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1.
Gen Physiol Biophys ; 38(6): 485-495, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31829306

RESUMO

Circular RNA-microRNA (circRNA-miR) node has recently been found to modulate cancer process. Here, we investigated whether circCDYL and miR-150-5p exerted biological function in colon cancer cells. Colon cancer tissues were collected and subjected to qRT-PCR assay for circCDYL and miR-150-5p. SW480 and SW620 cells were forced to overexpress circCDYL and miR-150-5p before subjected to viability, colony formation, apoptosis, migration, invasion and protein (associated with proliferation, apoptosis and signaling pathways) assays. To confirm the combined function, the cells were transfected to simultaneously overexpress circCDYL and miR-150-5p. We found circCDYL was generally decreased while miR-150-5p was increased in colon cancer tissues in parallel with the para-carcinoma tissues. In circCDYL-transfected SW480 and SW620 cells, circCDYL decreased viability and promoted apoptosis with down-regulation of c-Myc and cyclin D1, up-regulation of p53, and cleavage of caspase-3 and PARP. Besides, migration and invasion behaviors were impeded. By contrast, miR-150-5p showed a carcinogenesis. However, suppressive role of circCDYL in cellular growth and migration was restrained in the cells simultaneously transfected with circCDYL and miR-150-5p, which was companied by down-regulation of PTEN and phosphorylation of PI3K, AKT, JAK2 and STAT5. circCDYL overexpression repressed cellular growth and migration via repressing miR-150-5p in colon cancer cells.


Assuntos
Proteínas Correpressoras/genética , Neoplasias do Colo , Hidroliases/genética , MicroRNAs/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Humanos
2.
Chemistry ; 25(63): 14267-14272, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31603595

RESUMO

Bioinspired complexes employing the ligands 6-tert-butylpyridazine-3-thione (SPn) and pyridine-2-thione (SPy) were synthesized and fully characterized to mimic the tungstoenzyme acetylene hydratase (AH). The complexes [W(CO)(C2 H2 )(CHCH-SPy)(SPy)] (4) and [W(CO)(C2 H2 )(CHCH-SPn)(SPn)] (5) were formed by intramolecular nucleophilic attack of the nitrogen donors of the ligand on the coordinated C2 H2 molecule. Labelling experiments using C2 D2 with the SPy system revealed the insertion reaction proceeding via a bis-acetylene intermediate. The starting complex [W(CO)(C2 H2 )(SPy)2 ] (6) for these studies was accessed by the new acetylene precursor mixture [W(CO)(C2 H2 )n (MeCN)3-n Br2 ] (n=1 and 2; 7). All complexes represent rare examples in the field of W-C2 H2 chemistry with 4 and 5 being the first of their kind. In the ongoing debate on the enzymatic mechanism, the findings support activation of acetylene by the tungsten center.


Assuntos
Materiais Biomiméticos/química , Complexos de Coordenação/química , Tungstênio/química , Acetileno/química , Acetileno/metabolismo , Materiais Biomiméticos/metabolismo , Complexos de Coordenação/síntese química , Medição da Troca de Deutério , Hidroliases/química , Hidroliases/metabolismo , Espectroscopia de Ressonância Magnética , Conformação Molecular , Estereoisomerismo
3.
Plant Sci ; 289: 110223, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31623788

RESUMO

Phenylalanine is an aromatic amino acid that provides the carbon skeleton for the phenylpropanoid pathway, making many diverse chemicals used for structure, defense, and yet undiscovered functions. The identification of the arogenate dehydratase (ADT) enzymes in the genetic model Arabidopsis thaliana provided a platform to explore the roles of phenylalanine in all stages of life: germination, in the seed-to-seedling transition stage, organelle function, and in generation of defense mechanisms, enabling further studies in other plants. From the literature, data indicate that phenylalanine produced by ADT may have direct roles in organellar and tissue development. Recent studies implicate ADTs in cell division and protection from Reactive Oxygen Species, and in signaling and growth. Research in phenylalanine and subsequent phenylpropanoids also point to a role of phenylalanine as a purveyor of C and N nutrients. The understanding of phenylalanine action in plant cells is enhanced by recent research on phenylalanine in animal cells.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Fenilalanina/metabolismo , Arabidopsis/metabolismo , Germinação , Hidroliases/genética , Hidroliases/metabolismo , Fenilalanina/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Sementes/crescimento & desenvolvimento , Sementes/metabolismo
4.
Eur J Med Chem ; 182: 111656, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31494467

RESUMO

Chemical probes of epigenetic 'readers' of histone post-translational modifications (PTMs) have become powerful tools for mechanistic and functional studies of their target proteins in physiology and pathology. However, only limited 'reader' probes have been developed, which restricted our understanding towards these macromolecules and their roles in cells or animals. Here, we reported a structure-guided approach to develop and characterize benzo [d]oxazol-2(3H)-one analogs as the first potent and selective small-molecule inhibitors of chromodomain Y-like (CDYL), a histone methyllysine reader protein. The binding conformation between the chromodomain of CDYL and the modified peptidomimetics was studied via molecular docking and dynamic simulations, facilitating subsequent virtual screening of tens of hits from Specs chemical library validated by SPR technique (KD values: from 271.1 µM to 5.4 µM). Further design and synthesis of 43 compounds helped to interpret the structure-activity relationship (SAR) that lead to the discovery of novel small-molecule inhibitors of CDYL. Compound D03 (KD: 0.5 µM) was discovered and showed excellent selectivity among other chromodomain proteins, including CDYL2 (>140 folds), CDY1 (no observed binding) and CBX7 (>32 folds). Moreover, we demonstrated that D03 engaged with endogenous CDYL in a dose-dependent manner, and perturbed the recruitment of CDYL onto chromatin, resulting in transcriptional derepression of its target genes. Finally, the results showed that D03 promoted the development and branching of neurodendrites by inhibiting CDYL in hippocampal and cortical cultured neurons. This study not only discovers the first selective small-molecule inhibitors of CDYL, but provids a new chemical tool to intervene the dynamic nature of bio-macromolecules involved in epigenetic mechanism.


Assuntos
Benzoxazóis/farmacologia , Proteínas Correpressoras/antagonistas & inibidores , Hidroliases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Benzoxazóis/síntese química , Benzoxazóis/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Hidroliases/genética , Hidroliases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Neurônios/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
5.
Appl Microbiol Biotechnol ; 103(19): 8063-8074, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31482281

RESUMO

The capability of Escherichia coli to catabolize D-xylonate is a crucial component for building and optimizing the Dahms pathway. It relies on the inherent dehydratase and keto-acid aldolase activities of E. coli. Although the biochemical characteristics of these enzymes are known, their inherent expression regulation remains unclear. This knowledge is vital for the optimization of D-xylonate assimilation, especially in addressing the problem of D-xylonate accumulation, which hampers both cell growth and target product formation. In this report, molecular biology techniques and synthetic biology tools were combined to build a simple genetic switch controller for D-xylonate. First, quantitative and relative expression analysis of the gene clusters involved in D-xylonate catabolism were performed, revealing two D-xylonate-inducible operons, yagEF and yjhIHG. The 5'-flanking DNA sequence of these operons were then subjected to reporter gene assays which showed PyjhI to have low background activity and wide response range to D-xylonate. A PyjhI-driven synthetic genetic switch was then constructed containing feedback control to autoregulate D-xylonate accumulation and to activate the expression of the genes for 1,2,4-butanetriol (BTO) production. The genetic switch effectively reduced D-xylonate accumulation, which led to 31% BTO molar yield, the highest for direct microbial fermentation systems thus far. This genetic switch can be further modified and employed in the production of other compounds from D-xylose through the xylose oxidative pathway.


Assuntos
Butanóis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Engenharia Metabólica/métodos , Regiões Promotoras Genéticas/efeitos dos fármacos , Xilose/análogos & derivados , Aldeído Liases/genética , Aldeído Liases/metabolismo , Fusão Gênica Artificial , Perfilação da Expressão Gênica , Genes Reporter , Hidroliases/genética , Hidroliases/metabolismo , Xilose/metabolismo
6.
Nat Chem Biol ; 15(10): 966-974, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31477916

RESUMO

Pseudouridine (Ψ) is a post-transcriptional RNA modification that alters RNA-RNA and RNA-protein interactions that affect gene expression. Messenger RNA pseudouridylation was recently discovered as a widespread and conserved phenomenon, but the mechanisms responsible for selective, regulated pseudouridylation of specific sequences within mRNAs were unknown. Here, we have revealed mRNA targets for five pseudouridine synthases and probed the determinants of mRNA target recognition by the predominant mRNA pseudouridylating enzyme, Pus1, by developing high-throughput kinetic analysis of pseudouridylation in vitro. Combining computational prediction and rational mutational analysis revealed an RNA structural motif that is both necessary and sufficient for mRNA pseudouridylation. Applying this structural context information predicted hundreds of additional mRNA targets that were pseudouridylated in vivo. These results demonstrate a structure-dependent mode of mRNA target recognition by a conserved pseudouridine synthase and implicate modulation of RNA structure as the probable mechanism to regulate mRNA pseudouridylation.


Assuntos
Hidroliases/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Humanos , Mutação , Conformação de Ácido Nucleico , Saccharomyces cerevisiae/genética
7.
Plant Physiol Biochem ; 142: 43-52, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31272034

RESUMO

Yarrow (Achillea millefolium) is a medicinal plant from the Asteracea which biosynthesize different secondary metabolites especially terpenes and phenylpropanoids. To improve our understanding of the regulatory mechanisms behind the biosynthesis of these compounds we analyzed the expression of some genes associated with the biosynthesis of terpenes and phenylpropanoids in different tissues and in response to trans-cinnamic acid (tCA) as an inhibitor of PAL activity. Isolation and expression analysis of DXR, GPPS, PAL and CHS genes together with linalool synthase (LIS) as monoterpene synthase was conducted in different developmental stages of leaves, flowers and in response to trans-cinnamic acid (tCA). Differential expression of these genes observed in different tissues. tCA up-regulated the biosynthetic genes of monterpenes and down-regulated the biosynthetic genes of phenylpropanoids. Gene expression analysis in intact leaves and leaves without glandular trichomes showed that DXR, LIS, PAL and CHS are highly expressed in glandular trichomes while GPPS expressed ubiquitously. Analysis of essential oils composition showed that sesquiterpenes and monoterpenes are main compounds; in which from 57 identified compounds the highest were germacreneD (% 11.5), guaiol (%10.38), spatulenol (%8.73) and caryophyllene oxide (%7.48).


Assuntos
Achillea/genética , Achillea/metabolismo , Fenilpropionatos/metabolismo , Proteínas de Plantas/genética , Terpenos/metabolismo , Achillea/química , Achillea/efeitos dos fármacos , Aciltransferases/genética , Aciltransferases/metabolismo , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Vias Biossintéticas , Cinamatos/farmacologia , Farnesiltranstransferase/genética , Farnesiltranstransferase/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica de Plantas , Hidroliases/genética , Hidroliases/metabolismo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Tricomas/genética , Tricomas/metabolismo
8.
Molecules ; 24(11)2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31159367

RESUMO

Acyclic monoterpenes constitute a large and highly abundant class of secondary plant metabolites and are, therefore, attractive low-cost raw materials for the chemical industry. To date, numerous biocatalysts for their transformation are known, giving access to highly sought-after monoterpenoids. In view of the high selectivity associated with many of these reactions, the demand for enzymes generating commercially important target molecules is unabated. Here, linalool (de)hydratase-isomerase (Ldi, EC 4.2.1.127) from Castellaniella defragrans was examined for the regio- and stereoselective hydration of the acyclic monoterpene ß-myrcene to (S)-(+)-linalool. Expression of the native enzyme in Escherichia coli allowed for identification of bottlenecks limiting enzyme activity, which were investigated by mutating selected residues implied in enzyme assembly and function. Combining these analyses with the recently published 3D structures of Ldi highlighted the precisely coordinated reduction-oxidation state of two cysteine pairs in correct oligomeric assembly and the catalytic mechanism, respectively. Subcellular targeting studies upon fusion of Ldi to different signal sequences revealed the significance of periplasmic localization of the mature enzyme in the heterologous expression host. This study provides biochemical and mechanistic insight into the hydration of ß-myrcene, a nonfunctionalized terpene, and emphasizes its potential for access to scarcely available but commercially interesting tertiary alcohols.


Assuntos
Alcenos/metabolismo , Betaproteobacteria/metabolismo , Hidroliases/metabolismo , Monoterpenos/metabolismo , Álcoois/química , Álcoois/metabolismo , Alcenos/química , Catálise , Escherichia coli/metabolismo , Hidroliases/química , Hidrólise , Isomerases , Monoterpenos/química
9.
Biosci Biotechnol Biochem ; 83(10): 1884-1888, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31161894

RESUMO

Trans-o-hydroxybenzylidenepyruvate (tHBPA) hydratase-aldolase (RnoE) catalyzes the conversion of tHBPA to 2-hydroxybenzaldehyde and pyruvate. We purified RnoE from Rhodococcus opacus and characterized its enzymatic properties. It exhibited maximum enzyme activity at 60°C and catalyzed the reverse reaction, converting various aromatic benzaldehydes and pyruvate to benzylidenepyruvate, indicating that this enzyme can be adapted for the enzymatic synthesis of α, ß-unsaturated ketones.


Assuntos
Hidroliases/isolamento & purificação , Cetonas/metabolismo , Rhodococcus/enzimologia , Biodegradação Ambiental , Catálise , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Rhodococcus/metabolismo , Microbiologia do Solo
10.
PLoS Biol ; 17(6): e3000316, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31199794

RESUMO

Infections with human herpesviruses are ubiquitous and a public health concern worldwide. Current treatments reduce the severity of some symptoms associated to herpetic infections but neither remove the viral reservoir from the infected host nor protect from the recurrent symptom outbreaks that characterise herpetic infections. The difficulty in therapeutically tackling these viral systems stems in part from their remarkably large proteomes and the complex networks of physical and functional associations that they tailor. This study presents our efforts to unravel the complexity of the interactome of herpes simplex virus type 1 (HSV1), the prototypical herpesvirus species. Inspired by our previous work, we present an improved and more integrative computational pipeline for the protein-protein interaction (PPI) network reconstruction in HSV1, together with a newly developed consensus clustering framework, which allowed us to extend the analysis beyond binary physical interactions and revealed a system-level layout of higher-order functional associations in the virion proteome. Additionally, the analysis provided new functional annotation for the currently undercharacterised protein pUS10. In-depth bioinformatics sequence analysis unravelled structural features in pUS10 reminiscent of those observed in some capsid-associated proteins in tailed bacteriophages, with which herpesviruses are believed to share a common ancestry. Using immunoaffinity purification (IP)-mass spectrometry (MS), we obtained additional support for our bioinformatically predicted interaction between pUS10 and the inner tegument protein pUL37, which binds cytosolic capsids, contributing to initial tegumentation and eventually virion maturation. In summary, this study unveils new, to our knowledge, insights at both the system and molecular levels that can help us better understand the complexity behind herpesvirus infections.


Assuntos
Biologia Computacional/métodos , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/ultraestrutura , Animais , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Bases de Dados Factuais , Herpes Simples/metabolismo , Humanos , Hidroliases/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas , Relação Estrutura-Atividade , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , Vírion/metabolismo , Montagem de Vírus
11.
Appl Microbiol Biotechnol ; 103(17): 7151-7160, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31250059

RESUMO

10-hydroxy-cis-12 octadecenoic acid (10-HOE) is a type of octadecenoic acid with a hydroxyl on the C10 carbon. It is generated from linoleic acid (LA) catalyzed by linoleate hydratase in lactobacilli, which was initially named as myosin-cross-reactive antigen (MCRA). In lactobacilli, 10-HOE is the first intermediate in the production of conjugated LA (CLA). Although MCRA from bifidobacteria can generate 10-HOE, the precise role of 10-HOE in CLA production in bifidobacteria remains unknown. In the current work, 10-HOE and LA were added to the medium as the substrate both separately and synchronously to analyze their influence on CLA production. Using 10-HOE as the substrate, bifidobacteria were able to generate CLA by first converting it to LA, followed by CLA accumulation. Recombinant MCRA catalyzed the conversion of 10-HOE to LA, indicating that bifidobacterial MCRA can account for the reversible conversion between LA and 10-HOE. This is the first report to demonstrate the precise role of 10-HOE in the process of CLA production among bifidobacteria.


Assuntos
Bifidobacterium/metabolismo , Ácido Linoleico/metabolismo , Ácidos Oleicos/metabolismo , Proteínas de Bactérias/metabolismo , Bifidobacterium/enzimologia , Biotransformação , Hidroliases/metabolismo , Ácidos Linoleicos Conjugados/metabolismo , Especificidade da Espécie
12.
World J Gastroenterol ; 25(23): 2947-2960, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31249452

RESUMO

BACKGROUND: Changes in N-linked glycosylation have been observed in the circulation of individuals with hepatocellular carcinoma. In particular, an elevation in the level of core fucosylation has been observed. However, the mechanisms through which core fucose is increased are not well understood. We hypothesized that a review of the literature and related bioinformatic review regarding six genes known to be involved in the attachment of core fucosylation, the synthesis of the fucosylation substrate guanosine diphosphate (GDP)-fucose, or the transport of the substrate into the Golgi might offer mechanistic insight into the regulation of core fucose levels. AIM: To survey the literature to capture the involvement of genes regulating core N-linked fucosylation in hepatocellular carcinoma. METHODS: The PubMed biomedical literature database was searched for the association of hepatocellular carcinoma and each of the core fucose-related genes and their protein products. We also queried The Cancer Genome Atlas Liver hepatocellular carcinoma (LIHC) dataset for genetic, epigenetic and gene expression changes for the set of six genes using the tools at cBioportal. RESULTS: A total of 27 citations involving one or more of the core fucosylation-related genes (FPGT, FUK, FUT8, GMDS, SLC35C1, TSTA3) and hepatocellular carcinoma were identified. The same set of gene symbols was used to query the 371 patients with liver cancer in the LIHC dataset to identify the frequency of mRNA over or under expression, as well as non-synonymous mutations, copy number variation and methylation level. Although all six genes trended to more samples displaying over expression relative to under-expression, it was noted that a number of tumor samples had undergone amplification of the genes of the de novo synthesis pathway, GMDS (27 samples) and TSTA3 (78 samples). In contrast, the other four genes had undergone amplification in 2 or fewer samples. CONCLUSION: Amplification of genes involved in the de novo pathway for generation of GDP-fucose, GMDS and TSTA3, likely contributes to the elevated core fucose observed in hepatocellular carcinoma.


Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Redes e Vias Metabólicas/genética , Carboidratos Epimerases/metabolismo , Carcinoma Hepatocelular/patologia , Variações do Número de Cópias de DNA , Metilação de DNA , Glicoproteínas/metabolismo , Glicosilação , Guanosina Difosfato Fucose/metabolismo , Humanos , Hidroliases/metabolismo , Cetona Oxirredutases/metabolismo , Neoplasias Hepáticas/patologia , Mutação
13.
J Agric Food Chem ; 67(21): 5922-5931, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31067049

RESUMO

Neonicotinoid insecticide pollution in soil and water poses serious environmental risks. Microbial biodegradation is an important neonicotinoid insecticide degradation pathway in the environment. In this study, 70.0% of the acetamiprid in a 200 mg/L solution was degraded by actinomycetes Streptomyces canus CGMCC 13662 (isolated from soil) in 48 h, and the acetamiprid degradation half-life was 27.7 h. Acetamiprid was degraded to IM-1-2 (( E)-1-(1-(((6-chloropyridin-3-yl)methyl)(methyl) amino)ethylidene)urea) through hydrolysis of the cyanoimine moiety. Gene cloning and overexpression indicated that a novel nitrile hydratase with three unusual subunits (AnhD, AnhE, and AnhA) without accessory protein mediated IM-1-2 formation. The purified nitrile hydratase responsible for degrading acetamiprid had a Km of 5.85 mmol/L and a Vmax of 15.99 U/mg. A homology model suggested that AnhD-Glu56 and AnhE-His21 play important roles in the catalytic efficiency of the nitrile hydratase. S. canus CGMCC 13662 could be used to remediate environments contaminated with acetamiprid.


Assuntos
Actinobacteria/metabolismo , Proteínas de Bactérias/metabolismo , Hidroliases/metabolismo , Inseticidas/metabolismo , Neonicotinoides/metabolismo , Actinobacteria/enzimologia , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biodegradação Ambiental , Estabilidade Enzimática , Hidroliases/química , Hidroliases/genética , Inseticidas/química , Cinética , Neonicotinoides/química , Filogenia , Microbiologia do Solo
14.
J Microbiol ; 57(8): 694-703, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31079334

RESUMO

Dihydroxyacid dehydratase (DHAD), encoded by ILV3, catalyses the third step in the biosynthetic pathway of branched-chain amino acids (BCAAs), which include isoleucine (Ile), leucine (Leu), and valine (Val). Enzymes involved in BCAA biosynthesis exist in bacteria, plants, and fungi but not in mammals and are therefore attractive targets for antimicrobial or herbicide development. In this study, three paralogous ILV3 genes (FgILV3A, FgILV3B, and FgILV3C) were identified in the genome of Fusarium graminearum, the causal agent of Fusarium head blight (FHB). Deletion of FgILV3A alone or combined with FgILV3B or FgILV3C indicated an important role for FgILV3A in BCAA biosynthesis. FgILV3A deletion mutants lost the ability to grow on medium lacking amino acids. Exogenous supplementation of 1 mM Ile and Val rescued the auxotrophy of ΔFgIlv3A, though 5 mM was required to recover the growth defects in ΔFgIlv3AB and ΔFgIlv3AC strains, indicating that FgIlv3b and FgIlv3c exhibit redundant but accessory roles with FgIlv3a in BCAA biosynthesis. The auxotrophy of ΔFgIlv3A resulted in pleiotropic defects in aerial hyphal growth, in conidial formation and germination, and in aurofusarin accumulation. In addition, the mutants showed reduced virulence and deoxynivalenol production. Overall, our study demonstrates that FgIlv3a is crucial for BCAA biosynthesis in F. graminearum and a candidate fungicide target for FHB management.


Assuntos
Aminoácidos de Cadeia Ramificada/biossíntese , Fusarium/genética , Fusarium/patogenicidade , Hidroliases/genética , Proteínas Fúngicas/genética , Doenças das Plantas/microbiologia , Virulência
15.
World J Microbiol Biotechnol ; 35(5): 70, 2019 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-31011828

RESUMO

Cyanide is a nitrile which is used extensively in many industries like jewelry, mining, electroplating, plastics, dyes, paints, pharmaceuticals, food processing, and coal coking. Cyanides pose a serious health hazard due to their high affinity towards metals and cause malfunction of cellular respiration by inhibition of cytochrome c oxidase. This inhibition ultimately leads to histotoxic hypoxia, increased acidosis, reduced the functioning of the central nervous system and myocardial activity. Different physicochemical processes including oxidation by hydrogen peroxide, alkaline chlorination, and ozonization have been used to reduce cyanide waste from the environment. Microbial cyanide degradation which is considered as one the most successful techniques is used to take place through different biochemical/metabolic pathways involving reductive, oxidative, hydrolytic or substitution/transfer reactions. Groups of enzymes involved in microbial degradation are cyanidase, cyanide hydratase, formamidase, nitrilase, nitrile hydratase, cyanide dioxygenase, cyanide monooxygenase, cyanase and nitrogenase. In the future, more advancement of omics technologies and protein engineering will help us to recoup the environment from cyanide effluent. In this review, we have discussed the origin and environmental distribution of cyanide waste along with different bioremediation pathways and enzymes involved therein.


Assuntos
Bactérias/enzimologia , Cianetos/metabolismo , Fungos/enzimologia , Resíduos Industriais/análise , Plantas/enzimologia , Amidoidrolases , Aminoidrolases , Bactérias/classificação , Bactérias/metabolismo , Biodegradação Ambiental , Carbono-Nitrogênio Liases , Cianetos/toxicidade , Fungos/metabolismo , Hidroliases , Hidrolases , Microbiologia Industrial , Nitrogenase , Oxirredução , Plantas/classificação , Plantas/metabolismo , Sulfurtransferases , Tiossulfato Sulfurtransferase
16.
Med Oncol ; 36(6): 46, 2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31020480

RESUMO

This phase II clinical trial compared the efficacy and safety of second-line irinotecan and panitumumab treatment (IRI + Pmab) with that of irinotecan, fluoropyrimidines and panitumumab treatment (control) in patients with KRAS wild-type mCRC. The primary endpoint was progression-free survival. In addition, early predictive markers of treatment efficacy were explored. Eighty patients were planned to be recruited. Due to a slow accrual rate, only 48 patients were recruited from 2012 to 2016, of which 23 were allocated to the control group and 25 were allocated to the IRI + Pmab group. The median progression-free survival was 254 days (95% confidence interval, 159-306) for control, and 190 days (95% confidence interval, 159-213) for IRI + Pmab (log-rank test, P = 0.26). The response rate without confirmation was 21.7% (5/23) for control and 40.0% (10/25) for IRI + Pmab. Neutropenia, leukopenia, and anorexia were the most common Grade 3/4 adverse events, and several early drop-outs from the treatment protocol were observed in the control group. As for the biomarkers, carcinoembryonic antigen and lactate dehydrogenase (LDH) smoothly declined immediately after the initial dosing in patients with a partial response or stable disease. After starting treatment, LDH-1 and - 2 increased, while LDH-4 and - 5 decreased, irrespective of tumor response. However, exceptions were frequent. In conclusion, this study failed to prove the safety and efficacy of irinotecan and panitumumab treatment due to insufficient patient accrual. Although LDH and its isozymes changed after initiation of treatment, their ability to predict the tumor response may not surpass that of carcinoembryonic antigen levels.The University Hospital Medical Information Network Clinical Trial Registry: UMIN000007658.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Fluoruracila/administração & dosagem , Irinotecano/administração & dosagem , Panitumumabe/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Biomarcadores Tumorais/metabolismo , Antígeno Carcinoembrionário/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Fluoruracila/efeitos adversos , Humanos , Hidroliases/metabolismo , Irinotecano/efeitos adversos , Masculino , Pessoa de Meia-Idade , Panitumumabe/efeitos adversos , Proteínas Proto-Oncogênicas p21(ras)/genética
17.
PLoS Genet ; 15(4): e1008079, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30969963

RESUMO

Characterizing the fitness landscape, a representation of fitness for a large set of genotypes, is key to understanding how genetic information is interpreted to create functional organisms. Here we determined the evolutionarily-relevant segment of the fitness landscape of His3, a gene coding for an enzyme in the histidine synthesis pathway, focusing on combinations of amino acid states found at orthologous sites of extant species. Just 15% of amino acids found in yeast His3 orthologues were always neutral while the impact on fitness of the remaining 85% depended on the genetic background. Furthermore, at 67% of sites, amino acid replacements were under sign epistasis, having both strongly positive and negative effect in different genetic backgrounds. 46% of sites were under reciprocal sign epistasis. The fitness impact of amino acid replacements was influenced by only a few genetic backgrounds but involved interaction of multiple sites, shaping a rugged fitness landscape in which many of the shortest paths between highly fit genotypes are inaccessible.


Assuntos
Evolução Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Aptidão Genética , Leveduras/genética , Leveduras/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Epistasia Genética , Proteínas Fúngicas/química , Genes Fúngicos , Genótipo , Hidroliases/química , Hidroliases/genética , Hidroliases/metabolismo , Modelos Genéticos , Modelos Moleculares , Filogenia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
mSphere ; 4(1)2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30814316

RESUMO

Proteus mirabilis is a common cause of catheter-associated urinary tract infection (CAUTI) and secondary bacteremia, which are frequently polymicrobial. We previously utilized transposon insertion-site sequencing (Tn-Seq) to identify novel fitness factors for colonization of the catheterized urinary tract during single-species and polymicrobial infection, revealing numerous metabolic pathways that may contribute to P. mirabilis fitness regardless of the presence of other cocolonizing organisms. One such "core" fitness factor was d-serine utilization. In this study, we generated isogenic mutants in d-serine dehydratase (dsdA), d-serine permease (dsdX), and the divergently transcribed activator of the operon (dsdC) to characterize d-serine utilization in P. mirabilis and explore the contribution of this pathway to fitness during single-species and polymicrobial infection. P. mirabilis was capable of utilizing either d- or l-serine as a sole carbon or nitrogen source, and dsdA, dsdX, and dsdC were each specifically required for d-serine degradation. This capability was highly conserved among P. mirabilis isolates, although not universal among uropathogens: Escherichia coli and Morganella morganii utilized d-serine, while Providencia stuartii and Enterococcus faecalis did not. d-Serine utilization did not contribute to P. mirabilis growth in urine ex vivo during a 6-h time course but significantly contributed to fitness during single-species and polymicrobial CAUTI during a 96-h time course, regardless of d-serine utilization by the coinfecting isolate. d-Serine utilization also contributed to secondary bacteremia during CAUTI as well as survival in a direct bacteremia model. Thus, we propose d-serine utilization as a core fitness factor in P. mirabilis and a possible target for disruption of infection.IMPORTANCE Urinary tract infections are among the most common health care-associated infections worldwide, the majority of which involve a urinary catheter (CAUTI). Our recent investigation of CAUTIs in nursing home residents identified Proteus mirabilis, Enterococcus species, and Escherichia coli as the three most common organisms. These infections are also often polymicrobial, and we identified Morganella morganii, Enterococcus species, and Providencia stuartii as being more prevalent during polymicrobial CAUTI than single-species infection. Our research therefore focuses on identifying "core" fitness factors that are highly conserved in P. mirabilis and that contribute to infection regardless of the presence of these other organisms. In this study, we determined that the ability to degrade d-serine, the most abundant d-amino acid in urine and serum, strongly contributes to P. mirabilis fitness within the urinary tract, even when competing for nutrients with another organism. d-Serine uptake and degradation therefore represent potential targets for disruption of P. mirabilis infections.


Assuntos
Infecções Relacionadas a Cateter/microbiologia , Coinfecção , Aptidão Genética , Proteus mirabilis/enzimologia , Serina/metabolismo , Infecções Urinárias/microbiologia , Animais , Feminino , Hidroliases/genética , Camundongos , Mutação , Óperon , Infecções por Proteus/prevenção & controle , Proteus mirabilis/genética
19.
Proc Natl Acad Sci U S A ; 116(14): 6775-6783, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30872475

RESUMO

Fatty acid biosynthesis in α- and γ-proteobacteria requires two functionally distinct dehydratases, FabA and FabZ. Here, mechanistic cross-linking facilitates the structural characterization of a stable hexameric complex of six Escherichia coli FabZ dehydratase subunits with six AcpP acyl carrier proteins. The crystal structure sheds light on the divergent substrate selectivity of FabA and FabZ by revealing distinct architectures of the binding pocket. Molecular dynamics simulations demonstrate differential biasing of substrate orientations and conformations within the active sites of FabA and FabZ such that FabZ is preorganized to catalyze only dehydration, while FabA is primed for both dehydration and isomerization.


Assuntos
Proteína de Transporte de Acila/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Ácido Graxo Sintase Tipo II/química , Ácidos Graxos/química , Hidroliases/química , Simulação de Dinâmica Molecular , Complexos Multienzimáticos/química , Proteína de Transporte de Acila/genética , Proteína de Transporte de Acila/metabolismo , Catálise , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintase Tipo II/genética , Ácido Graxo Sintase Tipo II/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos/genética , Hidroliases/genética , Hidroliases/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo
20.
J Bacteriol ; 201(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30745367

RESUMO

Mycobacterium tuberculosis utilizes fatty acids of the host as the carbon source. Metabolism of odd-chain fatty acids by Mycobacterium tuberculosis produces propionyl coenzyme A (propionyl-CoA). The methylcitrate cycle is essential for mycobacteria to utilize the propionyl-CoA to persist and grow on these fatty acids. In M. smegmatis, methylcitrate synthase, methylcitrate dehydratase, and methylisocitrate lyase involved in the methylcitrate cycle are encoded by prpC, prpD, and prpB, respectively, in operon prpDBC In this study, we found that the nitrogen regulator GlnR directly binds to the promoter region of the prpDBC operon and inhibits its transcription. The binding motif of GlnR was identified by bioinformatic analysis and validated using DNase I footprinting and electrophoretic mobility shift assays. The GlnR-binding motif is separated by a 164-bp sequence from the binding site of PrpR, a pathway-specific transcriptional activator of methylcitrate cycle, but the binding affinity of GlnR to prpDBC is much stronger than that of PrpR. Deletion of glnR resulted in faster growth in propionate or cholesterol medium compared with the wild-type strain. The ΔglnR mutant strain also showed a higher survival rate in macrophages. These results illustrated that the nitrogen regulator GlnR regulates the methylcitrate cycle through direct repression of the transcription of the prpDBC operon. This finding not only suggests an unprecedented link between nitrogen metabolism and the methylcitrate pathway but also reveals a potential target for controlling the growth of pathogenic mycobacteria.IMPORTANCE The success of mycobacteria survival in macrophage depends on its ability to assimilate fatty acids and cholesterol from the host. The cholesterol and fatty acids are catabolized via ß-oxidation to generate propionyl coenzyme A (propionyl-CoA), which is then primarily metabolized via the methylcitrate cycle. Here, we found a typical GlnR binding box in the prp operon, and the affinity is much stronger than that of PrpR, a transcriptional activator of methylcitrate cycle. Furthermore, GlnR repressed the transcription of the prp operon. Deletion of glnR significantly enhanced the growth of Mycobacterium tuberculosis in propionate or cholesterol medium, as well as viability in macrophages. These findings provide new insights into the regulatory mechanisms underlying the cross talk of nitrogen and carbon metabolisms in mycobacteria.


Assuntos
Proteínas de Bactérias/biossíntese , Citratos/metabolismo , Regulação Bacteriana da Expressão Gênica , Redes e Vias Metabólicas/genética , Mycobacterium smegmatis/enzimologia , Proteínas Repressoras/metabolismo , Transcrição Genética , Sítios de Ligação , Carbono-Carbono Liases/biossíntese , Citrato (si)-Sintase/biossíntese , DNA Bacteriano/metabolismo , Deleção de Genes , Hidroliases/biossíntese , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/metabolismo , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/genética
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