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1.
Biomolecules ; 11(8)2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34439822

RESUMO

The genome of the halophilic archaea Haloferax mediterranei contains three ORFs that show homology with glutamine synthetase (GS) (glnA-1, glnA-2, and glnA-3). Previous studies have focused on the role of GlnA-1, suggesting that proteins GlnA-2 and GlnA-3 could play a different role to that of GS. Glutamine synthetase (EC 6.3.1.2) belongs to the class of ligases, including 20 subclasses of other different enzymes, such as aspartate-ammonia ligase (EC 6.3.1.1), glutamate-ethylamine ligase (EC 6.3.1.6), and glutamate-putrescine ligase (EC 6.3.1.11). The reaction catalyzed by glutamate-putrescine ligase is comparable to the reaction catalyzed by glutamine synthetase (GS). Both enzymes can bind a glutamate molecule to an amino group: ammonium (GS) or putrescine (glutamate-putrescine ligase). In addition, they present the characteristic catalytic domain of GS, showing significant similarities in their structure. Although these proteins are annotated as GS, the bioinformatics and experimental results obtained in this work indicate that the GlnA-2 protein (HFX_1688) is a glutamate-putrescine ligase, involved in polyamine catabolism. The most significant results are those related to glutamate-putrescine ligase's activity and the analysis of the transcriptional and translational expression of the glnA-2 gene in the presence of different nitrogen sources. This work confirms a new metabolic pathway in the Archaea domain which extends the knowledge regarding the utilization of alternative nitrogen sources in this domain.


Assuntos
Proteínas Arqueais/genética , Proteínas de Escherichia coli/genética , Regulação da Expressão Gênica em Archaea , Ácido Glutâmico/metabolismo , Haloferax mediterranei/enzimologia , Ligases/genética , Fixação de Nitrogênio/genética , Putrescina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Amônia/metabolismo , Proteínas Arqueais/metabolismo , Clonagem Molecular , Biologia Computacional/métodos , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Haloferax mediterranei/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Ligases/metabolismo , Filogenia , Biossíntese de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/enzimologia , Salmonella typhimurium/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Transcrição Genética
2.
Nat Commun ; 12(1): 2105, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33833232

RESUMO

Intestinal microbiota-derived metabolites have biological importance for the host. Polyamines, such as putrescine and spermidine, are produced by the intestinal microbiota and regulate multiple biological processes. Increased colonic luminal polyamines promote longevity in mice. However, no direct evidence has shown that microbial polyamines are incorporated into host cells to regulate cellular responses. Here, we show that microbial polyamines reinforce colonic epithelial proliferation and regulate macrophage differentiation. Colonisation by wild-type, but not polyamine biosynthesis-deficient, Escherichia coli in germ-free mice raises intracellular polyamine levels in colonocytes, accelerating epithelial renewal. Commensal bacterium-derived putrescine increases the abundance of anti-inflammatory macrophages in the colon. The bacterial polyamines ameliorate symptoms of dextran sulfate sodium-induced colitis in mice. These effects mainly result from enhanced hypusination of eukaryotic initiation translation factor. We conclude that bacterial putrescine functions as a substrate for symbiotic metabolism and is further absorbed and metabolised by the host, thus helping maintain mucosal homoeostasis in the intestine.


Assuntos
Colo/metabolismo , Escherichia coli/metabolismo , Mucosa Intestinal/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Putrescina/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Colite/induzido quimicamente , Colite/patologia , Sulfato de Dextrana/toxicidade , Células Epiteliais/metabolismo , Feminino , Microbioma Gastrointestinal/fisiologia , Homeostase , Mucosa Intestinal/citologia , Mucosa Intestinal/crescimento & desenvolvimento , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL
3.
Int J Mol Sci ; 22(8)2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33920993

RESUMO

Polyamines (PAs) dramatically affect root architecture and development, mainly by unknown mechanisms; however, accumulating evidence points to hormone signaling and reactive oxygen species (ROS) as candidate mechanisms. To test this hypothesis, PA levels were modified by progressively reducing ADC1/2 activity and Put levels, and then changes in root meristematic zone (MZ) size, ROS, and auxin and cytokinin (CK) signaling were investigated. Decreasing putrescine resulted in an interesting inverted-U-trend in primary root growth and a similar trend in MZ size, and differential changes in putrescine (Put), spermidine (Spd), and combined spermine (Spm) plus thermospermine (Tspm) levels. At low Put concentrations, ROS accumulation increased coincidently with decreasing MZ size, and treatment with ROS scavenger KI partially rescued this phenotype. Analysis of double AtrbohD/F loss-of-function mutants indicated that NADPH oxidases were not involved in H2O2 accumulation and that elevated ROS levels were due to changes in PA back-conversion, terminal catabolism, PA ROS scavenging, or another pathway. Decreasing Put resulted in a non-linear trend in auxin signaling, whereas CK signaling decreased, re-balancing auxin and CK signaling. Different levels of Put modulated the expression of PIN1 and PIN2 auxin transporters, indicating changes to auxin distribution. These data strongly suggest that PAs modulate MZ size through both hormone signaling and ROS accumulation in Arabidopsis.


Assuntos
Arabidopsis/anatomia & histologia , Citocininas/metabolismo , Ácidos Indolacéticos/metabolismo , Meristema/anatomia & histologia , Putrescina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arginina/farmacologia , Peróxido de Hidrogênio/metabolismo , Meristema/efeitos dos fármacos , Modelos Biológicos , Mutação/genética , NADPH Oxidases/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Fenótipo , Iodeto de Potássio/farmacologia , Transdução de Sinais/efeitos dos fármacos
4.
Food Microbiol ; 98: 103762, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33875200

RESUMO

Harmful levels of biogenic amines (BAs) are frequently identified in sufu. The microorganisms and mechanisms responsible for BA production in sufu, however, are not well documented. In this study, sufu samples were randomly obtained from various regions of China. Putrescine, tyramine, and histamine were quantitated as the most abundant BAs. According to the metagenome sequencing, the abundances and diversities of genes encoding the critical enzymes in BA production were acquired. The results showed that genes encoding arginine-, ornithine-, tryptophan-, and histidine decarboxylases were the predominant amino acid decarboxylase genes. Furthermore, 34 metagenome-assembled genomes (MAGs) were generated, of which 23 encoded at least one gene involved in BA production. Genetic analysis of MAGs indicated genera affiliated with Enterococcus, Lactobacillus-related, and Lactococcus were the major histamine-synthesizing bacteria, and tyrosine may be utilized by Bacillus, Chryseobacterium, Kurthia, Lysinibacillus, Macrococcus, and Streptococcus to product tyramine. The critical species involved in two putrescine-producing pathways were also explored. In the ornithine decarboxylase pathway, Lactobacillus-related and Veillonella were predicted to be the main performers, whereas Sphingobacterium and unclassified Flavobacteriaceae were the dominant executors in the agmatine deiminase pathway. The present study not only explained the BAs formation mechanism in sufu but also identified specific bacteria used to control BAs in fermented soybean products.


Assuntos
Bactérias/genética , Bactérias/metabolismo , Aminas Biogênicas/metabolismo , Alimentos de Soja/microbiologia , Bactérias/classificação , Bactérias/isolamento & purificação , Aminas Biogênicas/análise , China , Fermentação , Histamina/análise , Histamina/metabolismo , Metagenoma , Metagenômica , Putrescina/análise , Putrescina/metabolismo , Alimentos de Soja/análise , Soja/metabolismo , Soja/microbiologia , Tiramina/análise , Tiramina/metabolismo
5.
Biomolecules ; 11(4)2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33921742

RESUMO

Astrocytes serve essential roles in human brain function and diseases. Growing evidence indicates that astrocytes are central players of the feedback modulation of excitatory Glu signalling during epileptiform activity via Glu-GABA exchange. The underlying mechanism results in the increase of tonic inhibition by reverse operation of the astroglial GABA transporter, induced by Glu-Na+ symport. GABA, released from astrocytes, is synthesized from the polyamine (PA) putrescine and this process involves copper amino oxidase. Through this pathway, putrescine can be considered as an important source of inhibitory signaling that counterbalances epileptic discharges. Putrescine, however, is also a precursor for spermine that is known to enhance gap junction channel communication and, consequently, supports long-range Ca2+ signaling and contributes to spreading of excitatory activity through the astrocytic syncytium. Recently, we presented the possibility of neuron-glia redox coupling through copper (Cu+/Cu2+) signaling and oxidative putrescine catabolism. In the current work, we explore whether the Cu+/Cu2+ homeostasis is involved in astrocytic control on neuronal excitability by regulating PA catabolism. We provide supporting experimental data underlying this hypothesis. We show that the blockade of copper transporter (CTR1) by AgNO3 (3.6 µM) prevents GABA transporter-mediated tonic inhibitory currents, indicating causal relationship between copper (Cu+/Cu2+) uptake and the catabolism of putrescine to GABA in astrocytes. In addition, we show that MnCl2 (20 µM), an inhibitor of the divalent metal transporter DMT1, also prevents the astrocytic Glu-GABA exchange. Furthermore, we observed that facilitation of copper uptake by added CuCl2 (2 µM) boosts tonic inhibitory currents. These findings corroborate the hypothesis that modulation of neuron-glia coupling by copper uptake drives putrescine → GABA transformation, which leads to subsequent Glu-GABA exchange and tonic inhibition. Findings may in turn highlight the potential role of copper signaling in fine-tuning the activity of the tripartite synapse.


Assuntos
Astrócitos/metabolismo , Cobre/metabolismo , Neurônios/fisiologia , Putrescina/metabolismo , Animais , Células Cultivadas , Proteínas de Transporte de Cobre/antagonistas & inibidores , Proteínas de Transporte de Cobre/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Potenciais Pós-Sinápticos Inibidores , Camundongos , Neurônios/metabolismo , Ácido gama-Aminobutírico/metabolismo
6.
J Microbiol ; 59(2): 175-185, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33527317

RESUMO

Putrescine, a typical polyamine compound important for cell growth and stress resistance, can be utilized as an energy source. However, the regulation of its catabolism is unclear. Here the small RNA (sRNA) Spot 42, an essential regulator of carbon catabolite repression (CCR), was confirmed to participate in the post-transcriptional regulation of putrescine catabolism in Escherichia coli. Its encoding gene spf exclusively exists in the γ-proteobacteria and contains specific binding sites to the 5'-untranslated regions of the puuE gene, which encodes transaminase in the glutamylated putrescine pathway of putrescine catabolism converting γ-aminobutyrate (GABA) into succinate semialdehyde (SSA). The transcription of the spf gene was induced by glucose, inhibited by putrescine, and unaffected by PuuR, the repressor of puu genes. Excess Spot 42 repressed the expression of PuuE significantly in an antisense mechanism through the direct and specific base-pairing between the 51`-57 nt of Spot 42 and the 5'-UTR of puuE. Interestingly, Spot 42 mainly influenced the stability of the puuCBE transcript. This work revealed the regulatory role of Spot 42 in putrescine catabolism, in the switch between favorable and non-favorable carbon source utilization, and in the balance of metabolism of carbon and nitrogen sources.


Assuntos
Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Putrescina/metabolismo , RNA Bacteriano/metabolismo , Animais , Sítios de Ligação , Escherichia coli/genética , RNA Bacteriano/genética , Transcrição Genética
7.
Plant Cell Environ ; 44(5): 1565-1579, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33527435

RESUMO

Potassium (K) deficiency is a rather common situation that impacts negatively on biomass, photosynthesis and N assimilation, making K fertilization often unavoidable. Effects of K deficiency have been investigated for several decades and recently progress has been made in identifying metabolomics signatures thereby offering potential to monitor the K status of crops in the field. However, effects of low K conditions could also be due to the antagonism with other nutrients like calcium (Ca) and the well-known biomarker of K deficiency, putrescine, could be a response to Ca/K imbalance rather than K deficiency per se. To sort this out, we carried out experiments in sunflower grown at either low or high K, at high or low Ca, with or without putrescine added to the nutrient solution. Using metabolomics and proteomics analysis, we show that a significant part of the low K response, such as lower photosynthesis and N assimilation, is due to calcium and can be suppressed by low Ca conditions. Putrescine addition tends to restore photosynthesis and N assimilation but unlike low Ca does not suppress but aggravates the impact of low K conditions on catabolism, including the typical fall-over in pyruvate kinase. We conclude that (a) the effects of K deficiency on key metabolic processes can be partly alleviated by the use of low Ca and not only by K fertilization and (b) in addition to its role as a metabolite, putrescine participates in acclimation to low K via the regulation of the content in enzymes involved in carbon primary metabolism.


Assuntos
Cálcio/metabolismo , Helianthus/metabolismo , Potássio/metabolismo , Putrescina/metabolismo , Biomarcadores/metabolismo , Helianthus/fisiologia , Metabolômica , Nitrogênio/metabolismo , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Proteoma/metabolismo
8.
Plant Signal Behav ; 16(4): 1885187, 2021 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-33576705

RESUMO

Polyamines are known to accumulate in response to stress. Compelling evidence indicate a protective role for polyamines during defense. However, signaling pathways underlying polyamine functions have not been fully elucidated. We recently found that the polyamine putrescine (Put) accumulates during effector triggered immunity (ETI). Treatment with Put triggered local and systemic transcriptional reprogramming partly overlapping with systemic acquired resistance (SAR) responses. In addition, Put treatment led to local salicylic acid (SA) accumulation and systemic defenses against virulent bacteria. Consistent with this, we found that Put signaling is mainly ROS dependent and partly compromised by ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2) and NONEXPRESSOR of PR GENES1 (NPR1) loss-of-function mutations. Here, we propose a preliminary model by which putrescine contributes to local and systemic defenses in Arabidopsis thaliana.


Assuntos
Arabidopsis/imunologia , Putrescina/metabolismo , Arabidopsis/genética , Arabidopsis/microbiologia , Regulação da Expressão Gênica de Plantas , Modelos Biológicos , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal , Pseudomonas syringae , Ácido Salicílico/metabolismo
9.
Artigo em Inglês | MEDLINE | ID: mdl-33481671

RESUMO

Biogenic amines (BAs) are natural components of food produced mainly during metabolism in animals and plants. The determination of BAs is important because of their potential toxicity and their potential use as food spoilage indicators. In the present study, a method for the determination of six BAs (putrescine, cadaverine, histamine, ß-phenylethylamine, tyramine, and tryptamine) by Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS) with Atmospheric Pressure Chemical Ionisation (APCI) source has been used on trout samples (Salmo trutta) stored in ice for 15 days. The results showed that on day 15 quite large amounts of putrescine (76.530 mg/kg), cadaverine (85.530 mg/kg), tryptamine (25.210 mg/kg), and histamine (15.975mg/kg) were detected, while the other BAs remained low (ß-phenylethylamine: 3.230 mg/kg, tyramine: 0.165mg/kg). Furthermore, microbiological data (Total Vial Count- TVC, Pseudomonas spp, and Shewanella putrefaciens) showed that trout samples became organoleptically unacceptable on day 12, while volatile compound analysis showed a significant increase in total amounts of alcohols, aldehydes, and ketones on days 12 and 15.


Assuntos
Aminas Biogênicas/análise , Aminas Biogênicas/metabolismo , Truta/metabolismo , Compostos Orgânicos Voláteis/análise , Animais , Cadaverina/análise , Cadaverina/metabolismo , Contagem de Colônia Microbiana , Inocuidade dos Alimentos , Armazenamento de Alimentos , Histamina/análise , Histamina/metabolismo , Gelo , Fenetilaminas/análise , Fenetilaminas/metabolismo , Putrescina/análise , Putrescina/metabolismo , Alimentos Marinhos , Extração em Fase Sólida , Espectrometria de Massas em Tandem , Fatores de Tempo , Triptaminas/análise , Triptaminas/metabolismo , Tiramina/análise , Tiramina/metabolismo , Compostos Orgânicos Voláteis/metabolismo
10.
J Biol Chem ; 296: 100146, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33277357

RESUMO

The siderophore rhizoferrin (N1,N4-dicitrylputrescine) is produced in fungi and bacteria to scavenge iron. Putrescine-producing bacterium Ralstonia pickettii synthesizes rhizoferrin and encodes a single nonribosomal peptide synthetase-independent siderophore (NIS) synthetase. From biosynthetic logic, we hypothesized that this single enzyme is sufficient for rhizoferrin biosynthesis. We confirmed this by expression of R. pickettii NIS synthetase in Escherichia coli, resulting in rhizoferrin production. This was further confirmed in vitro using the recombinant NIS synthetase, synthesizing rhizoferrin from putrescine and citrate. Heterologous expression of homologous lbtA from Legionella pneumophila, required for rhizoferrin biosynthesis in that species, produced siderophore activity in E. coli. Rhizoferrin is also synthesized by Francisella tularensis and Francisella novicida, but unlike R. pickettii or L. pneumophila, Francisella species lack putrescine biosynthetic pathways because of genomic decay. Francisella encodes a NIS synthetase FslA/FigA and an ornithine decarboxylase homolog FslC/FigC, required for rhizoferrin biosynthesis. Ornithine decarboxylase produces putrescine from ornithine, but we show here in vitro that FigA synthesizes N-citrylornithine, and FigC is an N-citrylornithine decarboxylase that together synthesize rhizoferrin without using putrescine. We co-expressed F. novicida figA and figC in E. coli and produced rhizoferrin. A 2.1 Å X-ray crystal structure of the FigC N-citrylornithine decarboxylase reveals how the larger substrate is accommodated and how active site residues have changed to recognize N-citrylornithine. FigC belongs to a new subfamily of alanine racemase-fold PLP-dependent decarboxylases that are not involved in polyamine biosynthesis. These data reveal a natural product biosynthetic workaround that evolved to bypass a missing precursor and re-establish it in the final structure.


Assuntos
Proteínas de Bactérias/metabolismo , Compostos Férricos/metabolismo , Ferro/metabolismo , Peptídeo Sintases/metabolismo , Putrescina/metabolismo , Ralstonia pickettii/enzimologia , Sideróforos/metabolismo , Citratos/metabolismo , Francisella/enzimologia , Legionella pneumophila/enzimologia
11.
J Biol Chem ; 296: 100182, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33310703

RESUMO

Polyamines, such as putrescine, spermidine, and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system using CHO-MG cells, a widely used model to study alternative polyamine uptake routes and characterize polyamine transport inhibitors for therapy. CHO-MG cells present polyamine uptake deficiency and resistance to a toxic polyamine biosynthesis inhibitor methylglyoxal bis-(guanylhydrazone) (MGBG), but the molecular defects responsible for these cellular characteristics remain unknown. By genome sequencing of CHO-MG cells, we identified mutations in an unexplored gene, ATP13A3, and found disturbed mRNA and protein expression. ATP13A3 encodes for an orphan P5B-ATPase (ATP13A3), a P-type transport ATPase that represents a candidate polyamine transporter. Interestingly, ATP13A3 complemented the putrescine transport deficiency and MGBG resistance of CHO-MG cells, whereas its knockdown in WT cells induced a CHO-MG phenotype demonstrated as a decrease in putrescine uptake and MGBG sensitivity. Taken together, our findings identify ATP13A3, which has been previously genetically linked with pulmonary arterial hypertension, as a major component of the mammalian polyamine transport system that confers sensitivity to MGBG.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Poliaminas/metabolismo , Putrescina/metabolismo , Adenosina Trifosfatases/genética , Animais , Transporte Biológico , Células CHO , Cricetinae , Cricetulus , Inibidores Enzimáticos/farmacologia , Mitoguazona/farmacologia , Mutação , Sequenciamento Completo do Genoma/métodos
12.
J Food Sci ; 85(10): 3487-3497, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32893884

RESUMO

Seven candidates for starter cultures for cucumber fermentations belonging to the Lactobacillus pentosus and Lactobacillus plantarum species were characterized based on physiological features desired for pickling. The isolates presented variable carbohydrate utilization profile on API® 50CHL test strips. The L. pentosus strains were unable to utilize d-xylose in MRS broth or the M medium. The lactobacilli were unable to produce histamine, tyramine, putrescine, and cadaverine in biogenic amine broth containing the necessary precursors. Production of d-lactic acid by the lactobacilli, detected enzymatically, was stimulated by growth in MRS broth as compared to cucumber juice medium (CJM). The lactobacilli utilized malic acid in the malate decarboxylase medium. Exopolyssacharide biosynthesis related genes were amplified from the lactobacilli. A sugar type-dependent-ropy phenotype was apparent for all the cultures tested in MRS and CJM. The genes associated with bacteriocin production were detected in the lactobacilli, but not the respective phenotypes. The antibiotic susceptibility profile of the lactobacilli mimics that of other L. plantarum starter cultures. It is concluded that the lactobacilli strains studied here are suitable starter cultures for cucumber fermentation. PRACTICAL APPLICATION: The availability of such starter cultures enables the implementation of low salt cucumber fermentations that can generate products with consistent biochemistry and microbiological profile.


Assuntos
Cucumis sativus/microbiologia , Microbiologia de Alimentos/métodos , Lactobacillus pentosus/metabolismo , Lactobacillus plantarum/metabolismo , Cloreto de Sódio/análise , Bacteriocinas/análise , Bacteriocinas/metabolismo , Aminas Biogênicas/metabolismo , Cucumis sativus/química , Meios de Cultura/química , Meios de Cultura/metabolismo , Fermentação , Lactobacillus pentosus/crescimento & desenvolvimento , Lactobacillus plantarum/crescimento & desenvolvimento , Malatos/análise , Malatos/metabolismo , Putrescina/metabolismo , Cloreto de Sódio/metabolismo , Tiramina/análise , Tiramina/metabolismo
13.
Int J Mol Sci ; 21(16)2020 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-32824856

RESUMO

Carbonic anhydrase IX (CA9), a pH-regulating transmembrane protein, is highly expressed in solid tumors, and particularly in clear cell renal cell carcinoma (ccRCC). The catalytic mechanisms of CA9 are well defined, but its roles in mediating cell migration/invasion and survival in ccRCC remain to be determined. Here, we confirmed that the mRNA expression of CA9 in ccRCC was significantly higher than that in para-carcinoma tissues from analysis of the datasets in The Cancer Genome Atlas. CA9 knockdown upregulated oxidative phosphorylation-associated proteins and increased mitochondrial biogenesis, resulting in the reversal of the Warburg phenotype and the inhibition of cell growth. Our study revealed that CA9 knockdown upregulated mitochondrial arginase 2 (ARG2), leading to the accumulation of putrescine, which suppressed ccRCC proliferation. Surfaceomics analysis revealed that CA9 knockdown downregulated proteins associated with extracellular matrix (ECM)-receptor interaction and cell adhesion, resulting in decreased cell migration. CA9 silencing also downregulated amino acid transporters, leading to reduced cellular amino acids. Collectively, our data show that CA9 knockdown suppresses proliferation via metabolic reprogramming and reduced cell migration, reaffirming that CA9 is a potential therapeutic target for ccRCC treatment.


Assuntos
Antígenos de Neoplasias/metabolismo , Anidrase Carbônica IX/metabolismo , Carcinoma de Células Renais/metabolismo , Movimento Celular , Neoplasias Renais/metabolismo , Biogênese de Organelas , Putrescina/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Antígenos de Neoplasias/genética , Arginase/genética , Arginase/metabolismo , Anidrase Carbônica IX/genética , Linhagem Celular Tumoral , Proliferação de Células , Inativação Gênica , Células HEK293 , Humanos , Proteoma/genética , Proteoma/metabolismo , Putrescina/toxicidade , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Efeito Warburg em Oncologia
14.
Plant Physiol Biochem ; 154: 699-713, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32750647

RESUMO

Allantoin as a nitrogen metabolite can improve the salt tolerance in plants, but its mechanism of action remain elusive. Herein, the effects of pretreatment with exogenous allantoin in salt tolerance were investigated in sugar beet. The seedlings were subjected to salt stress (300 mM Na+) without or with different allantoin concentrations (0.01, 0.1, and 1 mM). The effects of allantoin on plant growth, homeostasis, oxidative damage, osmoregulation, and polyamine metabolism were studied. The results showed that salt stress inhibited the net photosynthetic rate and plant growth, and caused oxidative damage. However, these adverse effects were mitigated by exogenous allantoin in a dose-dependent manner, especially at 0.1 mM. Allantoin reduced the accumulation of ROS by increasing the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and AsA content. Under salt stress, allantoin reduced the root concentrations of free putrescine (Put) but increased the free spermine (Spm) in leaves and roots. Furthermore, allantoin decreased the Na+/K+ ratio and promoted the accumulation of betaine and soluble sugars in leaves and roots. Under salinity conditions, allantoin may enhance the antioxidant system and improve ion homeostasis by enhancing putrescine and/or spermine accumulation. In addition, Pearson's correlation and principal component analysis (PCA) established correlations between physiological parameters, and significant differences between different concentrations of allantoin were observed. In total, exogenous allantoin effectively reduced the oxidative damage and ion toxicity in sugar beet, caused by salinity, this finding would be helpful in improving salt tolerance in plant.


Assuntos
Alantoína/farmacologia , Antioxidantes/metabolismo , Beta vulgaris/fisiologia , Putrescina/metabolismo , Tolerância ao Sal , Beta vulgaris/efeitos dos fármacos , Plântula , Açúcares
15.
FASEB J ; 34(9): 10907-12921, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32767470

RESUMO

Metabolic reprograming is a hallmark of cancer, and the polyamine metabolic network is dysregulated in many cancers. Ornithine decarboxylase (ODC) is a rate-limiting enzyme for polyamine synthesis in the polyamine metabolic network. In many cancer cells, ODC is over-expressed, so this enzyme has been an attracting anti-cancer drug target. In the catalysis axis (pathway), ODC converts ornithine to putrescine. Meanwhile, ODC's activity is regulated by protein-protein interactions (PPIs), including the ODC-OAZ1-AZIN1 PPI axis and its monomer-dimer equilibrium. Previous studies showed that when ODC's activity is inhibited, the PPIs might counteract the inhibition efficiency. Therefore, we proposed that multipurpose inhibitors that can simultaneously inhibit ODC's activity and perturb the PPIs would be very valuable as drug candidates and molecular tools. To discover multipurpose ODC inhibitors, we established a computational pipeline by combining positive screening and negative screening. We used this pipeline for the forward screening of multipurpose ligands that might inhibit ODC's activity, block ODC-OAZ1 interaction and enhance ODC non-functional dimerization. With a combination of different experimental assays, we identified three multipurpose ODC inhibitors. At last, we showed that one of these inhibitors is a promising drug candidate. This work demonstrated that our computational pipeline is useful for discovering multipurpose ODC inhibitors, and multipurpose inhibitors would be very valuable. Similar with ODC, there are a lot of proteins in human proteome that act as both enzymes and PPI components. Therefore, this work is not only presenting new molecular tools for polyamine study, but also providing potential insights and protocols for discovering multipurpose inhibitors to target more important protein targets.


Assuntos
Inibidores da Ornitina Descarboxilase/farmacologia , Ornitina Descarboxilase/metabolismo , Ornitina/metabolismo , Putrescina/metabolismo , Células A549 , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Biocatálise/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Ensaios de Triagem em Larga Escala/métodos , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Ornitina Descarboxilase/química , Inibidores da Ornitina Descarboxilase/química , Inibidores da Ornitina Descarboxilase/metabolismo , Ligação Proteica/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
16.
Sci Rep ; 10(1): 12240, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699288

RESUMO

Tea plant often suffers from low temperature induced damage during its growth. How to improve the cold resistance of tea plant is an urgent problem to be solved. Nitric oxide (NO), γ-aminobutyric acid (GABA) and proline have been proved that can improve the cold resistance of tea plants, and signal transfer and biosynthesis link between them may enhance their function. NO is an important gas signal material in plant growth, but our understanding of the effects of NO on the GABA shunt, proline and NO biosynthesis are limited. In this study, the tea roots were treated with a NO donor (SNAP), NO scavenger (PTIO), and NO synthase inhibitor (L-NNA). SNAP could improve activities of arginine decarboxylase, ornithine decarboxylase, glutamate decarboxylase, GABA transaminase and Δ1-pyrroline-5-carboxylate synthetase and the expression level of related genes during the treatments. The contents of putrescine and spermidine under SNAP treatment were 45.3% and 37.3% higher compared to control at 24 h, and the spermine content under PTIO treatment were 57.6% lower compare to control at 12 h. Accumulation of proline of SNAP and L-NNA treatments was 52.2% and 43.2% higher than control at 48 h, indicating other pathway of NO biosynthesis in tea roots. In addition, the NO accelerated the consumption of GABA during cold storage. These facts indicate that NO enhanced the cold tolerance of tea, which might regulate the metabolism of the GABA shunt and of proline, associated with NO biosynthesis.


Assuntos
Camellia sinensis/metabolismo , Óxido Nítrico/metabolismo , Raízes de Plantas/metabolismo , Poliaminas/metabolismo , Prolina/metabolismo , Chá/metabolismo , Ácido gama-Aminobutírico/metabolismo , Carboxiliases/metabolismo , Temperatura Baixa , Resposta ao Choque Frio/fisiologia , Óxidos N-Cíclicos/metabolismo , Glutamato Descarboxilase/metabolismo , Imidazóis/metabolismo , Doadores de Óxido Nítrico/metabolismo , Ornitina Descarboxilase/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Putrescina/metabolismo , S-Nitroso-N-Acetilpenicilamina/metabolismo , Espermidina/metabolismo , Espermina/metabolismo
17.
Inflamm Res ; 69(9): 937-950, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32488317

RESUMO

OBJECTIVE: To measure diamine oxidase (DAO) activity with high sensitivity in complex matrices like plasma or tissue extracts radioactive putrescine or horseradish peroxidase (HRP)/hydrogen peroxide (H2O2) coupling must be used. The use of radioactive material should be avoided and HRP/H2O2 coupling is compromised by antioxidants. METHODS AND RESULTS: Condensation of ortho-aminobenzaldehyde (oABA) with delta-1-pyrroline and delta-1-piperideine, the autocyclization products of the DAO-oxidized natural substrates putrescine and cadaverine, generates new quinazoline fluorophores with absorption and excitation maxima of 430 and 460 nm, respectively, and peak emission at 620 nm. Fluorescent-based detection limits are 20-40 times lower compared to absorption measurements. This assay can be used to measure DAO activity in human plasma after spiking recombinant human (rh)DAO, in rat plasma after intravenous rhDAO administration, in pregnancy plasma and in tissue extracts of DAO wild-type and knock-out mice. Using rat plasma the correlation between rhDAO activity and ELISA data is 99%. Human and rat plasma without DAO spiking and tissue extracts from DAO knock-out mice showed stable and low fluorescence in the presence of high substrate concentrations. CONCLUSIONS: Incubation of DAO with the natural substrates putrescine and cadaverine and oABA generates novel fluorophores increasing the detection limit compared to absorption measurements at least tenfold. This simple, sensitive and specific assay allows the non-radioactive quantification of DAO activity in complex matrices like plasma and tissue extracts without interference by antioxidants.


Assuntos
Amina Oxidase (contendo Cobre)/metabolismo , Corantes Fluorescentes , Animais , Cadaverina/metabolismo , Humanos , Camundongos , Putrescina/metabolismo , Ratos
18.
Int J Mycobacteriol ; 9(2): 138-143, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32474534

RESUMO

Background: Polyamines are widespread intracellular molecules able to influence antibiotic susceptibility, but almost nothing is known on their occurrence and physiological role in mycobacteria. Methods: here, we analyzed transcriptomic, proteomic and biochemical data and obtained the first evidence for the post-transcriptional expression of some genes attributed to polyamine metabolism and polyamine transport in Mycolicibacterium smegmatis (basionym Mycobacterium smegmatis). Results: in our experiments, exponentially growing cells demonstrated transcription of 21 polyamine-associated genes and possessed 7 enzymes of polyamine metabolism and 2 polyamine transport proteins. Conclusion: Mycolicibacterium smegmatis putrescine synthesizing enzyme agmatinase SpeB was originally shown to catalyze agmatine conversion to putrescine in vitro. Nevertheless, we have not found any polyamines in mycobacterial cells.


Assuntos
Mycobacterium smegmatis/química , Mycobacterium smegmatis/enzimologia , Poliaminas/análise , Ureo-Hidrolases/metabolismo , Agmatina/metabolismo , Perfilação da Expressão Gênica , Mycobacterium smegmatis/genética , Proteômica , Putrescina/metabolismo , Ureo-Hidrolases/genética
19.
Sci Rep ; 10(1): 9703, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32546787

RESUMO

Many women suffer from urinary tract infections (UTIs). In addition to pain and increased urgency to urinate, malodour is a significant issue for these patients. The specific factors causing this malodour are unclear, and there are no targeted treatment options to counteract it effectively. We used a metabolomics approach to compare the chemical composition of metabolites in the urine of women with E. coli UTIs (n = 15) and those who are healthy (n = 10). The biogenic amines trimethylamine and putrescine, which cause malodour in other urogenital conditions, were significantly increased in UTI patients. Conversely, the precursor of trimethylamine, trimethylamine N-oxide, was lower. To further confirm the source of the malodorous compounds, in vitro experiments were conducted by incubating strains of uropathogenic E. coli in sterilized urine from healthy women. All tested strains accumulated trimethylamine and putrescine. Notably, cadaverine was also produced by E. coli strains in vitro; however, it was not significantly different between both groups. We confirmed that the malodorous amines TMA and putrescine are found in higher concentrations in the urine of patients with an E.coli-caused UTI.


Assuntos
Aminas Biogênicas/metabolismo , Infecções por Escherichia coli/microbiologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/metabolismo , Cadaverina/metabolismo , Infecções por Escherichia coli/metabolismo , Metabolômica , Metilaminas/metabolismo , Odorantes , Putrescina/metabolismo , Tiramina/metabolismo
20.
Food Chem ; 331: 127347, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32574945

RESUMO

Biogenic amines (BAs) are a class of bioactive organics produced during the fermentation of soy sauce. A high concentration of BAs may bring about serious physiological and toxicological effects on the human body. In this study, we reported an optimized process to produce soy sauce with lower BA concentration and found the contents of putrescine, cadaverine and histamine increased with the increase of fermentation temperature but decreased with the increase of NaCl concentration. The final content of total BAs with improved fermentation was 105.56 ± 0.13 mg/L, which was reduced by 89.11% compared to traditional brewing. Besides, the pilot production test was performed to verify the optimized conditions and physicochemical indexes were measured to better understand the change principle of the chemical compounds. Taken together, we present an effective process to inhibit the formation of BAs while ensuring that characteristic nutrients are not lost.


Assuntos
Aminas Biogênicas/biossíntese , Alimentos de Soja , Aminas Biogênicas/análise , Cadaverina/análise , Cadaverina/metabolismo , Fermentação , Indústria de Processamento de Alimentos/métodos , Histamina/análise , Histamina/metabolismo , Putrescina/análise , Putrescina/metabolismo , Cloreto de Sódio , Alimentos de Soja/análise , Temperatura
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