RESUMEN
The biogenic polyamines, spermidine (Spd) and spermine (Spm), are present at millimolar concentrations in all eukaryotic cells, where they participate in the regulation of vitally important cellular functions. Polyamine analogs and derivatives are a traditional and important instrument for the investigation of the cellular functions of polyamines, enzymes of their metabolism, and the regulation of the biosynthesis of antizyme-a key downregulator of polyamine homeostasis. Here, we describe convenient gram-scale syntheses of a set of C-methylated analogs of Spd. The biochemical properties of these compounds and the possibility for the regulation of their activity by moving a methyl group along the polyamine backbone and by changing the stereochemistry of the chiral center(s) are discussed.
Asunto(s)
Poliaminas Biogénicas , Espermidina , Poliaminas/metabolismo , Espermina/metabolismo , HomeostasisRESUMEN
Orthosteric binding sites of olfactory receptors have been well understood for ligand-receptor interactions. However, a lack of explanation for subtle differences in ligand profile of olfactory receptors even with similar orthosteric binding sites promotes more exploration into the entry tunnels of the receptors. An important question regarding entry tunnels is the number of entry tunnels, which was previously believed to be one. Here, we used TAAR9 that recognizes important biogenic amines such as cadaverine, spermine, and spermidine as a model for entry tunnel study. We identified two entry tunnels in TAAR9 and described the residues that form the tunnels. In addition, we found two vestibular binding pockets, each located in one tunnel. We further confirmed the function of two tunnels through site-directed mutagenesis. Our study challenged the existing views regarding the number of entry tunnels in the subfamily of olfactory receptors and demonstrated the possible mechanism how the entry tunnels function in odorant recognition.
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Neuronas Receptoras Olfatorias , Receptores Acoplados a Proteínas G/química , Receptores Odorantes/química , Animales , Sitios de Unión , Poliaminas Biogénicas/química , Ratones , Mutagénesis Sitio-Dirigida , Receptores Acoplados a Proteínas G/genética , Receptores Odorantes/metabolismoRESUMEN
Biogenic amines activate G-protein-coupled receptors (GPCRs) in the central nervous system in vertebrate animals. Several biogenic amines, when excreted, stimulate trace amine-associated receptors (TAARs), a group of GPCRs in the main olfactory epithelium, and elicit innate behaviors. How TAARs recognize amines with varying numbers of amino groups is largely unknown. We reasoned that a comparison between lamprey and mammalian olfactory TAARs, which are thought to have evolved independently but show convergent responses to polyamines, may reveal structural determinants of amine recognition. Here, we demonstrate that sea lamprey TAAR365 (sTAAR365) responds strongly to biogenic polyamines cadaverine, putrescine, and spermine, and shares a similar response profile as a mammalian TAAR (mTAAR9). Docking and site-directed mutagenesis analyses show that both sTAAR365 and mTAAR9 recognize the two amino groups of cadaverine with the conserved Asp3.32 and Tyr6.51 residues. sTAAR365, which has remarkable sensitivity for cadaverine (EC50 = 4 nM), uses an extra residue, Thr7.42, to stabilize ligand binding. These cadaverine recognition sites also interact with amines with four and three amino groups (spermine and spermidine, respectively). Glu7.36 of sTAAR365 cooperates with Asp3.32 and Thr7.42 to recognize spermine, whereas mTAAR9 recognizes spermidine through an additional aromatic residue, Tyr7.43. These results suggest a conserved mechanism whereby independently evolved TAAR receptors recognize amines with two, three, or four amino groups using the same recognition sites, at which sTAAR365 and mTAAR9 evolved distinct motifs. These motifs interact directly with the amino groups of the polyamines, a class of potent and ecologically important odorants, mediating olfactory signaling.
Asunto(s)
Poliaminas Biogénicas/química , Proteínas de Peces/química , Simulación del Acoplamiento Molecular , Receptores Odorantes/química , Secuencias de Aminoácidos , Animales , Sitios de Unión , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Células HEK293 , Humanos , Lampreas , Ratones , Mutagénesis Sitio-Dirigida , Receptores Odorantes/genética , Receptores Odorantes/metabolismoRESUMEN
Polyamines are fundamental molecules of life, and their deep evolutionary history is reflected in extensive biosynthetic diversification. The polyamines putrescine, agmatine, and cadaverine are produced by pyridoxal 5'-phosphate-dependent L-ornithine, L-arginine, and L-lysine decarboxylases (ODC, ADC, LDC), respectively, from both the alanine racemase (AR) and aspartate aminotransferase (AAT) folds. Two homologous forms of AAT-fold decarboxylase are present in bacteria: an ancestral form and a derived, acid-inducible extended form containing an N-terminal fusion to the receiver-like domain of a bacterial response regulator. Only ADC was known from the ancestral form and limited to the Firmicutes phylum, whereas extended forms of ADC, ODC, and LDC are present in Proteobacteria and Firmicutes. Here, we report the discovery of ancestral form ODC, LDC, and bifunctional O/LDC and extend the phylogenetic diversity of functionally characterized ancestral ADC, ODC, and LDC to include phyla Fusobacteria, Caldiserica, Nitrospirae, and Euryarchaeota. Using purified recombinant enzymes, we show that these ancestral forms have a nascent ability to decarboxylate kinetically less preferred amino acid substrates with low efficiency, and that product inhibition primarily affects preferred substrates. We also note a correlation between the presence of ancestral ODC and ornithine/arginine auxotrophy and link this with a known symbiotic dependence on exogenous ornithine produced by species using the arginine deiminase system. Finally, we show that ADC, ODC, and LDC activities emerged independently, in parallel, in the homologous AAT-fold ancestral and extended forms. The emergence of the same ODC, ADC, and LDC activities in the nonhomologous AR-fold suggests that polyamine biosynthesis may be inevitable.
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Proteínas Arqueales , Bacterias , Proteínas Bacterianas , Poliaminas Biogénicas , Carboxiliasas , Euryarchaeota , Evolución Molecular , Ornitina Descarboxilasa , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Bacterias/enzimología , Bacterias/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Poliaminas Biogénicas/biosíntesis , Poliaminas Biogénicas/química , Carboxiliasas/química , Carboxiliasas/genética , Carboxiliasas/metabolismo , Euryarchaeota/enzimología , Euryarchaeota/genética , Ornitina Descarboxilasa/química , Ornitina Descarboxilasa/genética , Ornitina Descarboxilasa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Polyamine detection and depletion have been extensively investigated for cancer prevention and treatment. However, the therapeutic efficacy is far from satisfactory, mainly due to a polyamine compensation mechanism from the systemic circulation in the tumor environment. Herein, we explore a new solution for improving polyamine detection as well as a possible consumption therapy based on a new photosensitizer that can efficiently consume polyamines via an irreversible chemical reaction. The new photosensitizer is pyrrolopyrroleaza-BODIPY pyridinium salt (PPAB-PyS) nanoparticles that can react with the over-expressed polyamine in cancer cells and produce two photosensitizers with enhanced phototoxicity on cancer destruction. Meanwhile, PPAB-PyS nanoparticles provide a simultaneous ratiometric fluorescence imaging of intracellular polyamine. This combination polyamine consumption with a chemical reaction provides a new modality to enable polyamine detection along with photodynamic therapy as well as a putative depletion of polyamines for cancer treatment and prevention.
Asunto(s)
Poliaminas Biogénicas/análisis , Nanopartículas/química , Neoplasias/tratamiento farmacológico , Fotoquimioterapia/métodos , Poliaminas Biogénicas/química , Línea Celular Tumoral , Humanos , Neoplasias/química , Neoplasias/prevención & control , Imagen ÓpticaRESUMEN
Tauopathies display a spectrum of phenotypes from cognitive to affective behavioral impairments; however, mechanisms promoting tau pathology and how tau elicits behavioral impairment remain unclear. We report a unique interaction between polyamine metabolism, behavioral impairment, and tau fate. Polyamines are ubiquitous aliphatic molecules that support neuronal function, axonal integrity, and cognitive processing. Transient increases in polyamine metabolism hallmark the cell's response to various insults, known as the polyamine stress response (PSR). Dysregulation of gene transcripts associated with polyamine metabolism in Alzheimer's disease (AD) brains were observed, and we found that ornithine decarboxylase antizyme inhibitor 2 (AZIN2) increased to the greatest extent. We showed that sustained AZIN2 overexpression elicited a maladaptive PSR in mice with underlying tauopathy (MAPT P301S; PS19). AZIN2 also increased acetylpolyamines, augmented tau deposition, and promoted cognitive and affective behavioral impairments. Higher-order polyamines displaced microtubule-associated tau to facilitate polymerization but also decreased tau seeding and oligomerization. Conversely, acetylpolyamines promoted tau seeding and oligomers. These data suggest that tauopathies launch an altered enzymatic signature that endorses a feed-forward cycle of disease progression. Taken together, the tau-induced PSR affects behavior and disease continuance, but may also position the polyamine pathway as a potential entry point for plausible targets and treatments of tauopathy, including AD.
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Enfermedad de Alzheimer/metabolismo , Poliaminas Biogénicas/metabolismo , Carboxiliasas/metabolismo , Proteínas Portadoras/metabolismo , Hipocampo/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Carboxiliasas/genética , Proteínas Portadoras/genética , Femenino , Hipocampo/patología , Humanos , Masculino , Ratones , Ratones Transgénicos , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
The specific regulation of cell metabolism is one of cornerstones of biochemistry [...].
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Poliaminas Biogénicas/metabolismo , Animales , HumanosRESUMEN
A nanoprobe array based on fluorescent nitrogen-rich carbon dots (N-CDs) and Ag+ was constructed for simultaneous qualitative and quantitative determination of seven kinds of biogenic polyamines (BAs), including tryptamine (Try), histamine (His), putrescine (Put), cadaverine (Cad), spermine (Spm), spermidine (Spd), and agmatine (Agm). Ag+ can specifically bind to the N-CDs and quench the fluorescence of the N-CDs through a static mechanism. BAs further statically quench the fluorescence of the N-CD@Ag+ composite by bridging two Ag+ centers of the N-CD@Ag+. The nanoprobe array was constructed based on the differential fluorescence response arising from the differential binding affinity of various BAs. BAs can be differentiated and analyzed by the nanoprobe array within the concentration range 0.5-500 µM. The preliminary diluted and artificially spiked commercial human serum was utilized to simulate the serum environment for assessing the performance of the nanoprobe array in real samples. The N-CD@Ag+ system can recognize BAs with 100% accuracy in simulated human serum samples. The quantitative determination of BAs - no matter in a one-component system or a three-component system - was also realized by using the N-CD@Ag+ system even in the simulated serum environment. The recovery rates from spiked serum samples were higher 99%, and the relative standard deviation (RSD) was less than 3%. Based on the excellent multi-BA determination performance, a BA-related disease model about cerebral ischemia was constructed. Healthy cases as well as mild, moderate, and severe cerebral ischemia cases can be well identified from the disease model based on the N-CD@Ag+ nanoprobe array. Schematic representation of fluorescent nanoprobe array constructed by carbon nanodots (N-CDs) and Ag+ for qualitative and quantitative analyses of biogenic polyamines (BAs) and diagnosis of cerebral ischemia (CI) through linear discriminant analysis (LDA) and support vector machine (SVM).
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Poliaminas Biogénicas/sangre , Colorantes Fluorescentes/química , Puntos Cuánticos/química , Biomarcadores/sangre , Carbono/química , Humanos , Modelos Lineales , Plata/química , Espectrometría de Fluorescencia/métodosRESUMEN
BACKGROUND: Pheochromocytomas (PCCs) and paragangliomas (PGLs) are neuroendocrine tumors that are mostly benign. Metastatic disease does occur in about 10% of cases of PCC and up to 25% of PGL, and for these patients no effective therapies are available. Patients with mutations in the succinate dehydrogenase subunit B (SDHB) gene tend to have metastatic disease. We hypothesized that a down-regulation in the active succinate dehydrogenase B subunit should result in notable changes in cellular metabolic profile and could present a vulnerability point for successful pharmacological targeting. METHODS: Metabolomic analysis was performed on human hPheo1 cells and shRNA SDHB knockdown hPheo1 (hPheo1 SDHB KD) cells. Additional analysis of 115 human fresh frozen samples was conducted. In vitro studies using N1,N11-diethylnorspermine (DENSPM) and N1,N12- diethylspermine (DESPM) treatments were carried out. DENSPM efficacy was assessed in human cell line derived mouse xenografts. RESULTS: Components of the polyamine pathway were elevated in hPheo1 SDHB KD cells compared to wild-type cells. A similar observation was noted in SDHx PCC/PGLs tissues compared to their non-mutated counterparts. Specifically, spermidine, and spermine were significantly elevated in SDHx-mutated PCC/PGLs, with a similar trend in hPheo1 SDHB KD cells. Polyamine pathway inhibitors DENSPM and DESPM effectively inhibited growth of hPheo1 cells in vitro as well in mouse xenografts. CONCLUSIONS: This study demonstrates overactive polyamine pathway in PCC/PGL with SDHB mutations. Treatment with polyamine pathway inhibitors significantly inhibited hPheo1 cell growth and led to growth suppression in xenograft mice treated with DENSPM. These studies strongly implicate the polyamine pathway in PCC/PGL pathophysiology and provide new foundation for exploring the role for polyamine analogue inhibitors in treating metastatic PCC/PGL. PRéCIS: Cell line metabolomics on hPheo1 cells and PCC/PGL tumor tissue indicate that the polyamine pathway is activated. Polyamine inhibitors in vitro and in vivo demonstrate that polyamine inhibitors are promising for malignant PCC/PGL treatment. However, further research is warranted.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/tratamiento farmacológico , Poliaminas Biogénicas/antagonistas & inhibidores , Paraganglioma/tratamiento farmacológico , Feocromocitoma/tratamiento farmacológico , Neoplasias de las Glándulas Suprarrenales/genética , Neoplasias de las Glándulas Suprarrenales/metabolismo , Animales , Poliaminas Biogénicas/metabolismo , Línea Celular Tumoral , Humanos , Masculino , Metabolómica , Ratones , Mutación , Paraganglioma/genética , Paraganglioma/metabolismo , Feocromocitoma/genética , Feocromocitoma/metabolismo , Succinato Deshidrogenasa/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Many gaps in our understanding of Alzheimer's disease remain despite intense research efforts. One such prominent gap is the mechanism of Tau condensation and fibrillization. One viewpoint is that positively charged Tau is condensed by cytosolic polyanions. However, this hypothesis is likely based on an overestimation of the abundance and stability of cytosolic polyanions and an underestimation of crucial intracellular constituents - the cationic polyamines. Here, we propose an alternative mechanism grounded in cellular biology. We describe extensive molecular dynamics simulations and analysis on physiologically relevant model systems, which suggest that it is not positively charged, unmodified Tau that is condensed by cytosolic polyanions but negatively charged, hyperphosphorylated Tau that is condensed by cytosolic polycations. Our work has broad implications for anti-Alzheimer's research and drug development and the broader field of tauopathies in general, potentially paving the way to future etiologic therapies.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Poliaminas Biogénicas/efectos adversos , Proteínas tau/metabolismo , Poliaminas Biogénicas/química , Citosol/metabolismo , Humanos , Modelos Biológicos , Simulación de Dinámica Molecular , Fosforilación , Poliaminas/metabolismo , Polielectrolitos/metabolismo , Agregación Patológica de Proteínas/etiología , Agregación Patológica de Proteínas/metabolismo , Tauopatías , Proteínas tau/efectos de los fármacosRESUMEN
Hyperpigmentary conditions can arise when melanogenesis in the epidermis is misregulated. Understanding the pathways underlying melanogenesis is essential for the development of effective treatments. Here, we report that a group of metabolites called polyamines are important in the control of melanogenesis in human skin. Polyamines are cationic molecules present in all cells and are essential for cellular function. We report that polyamine regulator ODC1 is upregulated in melanocytes from melasma lesional skin. We report that the polyamine putrescine can promote pigmentation in human skin explants and primary normal human epidermal melanocytes through induction of tyrosinase which is rate-limiting for the synthesis of melanin. Putrescine supplementation on normal human epidermal melanocytes results in the activation of polyamine catabolism, which results in increased intracellular H2O2. Polyamine catabolism is also increased in human skin explants that have been treated with putrescine. We further report that inhibition of polyamine catabolism prevents putrescine-induced promotion of tyrosinase levels and pigmentation in normal human epidermal melanocytes, showing that polyamine catabolism is responsible for the putrescine induction of melanogenesis. Our data showing that putrescine promotes pigmentation has important consequences for hyperpigmented and hypopigmented conditions. Further understanding of how polyamines control epidermal pigmentation could open the door for the development of new therapeutics.
Asunto(s)
Epidermis/efectos de los fármacos , Melaninas/biosíntesis , Putrescina/farmacología , Poliaminas Biogénicas/metabolismo , Células Cultivadas , Transportadores de Ácidos Dicarboxílicos/fisiología , Epidermis/metabolismo , Humanos , Melanocitos/efectos de los fármacos , Melanocitos/metabolismo , Persona de Mediana Edad , Proteínas de Transporte de Membrana Mitocondrial/fisiología , Putrescina/análogos & derivados , Pigmentación de la Piel/efectos de los fármacosRESUMEN
Treatment of patients with triple-negative breast cancer (TNBC) is limited by a lack of effective molecular therapies targeting this disease. Recent studies have identified metabolic alterations in cancer cells that can be targeted to improve responses to standard-of-care chemotherapy regimens. Using MDA-MB-468 and SUM-159PT TNBC cells, along with LC-MS/MS and HPLC metabolomics profiling, we found here that exposure of TNBC cells to the cytotoxic chemotherapy drugs cisplatin and doxorubicin alter arginine and polyamine metabolites. This alteration was because of a reduction in the levels and activity of a rate-limiting polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). Using gene silencing and inhibitor treatments, we determined that the reduction in ODC was mediated by its negative regulator antizyme, targeting ODC to the proteasome for degradation. Treatment with the ODC inhibitor difluoromethylornithine (DFMO) sensitized TNBC cells to chemotherapy, but this was not observed in receptor-positive breast cancer cells. Moreover, TNBC cell lines had greater sensitivity to single-agent DFMO, and ODC levels were elevated in TNBC patient samples. The alterations in polyamine metabolism in response to chemotherapy, as well as DFMO-induced preferential sensitization of TNBC cells to chemotherapy, reported here suggest that ODC may be a targetable metabolic vulnerability in TNBC.
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Poliaminas Biogénicas/biosíntesis , Citotoxinas/farmacología , Eflornitina/farmacología , Proteínas de Neoplasias , Inhibidores de la Ornitina Descarboxilasa/farmacología , Ornitina Descarboxilasa/metabolismo , Neoplasias de la Mama Triple Negativas , Línea Celular Tumoral , Femenino , Humanos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas/metabolismo , Proteolisis/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
BACKGROUND/AIM: Polyamines are important for the growth of eukaryotic cells. At high levels, they promote proliferation, invasion and migration of tumour cells. Polyamine metabolism is an important new target for anticancer therapy. Some polyamine analogues can have an inhibitory effect on tumour cells. The aim of this study was to explore the potential of certain butylated derivatives of propanediamine for prostate cancer chemotherapy. MATERIALS AND METHODS: Human prostate cancer cells, LNCaP, were used for the evaluation of the antiproliferative activity of polyamine analogs and their influence on spermine oxidase. RESULTS: Tetrabutyl propanediamine and two new polyamine analogues inhibited the growth of LNCaP cells. At the same time, a strong activation of spermine oxidase was observed. CONCLUSION: The investigated compounds demonstrated their potential value in the therapy of human prostate cancer. Their effect might be attributed to the activation of the polyamine catabolic pathway.
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Diaminas/farmacología , Poliaminas/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Poliaminas Biogénicas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Masculino , Redes y Vías Metabólicas , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Poliamino OxidasaRESUMEN
BACKGROUND: Polyamines can induce protein aggregation that can be related to the physiology of the cellular function. Polyamines have been implicated in protein aggregation which may lead to neuropathic and non neuropathic amyloidosis. SCOPE OF REVIEW: Change in the level of polyamine concentration has been associated with ageing and neurodegeneration such as Parkinson's disease, Alzheimer's disease. Lysozyme aggregation in the presence of polyamines leads to non neuropathic amyloidosis. Polyamine analogues can suppress or inhibit protein aggregation suggesting their efficacy against amyloidogenic protein aggregates. MAJOR CONCLUSIONS: In this study we report the comparative interactions of lysozyme with the polyamine analogue, 1-naphthyl acetyl spermine in comparison with the biogenic polyamines through spectroscopy, calorimetry, imaging and docking techniques. The findings revealed that the affinity of binding varied as spermidine > 1-naphthyl acetyl spermine > spermine. The biogenic polyamines accelerated the rate of fibrillation significantly, whereas the analogue inhibited the rate of fibrillation to a considerable extent. The polyamines bind near the catalytic diad residues viz. Glu35 and Asp52, and in close proximity of Trp62 residue. However, the analogue showed dual nature of interaction where its alkyl amine region bind in same way as the biogenic polyamines bind to the catalytic site, while the naphthyl group makes hydrophobic contacts with Trp62 and Trp63, thereby suggesting its direct influence on fibrillation. GENERAL SIGNIFICANCE: This study, thus, potentiates, the development of a polyamine analogue that can perform as an effective inhibitor targeted towards aggregation of amyloidogenic proteins.
Asunto(s)
Amiloide/metabolismo , Proteínas Aviares/metabolismo , Pollos/metabolismo , Muramidasa/metabolismo , Espermidina/metabolismo , Espermina/análogos & derivados , Amiloidosis/metabolismo , Animales , Poliaminas Biogénicas/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Simulación del Acoplamiento Molecular , Agregado de Proteínas , Agregación Patológica de Proteínas/metabolismo , Espermina/metabolismoRESUMEN
Adverse conditions and biotic strain can lead to significant losses and impose limitations on plant yield. Polyamines (PAs) serve as regulatory molecules for both abiotic/biotic stress responses and cell protection in unfavourable environments. In this work, the transcription pattern of 24 genes orchestrating PA metabolism was investigated in Cucumber Mosaic Virus or Potato Virus Y infected and cold stressed tomato plants. Expression analysis revealed a differential/pleiotropic pattern of gene regulation in PA homeostasis upon biotic, abiotic or combined stress stimuli, thus revealing a discrete response specific to diverse stimuli: (i) biotic stress-influenced genes, (ii) abiotic stress-influenced genes, and (iii) concurrent biotic/abiotic stress-regulated genes. The results support different roles for PAs against abiotic and biotic stress. The expression of several genes, significantly induced under cold stress conditions, is mitigated by a previous viral infection, indicating a possible priming-like mechanism in tomato plants pointing to crosstalk among stress signalling. Several genes and resulting enzymes of PA catabolism were stimulated upon viral infection. Hence, we suggest that PA catabolism resulting in elevated H2O2 levels could mediate defence against viral infection. However, after chilling, the activities of enzymes implicated in PA catabolism remained relatively stable or slightly reduced. This correlates to an increase in free PA content, designating a per se protective role of these compounds against abiotic stress.
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Poliaminas Biogénicas/metabolismo , Solanum lycopersicum/genética , Estrés Fisiológico/genética , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica de las Plantas/genética , Homeostasis , Peróxido de Hidrógeno/metabolismo , Solanum lycopersicum/metabolismo , Proteínas de Plantas/genética , Poliaminas/metabolismo , Estrés Fisiológico/fisiologíaRESUMEN
Polyamines are positively charged small molecules ubiquitously existing in all living organisms, and they are considered as one kind of the most ancient cellular components. The most common polyamines are spermidine, spermine, and their precursor putrescine generated from ornithine. Polyamines play critical roles in cells by stabilizing chromatin structure, regulating DNA replication, modulating gene expression, etc., and they also affect the structure and function of proteins. A few studies have investigated the impact of polyamines on protein structure and function previously, but no reports have focused on a protein-based biological module with a dedicated function. In this report, we investigated the impact of polyamines (putrescine, spermidine, and spermine) on the cyanobacterial KaiABC circadian oscillator. Using an established in vitro reconstitution system, we noticed that polyamines could disrupt the robustness of the KaiABC oscillator by inducing the denaturation of the Kai proteins (KaiA, KaiB, and KaiC). Further experiments showed that the denaturation was likely due to the induced change of the thermal stability of the clock proteins. Our study revealed an intriguing role of polyamines as a component in complex cellular environments and would be of great importance for elucidating the biological function of polyamines in future.
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Proteínas Bacterianas/química , Poliaminas Biogénicas/química , Relojes Biológicos , Péptidos y Proteínas de Señalización del Ritmo Circadiano/química , Desnaturalización Proteica , Synechococcus/químicaRESUMEN
Several host and viral processes contribute to forming infectious virions. Polyamines are small host molecules that play diverse roles in viral replication. We previously demonstrated that polyamines are crucial for RNA viruses; however, the mechanisms by which polyamines function remain unknown. Here, we investigated the role of polyamines in the replication of the bunyaviruses Rift Valley fever virus (vaccine strain MP-12) and La Crosse virus (LACV). We found that polyamine depletion did not impact viral RNA or protein accumulation, despite significant decreases in titer. Viral particles demonstrated no change in morphology, size, or density. Thus, polyamine depletion promotes the formation of noninfectious particles. These particles interfere with virus replication and stimulate innate immune responses. We extended this phenotype to Zika virus; however, coxsackievirus did not similarly produce noninfectious particles. In sum, polyamine depletion results in the accumulation of noninfectious particles that interfere with replication and stimulate immune signaling, with important implications for targeting polyamines therapeutically, as well as for vaccine strategies.IMPORTANCE Bunyaviruses are emerging viral pathogens that cause encephalitis, hemorrhagic fevers, and meningitis. We have uncovered that diverse bunyaviruses require polyamines for productive infection. Polyamines are small, positively charged host-derived molecules that play diverse roles in human cells and in infection. In polyamine-depleted cells, bunyaviruses produce an overabundance of noninfectious particles that are indistinguishable from infectious particles. However, these particles interfere with productive infection and stimulate antiviral signaling pathways. We further find that additional enveloped viruses are similarly sensitive to polyamine depletion but that a nonenveloped enterovirus is not. We posit that polyamines are required to maintain bunyavirus infectivity and that polyamine depletion results in the accumulation of interfering noninfectious particles that limit infectivity. These results highlight a novel means by which bunyaviruses use polyamines for replication and suggest promising means to target host polyamines to reduce virus replication.
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Poliaminas Biogénicas/inmunología , Infecciones por Bunyaviridae/inmunología , Virus Defectuosos/fisiología , Virus de la Encefalitis de California/fisiología , Virus de la Fiebre del Valle del Rift/fisiología , Virión/fisiología , Replicación Viral/inmunología , Infecciones por Bunyaviridae/genética , Infecciones por Bunyaviridae/patología , Línea Celular Tumoral , HumanosRESUMEN
Previous studies have already highlighted the correlation between Sporisorium scitamineum pathogenicity and sugarcane polyamine accumulation. It was shown that high infectivity correlates with an increase in the amount of spermidine, spermine and cadaverine conjugated to phenols in the sensitive cultivars whereas resistant plants mainly produce free putrescine. However, these previous studies did not clarify the role of these polyamides in the disorders caused to the plant. Therefore, the purpose of this research is to clarify the effect of polyamines on the development of smut disease. In this paper, commercial polyamines were firstly assayed on smut teliospores germination. Secondly, effects were correlated to changes in endogenous polyamines after contact with defense sugarcane glycoproteins. Low concentrations of spermidine significantly activated teliospore germination, while putrescine had no activating effect on germination. Interestingly, it was observed that the diamine caused nuclear decondensation and breakage of the teliospore cell wall whereas the treatment of teliospores with spermidine did not induce nuclear decondensation or cell wall breakdown. Moreover, the number of polymerized microtubules increased in the presence of 7.5 mM spermidine but it decreased with putrescine which indicates that polyamines effects on Sporisorium scitamineum teliospore germination could be mediated through microtubules interaction. An increased production of polyamines in smut teliospores has been related to sugarcane resistance to the disease. Teliospores incubation with high molecular mass glycoproteins (HMMG) from the uninoculated resistant variety of sugarcane, Mayari 55-14, caused an increase of the insoluble fraction of putrescine, spermidine and spermine inside the teliospore cells. Moreover, the level of the soluble fraction of spermidine (S fraction) increased inside teliospores and the excess was released to the medium. The HMMG glycoproteins purified from Mayarí 55-14 plants previously inoculated with the pathogen significantly increased the levels of both retained and secreted soluble putrescine and spermidine. Polyamines levels did not increase in teliospores after incubation with HMMG produced by non resistant variety Barbados 42231 which could be related to the incapacity of these plants to defend themselves against smut disease. Thus, a hypothesis about the role of polyamines in sugarcane-smut interaction is explained.
Asunto(s)
Poliaminas Biogénicas/metabolismo , Glicoproteínas/metabolismo , Inmunidad de la Planta , Saccharum/microbiología , Esporas Fúngicas/metabolismo , Ustilaginales/metabolismo , Poliaminas Biogénicas/fisiología , Glicoproteínas/fisiología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Putrescina/metabolismo , Putrescina/fisiología , Saccharum/metabolismo , Espermidina/metabolismo , Espermidina/fisiología , Espermina/metabolismo , Espermina/fisiología , Ustilaginales/fisiologíaRESUMEN
In this work, the effect of two organic polyamines (spermine and spermidine) on the fluorescence intensity and activity of bovine intestinal alkaline phosphatase (BIALP) are investigated. The interaction of BIALP with spermine and spermidine was studied in a diethanolamine buffer with 0.5 mM magnesium chloride (pH 9.8) and at two temperatures by using the fluorescence quenching method. Furthermore, the activity of enzyme was studied using UV-Vis spectrophotometry in a diethanolamine buffer with 0.5 mM magnesium chloride, at 37 °C, in the absence and presence of different concentrations of each polyamine (0-5 mM). It was demonstrated that both polyamines quenched the intrinsic fluorescence of BIALP by the static quenching process. Based on these results, the values of the binding site for both polyamines were close to each other and decreased by increasing the temperature. The calculated thermodynamic parameters (ΔH° < 0 and ΔS° < 0) also showed that the acting forces in the formation of the complex between BIALP and polyamines were hydrogen bonds and van der Waals forces with an overall favorable Gibbs free energy change (∆G° < 0). In addition, kinetic studies revealed that these polyamines enhanced the enzyme activity of BIALP in a concentration-dependent manner. This result also indicated that spermine had more of an effect on BIALP activity in the same condition. Also, molecular docking as well as thermodynamic parameters showed that hydrogen bonds and van der Waals forces played an important role in the stabilization of BIALP-polyamine complexes.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Poliaminas Biogénicas/metabolismo , Intestinos/enzimología , Simulación del Acoplamiento Molecular , Análisis Espectral , Fosfatasa Alcalina/química , Animales , Bovinos , Enlace de Hidrógeno , Cinética , Unión Proteica , Conformación Proteica , TermodinámicaRESUMEN
Leukemic cells from different patients exhibit different sensitivity to anticancer drugs including doxorubicin (DOX). Resistance to chemotherapy decreases efficacy of the treatment and promotes cancer recurrence and metastases. One of the approaches to overcome drug resistance includes E2F1-mediated regulation of the Ñ73 protein that belongs to the Ñ53 family. Its ΔNp73 isoform exhibits pro-oncogenic effects, and TAp73 - anti-oncogenic effects. Human cytomegalovirus (HCMV), often found in tumors, suppresses pro-apoptotic pathways and E2F1/p73 in particular. The activity of E2F1 and p73 transcription factors is linked to metabolism of biogenic polyamines. Therefore, it could be suggested that compounds that target polyamine-metabolizing enzymes can sensitize HCMV-infected hematological malignancies to doxorubicin. Here we report that HCMV infection of ТÐÐ -1 monocytic leukemic cells considerably elevates E2F1 levels and shifts the balance between the Ñ73 isoforms towards ΔNp73 leading to survival of DOX-treated leukemic cells. In contrast, MDL72.527, an inhibitor of polyamine catabolism, decreases ΔNp73/ТÐÑ73 ratio and thus restores sensitivity of the cells to DOX. Our findings indicate the combination of doxorubicin and MDL72.527 may present a novel strategy for therapy of leukemia in patients with and without HCMV infection.