coupled receptors (GPCRs) to many SH3 domain-containing proteins (SH3-CPs) regulate important physiological functions. These GPCRs may share a common pathway by signaling to SH3-CPs via agonist-dependent arrestin recruitment rather than through direct interactions. In the present study, 19F-NMR and cellular studies revealed that downstream of GPCR activation engagement of the receptor-phospho-tail with arrestin allosterically regulates the specific conformational states and functional outcomes of remote ß-arrestin 1 proline regions (PRs). The observed NMR chemical shifts of arrestin PRs were consistent with the intrinsic efficacy and specificity of SH3 domain recruitment, which was controlled by defined propagation pathways. Moreover, in vitro reconstitution experiments and biophysical results showed that the receptor-arrestin complex promoted SRC kinase activity through an allosteric mechanism. Thus, allosteric regulation of the conformational states of ß-arrestin 1 PRs by GPCRs and the allosteric activation of downstream effectors by arrestin are two important mechanisms underlying GPCR-to-SH3-CP signaling.


Assuntos
Regulação Alostérica , Arrestina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Domínios de Homologia de src , Células Cultivadas , Células HEK293 , Humanos
3.
J Nat Prod ; 83(9): 2803-2808, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32915576

RESUMO

Polycyclic tetramate macrolactams (PoTeMs) are a group of hybrid PK-NRP natural products having a variable set of carbocyclic rings, a conserved assembly pathway, and diverse bioactivities. We report here the identification of seven new PoTeMs, clifednamides D-J (3-9), along with the known clifednamides A (1) and B (2) through rational pathway refactoring and heterologous expression. Remarkably, clifednamides D (3), G (6), and H (7) feature an unprecedented 27,28-seco skeleton. The cytotoxic activities of compounds 1-9 indicated that the hydroxy group of C-25, the methyl group of C-30, the inner five-membered ring, and the intact macrocycle are all critical for the activities. Meanwhile, the cytochrome P450 enzyme CftS023A and the hydroxylase CftS023E involved in oxidative tailoring of clifednamides were found to decorate the fused 5-6 bicyclic intermediates. Accordingly, the biosynthetic pathway for clifednamides was proposed.


Assuntos
Antibacterianos/biossíntese , Antibacterianos/química , Streptomyces/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Vias Biossintéticas , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Oxigenases de Função Mista/metabolismo , Estrutura Molecular , Oxirredução , Microbiologia do Solo , Streptomyces/metabolismo
4.
J Neurochem ; 148(4): 550-560, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30451284

RESUMO

Protein Phosphatase Mg2+ /Mn2+ -Dependent 1K (PPM1K),also named as PP2Cm or branched-chain α-ketoacid dehydrogenase complex phosphatase, is a member of the metal-dependent phosphatase family and an important metabolic regulator. Single nucleotide polymorphisms (SNPs) in PPM1K contributing to protein functional defects have been found to be associated with numerous human diseases, such as cardiovascular disease, maple syrup urine disease, type 2 diabetes, and neurological disease. PPM1K N94K is an identified missense mutant produced by one of the SNPs in the human PPM1K coding sequence. However, the effects of the N94K mutant on its activity and structural property have not been defined. Here, we performed a detailed enzymological study using steady-state kinetics in the presence of pNPP or phospho-peptide substrates and crystallographic analyses of the wild-type and N94K PPM1K. The PPM1K-N94K significantly impaired its Mg2+ -dependent catalytic activity and structural analysis demonstrated that the N94K mutation induced a conformational change in the key residue in coordinating the Mg2+ in the active site. Specifically, three Mg2+ were located in the active site of the PPM1K N94K instead of two Mg2+ in the PPM1K wild type. Therefore, our results provide a structure basis for the metal ion-dependent PPM1K-N94K phosphatase activity.


Assuntos
Proteína Fosfatase 2C/química , Proteína Fosfatase 2C/genética , Biocatálise , Humanos , Mutação , Relação Estrutura-Atividade
5.
Acta Pharmacol Sin ; 38(4): 524-538, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28260799

RESUMO

Bisindolylmaleimides, a series of derivatives of a PKC inhibitor staurosporine, exhibit potential as anti-cancer drugs and have received considerable attention in clinical trials. This study aims to investigate the effects of a bisindolylmaleimide alkaloid 155Cl (BMA-155Cl) with a novel structure on autophagy and apoptosis in human hepatocarcinoma HepG-2 cells in vitro and in vivo. The cell poliferation was assessed with a MTT assay. Autophagy was evaluated by MDC staining and TEM analysis. Apoptosis was investigated using Annexin V-FITC/PI and DAPI staining. The antitumor effects were further evaluated in nude mice bearing HepG-2 xenografts, which received BMA-155Cl (10, 20 mg/kg, ip) for 18 days. Autophagy- and apoptosis-associated proteins and their mRNA levels were examined with Western blotting, immunohistochemistry, and RT-PCR. BMA-155Cl (2.5-20 µmol/L) inhibited the growth of HepG-2 cells with IC50 values of 16.62±1.34, 12.21±0.83, and 8.44±1.82 µmol/L at 24, 48, and 72 h, respectively. Furthermore, BMA-155Cl (5-20 µmol/L) dose-dependently induced autophagy and apoptosis in HepG-2 cells. The formation of autophagic vacuoles was induced by BMA-155Cl (10 µmol/L) at approximately 6 h and peaked at approximately 15 h. Pretreatment with 3-MA potentiated BMA-155Cl-mediated apoptotic cell death. This compound dose-dependently increased the mRNA and protein levels of Beclin-1, NF-κB p65, p53, and Bax, but decreased the expression of IκB and Bcl-2. Pretreatment with BAY 11-7082, a specific inhibitor of NF-κB p65, blocked BMA-155Cl-induced expression of autophagy- and apoptosis-associated proteins. BMA-155Cl administration effectively suppressed the growth of HepG-2 xenografts in vivo, and increased the protein expression levels of LC3B, Beclin-1, NF-κB p65, and Bax in vivo. We conclude that the NF-κB p65 pathway is involved in BMA-155Cl-triggered autophagy, followed by apoptosis in HepG-2 cells in vitro and in vivo. Hence, BMA-155Cl could be a promising pro-apoptotic candidate for developing as a novel anti-cancer drug.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Alcaloides Indólicos/uso terapêutico , Indóis/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Maleimidas/uso terapêutico , Animais , Proteína Beclina-1/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Células Hep G2 , Humanos , Proteínas I-kappa B/metabolismo , Alcaloides Indólicos/farmacologia , Indóis/farmacologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Maleimidas/farmacologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Associadas aos Microtúbulos/metabolismo , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismo
6.
Bioorg Med Chem ; 24(9): 2125-36, 2016 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-27039251

RESUMO

As our ongoing work on research of gelatinase inhibitors, an array of hydrazide-containing peptidomimetic derivatives bearing quinoxalinone as well as spiro-heterocyclic backbones were designed, synthesized, and assayed for their in vitro enzymatic inhibitory effects. The results demonstrated that both the quinoxalinone (series I and II) and 1,4-dithia-7-azaspiro[4,4]nonane-based hydrazide peptidomimetics (series III) displayed remarkably selectivity towards gelatinase A as compared to APN, with IC50 values in the micromole range. Structure-activity relationships were herein briefly discussed. Given evidences have validated that gelatinase inhibition may be contributable to the therapy of HIV-1 infection, all the target compounds were also submitted to the preliminary in vitro anti-HIV-1 evaluation. It resulted that gelatinase inhibition really has positive correlation with anti-HIV-1 activity, especially compounds 4m and 7h, which gave enhanced gelatinase inhibition in comparison with the positive control LY52, and also decent anti-HIV-1 potencies. The FlexX docking results provided a straightforward insight into the binding pattern between inhibitors and gelatinase, as well as the selective inhibition towards gelatinase over APN. Collectively, our research encouraged potent gelatinase inhibitors might be used in the development of anti-HIV-1 agents. And else, compounds 4m and 7h might be promising candidates to be considered for further chemical optimization.


Assuntos
Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Azidas/farmacologia , Gelatinases/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Peptidomiméticos , Fármacos Anti-HIV/química , Desenho de Fármacos
7.
Beilstein J Org Chem ; 10: 858-862, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24778741

RESUMO

Two pairs of geometrical isomers - cuevaenes A (1) and C (3) as well as cuevaenes D (4) and E (5) - and cuevaene B (2) were isolated from gdmAI-disrupted Streptomyces sp. LZ35. The constitution of cuevaene C (3) was found to be identical to cuevaene A (1) by means of NMR spectroscopy and high resolution mass spectrometry. However, the relative configurations of the triene side chain moieties were determined to be different. It was established on the basis of spectroscopic data that cuevaenes D (4) and E (5) are amides and geometrical isomers. Cuevaenes A-C (1-3) displayed moderate activity against Gram-positive bacteria (e.g., Bacillus subtilis strain ATCC 11060) and modest activity against fungi (e.g., Fusarium verticillioides strain S68 and Rhizoctonia solani strain GXE4). However, cuevaenes D (4) and E (5) showed no inhibitory activity against any of the tested microbes.

8.
J Asian Nat Prod Res ; 14(7): 613-7, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22574939

RESUMO

Two new and three known abscisic acid-related metabolites were obtained from the potato dextrose agar culture of Phellinus vaninii YB2005. Their structures were established on the basis of detailed spectroscopic analyses, including 1D NMR, 2D NMR, and HR-Q-TOF-MS techniques. The putative biosynthesis pathway of these secondary metabolites would decipher the mechanism of the symbiosis between plant and fungi from the view of chemistry.


Assuntos
Ácido Abscísico , Basidiomycota/química , Ácido Abscísico/análogos & derivados , Ácido Abscísico/química , Ácido Abscísico/isolamento & purificação , Ácido Abscísico/metabolismo , China , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular
9.
Nucleic Acids Res ; 37(3): 832-48, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19095693

RESUMO

PRMT1, an arginine methyltransferase, plays an important role in numerous cellular processes. In this study, we demonstrate a feedback regulatory loop between PRMT1 and the orphan receptor TR3. Unlike another orphan receptor HNF4, TR3 is not methylated by PRMT1 although they physically interact with each other. By delaying the TR3 protein degradation, PRMT1 binding leads to the elevation of TR3 cellular protein level, thereby enhances the DNA binding and transactivation activity of TR3 in a non-methyltransferase manner. Another coactivator SRC-2 acts synergistically with PRMT1 to regulate TR3 functions. In turn, TR3 binding to the catalytic domain of PRMT1 causes an inhibition of the PRMT1 methyltransferase activity. This repression results in the functional changes in some of PRMT1 substrates, including STAT3 and Sam68. The negative regulation of PRMT1 by TR3 was further confirmed in both TR3-knockdown cells and TR3-knockout mice with the use of an agonist for TR3. Taken together, our study not only identifies a regulatory role of PRMT1, independent on methyltransferase activity, in TR3 transactivation, but also characterizes a novel function of TR3 in the repression of PRMT1 methyltransferase activity.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Retroalimentação Fisiológica , Proteína-Arginina N-Metiltransferases/metabolismo , Receptores de Esteroides/metabolismo , Proteínas Repressoras/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/agonistas , Humanos , Camundongos , Camundongos Knockout , Coativador 2 de Receptor Nuclear/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Receptores de Esteroides/agonistas , Proteínas Repressoras/antagonistas & inibidores
10.
Phytochemistry ; 192: 112929, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34481176

RESUMO

From the aerial extracts of Coptosapelta diffusa (Champ. ex Benth.) Steenis, twenty-one compounds were isolated and identified by means of column chromatography and NMR and MS techniques, respectively. Amongst, ten ones were determined to be undescribed compounds including six seco-iridoid glucosides (1-6), 2-(hydroxymethyl)-1,2,3,4-tetrahydroanthracene-9,10-dione (7) and three guaiane-type sesquiterpenes (15-17). Compounds 7, 8 and 9 exhibited inhibitory activities against Staphylococcus aureus ATCC25923 with MIC of 8, 4 and 8 µg/mL. The use of 1-6 (iridoids), 7-14 (anthraquinones) and 15-17 (sesquiterpenes) as chemotaxonomic markers for this species was evidenced. Structurally, 7-14 are similar to those anthraquinones isolated from other species of the family Rubiaceae, confirming their close phylogenetic relationship. Whereas, these iridoids and sesquiterpenes with unique structures provided chemotaxonomic evidence to support the genus Coptosapelta (the tribe Coptosapelteae) as a sister of the subfamily Rubioideae. These results contrast with the general producing tendency of indole alkaloids by the species of the subfamily Cinchonoideae, and merit chemotaxonomic significance for the delimitation of Coptosapelta.


Assuntos
Rubiaceae , Antraquinonas , Glucosídeos Iridoides , Iridoides , Filogenia , Extratos Vegetais
11.
J Cell Mol Med ; 14(9): 2281-91, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19538463

RESUMO

The macrolide compound MFTZ-1 has been identified as a novel topoisomerase II (Top2) inhibitor with potent in vitro and in vivo anti-tumour activities. In this study, we further examined the effects of MFTZ-1 on hypoxia-inducible factor-1α (HIF-1α) accumulation, vascular endothelial growth factor (VEGF) secretion and angiogenesis. MFTZ-1 reduced HIF-1α accumulation driven by hypoxia or growth factors in human cancer cells. Mechanistic studies revealed that MFTZ-1 did not affect the degradation of HIF-1α protein or the level of HIF-1α mRNA. By contrast, MFTZ-1 apparently inhibited constitutive and inducible activation of both phosphatidylinositol-3-kinase (PI3K)-Akt and p42/p44 mitogen-activated protein kinase (MAPK) pathways. Further studies revealed that MFTZ-1 abrogated the HIF-1α-driven increase in VEGF mRNA and protein secretion. MFTZ-1 also lowered the basal level of VEGF secretion. The results reveal an important feature that MFTZ-1 can reduce constitutive, HIF-1α-independent VEGF secretion and concurrently antagonize inducible, HIF-1α-dependent VEGF secretion. Moreover, MFTZ-1 disrupted tube formation of human umbilical vein endothelial cells (HUVECs) stimulated by hypoxia with low-concentration serum or by serum at normoxia, and inhibited HUVECs migration at normoxia. MFTZ-1 also prevented microvessel outgrowth from rat aortic ring. These data reflect the potent anti-angiogenesis of MFTZ-1 under different conditions. Furthermore, using specific small interfering RNA targeting Top2α or Top2-defective HL60/MX2 cells, we showed that MFTZ-1 affected HIF-1α accumulation and HUVECs tube formation irrelevant to its Top2 inhibition. Taken together, our data collectively reveal that MFTZ-1 reduces constitutive and inducible HIF-1α accumulation and VEGF secretion possibly via PI3K-Akt and MAPK pathways, eliciting anti-angiogenesis independently of its Top2 inhibition.


Assuntos
DNA Topoisomerases Tipo II/metabolismo , Eicosanoides/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Macrolídeos/farmacologia , Inibidores da Topoisomerase II/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular Tumoral , Eicosanoides/uso terapêutico , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Macrolídeos/uso terapêutico , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neovascularização Patológica/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos
12.
Nat Prod Res ; 34(15): 2219-2224, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31184497

RESUMO

Strain HT88 was isolated from the fresh stems of Mallotus nudiflorus L, and it was identified as Nocardiopsis sp. by analyzing its morphology and the 16S rRNA sequence. The extracts of fermented HT88 showed potent antimicrobial activities. Bioassay guided separation of extracts led to eight proline (or hydroxyproline, Hyp)-containing cyclic dipeptides. Their structures were determined by 1D and 2D NMR spectroscopy and ESI mass spectrometry and further comparison with existing 1H and 13C NMR, melting points and specific rotation data. The eight 2,5-diketopiperazines (DKPs) were identified as cyclo(L-Pro-L-Leu) (1), cyclo(Pro-Leu) (2), cyclo(L-trans-Hyp-L-Leu) (3), cyclo(D-trans-Hyp-D-Leu) (4), and cyclo(D-Pro-L-Phe) (5), cyclo(L-Pro-L-Phe) (6), and cyclo(D-cis-Hyp-L-Phe) (7), cyclo(L-trans-Hyp-L-Phe) (8), respectively. Up to date, this is the first isolation of four pairs of proline based DKPs from Nocardiopsis sp.


Assuntos
Dipeptídeos/isolamento & purificação , Mallotus (Planta)/microbiologia , Nocardia/química , Prolina , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Dicetopiperazinas/química , Dicetopiperazinas/isolamento & purificação , Dipeptídeos/química , Dipeptídeos/farmacologia , Hidroxiprolina , Testes de Sensibilidade Microbiana , Peptídeos Cíclicos
13.
Chin J Nat Med ; 18(12): 952-956, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33357726

RESUMO

Constitutively expression of the pathway-specific activators is an effective method to activate silent gene clusters and improve natural product production. In this study, nine shunt products of aminoansamycins (1-9) were identified from a recombinant mutant strain S35-LAL by overexpressed the large-ATP-binding regulator of the LuxR family (LAL) gene aas1 in Streptomyces sp. S35. All the compounds showed no anti-microbial, anti-T3SS and cytotoxic activities.


Assuntos
Produtos Biológicos/metabolismo , Lactamas Macrocíclicas/metabolismo , Família Multigênica , Streptomyces/genética , Organismos Geneticamente Modificados , Streptomyces/metabolismo
14.
Chembiochem ; 10(7): 1243-51, 2009 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-19308924

RESUMO

Tailor made: We report the rational biosynthesis of C15 hydroxylated non-quinone geldanamycin analogues by site-directed mutagenesis of the geldanamycin polyketide synthase (PKS), together with a combination of post-PKS tailoring genes. Rational biosynthetic engineering allowed the generation of geldanamycin derivatives, such as DHQ3 illustrated in the figure, which had superior pharmacological properties in comparison to the parent compound. A rational biosynthetic engineering approach was applied to the optimization of the pharmacological properties of the benzoquinone ansamycin, geldanamycin. Geldanamycin and its natural or semisynthetic derivatives have the potential to serve as anticancer chemotherapeutic agents. However, these first-generation Hsp90 inhibitors share an unfavorable structural feature that causes both reduced efficacy and toxicity during clinical evaluation. We report the rationally designed biosynthesis of C15 hydroxylated non-quinone geldanamycin analogues by site-directed mutagenesis of the geldanamycin polyketide synthase (PKS), together with a combination of post-PKS tailoring genes. A 15-hydroxyl-17-demethoxy non-quinone analogue, DHQ3, exhibited stronger inhibition of Hsp90 ATPase activity (4.6-fold) than geldanamycin. Taken together, the results of the present study indicate that rational biosynthetic engineering allows the generation of derivatives of geldanamycin with superior pharmacological properties.


Assuntos
Antibióticos Antineoplásicos/química , Benzoquinonas/química , Benzoquinonas/metabolismo , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Engenharia Genética , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Lactamas Macrocíclicas/farmacologia , Dados de Sequência Molecular , Família Multigênica , Mutagênese Sítio-Dirigida , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Alinhamento de Sequência
15.
J Nat Prod ; 72(4): 645-9, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19275222

RESUMO

To determine the biosynthesis pathway of the atisine-type diterpenoid alkaloids spiramines A/B and C/D, feeding experiments in in vitro cultured plantlets and enzymatic transformations in cell-free extracts were performed in combination with LCMS and tandem MS analyses. L-[2-(13)C,(15)N]Serine was used in the feeding experiments and enzymatic transformations, and the diterpene spiraminol was identified as a biosynthetic precursor of spiramine alkaloids. The LCMS and tandem MS spectra of the extracts from these experiments indicated that L-[2-(13)C,(15)N]serine was incorporated into spiramines A/B and C/D. The labeled reaction products show that l-serine is the one possible nitrogen source involved in the biosynthesis of atisine-type DAs.


Assuntos
Alcaloides/metabolismo , Diterpenos/metabolismo , Medicamentos de Ervas Chinesas/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Spiraea/química , Alcaloides/química , Diterpenos/química , Medicamentos de Ervas Chinesas/química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Estrutura Molecular , Serina/química , Serina/metabolismo
16.
Nucleic Acids Res ; 35(21): 7348-59, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17962304

RESUMO

p300 regulates the transcriptional activity of a variety of transcription factors by forming an activation complex and/or promoting histone acetylation. Here, we show a unique characteristic of orphan receptor TR3 in negatively regulating the function of p300. TR3 was found to interact with p300 and inhibited the acetylation of transcription factors induced by p300, resulting in the repression of their transcriptional activity. Further analysis revealed that both a conserved transcriptional adapter motif (TRAM) in p300 and a specific sequence FLELFIL in TR3 were critical for their interaction. TR3 binding completely covered the histone acetyltransferase (HAT) domain of p300 and resulted in suppression of the HAT activity, as the p300-induced histone H3 acetylation and transcription were inhibited with the presence TR3. Furthermore, an agonist of TR3, a natural octaketide isolated from Dothiorella sp. HTF3 of an endophytical fungus, was shown to be a potent compound for inhibiting p300 HAT activity (IC(50) = 1.5 microg/ml) in vivo. More importantly, this agonist could repress the transcriptional activity of transcription factors, and proliferation of cancer cells. Taken together, our results not only delineate a novel transcriptional repressor function for TR3, but also reveal its modulation on p300 HAT activity as the underlying mechanism.


Assuntos
Receptores de Esteroides/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição de p300-CBP/antagonistas & inibidores , Acetilação , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Regulação para Baixo , Regulação da Expressão Gênica , Humanos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Receptores de Esteroides/agonistas , Receptores de Esteroides/química , Receptores dos Hormônios Tireóideos/agonistas , Receptores dos Hormônios Tireóideos/química , Proteínas Repressoras/agonistas , Proteínas Repressoras/química , Fatores de Transcrição de p300-CBP/química , Fatores de Transcrição de p300-CBP/metabolismo
17.
Arch Pharm Res ; 32(1): 3-5, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19183870

RESUMO

A new aporphine alkaloid, beta-magnoflorine (1), together with a known aporphine alkaloid, alpha-magnoflorine (2), were isolated from the aerial parts of Clematis parviloba. Their structures were elucidated on the basis of comprehensive spectroscopic techniques. In addition, both compounds showed potent antifungal activities against Penicillium avellaneum UC-4376.


Assuntos
Antifúngicos/farmacologia , Aporfinas/farmacologia , Clematis , Penicillium/efeitos dos fármacos , Antifúngicos/química , Antifúngicos/isolamento & purificação , Aporfinas/química , Aporfinas/isolamento & purificação , Clematis/química , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Estrutura Molecular , Penicillium/crescimento & desenvolvimento , Componentes Aéreos da Planta
18.
Structure ; 27(7): 1162-1170.e3, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31080119

RESUMO

Arrestins, in addition to desensitizing GPCR-induced G protein activation, also mediate G protein-independent signaling by interacting with various signaling proteins. Among these, arrestins regulate MAPK signal transduction by scaffolding mitogen-activated protein kinase (MAPK) signaling components such as MAPKKK, MAPKK, and MAPK. In this study, we investigated the binding mode and interfaces between arrestin-3 and JNK3 using hydrogen/deuterium exchange mass spectrometry, 19F-NMR, and tryptophan-induced Atto 655 fluorescence-quenching techniques. Results suggested that the ß1 strand of arrestin-3 is the major and potentially only interaction site with JNK3. The results also suggested that C-lobe regions near the activation loop of JNK3 form the potential binding interface, which is variable depending on the ATP binding status. Because the ß1 strand of arrestin-3 is buried by the C-terminal strand in its basal state, C-terminal truncation (i.e., pre-activation) of arrestin-3 facilitates the arrestin-3/JNK3 interaction.


Assuntos
Trifosfato de Adenosina/química , Arrestinas/química , Proteína Quinase 10 Ativada por Mitógeno/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Arrestinas/genética , Arrestinas/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/genética , Proteína Quinase 10 Ativada por Mitógeno/genética , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
19.
Environ Microbiol ; 10(10): 2684-91, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18631363

RESUMO

Plant microbiota (the microorganisms that live in any associations with plant tissues) represents a rather unexplored area of metagenomic research compared with soils and oceans. Constructing a metagenomic library for plant microbiota is technically challenging. Using all the biomass without pre-enrichment could lead to vast proportions of the host plant DNA in the metagenomic library, doubtless obliterating the microbial contribution. Therefore, the first and essential step is to enrich for the constituent microorganisms from plant tissues. Here, a strong enrichment for plant microbiota was achieved by coupling SDS (sodium dodecyl sulfate) with NaCl, creating a predominantly microbial metagenomic library that contains 88% bacterial inserts. 16S rDNA sequence analysis revealed that the metagenomic DNA of enrichments originates from very diverse microorganisms. At least 74 distinct ribotypes (at a 97% threshold) from seven different bacterial phyla were identified and mainly distributed among Actinobacteria and Proteobacteria. Additionally, a simplified version of Amplified Ribosomal DNA Restriction Analysis (ARDRA) was developed for a quick and efficient assessment of the enriching procedures. This work opens further insight into the great biotechnical potential of plant microbiota, holding more potential for drug discovery through a metagenomic strategy, and paving the way for recovery and biochemical characterization of functional gene repertoire from plant microbiota.


Assuntos
Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , DNA Bacteriano/genética , Biblioteca Gênica , Plantas/microbiologia , Biodiversidade , DNA Bacteriano/química , DNA Ribossômico/química , DNA Ribossômico/genética , Genes de RNAr , Genótipo , Dados de Sequência Molecular , Filogenia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Ribotipagem , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico
20.
J Microbiol Biotechnol ; 18(6): 1101-8, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18600054

RESUMO

Geldanamycin and its analogs are important anticancer agents that inhibit the newly targeted, heat-shock protein (Hsp) 90, which is a chaperone protein in eukaryotic cells. To resolve which geldanamycin biosynthetic genes are responsible for particular post-polyketide synthase (PKS) processing steps and in which order the reactions occur, we individually inactivated candidate genes in Streptomyces hygroscopicus subsp. duamyceticus JCM4427, and isolated and elucidated the structures of intermediates from each mutant. The results indicated that gel7 governs at least one of the benzoquinone ring oxidation steps. In addition, gel16 was found to be involved in double-bond formation between C-4 and C-5 of 4,5-dihydrogeldanamycin, which confirmed our previous findings that this double bond reduced during the post-PKS modification of the polyketide assembly. In addition, pro-geldanamycin, which does not possess a double bond at C-4/5, was purified from the gel7 and 8 double-gene-inactivated mutant.


Assuntos
Proteínas de Bactérias/metabolismo , Benzoquinonas/metabolismo , Lactamas Macrocíclicas/metabolismo , Família Multigênica , Policetídeo Sintases/metabolismo , Streptomyces/genética , Sequência de Aminoácidos , Clonagem Molecular , Genes Bacterianos , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese Insercional , Streptomyces/metabolismo
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