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1.
Cells ; 10(8)2021 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-34440719

RESUMO

Neuroblastoma (NB) is a common malignant solid tumor in children and accounts for 15% of childhood cancer mortality. Amplification of the N-Myc oncogene is a well-established poor prognostic marker in NB patients and strongly correlates with higher tumor aggression and resistance to treatment. New therapies for patients with N-Myc-amplified NB need to be developed. After treating NB cells with BSAO/SPM, the detection of apoptosis was determined after annexin V-FITC labeling and DNA staining with propidium iodide. The mitochondrial membrane potential activity was checked, labeling cells with the probe JC-1 dye. We analyzed, by real-time RT-PCR, the transcript of genes involved in the apoptotic process, to determine possible down- or upregulation of mRNAs after the treatment on SJNKP and the N-Myc-amplified IMR5 cell lines with BSAO/SPM. The experiments were carried out considering the proapoptotic genes Tp53 and caspase-3. After treatment with BSAO/SPM, both cell lines displayed increased mRNA levels for all these proapoptotic genes. Western blotting analysis with PARP and caspase-3 antibody support that BSAO/SPM treatment induces high levels of apoptosis in cells. The major conclusion is that BSAO/SPM treatment leads to antiproliferative and cytotoxic activity of both NB cell lines, associated with activation of apoptosis.


Assuntos
Amina Oxidase (contendo Cobre)/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , MicroRNAs/metabolismo , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/tratamento farmacológico , Espermina/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Caspase 3/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , MicroRNAs/genética , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/enzimologia , Neuroblastoma/genética , Ratos Wistar , Transdução de Sinais , Espermina/metabolismo , Proteína Supressora de Tumor p53/genética
2.
Biomolecules ; 11(8)2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34439853

RESUMO

Polyamines (PAs) are polycationic biomolecules containing multiple amino groups. Patients with HIV-associated neurocognitive disorder (HAND) have high concentrations of the polyamine N-acetylated spermine in their brain and cerebral spinal fluid (CSF) and have increased PA release from astrocytes. These effects are due to the exposure to HIV-Tat. In healthy adult brain, PAs are accumulated but not synthesized in astrocytes, suggesting that PAs must enter astrocytes to be N-acetylated and released. Therefore, we tested if Cx43 hemichannels (Cx43-HCs) are pathways for PA flux in control and HIV-Tat-treated astrocytes. We used biotinylated spermine (b-SPM) to examine polyamine uptake. We found that control astrocytes and those treated with siRNA-Cx43 took up b-SPM, similarly suggesting that PA uptake is via a transporter/channel other than Cx43-HCs. Surprisingly, astrocytes pretreated with both HIV-Tat and siRNA-Cx43 showed increased accumulation of b-SPM. Using a novel polyamine transport inhibitor (PTI), trimer 44NMe, we blocked b-SPM uptake, showing that PA uptake is via a PTI-sensitive transport mechanism such as organic cation transporter. Our data suggest that Cx43 HCs are not a major pathway for b-SPM uptake in the condition of normal extracellular calcium concentration but may be involved in the release of PAs to the extracellular space during viral infection.


Assuntos
Astrócitos/metabolismo , Transporte Biológico/efeitos dos fármacos , Conexina 43/metabolismo , Infecções por HIV/metabolismo , Espermina/metabolismo , Animais , Astrócitos/virologia , HIV-1 , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células
3.
Nat Commun ; 12(1): 3973, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172751

RESUMO

In human cells, P5B-ATPases execute the active export of physiologically important polyamines such as spermine from lysosomes to the cytosol, a function linked to a palette of disorders. Yet, the overall shape of P5B-ATPases and the mechanisms of polyamine recognition, uptake and transport remain elusive. Here we describe a series of cryo-electron microscopy structures of a yeast homolog of human ATP13A2-5, Ypk9, determined at resolutions reaching 3.4 Å, and depicting three separate transport cycle intermediates, including spermine-bound conformations. Surprisingly, in the absence of cargo, Ypk9 rests in a phosphorylated conformation auto-inhibited by the N-terminus. Spermine uptake is accomplished through an electronegative cleft lined by transmembrane segments 2, 4 and 6. Despite the dramatically different nature of the transported cargo, these findings pinpoint shared principles of transport and regulation among the evolutionary related P4-, P5A- and P5B-ATPases. The data also provide a framework for analysis of associated maladies, such as Parkinson's disease.


Assuntos
ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Espermina/metabolismo , Transporte Biológico , Chaetomium/química , Microscopia Crioeletrônica , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Fosforilação , Conformação Proteica , ATPases Translocadoras de Prótons/genética
4.
DNA Cell Biol ; 40(9): 1144-1157, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34165351

RESUMO

Polyamines (PAs), especially spermidine and spermine (which are involved in various types of abiotic stress tolerance), have been reported in many plant species. In this study, we identified 14 putative S-adenosylmethionine decarboxylase genes (GhSAMDC1-14) in upland cotton. Based on phylogenetic and expression analyses conducted under different abiotic stresses, we selected and transferred GhSAMDC3 into Arabidopsis thaliana. Compared to the wild type, transgenic plants displayed rapid growth and increases in average leaf area and leaf number of 52% and 36%, respectively. In transgenic plants, the germination vigor and rate were markedly enhanced under NaCl treatment, and the plant survival rate increased by 50% under 300 mM NaCl treatment. The spermidine content was significantly increased, possibly due to the synthesis of a series of PAs and oxidant and antioxidant genes, resulting in improved salinity tolerance in Arabidopsis. Various salinity resistance-related genes were upregulated in transgenic plants. Together, these results indicate that ectopic expression of GhSAMDC3 raised salinity tolerance by the accumulation of spermidine and activation of salinity tolerance-related genes in A. thaliana.


Assuntos
Adenosilmetionina Descarboxilase/genética , Proteínas de Arabidopsis/genética , Arabidopsis , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas , Tolerância ao Sal/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Expressão Ectópica do Gene , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Espermidina/metabolismo , Espermina/metabolismo
5.
Sci Rep ; 11(1): 10669, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021188

RESUMO

Rice which belongs to the grass family is vulnerable to water stress. As water resources get limited, the productivity of rice is affected especially in granaries located at drought prone areas. It would be even worse in granaries located in drought prone areas such as KADA that receives the lowest rainfall in Malaysia. Spermine (SPM), a polyamine compound that is found ubiquitiosly in plants is involved in adaptation of biotic and abiotic stresses. The effect of SPM on growth,grain filling and yield of rice at three main granaries namely, IADA BLS, MADA and KADA representing unlimited water, limited water and water stress conditions respectively, were tested during the main season. Additinally, the growth enhancer was also tested during off season at KADA. Spermine increased plant height, number of tillers per hill and chlorophyll content in all three granaries. Application of SPM improved yield by 38, 29 and 20% in MADA, KADA and IADA BLS, respectively. Harvest index showed 2.6, 6 and 16% increases at IADA BLS, KADA and MADA, respectively in SPM treated plants as compared to untreated. Except for KADA which showed a reduction in yield at 2.54 tha-1, SPM improved yield at MADA, 7.21 tha-1 and IADA BLS, 9.13 tha-1 as compared to the average yield at these respective granaries. In the second trial, SPM increased the yield to 7.0 and 6.4 tha-1 during main and off seasons, respectively, indicating that it was significantly higher than control and the average yield reported by KADA. The yield of SPM treatments improved by 25 and 33% with an increment of farmer's income at main and off seasons, respectively. Stomatal width was significantly higher than control at 11.89 µm. In conclusion, irrespective of the tested granaries and rice variety, spermine mediated plots displayed increment in grain yield.


Assuntos
Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Desenvolvimento Vegetal/fisiologia , Espermina/metabolismo , Adaptação Fisiológica , Desidratação , Secas , Fenótipo , Água/metabolismo
6.
Int J Mol Sci ; 22(9)2021 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-33922247

RESUMO

Spermine, a member of polyamines, exists in all organisms and is essential for normal cell growth and function. It is highly expressed in the prostate compared with other organs and is detectable in urine, tissue, expressed prostatic secretions, and erythrocyte. A significant reduction of spermine level was observed in prostate cancer (PCa) tissue compared with benign prostate tissue, and the level of urinary spermine was also significantly lower in men with PCa. Decreased spermine level may be used as an indicator of malignant phenotype transformation from normal to malignant tissue in prostate. Studies targeting polyamines and key rate-limiting enzymes associated with spermine metabolism as a tool for PCa therapy and chemoprevention have been conducted with various polyamine biosynthesis inhibitors and polyamine analogues. The mechanism between spermine and PCa development are possibly related to the regulation of polyamine metabolism, cancer-driving pathways, oxidative stress, anticancer immunosurveillance, and apoptosis regulation. Although the specific mechanism of spermine in PCa development is still unclear, ongoing research in spermine metabolism and its association with PCa pathophysiology opens up new opportunities in the diagnostic and therapeutic roles of spermine in PCa management.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/tratamento farmacológico , Espermina/metabolismo , Animais , Humanos , Masculino , Neoplasias da Próstata/metabolismo
7.
Int J Mol Sci ; 22(5)2021 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-33799982

RESUMO

ATP13A2, a late endo-/lysosomal polyamine transporter, is implicated in a variety of neurodegenerative diseases, including Parkinson's disease and Kufor-Rakeb syndrome, an early-onset atypical form of parkinsonism. Loss-of-function mutations in ATP13A2 result in lysosomal deficiency as a consequence of impaired lysosomal export of the polyamines spermine/spermidine. Furthermore, accumulating evidence suggests the involvement of ATP13A2 in regulating the fate of α-synuclein, such as cytoplasmic accumulation and external release. However, no consensus has yet been reached on the mechanisms underlying these effects. Here, we aimed to gain more insight into how ATP13A2 is linked to α-synuclein biology in cell models with modified ATP13A2 activity. We found that loss of ATP13A2 impairs lysosomal membrane integrity and induces α-synuclein multimerization at the membrane, which is enhanced in conditions of oxidative stress or exposure to spermine. In contrast, overexpression of ATP13A2 wildtype (WT) had a protective effect on α-synuclein multimerization, which corresponded with reduced αsyn membrane association and stimulation of the ubiquitin-proteasome system. We also found that ATP13A2 promoted the secretion of α-synuclein through nanovesicles. Interestingly, the catalytically inactive ATP13A2 D508N mutant also affected polyubiquitination and externalization of α-synuclein multimers, suggesting a regulatory function independent of the ATPase and transport activity. In conclusion, our study demonstrates the impact of ATP13A2 on α-synuclein multimerization via polyamine transport dependent and independent functions.


Assuntos
ATPases Translocadoras de Prótons/metabolismo , alfa-Sinucleína/metabolismo , Linhagem Celular Tumoral , Exocitose , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Mutação , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/metabolismo , Multimerização Proteica , ATPases Translocadoras de Prótons/genética , Espermina/metabolismo , Ubiquitina/metabolismo
8.
Int J Mol Sci ; 22(5)2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33652986

RESUMO

Polyamines are involved in various biological functions, including cell proliferation, differentiation, gene regulation, etc. Recently, it was found that polyamines exhibit biphasic effects on gene expression: promotion and inhibition at low and high concentrations, respectively. Here, we compared the effects of three naturally occurring tetravalent polyamines, spermine (SPM), thermospermine (TSPM), and N4-aminopropylspermidine (BSPD). Based on the single DNA observation with fluorescence microscopy together with measurements by atomic force microscopy revealed that these polyamines induce shrinkage and then compaction of DNA molecules, at low and high concentrations, respectively. We also performed the observation to evaluate the effects of these polyamine isomers on the activity of gene expression by adapting a cell-free luciferase assay. Interestingly, the potency of their effects on the DNA conformation and also on the inhibition of gene expression activity indicates the highest for TSPM among spermine isomers. A numerical evaluation of the strength of the interaction of these polyamines with negatively charged double-strand DNA revealed that this ordering of the potency corresponds to the order of the strength of the attractive interaction between phosphate groups of DNA and positively charged amino groups of the polyamines.


Assuntos
Bacteriófago T4/genética , Regulação Viral da Expressão Gênica , Espermina/análogos & derivados , Espermina/metabolismo , Bacteriófago T4/química , Bacteriófago T4/metabolismo , DNA Viral/química , DNA Viral/genética , DNA Viral/metabolismo , Isomerismo , Modelos Moleculares , Conformação de Ácido Nucleico , Espermina/química
9.
Circ Heart Fail ; 14(1): e007275, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33464957

RESUMO

BACKGROUND: Heart failure (HF) is a heterogeneous disease characterized by significant metabolic disturbances; however, the breadth of metabolic dysfunction before the onset of overt disease is not well understood. The purpose of this study was to determine the association of circulating metabolites with incident HF to uncover novel metabolic pathways to disease. METHODS: We performed targeted plasma metabolomic profiling in a deeply phenotyped group of Black adults from the JHS (Jackson Heart Study; n=2199). We related metabolites associated with incident HF to established etiological mechanisms, including increased left ventricular mass index and incident coronary heart disease. Furthermore, we evaluated differential associations of metabolites with HF with preserved ejection fraction versus HF with reduced ejection fraction. RESULTS: Metabolites associated with incident HF included products of posttranscriptional modifications of RNA, as well as polyamine and nitric oxide metabolism. A subset of metabolite-HF associations was independent of well-established HF pathways such as increased left ventricular mass index and incident coronary heart disease and included homoarginine (per 1 SD increase in metabolite level, hazard ratio, 0.77; P=1.2×10-3), diacetylspermine (hazard ratio, 1.34; P=3.4×10-3), and uridine (hazard ratio, 0.79; P, 3×10-4). Furthermore, metabolites involved in pyrimidine metabolism (orotic acid) and collagen turnover (N-methylproline) among others were part of a distinct metabolic signature that differentiated individuals with HF with preserved ejection fraction versus HF with reduced ejection fraction. CONCLUSIONS: The integration of clinical phenotyping with plasma metabolomic profiling uncovered novel metabolic processes in nontraditional disease pathways underlying the heterogeneity of HF development in Black adults.


Assuntos
Afro-Americanos , Doença das Coronárias/metabolismo , Insuficiência Cardíaca/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Metabolômica , Adulto , Idoso , Estudos de Casos e Controles , Colágeno/metabolismo , Doença das Coronárias/epidemiologia , Modificador do Efeito Epidemiológico , Grupo com Ancestrais do Continente Europeu , Feminino , Fatores de Risco de Doenças Cardíacas , Insuficiência Cardíaca/epidemiologia , Insuficiência Cardíaca/fisiopatologia , Homoarginina/metabolismo , Humanos , Hipertrofia Ventricular Esquerda/epidemiologia , Incidência , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Ácido Orótico/metabolismo , Poliaminas/metabolismo , Prolina/análogos & derivados , Prolina/metabolismo , Modelos de Riscos Proporcionais , Pirimidinas/metabolismo , Processamento Pós-Transcricional do RNA , Risco , Espermina/análogos & derivados , Espermina/metabolismo , Volume Sistólico/fisiologia , Uridina/metabolismo
10.
Invest Ophthalmol Vis Sci ; 62(1): 15, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33439228

RESUMO

Purpose: To identify tissue metabolomic profiles in biopsy specimens with IgG4-related ophthalmic disease (IgG4-ROD) and mucosa-associated lymphoid tissue (MALT) lymphoma and investigate their potential implication in the disease pathogenesis and biomarkers. Methods: We conducted a comprehensive analysis of the metabolomes and lipidomes of biopsy-proven IgG4-ROD (n = 22) and orbital MALT lymphoma (n = 21) specimens and matched adjacent microscopically normal adipose tissues using liquid chromatography time-of-flight mass spectrometry. The altered metabolomic profiles were visualized by heat map and principal component analysis. Metabolic pathway analysis was performed by Metabo Analyst 4.0 using differentially expressed metabolites. The diagnostic performance of the metabolic markers was evaluated using receiver operating characteristic curves. Machine learning algorithms were implemented by random forest using the R environment. Finally, an independent set of 18 IgG4-ROD and 17 orbital MALT lymphoma specimens were used to validate the identified biomarkers. Results: The principal component analysis showed a significant difference of both IgG4-ROD and orbital MALT lymphoma for biopsy specimens and controls. Interestingly, lesions in IgG4-ROD were uniquely enriched in arachidonic metabolism, whereas those in orbital MALT lymphoma were enriched in tricarboxylic acid cycle metabolism. We identified spermine as the best discriminator between IgG4-ROD and orbital MALT lymphoma, and the area under the receiver operating characteristic curve of the spermine to discriminate between the two diseases was 0.89 (95% confidence interval, 0.803-0.984). A random forest model incorporating a panel of five metabolites showed a high area under the receiver operating characteristic curve value of 0.983 (95% confidence interval, 0.981-0.984). The results of validation revealed that four tissue metabolites: N1,N12-diacetylspermine, spermine, malate, and glycolate, had statistically significant differences between IgG4-ROD and orbital MALT lymphoma with receiver operating characteristic values from 0.708 to 0.863. Conclusions: These data revealed the characteristic differences in metabolomic profiles between IgG4-ROD and orbital MALT lymphoma, which may be useful for developing new diagnostic biomarkers and elucidating the pathogenic mechanisms of these common orbital lymphoproliferative disorders.


Assuntos
Biomarcadores Tumorais/metabolismo , Imunoglobulina G/sangue , Linfoma de Zona Marginal Tipo Células B/metabolismo , Metaboloma/fisiologia , Doenças Orbitárias/metabolismo , Neoplasias Orbitárias/metabolismo , Paraproteinemias/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Cromatografia Líquida de Alta Pressão , Feminino , Glicolatos/metabolismo , Humanos , Linfoma de Zona Marginal Tipo Células B/diagnóstico , Malatos/metabolismo , Masculino , Espectrometria de Massas , Metabolômica , Pessoa de Meia-Idade , Doenças Orbitárias/diagnóstico , Neoplasias Orbitárias/diagnóstico , Paraproteinemias/diagnóstico , Análise de Componente Principal , Curva ROC , Estudos Retrospectivos , Espermina/análogos & derivados , Espermina/metabolismo
11.
J Mol Neurosci ; 71(3): 625-637, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32816235

RESUMO

The choroid plexus (CP) constitutes a barrier between the blood and the cerebrospinal fluid (CSF) which regulates the exchange of substances between these two fluids through mechanisms that are not completely understood. Polyamines as spermine, spermidine and putrescine are produced by all cells and are present in the CSF. Interestingly, their levels are altered in some neuronal disorders as Alzheimer's and Parkinson's diseases, thus increasing the interest in their signalling in the central nervous system (CNS). Cadaverine, on the other hand, is synthetized by the intestinal microbiome, suggesting that the presence of this bacterial metabolite in the CSF requires that it is up taken to the CNS across brain barriers. We knew that polyamines are detected by the olfactory signalling cascade operating at the CP, but the receptor involved had not been identified. The zebrafish TAAR13c was the only receptor known to bind a polyamine-cadaverine. Thus, we searched for a human receptor with homology to TAAR13c and found that some human TAARs including TAAR1 showed great homology. Then, we confirmed the expression of TAAR1 mRNA and protein in a human cell line of the CP, and in human CP samples. Calcium imaging assays after TAAR1 knockdown in these cells with a specific siRNA against TAAR1 showed a consistent reduction in the responses of these cells to cadaverine and spermidine, but not to spermine, suggesting that TAAR1 is activated by cadaverine and spermidine, but not spermine.


Assuntos
Cadaverina/metabolismo , Cálcio/metabolismo , Plexo Corióideo/citologia , Receptores Acoplados a Proteínas G/metabolismo , Espermina/metabolismo , Células Cultivadas , Humanos , Receptores Acoplados a Proteínas G/genética
12.
Proc Natl Acad Sci U S A ; 117(49): 31198-31207, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33229544

RESUMO

Recessive loss-of-function mutations in ATP13A2 (PARK9) are associated with a spectrum of neurodegenerative disorders, including Parkinson's disease (PD). We recently revealed that the late endo-lysosomal transporter ATP13A2 pumps polyamines like spermine into the cytosol, whereas ATP13A2 dysfunction causes lysosomal polyamine accumulation and rupture. Here, we investigate how ATP13A2 provides protection against mitochondrial toxins such as rotenone, an environmental PD risk factor. Rotenone promoted mitochondrial-generated superoxide (MitoROS), which was exacerbated by ATP13A2 deficiency in SH-SY5Y cells and patient-derived fibroblasts, disturbing mitochondrial functionality and inducing toxicity and cell death. Moreover, ATP13A2 knockdown induced an ATF4-CHOP-dependent stress response following rotenone exposure. MitoROS and ATF4-CHOP were blocked by MitoTEMPO, a mitochondrial antioxidant, suggesting that the impact of ATP13A2 on MitoROS may relate to the antioxidant properties of spermine. Pharmacological inhibition of intracellular polyamine synthesis with α-difluoromethylornithine (DFMO) also increased MitoROS and ATF4 when ATP13A2 was deficient. The polyamine transport activity of ATP13A2 was required for lowering rotenone/DFMO-induced MitoROS, whereas exogenous spermine quenched rotenone-induced MitoROS via ATP13A2. Interestingly, fluorescently labeled spermine uptake in the mitochondria dropped as a consequence of ATP13A2 transport deficiency. Our cellular observations were recapitulated in vivo, in a Caenorhabditis elegans strain deficient in the ATP13A2 ortholog catp-6 These animals exhibited a basal elevated MitoROS level, mitochondrial dysfunction, and enhanced stress response regulated by atfs-1, the C. elegans ortholog of ATF4, causing hypersensitivity to rotenone, which was reversible with MitoTEMPO. Together, our study reveals a conserved cell protective pathway that counters mitochondrial oxidative stress via ATP13A2-mediated lysosomal spermine export.


Assuntos
Fator 4 Ativador da Transcrição/genética , Adenosina Trifosfatases/genética , Proteínas de Caenorhabditis elegans/genética , Mitocôndrias/genética , ATPases Translocadoras de Prótons/genética , Fatores de Transcrição/genética , Animais , Caenorhabditis elegans , Eflornitina/farmacologia , Fibroblastos/efeitos dos fármacos , Lisossomos/genética , Lisossomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Mutação/genética , Estresse Oxidativo/efeitos dos fármacos , Doença de Parkinson/genética , Doença de Parkinson/patologia , Poliaminas/metabolismo , Rotenona/farmacologia , Espermina/metabolismo , Fator de Transcrição CHOP/genética
13.
Vet Microbiol ; 250: 108839, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33002680

RESUMO

Like obligate intracellular parasites, viruses co-opt host cell resources to establish productive infections. Polyamines are key aliphatic molecules that perform important roles in cellular growth and proliferation. They are also needed for the successful multiplication of various viruses. Little is known about the effects of polyamines on Arteriviridae infections. Here, porcine reproductive and respiratory syndrome virus (PRRSV), an economically prominent porcine virus, was used to investigate virus-polyamine interactions. We found that PRRSV infection significantly downregulated the levels of cellular polyamines. Using an inhibitor or specific short interfering RNAs (siRNAs) of ornithine decarboxylase 1, a key anabolic enzyme involved in the classical de novo biosynthesis of polyamines, we found that polyamine depletion abrogated PRRSV proliferation, and this effect was recoverable by adding exogenous spermidine and spermine, but not putrescine to the cells, suggesting that the host inhibits polyamine biosynthesis to restrict PRRSV proliferation. Further analysis revealed that the expression level of spermidine-spermine acetyltransferase 1 (SAT1), a catabolic enzyme that reduces spermidine and spermine levels, was upregulated during PRRSV infection, but conversely, SAT1 had an inhibitory effect on PRRSV reproduction. Our data show that polyamines are important molecules during PRRSV-host interactions, and polyamines and their biosynthetic pathways are potential therapeutic targets against PRRSV infection.


Assuntos
Acetiltransferases/genética , Interações entre Hospedeiro e Microrganismos , Poliaminas/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Espermidina/metabolismo , Espermina/metabolismo , Acetiltransferases/metabolismo , Animais , Linhagem Celular , Regulação para Baixo , Regulação da Expressão Gênica , Rim/citologia , Suínos , Regulação para Cima , Replicação Viral
14.
Nat Microbiol ; 5(12): 1472-1480, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32929190

RESUMO

Cellular adhesion mediates many important plant-microbe interactions. In the devastating blast fungus Magnaporthe oryzae1, powerful glycoprotein-rich mucilage adhesives2 cement melanized and pressurized dome-shaped infection cells-appressoria-to host rice leaf surfaces. Enormous internal turgor pressure is directed onto a penetration peg emerging from the unmelanized, thin-walled pore at the appressorial base1-4, forcing it through the leaf cuticle where it elongates invasive hyphae in underlying epidermal cells5. Mucilage sealing around the appressorial pore facilitates turgor build-up2, but the molecular underpinnings of mucilage secretion and appressorial adhesion are unknown. Here, we discovered an unanticipated and sole role for spermine in facilitating mucilage production by mitigating endoplasmic reticulum (ER) stress in the developing appressorium. Mutant strains lacking the spermine synthase-encoding gene SPS1 progressed through all stages of appressorial development, including penetration peg formation, but cuticle penetration was unsuccessful due to reduced appressorial adhesion, which led to solute leakage. Mechanistically, spermine neutralized off-target oxygen free radicals produced by NADPH oxidase-1 (Nox1)3,6 that otherwise elicited ER stress and the unfolded protein response, thereby critically reducing mucilage secretion. Our study reveals that spermine metabolism via redox buffering of the ER underpins appressorial adhesion and rice cell invasion and provides insights into a process that is fundamental to host plant infection.


Assuntos
Ascomicetos/metabolismo , Oryza/microbiologia , Doenças das Plantas/virologia , Espermina/metabolismo , Ascomicetos/genética , Ascomicetos/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hifas/genética , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Espermina Sintase/genética , Espermina Sintase/metabolismo
15.
Sci Rep ; 10(1): 14418, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32879344

RESUMO

Polyamines play essential roles in plant development and various stress responses. In this study, one of the cotton S-adenosylmethionine decarboxylase (SAMDC) genes, GhSAMDC1, was constructed in the pGWB17 vector and overexpressed in tobacco. Leaf area and plant height increased 25.9-36.6% and 15.0-27.0%, respectively, compared to the wild type, and flowering time was advanced by 5 days in transgenic tobacco lines. Polyamine and gene expression analyses demonstrated that a decrease in spermidine and an increase in total polyamines and spermine might be regulated by NtSPDS4 and NtSPMS in transgenic plants. Furthermore, exogenous spermidine, spermine and spermidine synthesis inhibitor dicyclohexylamine were used for complementary tests, which resulted in small leaves and dwarf plants, big leaves and early flowering, and big leaves and dwarf plants, respectively. These results indicate that spermidine and spermine are mainly involved in the vegetative growth and early flowering stages, respectively. Expression analysis of flowering-related genes suggested that NtSOC1, NtAP1, NtNFL1 and NtFT4 were upregulated in transgenic plants. In conclusion, ectopic GhSAMDC1 is involved in the conversion of spermidine to spermine, resulting in rapid vegetative growth and early flowering in tobacco, which could be applied to genetically improve plants.


Assuntos
Adenosilmetionina Descarboxilase/genética , Flores/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Tabaco/genética , Adenosilmetionina Descarboxilase/metabolismo , Flores/crescimento & desenvolvimento , Gossypium/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Espermina/metabolismo , Tabaco/crescimento & desenvolvimento , Tabaco/metabolismo
16.
Amino Acids ; 52(8): 1169-1180, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32816168

RESUMO

Polyamines (PAs), such as spermidine (SPD) and spermine (SPM), are essential to promote cell growth, survival, proliferation, and longevity. In the adult central nervous system (CNS), SPD and SPM are accumulated predominantly in healthy adult glial cells where PA synthesis is not present. To date, the accumulation and biosynthesis of PAs in developing astrocytes are not well understood. The purpose of the present study was to determine the contribution of uptake and/or synthesis of PAs using proliferation of neonatal astrocytes as an endpoint. We inhibited synthesis of PAs using α-difluoromethylornithine (DFMO; an inhibitor of the PA biosynthetic enzyme ornithine decarboxylase (ODC)) and inhibited uptake of PAs using trimer44NMe (PTI; a novel polyamine transport inhibitor). DFMO, but not PTI alone, blocked proliferation, suggesting that PA biosynthesis was present. Furthermore, exogenous administration of SPD rescued cell proliferation when PA synthesis was blocked by DFMO. When both synthesis and uptake of PAs were inhibited (DFMO + PTI), exogenous SPD no longer supported proliferation. These data indicate that neonatal astrocytes synthesize sufficient quantities of PAs de novo to support cell proliferation, but are also able to import exogenous PAs. This suggests that the PA uptake mechanism is present in both neonates as well as in adults and can support cell proliferation in neonatal astrocytes when ODC is blocked.


Assuntos
Astrócitos/metabolismo , Poliaminas/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Eflornitina , Poliaminas/antagonistas & inibidores , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Espermidina/metabolismo , Espermina/metabolismo
17.
Anal Biochem ; 607: 113831, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32739346

RESUMO

We developed a new procedure for the comprehensive analysis of metabolites and enzymes involved in polyamine metabolism pathways. The procedure utilizes stable isotope-labeled polyamines and directly and precisely determines labeled products from enzymatic reactions by ESI-Q-TOF-MS. The activity of different enzymes could be determined in essentially the same manner by suitably adjusting the reaction conditions for each individual enzyme. We applied the procedure to extracts of regenerating rat liver and analyzed the changes in polyamine-metabolizing enzymes and polyamine contents during recovery from partial hepatectomy. A general outline of polyamine metabolism and information of polyamine dynamics were obtained. This kind of comprehensive information would be valuable in unifying detailed but fragmentary information obtained through conventional analyses focusing on one or a few enzymes and on a limited aspect of polyamine metabolic pathway.


Assuntos
Enzimas/metabolismo , Poliaminas/análise , Poliaminas/metabolismo , Animais , Isótopos de Carbono/química , Ativação Enzimática , Marcação por Isótopo , Fígado/metabolismo , Masculino , Metionina/química , Ratos , Espectrometria de Massas por Ionização por Electrospray , Espermidina/química , Espermidina/metabolismo , Espermina/química , Espermina/metabolismo
18.
Plant J ; 104(2): 416-432, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32666545

RESUMO

Polyamines, such as putrescine, spermidine and spermine (Spm), are low-molecular-weight polycationic molecules present in all living organisms. Despite their implication in plant cellular processes, little is known about their molecular mode of action. Here, we demonstrate that polyamines trigger a rapid increase in the regulatory membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2 ), and that this increase is required for polyamine effects on K+ efflux in Arabidopsis roots. Using in vivo 32 Pi -labelling of Arabidopsis seedlings, low physiological (µm) concentrations of Spm were found to promote a rapid PIP2 increase in roots that was time- and dose-dependent. Confocal imaging of a genetically encoded PIP2 biosensor revealed that this increase was triggered at the plasma membrane. Differential 32 Pi -labelling suggested that the increase in PIP2 was generated through activation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity rather than inhibition of a phospholipase C or PIP2 5-phosphatase activity. Systematic analysis of transfer DNA insertion mutants identified PIP5K7 and PIP5K9 as the main candidates involved in the Spm-induced PIP2 response. Using non-invasive microelectrode ion flux estimation, we discovered that the Spm-triggered K+ efflux response was strongly reduced in pip5k7 pip5k9 seedlings. Together, our results provide biochemical and genetic evidence for a physiological role of PIP2 in polyamine-mediated signalling controlling K+ flux in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Raízes de Plantas/metabolismo , Potássio/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Raízes de Plantas/efeitos dos fármacos , Plantas Geneticamente Modificadas , Poliaminas/metabolismo , Poliaminas/farmacologia , Espermina/metabolismo
19.
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
20.
Anal Chem ; 92(16): 11242-11249, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32672445

RESUMO

There is growing interest in the characterization of protein complexes and their interactions with ligands using native ion mobility mass spectrometry. A particular challenge, especially for membrane proteins, is preserving noncovalent interactions and maintaining native-like structures. Different approaches have been developed to minimize activation of protein complexes by manipulating charge on protein complexes in solution and the gas-phase. Here, we report the utility of polyamines that have exceptionally high charge-reducing potencies with some molecules requiring 5-fold less than trimethylamine oxide to elicit the same effect. The charge-reducing molecules do not adduct to membrane protein complexes and are also compatible with ion-mobility mass spectrometry, paving the way for improved methods of charge reduction.


Assuntos
Proteínas de Transporte de Cátions/análise , Proteínas de Escherichia coli/análise , Histamina/química , Metilaminas/química , Espermidina/química , Espermina/química , Proteínas de Transporte de Cátions/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Histamina/metabolismo , Ligantes , Espectrometria de Massas/métodos , Metilaminas/metabolismo , Ligação Proteica , Espermidina/metabolismo , Espermina/metabolismo , Eletricidade Estática
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