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1.
J Cancer Res Clin Oncol ; 146(1): 75-86, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31754833

RESUMO

PURPOSE: The enzymes gamma-glutamyl hydrolase (GGH) and folylpolyglutamate synthetase (FPGS) regulate intracellular folate concentrations needed for cell proliferation, DNA synthesis, and repair. High GGH expression affects 5-FU thymidylate synthase (TS) inhibition and is a risk factor for various malignancies. Here, the clinical significance of GGH and FPGS expression was investigated in Stage II/III gastric cancer patients undergoing postoperative adjuvant chemotherapy with S-1. METHODS: Surgical specimens of cancer tissue and adjacent normal mucosa, obtained from 253 patients with previously untreated gastric cancer, were examined. GGH and FPGS mRNA expression was measured by qPCR to evaluate their clinicopathological significance in gastric cancer patients after curative resection. RESULTS: While FPGS expression showed no significant differences between the cancerous and normal samples, GGH expression was higher in cancer tissue than in adjacent normal mucosa. High GGH expression was correlated with age, histological type, and vascular invasion. Overall survival (OS) of patients with high GGH mRNA expression was significantly poorer than of patients with low GGH expression. Multivariate analysis showed that high GGH expression was an independent prognostic factor of OS (HR: 2.58, 95% CI 1.29-5.16). Patients who received S-1 adjuvant treatment showed a significantly poor OS between high GGH/low FPGS and low GGH/high FPGS. Patients without adjuvant treatment showed no significant difference. CONCLUSION: GGH expression was significantly higher in gastric cancer tissue than in adjacent normal mucosa. High GGH and low FPGS expression is a useful independent predictor of poor outcomes in stage II/III gastric cancer patients undergoing postoperative adjuvant chemotherapy with S-1.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/biossíntese , Peptídeo Sintases/biossíntese , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/enzimologia , gama-Glutamil Hidrolase/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Quimioterapia Adjuvante , Combinação de Medicamentos , Feminino , Mucosa Gástrica/enzimologia , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Ácido Oxônico/administração & dosagem , Peptídeo Sintases/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/cirurgia , Tegafur/administração & dosagem , gama-Glutamil Hidrolase/genética
2.
Nat Chem ; 11(7): 653-661, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31182822

RESUMO

Non-ribosomal peptide synthetases (NRPSs) are giant enzyme machines that activate amino acids in an assembly line fashion. As NRPSs are not restricted to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would enable microbial production of a diverse range of peptides; however, the structural requirements for reprogramming NRPSs to facilitate the production of new peptides are not clear. Here we describe a new fusion point inside the condensation domains of NRPSs that results in the development of the exchange unit condensation domain (XUC) concept, which enables the efficient production of peptides, even containing non-natural amino acids, in yields up to 280 mg l-1. This allows the generation of more specific NRPSs, reducing the number of unwanted peptide derivatives, but also the generation of peptide libraries. The XUC might therefore be suitable for the future optimization of peptide production and the identification of bioactive peptide derivatives for pharmaceutical and other applications.


Assuntos
Peptídeo Sintases/biossíntese , Engenharia de Proteínas/métodos , Aminoácidos/química , Bacillus/genética , Sequência de Bases , Escherichia coli/genética , Família Multigênica , Biblioteca de Peptídeos , Peptídeo Sintases/química , Peptídeo Sintases/genética , Photorhabdus/enzimologia , Domínios Proteicos/genética , Especificidade por Substrato , Xenorhabdus/genética
3.
J Basic Microbiol ; 59(2): 148-157, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30238507

RESUMO

Actinomycin peptide synthetase genes constitute two oppositely oriented transcriptional units, acmADR, and acmBC, separated by a non-coding intergenic region. Gene constructs of the intergenic region together with its adjoining gene acmA or acmB from the actinomycin biosynthetic gene cluster of Streptomyces chrysomallus were transferred into Streptomyces lividans TK64. Each construct expressed the respective synthetase indicating divergent promoters. Primer extension revealed for both directions -10 and -35 boxes similar to σ70 -dependent promoters from Streptomyces and E. coli. No conspicuous regulatory sequences were detected. Accordingly, S. chrysomallus-grown in glucose-containing medium-produced the peptide synthetases AcmA and AcmB/C as well as actinomycin during logarithmic growth phase. Alignments with the corresponding intergenic region of the actinomycin biosynthetic gene cluster in Streptomyces antibioticus identified analogous -10 and -35 boxes of σ70 consensus sequence. However, in S. antibioticus-cultivated in the same conditions-AcmA and AcmB/C were at maximum activity in late log phase and actinomycin formation peaked in stationary phase. The different patterns of formation of actinomycin and its peptide synthetases encoded by the highly homologous actinomycin biosynthetic gene clusters in S. chrysomallus and S. antibioticus suggest strain-specific control of biosynthesis in agreement with absence of pathway-specific regulatory genes.


Assuntos
Dactinomicina/biossíntese , Peptídeo Sintases/biossíntese , Streptomyces antibioticus/metabolismo , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Meios de Cultura/química , Dactinomicina/química , Escherichia coli/genética , Genes Bacterianos/genética , Vetores Genéticos , Glucose/metabolismo , Redes e Vias Metabólicas/genética , Família Multigênica , Peptídeo Sintases/genética , Regiões Promotoras Genéticas , Streptomyces/genética , Streptomyces/crescimento & desenvolvimento , Streptomyces antibioticus/genética , Streptomyces antibioticus/crescimento & desenvolvimento , Transcrição Genética
4.
Biomed Pharmacother ; 109: 716-725, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30551524

RESUMO

OBJECTIVE: Coronary heart disease is a common cause of death and disability worldwide and mainly results from myocardial ischemia-reperfusion (I/R) injury. This study aimed to elucidate the roles and possible mechanism of long noncoding RNA Component Of Mitochondrial RNA Processing Endoribonuclease (RMRP) in protecting against ischemic myocardial injury. MATERIAL AND METHODS: The H9c2 cardiomyocytes were cultured under hypoxia condition to induce myocardial injury. The RMRP expression under hypoxia condition was determined, followed by investigation of the effects of RMRP dysregulation on hypoxia-induced injury in H9c2 cells. In addition, the regulatory relationship between RMRP and miR-206 was detected, and the potential target of miR-206 was identified. Besides, the association of RMRP and activation of PI3K/AKT/mTOR signaling pathway was explored. Furthermore, an in vivo rat model of myocardial I/R injury was established by subjection to 60 min ischemia and subsequently 24 h reperfusion for validation of the role of RMRP in regulating myocardial I/R injury in vivo. RESULTS: The results showed that overexpression of RMRP aggravated hypoxia-induced injury in H9c2 cells. Moreover, miR-206 was negatively regulated by RMRP and overexpression of RMRP aggravated hypoxia injury by downregulation of miR-206. Furthermore, ATG3 was a target of miR-206, and he effects of miR-206 on hypoxia injury were through targeting ATG3. Besides, overexpression of RMRP activated PI3K/AKT/mTOR pathway in hypoxia-treated H9c2 cells, which were reversed by miR-206 overexpression at the same time. Furthermore, in an in vivo rat model of myocardial I/R injury, suppression of RMPR improved cardiac function and inhibited apoptosis after myocardial I/R injury. CONCLUSIONS: Our findings reveal that upregulation of RMRP may aggravate myocardial I/R injury possible by downregulation of miR-206 and subsequently upregulation of ATG3. Activation of PI3K/Akt/mTOR pathway may be a key downstream mechanism mediating the cardioprotection of RMPR/miR-206/ATG3 axis against myocardial I/R injury.


Assuntos
Proteínas Relacionadas à Autofagia/biossíntese , MicroRNAs/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Peptídeo Sintases/biossíntese , RNA Longo não Codificante/biossíntese , Regulação para Cima/fisiologia , Animais , Proteínas Relacionadas à Autofagia/genética , Linhagem Celular , Expressão Gênica , Marcação de Genes/métodos , Masculino , MicroRNAs/genética , Traumatismo por Reperfusão Miocárdica/genética , Peptídeo Sintases/genética , RNA Longo não Codificante/genética , Ratos , Ratos Sprague-Dawley
5.
Int J Food Microbiol ; 284: 1-10, 2018 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29990634

RESUMO

Many foods and beverages in temperate and tropical regions are prone to contamination by ochratoxin A (OTA), one of the most harmful mycotoxins for human and animal health. Aspergillus ochraceus and Aspergillus carbonarius are considered among the main responsible for OTA contamination. We have previously demonstrated that four low or non- fermenting yeasts are able to control the growth and sporulation of OTA-producing Aspergilli both in vitro and on detached grape berries: the biocontrol effect was partly due to the release of volatile organic compounds (VOCs). Aiming to further characterise the effect of VOCs produced by biocontrol yeast strains, we observed that, beside vegetative growth and sporulation, the volatile compounds significantly reduced the production of OTA by two A. carbonarius and A. ochraceus isolates. Exposure to yeast VOCs also affected gene expression in both species, as confirmed by downregulation of polyketide synthase, non-ribosomal peptide synthase, monooxygenase, and the regulatory genes laeA and veA. The main compound of yeast VOCs was 2-phenylethanol, as detected by Headspace-Solid Phase Microextraction-Gas Chromatography-Tandem Mass Spectrometry (HS-SPME-GC-MS) analysis. Yeast VOCs represent a promising tool for the containment of growth and development of mycotoxigenic fungi, and a valuable aid to guarantee food safety and quality.


Assuntos
Aspergillus/crescimento & desenvolvimento , Aspergillus/metabolismo , Agentes de Controle Biológico/metabolismo , Interações Microbianas/fisiologia , Micotoxinas/biossíntese , Ocratoxinas/biossíntese , Compostos Orgânicos Voláteis/metabolismo , Aspergillus/genética , Frutas/microbiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Humanos , Oxigenases de Função Mista/biossíntese , Peptídeo Sintases/biossíntese , Álcool Feniletílico/isolamento & purificação , Policetídeo Sintases/biossíntese , Esporos Fúngicos/crescimento & desenvolvimento , Vitis/microbiologia
6.
J Neuroimmune Pharmacol ; 13(3): 383-395, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29790105

RESUMO

Tissue damage and pathogen invasion during surgical trauma have been identified as contributing factors leading to neuroinflammation in the hippocampus, which can be protected by stimulation of the cholinergic anti-inflammatory pathway using the acetylcholinesterase inhibitor physostigmine. Macroautophagy, an intracellular degradation pathway used to recycle and eliminate damaged proteins and organelles by lysosomal digestion, seems to be important for cell survival under stress conditions. This study aimed to examine the role of autophagy in physostigmine-mediated hippocampal cell protection in a rat model of surgery stress. In the presence or absence of physostigmine, adult Wistar rats underwent surgery in combination with lipopolysaccharide (LPS). Activated microglia, apoptosis-, autophagy-, and anti-inflammatory-related genes and -proteins in the hippocampus were determined by Real-Time PCR, Western blot and fluorescence microscopy after 1 h, 24 h and 3 d. Surgery combined with LPS-treatment led to microglia activation after 1 h and 24 h which was accompanied by apoptotic cell death after 24 h in the hippocampus. Furthermore, it led to a decreased expression of ATG-3 after 24 h and an increased expression of p62/ SQSTM1 after 1 h and 24 h. Administration of physostigmine significantly increased autophagy related markers and restored the autophagic flux after surgery stress, detected by increased degradation of p62/ SQSTM1 in the hippocampus after 1 h and 24 h. Furthermore, physostigmine reduced activated microglia and apoptosis relevant proteins and elevated the increased expression of TGF-beta1 and MFG-E8 after surgery stress. In conclusion, activation of autophagy may be essential in physostigmine-induced neuroprotection against surgery stress.


Assuntos
Autofagia/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Lipopolissacarídeos/antagonistas & inibidores , Fármacos Neuroprotetores/farmacologia , Fisostigmina/farmacologia , Estresse Fisiológico , Animais , Apoptose/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/biossíntese , Proteína Beclina-1/metabolismo , Inflamação/genética , Inflamação/patologia , Inflamação/psicologia , Lipopolissacarídeos/toxicidade , Ativação de Macrófagos/efeitos dos fármacos , Masculino , Microglia/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Peptídeo Sintases/biossíntese , Período Pós-Operatório , Ratos , Ratos Wistar , Proteína Sequestossoma-1/biossíntese
7.
Microbiologyopen ; 7(5): e00598, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29575742

RESUMO

The Pc21 g14570 gene of Penicillium chrysogenum encodes an ortholog of a class 2 histone deacetylase termed HdaA which may play a role in epigenetic regulation of secondary metabolism. Deletion of the hdaA gene induces a significant pleiotropic effect on the expression of a set of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS)-encoding genes. The deletion mutant exhibits a decreased conidial pigmentation that is related to a reduced expression of the PKS gene Pc21 g16000 (pks17) responsible for the production of the pigment precursor naphtha-γ-pyrone. Moreover, the hdaA deletion caused decreased levels of the yellow pigment chrysogine that is associated with the downregulation of the NRPS-encoding gene Pc21 g12630 and associated biosynthetic gene cluster. In contrast, transcriptional activation of the sorbicillinoids biosynthetic gene cluster occurred concomitantly with the overproduction of associated compounds . A new compound was detected in the deletion strain that was observed only under conditions of sorbicillinoids production, suggesting crosstalk between biosynthetic gene clusters. Our present results show that an epigenomic approach can be successfully applied for the activation of secondary metabolism in industrial strains of P. chrysogenum.


Assuntos
Regulação Fúngica da Expressão Gênica , Histona Desacetilases/deficiência , Penicillium chrysogenum/genética , Penicillium chrysogenum/metabolismo , Metabolismo Secundário , Vias Biossintéticas , Deleção de Genes , Peptídeo Sintases/biossíntese , Pigmentos Biológicos/metabolismo , Policetídeo Sintases/biossíntese , Esporos Fúngicos/metabolismo
8.
Peptides ; 101: 60-68, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29289698

RESUMO

Kyotorphin (KTP; L-tyrosyl-l-arginine), an opioid-like analgesic discovered in the bovine brain, is potentially a neuromodulator because of its localization in synaptosomes, the existence of a specific KTP receptor, and the presence of its biosynthetic enzyme in the brain. KTP is formed in the brain from its constituent amino acids, L-tyrosine and L-arginine, by an enzyme termed KTP synthetase. However, the latter has never been identified. We aimed to test the hypothesis that tyrosyl-tRNA synthetase (TyrRS) is also KTP synthetase. We found that recombinant hTyrRS synthesizes KTP from tyrosine, arginine, and ATP, with Km = 1400 µM and 200 µM for arginine and tyrosine, respectively. TyrRS knockdown of PC12 cells with a small interfering RNA (siRNA) in the presence of 1.6 mM tyrosine, arginine, proline, or tryptophan significantly reduced the level of KTP, but not those of tyrosine-tyrosine, tyrosine-proline, or tyrosine-tryptophan. siRNA treatment did not affect cell survival or proliferation. In mice, TyrRS levels were found to be greater in the midbrain and medulla oblongata than in other brain regions. When arginine was administered 2 h prior to brain dissection, the KTP levels in these regions plus olfactory bulb significantly increased, although basal brain KTP levels remained relatively even. Our conclusion is further supported by a positive correlation across brain regions between TyrRS expression and arginine-accelerated KTP production.


Assuntos
Endorfinas/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Bulbo/enzimologia , Mesencéfalo/enzimologia , Peptídeo Sintases/biossíntese , Tirosina-tRNA Ligase/biossíntese , Animais , Endorfinas/genética , Técnicas de Silenciamento de Genes , Humanos , Masculino , Bulbo/citologia , Mesencéfalo/citologia , Camundongos , Células PC12 , Peptídeo Sintases/genética , Ratos , Tirosina-tRNA Ligase/genética
9.
Fungal Genet Biol ; 111: 60-72, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29155067

RESUMO

The symbiosis between Epichloë festucae and its host perennial ryegrass (Lolium perenne) is a model system for mutualistic interactions in which the fungal endophyte grows between plant shoot cells and acquires host nutrients to survive. E. festucae synthesises the siderophore epichloënin A (EA) via SidN, a non-ribosomal peptide synthetase (NRPS). EA is involved in the acquisition of iron, an essential micronutrient, as part of the process of maintaining a stable symbiotic interaction. Here, we mutated a different NRPS gene sidC and showed that it is required for production of a second siderophore ferricrocin (FC). Furthermore mutations in sidA, encoding an l-ornithine N5-monooxygenase, abolished both EA and FC production. Axenic growth phenotypes of the siderophore mutants were altered relative to wild-type (WT) providing insights into the roles of E. festucae siderophores in iron trafficking and consequently in growth and morphogenesis. During iron-limitation, EA is the predominant siderophore and in addition to its role in iron acquisition it appears to play roles in intracellular iron sequestration and oxidative stress tolerance. FC in contrast is exclusively located intracellularly and is the dominant siderophore under conditions of iron sufficiency when it is likely to have roles in iron storage and iron transport. Intriguingly, EA acts to promote but may also moderate E. festucae growth (depending on the amount of available iron). We therefore hypothesise that coordinated cellular iron sequestration through FC and EA may be one of the mechanisms that E. festucae employs to manage and restrain its growth in response to iron fluxes and ultimately persist as a controlled symbiont.


Assuntos
Epichloe/fisiologia , Ferro/metabolismo , Peptídeo Sintases/fisiologia , Sideróforos/fisiologia , Epichloe/enzimologia , Epichloe/genética , Genes Fúngicos , Homeostase , Lolium/microbiologia , Mutagênese , Estresse Oxidativo , Peptídeo Sintases/biossíntese , Peptídeo Sintases/genética , Sideróforos/biossíntese , Sideróforos/genética
10.
J Bacteriol ; 199(21)2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28784817

RESUMO

Hemolysins are important virulence factors for many bacterial pathogens, including Serratia marcescens The role of the major hemolysin gene in the insect pathogen Serratia sp. strain SCBI was investigated using both forward and reverse-genetics approaches. Introduction of the major hemolysin gene into Escherichia coli resulted in a gain of both virulence and hemolytic activity. Inactivation of this hemolysin in Serratia sp. SCBI resulted in a loss of hemolysis but did not attenuate insecticidal activity. Unexpectedly, inactivation of the hemolysin gene in Serratia sp. SCBI resulted in significantly increased motility and increased antimicrobial activity. Reverse transcription-quantitative PCR (qRT-PCR) analysis of mutants with a disrupted hemolysin gene showed a dramatic increase in mRNA levels of a nonribosomal peptide synthetase gene, swrA, which produces the surfactant serrawettin W2. Mutation of the swrA gene in Serratia sp. SCBI resulted in highly varied antibiotic activity, motility, virulence, and hemolysis phenotypes that were dependent on the site of disruption within this 17.75-kb gene. When introduced into E. coli, swrA increases rates of motility and confers antimicrobial activity. While it is unclear how inactivation of the major hemolysin gene influences the expression of swrA, these results suggest that swrA plays an important role in motility and antimicrobial activity in Serratia sp. SCBI.IMPORTANCE The opportunistic Gram-negative bacteria of the genus Serratia are widespread in the environment and can cause human illness. A comparative genomics analysis between Serratia marcescens and a new Serratia species from South Africa, termed Serratia sp. strain SCBI, shows that these two organisms are closely related but differ in pathogenesis. S. marcescens kills Caenorhabditis nematodes, while Serratia sp. SCBI is not harmful and forms a beneficial association with them. This distinction presented the opportunity to investigate potential differences in regulation of common virulence mechanisms between these two species. With the emergence of antibiotic-resistant microorganisms, there is a widespread need to understand the regulation of pathogenesis. The significance of this study is the presentation of evidence for cross-pathway regulation of virulence factors and how the elimination of one mechanism may be compensated for by the upregulation of others.


Assuntos
Regulação Bacteriana da Expressão Gênica , Inativação Gênica , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Peptídeo Sintases/biossíntese , Serratia/genética , Serratia/metabolismo , Animais , Anti-Infecciosos/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Insetos/microbiologia , Insetos/fisiologia , Locomoção , Peptídeo Sintases/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Serratia/enzimologia , Serratia/patogenicidade , Análise de Sobrevida , Virulência
11.
J Biosci ; 42(1): 175-187, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28229977

RESUMO

Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) present in bacteria and fungi are the major multi-modular enzyme complexes which synthesize secondary metabolites like the pharmacologically important antibiotics and siderophores. Each of the multiple modules of an NRPS activates a different amino or aryl acid, followed by their condensation to synthesize a linear or cyclic natural product. The studies on NRPS domains, the knowledge of their gene cluster architecture and tailoring enzymes have helped in the in silico genetic screening of the ever-expanding sequenced microbial genomic data for the identification of novel NRPS/PKS clusters and thus deciphering novel non-ribosomal peptides (NRPs). Adenylation domain is an integral part of the NRPSs and is the substrate selecting unit for the final assembled NRP. In some cases, it also requires a small protein, the MbtH homolog, for its optimum activity. The presence of putative adenylation domain and MbtH homologs in a sequenced genome can help identify the novel secondary metabolite producers. The role of the adenylation domain in the NRPS gene clusters and its characterization as a tool for the discovery of novel cryptic NRPS gene clusters are discussed.


Assuntos
Antibacterianos/biossíntese , Peptídeo Sintases/biossíntese , Policetídeo Sintases/biossíntese , Sideróforos/biossíntese , Bactérias/química , Bactérias/metabolismo , Produtos Biológicos/química , Fungos/química , Fungos/metabolismo , Humanos , Família Multigênica , Peptídeo Sintases/genética , Policetídeo Sintases/genética , Sideróforos/genética
12.
Prostate ; 77(1): 10-21, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27550065

RESUMO

BACKGROUND: Our goal was to investigate de novo purine biosynthetic gene PAICS expression and evaluate its role in prostate cancer progression. METHODS: Next-generation sequencing, qRTPCR and immunoblot analysis revealed an elevated expression of a de novo purine biosynthetic gene, Phosphoribosylaminoimidazole Carboxylase, Phosphoribosylaminoimidazole Succinocarboxamide Synthetase (PAICS) in a progressive manner in prostate cancer. Functional analyses were performed using prostate cancer cell lines- DU145, PC3, LnCaP, and VCaP. The oncogenic properties of PAICS were studied both by transient and stable knockdown strategies, in vivo chicken chorioallantoic membrane (CAM) and murine xenograft models. Effect of BET bromodomain inhibitor JQ1 on the expression level of PAICS was also studied. RESULTS: Molecular staging of prostate cancer is important factor in effective diagnosis, prognosis and therapy. In this study, we identified a de novo purine biosynthetic gene; PAICS is overexpressed in PCa and its expression correlated with disease aggressiveness. Through several in vitro and in vivo functional studies, we show that PAICS is necessary for proliferation and invasion in prostate cancer cells. We identified JQ1, a BET bromodomain inhibitor previously implicated in regulating MYC expression and demonstrated role in prostate cancer, abrogates PAICS expression in several prostate cancer cells. Furthermore, we observe loss of MYC occupancy on PAICS promoter in presence of JQ1. CONCLUSIONS: Here, we report that evaluation of PAICS in prostate cancer progression and its role in prostate cancer cell proliferation and invasion and suggest it as a valid therapeutic target. We suggest JQ1, a BET-domain inhibitor, as possible therapeutic option in targeting PAICS in prostate cancer. Prostate 77:10-21, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Biomarcadores Tumorais/biossíntese , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Peptídeo Sintases/biossíntese , Neoplasias da Próstata/enzimologia , Purinas/biossíntese , Animais , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Galinhas , Humanos , Masculino , Camundongos , Camundongos Nus , Invasividade Neoplásica/patologia , Peptídeo Sintases/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Biossíntese de Proteínas/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
13.
Appl Biochem Biotechnol ; 182(1): 155-167, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27854037

RESUMO

This study aimed to develop biocontrol Bacillus and explore bacterial biocontrol substances. According to the blood agar test, strain FJAT-14262 was screened as a biosurfactant-producer. The biosurfactant-producing ability of FJAT-14262 was further confirmed by the oil spreading tests because of its amphipathic character. Furthermore, its fermentation supernatant could decrease the surface tension from 74.1 to 32.7 mN m-1. Fourier transform infrared spectroscopy (FT-IR) analysis indicated that the biosurfactant produced by the strain FJAT-14262 was a kind of lipopeptides. Reverse-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC-MS) analysis demonstrated that this lipopeptide contained surfactin with polar amino acids and hydrophobic fatty acid chains. Moreover, bioinformatic analysis revealed that the nonribosomal peptide synthetases genes srfAA, srfAB, and srfAC were structurally conserved in the FJAT-14262 genome. Importantly, the crude surfactant exhibited strong inhibitory activities against Fusarium oxysporum, suggesting that strain FJAT-14262 could be a potential biological control agent against Fusarium wilt.


Assuntos
Antifúngicos/metabolismo , Bacillus/química , Proteínas de Bactérias/biossíntese , Genoma Bacteriano , Lipopeptídeos/biossíntese , Tensoativos/metabolismo , Aminoácidos/química , Antifúngicos/química , Antifúngicos/farmacologia , Bacillus/genética , Bacillus/isolamento & purificação , Bacillus/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Fermentação , Fusarium/efeitos dos fármacos , Fusarium/crescimento & desenvolvimento , Expressão Gênica , Interações Hidrofóbicas e Hidrofílicas , Isoenzimas/biossíntese , Isoenzimas/genética , Lipopeptídeos/química , Lipopeptídeos/farmacologia , Orchidaceae/microbiologia , Peptídeo Sintases/biossíntese , Peptídeo Sintases/genética , Plantas Medicinais/microbiologia , Rizosfera , Tensão Superficial , Tensoativos/química , Tensoativos/farmacologia
14.
PLoS One ; 11(8): e0161199, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27551732

RESUMO

Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) each give rise to a vast array of complex bioactive molecules with further complexity added by the existence of natural PKS-NRPS fusions. Rational genetic engineering for the production of natural product derivatives is desirable for the purpose of incorporating new functionalities into pre-existing molecules, or for optimization of known bioactivities. We sought to expand the range of natural product diversity by combining modules of PKS-NRPS hybrids from different hosts, hereby producing novel synthetic natural products. We succeeded in the construction of a functional cross-species chimeric PKS-NRPS expressed in Aspergillus nidulans. Module swapping of the two PKS-NRPS natural hybrids CcsA from Aspergillus clavatus involved in the biosynthesis of cytochalasin E and related Syn2 from rice plant pathogen Magnaporthe oryzae lead to production of novel hybrid products, demonstrating that the rational re-design of these fungal natural product enzymes is feasible. We also report the structure of four novel pseudo pre-cytochalasin intermediates, niduclavin and niduporthin along with the chimeric compounds niduchimaeralin A and B, all indicating that PKS-NRPS activity alone is insufficient for proper assembly of the cytochalasin core structure. Future success in the field of biocombinatorial synthesis of hybrid polyketide-nonribosomal peptides relies on the understanding of the fundamental mechanisms of inter-modular polyketide chain transfer. Therefore, we expressed several PKS-NRPS linker-modified variants. Intriguingly, the linker anatomy is less complex than expected, as these variants displayed great tolerance with regards to content and length, showing a hitherto unreported flexibility in PKS-NRPS hybrids, with great potential for synthetic biology-driven biocombinatorial chemistry.


Assuntos
Aspergillus nidulans/genética , Engenharia Genética , Peptídeo Sintases/genética , Policetídeo Sintases/genética , Aspergillus nidulans/enzimologia , Produtos Biológicos , Citocalasinas/biossíntese , Regulação Enzimológica da Expressão Gênica , Genes Sintéticos/genética , Magnaporthe/enzimologia , Magnaporthe/genética , Peptídeo Sintases/biossíntese , Policetídeo Sintases/biossíntese , Especificidade por Substrato
15.
J Pept Sci ; 22(9): 564-70, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27465074

RESUMO

From the evolutionary melting pot of natural product synthetase genes, microorganisms elicit antibiotics, communication tools, and iron scavengers. Chemical biologists manipulate these genes to recreate similarly diverse and potent biological activities not on evolutionary time scales but within months. Enzyme engineering has progressed considerably in recent years and offers new screening, modelling, and design tools for natural product designers. Here, recent advances in enzyme engineering and their application to nonribosomal peptide synthetases are reviewed. Among the nonribosomal peptides that have been subjected to biosynthetic engineering are the antibiotics daptomycin, calcium-dependent antibiotic, and gramicidin S. With these peptides, incorporation of unnatural building blocks and modulation of bioactivities via various structural modifications have been successfully demonstrated. Natural product engineering on the biosynthetic level is not a reliable method yet. However, progress in the understanding and manipulation of biosynthetic pathways may enable the routine production of optimized peptide drugs in the near future. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.


Assuntos
Antibacterianos/biossíntese , Daptomicina/biossíntese , Gramicidina/biossíntese , Peptídeo Sintases/biossíntese , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Antibacterianos/química , Química Click , Daptomicina/química , Evolução Molecular Direcionada , Desenho de Drogas , Expressão Gênica , Gramicidina/química , Mutação , Peptídeo Sintases/química , Peptídeo Sintases/genética , Peptídeos/química , Peptídeos/genética , Domínios Proteicos
16.
J Clin Pharmacol ; 56(12): 1563-1569, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27146084

RESUMO

Sex differences in the prevalence of autoimmune diseases such as rheumatoid arthritis (RA) are well known, but little is known about those differences in relation to therapeutic response. Reduced folate carrier-1 (RFC-1), folypolyformyl glutamate synthase (FPGS), and γ-glutamyl hydrolase (GGH) are important transporters and enzymes that convert methotrexate (MTX) in the body. This study investigated the sex differences in mRNA expression of RFC-1, FPGS, and GGH in 190 unrelated healthy Japanese people. The genotypes and mRNA expression were determined using the real-time PCR method. Significant differences between men and women were observed in RFC-1, FPGS, and GGH mRNA expression. The mRNA expression of FPGS and GGH was greater in women than that in men, but the expression of RFC-1 was less in the former than the latter. In stratified analysis by genotype, significant differences in sex-specific mRNA expression were observed in G/G of FPGS, C/C of GGH 452, and C/C of GGH -401. All showed greater mRNA expression in women than in men. In the 5 single-nucleotide polymorphisms RFC-1 80G>A, RFC-1 -43T>C, FPGS 1994G>A, GGH 452C>T, and GGH -401C>T examined, the FPGS 1994 G/G (1.46-fold), GGH 452 C/C (2.16-fold), and GGH -401 C/C (2.68-fold) genotypes showed significantly higher mRNA expression in women than in men. Healthy Japanese adults in this study showed sex-specific differences in mRNA expression that differed among RFC-1, FPGS, and GGH. Therefore, the relationship between genetic polymorphisms and mRNA expression including sex differences might contribute to the variation in the efficacy/toxicity of MTX in patients with RA.


Assuntos
Grupo com Ancestrais do Continente Asiático , Proteínas de Membrana Transportadoras/biossíntese , Peptídeo Sintases/biossíntese , RNA Mensageiro/biossíntese , Caracteres Sexuais , gama-Glutamil Hidrolase/biossíntese , Adulto , Grupo com Ancestrais do Continente Asiático/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Masculino , Proteínas de Membrana Transportadoras/genética , Peptídeo Sintases/genética , Vigilância da População , RNA Mensageiro/genética , Adulto Jovem , gama-Glutamil Hidrolase/genética
17.
Biomed Res Int ; 2016: 3073949, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27073802

RESUMO

To clone and express the γ-polyglutamic acid (γ-PGA) synthetase gene pgsBCA in Bacillus subtilis, a pWB980 plasmid was used to construct and transfect the recombinant expression vector pWB980-pgsBCA into Bacillus subtilis WB600. PgsBCA was expressed under the action of a P43 promoter in the pWB980 plasmid. Our results showed that the recombinant bacteria had the capacity to synthesize γ-PGA. The expression product was secreted extracellularly into the fermentation broth, with a product yield of 1.74 g/L or higher. γ-PGA samples from the fermentation broth were purified and characterized. Hydrolysates of γ-PGA presented in single form, constituting simple glutamic acid only, which matched the characteristics of the infrared spectra of the γ-PGA standard, and presented as multimolecular aggregates with a molecular weight within the range of 500-600 kDa. Expressing the γ-PGA synthetase gene pgsBCA in B. subtilis system has potential industrial applications.


Assuntos
Bacillus subtilis/enzimologia , Escherichia coli/genética , Peptídeo Sintases/genética , Bacillus subtilis/genética , Clonagem Molecular , Fermentação , Regulação Bacteriana da Expressão Gênica , Peptídeo Sintases/biossíntese , Plasmídeos/biossíntese , Plasmídeos/genética
18.
Appl Microbiol Biotechnol ; 100(4): 1559-1565, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26658983

RESUMO

Cyanophycin (CP) can be successfully produced in plants by the ectopic expression of the CphA synthetase from Thermosynechococcus elongatus BP-1 (Berg et al. 2000), yielding up to 6.8 % of dry weight (DW) in tobacco leaf tissue and 7.5 % in potato tubers (Huehns et al. 2008, 2009). Though, high amounts of the polymer lead to phenotypical abnormalities in both crops. The extension of abnormalities and the maximum amount of CP tolerated depend on the compartment that CP production is localized at the tissue/crop in which CP was produced (Huehns et al. 2008, 2009; Neumann et al. 2005). It cannot be ascribed to a depletion of arginine, lysine, or aspartate, the substrates for CP synthesis.


Assuntos
Proteínas de Bactérias/biossíntese , Cianobactérias/enzimologia , Expressão Gênica , Peptídeo Sintases/biossíntese , Proteínas Recombinantes/biossíntese , Solanum tuberosum/metabolismo , Tabaco/metabolismo , Proteínas de Bactérias/genética , Cianobactérias/genética , Peptídeo Sintases/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Tubérculos/genética , Tubérculos/metabolismo , Proteínas Recombinantes/genética , Solanum tuberosum/genética , Tabaco/genética
19.
Int J Mol Med ; 37(2): 319-28, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26676887

RESUMO

To establish the individualized treatment of patients with colorectal cancer, factors associated with chemotherapeutic effects should be identified. However, to the best of our knowledge, few studies are available on this topic, although it is known that the prognosis of patients and sensitivity to chemotherapy depend on the location of the tumor and that the tumor location is important for individualized treatment. In this study, primary tumors obtained from 1,129 patients with colorectal cancer were used to measure the mRNA expression levels of the following genes associated with the effects of standard chemotherapy for colorectal cancer: 5-fluorouracil (5-FU)-related thymidylate synthase (TYMS), dihydropyrimidine dehydrogenase (DPYD) and thymidine phosphorylase (TYMP); folate-related dihydrofolate reductase (DHFR), folylpolyglutamate synthase (FPGS) and gamma-glutamyl hydrolase (GGH); irinotecan-related topoisomerase I (TOP1); oxaliplatin-related excision repair cross-complementing 1 (ERCC1); biologic agent-related vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR). Large-scale population analysis was performed to determine the association of gene expression with the clinicopathological features, in particular, the location of the colorectal cancer. From the results of our analysis of the mRNA expression of these 10 genes, we noted the strongest correlation between DPYD and TYMP, followed by TYMS and DHFR. The location of the colorectal cancer was classified into 4 regions (the right­ and left-sided colon, rectosigmoid and rectum) and was compared with gene expression. A significant difference in all genes, apart from VEGF, was noted. Of the remaining 9 genes, the highest expression of TYMS and DPYD was observed in the right­sided colon; the highest expression of GGH and EGFR was noted in the left-sided colon; the highest expression of DHFR, FPGS, TOP1 and ERCC1 was noted in the rectosigmoid, whereas TYMP expression was approximately equivalent in the right-sided colon and rectum, and higher than that in other locations. The data generated from this study may prove to be useful for the development of individualized chemotherapeutic treatments for patients with colorectal cancer, and will mean that the tumor location is taken into account.


Assuntos
Neoplasias Colorretais/genética , DNA Topoisomerases Tipo I/biossíntese , Proteínas de Ligação a DNA/biossíntese , Endonucleases/biossíntese , Receptores ErbB/biossíntese , Peptídeo Sintases/biossíntese , Tetra-Hidrofolato Desidrogenase/biossíntese , Timidina Fosforilase/biossíntese , gama-Glutamil Hidrolase/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , DNA Topoisomerases Tipo I/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Receptores ErbB/genética , Feminino , Fluoruracila/administração & dosagem , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Irinotecano , Masculino , Pessoa de Meia-Idade , Compostos Organoplatínicos/administração & dosagem , Oxaliplatina , Peptídeo Sintases/genética , Prognóstico , RNA Mensageiro/biossíntese , Tetra-Hidrofolato Desidrogenase/genética , Timidina Fosforilase/genética , gama-Glutamil Hidrolase/genética
20.
Nat Chem Biol ; 11(9): 721-7, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26236937

RESUMO

Cyclodipeptide synthases (CDPSs) constitute a family of peptide bond-forming enzymes that use aminoacyl-tRNAs for the synthesis of cyclodipeptides. Here, we describe the activity of 41 new CDPSs. We also show that CDPSs can be classified into two main phylogenetically distinct subfamilies characterized by specific functional subsequence signatures, named NYH and XYP. All 11 previously characterized CDPSs belong to the NYH subfamily, suggesting that further special features may be yet to be discovered in the other subfamily. CDPSs synthesize a large diversity of cyclodipeptides made up of 17 proteinogenic amino acids. The identification of several CDPSs having the same specificity led us to determine specificity sequence motifs that, in combination with the phylogenetic distribution of CDPSs, provide a first step toward being able to predict the cyclodipeptides synthesized by newly discovered CDPSs. The determination of the activity of ten more CDPSs with predicted functions constitutes a first experimental validation of this predictive approach.


Assuntos
Proteínas de Bactérias/química , Dipeptídeos/química , Proteínas Fúngicas/química , Peptídeo Sintases/química , Peptídeos Cíclicos/química , Motivos de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Biologia Computacional , Ciclização , Bases de Dados Genéticas , Dipeptídeos/biossíntese , Dipeptídeos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Expressão Gênica , Dados de Sequência Molecular , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Peptídeo Sintases/biossíntese , Peptídeo Sintases/genética , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/genética , Filogenia , Estrutura Terciária de Proteína , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato
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