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1.
PLoS One ; 17(5): e0269276, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35639710

RESUMO

Positive and counter-selectable markers have been successfully integrated as a part of numerous genetic assays in many model organisms. In this study, we investigate the mechanism of resistance to arginine analog canavanine and its applicability for genetic selection in Schizosaccharomyces pombe. Deletion of both the arginine permease gene cat1 and SPBC18H10.16/vhc1 (formerly mistakenly called can1) provides strong drug resistance, while the single SPBC18H10.16/vhc1 deletion does not have an impact on canavanine resistance. Surprisingly, the widely used can1-1 allele does not encode for a defective arginine permease but rather corresponds to the any1-523C>T allele. The strong canavanine-resistance conferred by this allele arises from an inability to deposit basic amino acid transporters on the cellular membrane. any1-523C>T leads to reduced post-translational modifications of Any1 regulated by the Tor2 kinase. We also demonstrate that any1-523C>T is a dominate allele. Our results uncover the mechanisms of canavanine-resistance in fission yeast and open the opportunity of using cat1, vhc1 and any1 mutant alleles in genetic assays.


Assuntos
Sistemas de Transporte de Aminoácidos , Arrestinas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Simportadores , Alelos , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Arginina/metabolismo , Arrestinas/genética , Arrestinas/metabolismo , Canavanina/metabolismo , Mutação , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Simportadores/genética , Simportadores/metabolismo
2.
Environ Microbiol ; 23(10): 5823-5836, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33830599

RESUMO

The bacterial cell wall is made of peptidoglycan (PG), a polymer that is essential for maintenance of cell shape and survival. Many bacteria alter their PG chemistry as a strategy to adapt their cell wall to external challenges. Therefore, identifying these environmental cues is important to better understand the interplay between microbes and their habitat. Here, we used the soil bacterium Pseudomonas putida to uncover cell wall modulators from plant extracts and found canavanine (CAN), a non-proteinogenic amino acid. We demonstrated that cell wall chemical editing by CAN is licensed by P. putida BSAR, a broad-spectrum racemase which catalyses production of dl-CAN from l-CAN, which is produced by many legumes. Importantly, d-CAN diffuses to the extracellular milieu thereby having a potential impact on other organisms inhabiting the same niche. Our results show that d-CAN alters dramatically the PG structure of Rhizobiales (e.g., Agrobacterium tumefaciens, Sinorhizobium meliloti), impairing PG crosslinkage and cell division. Using A. tumefaciens, we demonstrated that the detrimental effect of d-CAN is suppressed by a single amino acid substitution in the cell division PG transpeptidase penicillin binding protein 3a. Collectively, this work highlights the role of amino acid racemization in cell wall chemical editing and fitness.


Assuntos
Alphaproteobacteria , Peptidoglicano , Alphaproteobacteria/metabolismo , Proteínas de Bactérias/metabolismo , Canavanina/análise , Canavanina/metabolismo , Parede Celular/metabolismo , Morfogênese , Peptidoglicano/metabolismo
3.
PLoS One ; 16(3): e0235303, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33730086

RESUMO

Continuous culture systems allow for the controlled growth of microorganisms over a long period of time. Here, we develop a novel test for mutagenicity that involves growing yeast in continuous culture systems exposed to low levels of mutagen for a period of approximately 20 days. In contrast, most microorganism-based tests for mutagenicity expose the potential mutagen to the biological reporter at a high concentration of mutagen for a short period of time. Our test improves upon the sensitivity of the well-established Ames test by at least 20-fold for each of two mutagens that act by different mechanisms (the intercalator ethidium bromide and alkylating agent methyl methanesulfonate). To conduct the tests, cultures were grown in small, inexpensive continuous culture systems in media containing (potential) mutagen, and the resulting mutagenicity of the added compound was assessed via two methods: a canavanine-based plate assay and whole genome sequencing. In the canavanine-based plate assay, we were able to detect a clear relationship between the amount of mutagen and the number of canavanine-resistant mutant colonies over a period of one to three weeks of exposure. Whole genome sequencing of yeast grown in continuous culture systems exposed to methyl methanesulfonate demonstrated that quantification of mutations is possible by identifying the number of unique variants across each strain. However, this method had lower sensitivity than the plate-based assay and failed to distinguish the different concentrations of mutagen. In conclusion, we propose that yeast grown in continuous culture systems can provide an improved and more sensitive test for mutagenicity.


Assuntos
Etídio/farmacologia , Metanossulfonato de Metila/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Canavanina/farmacologia , Meios de Cultura/química , DNA Fúngico/química , DNA Fúngico/metabolismo , Testes de Mutagenicidade/instrumentação , Testes de Mutagenicidade/métodos , Saccharomyces cerevisiae/genética , Sequenciamento Completo do Genoma
4.
Cell Mol Life Sci ; 78(6): 3021-3044, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33230565

RESUMO

Arginine deprivation therapy (ADT) is a new metabolic targeting approach with high therapeutic potential for various solid cancers. Combination of ADT with low doses of the natural arginine analog canavanine effectively sensitizes malignant cells to irradiation. However, the molecular mechanisms determining the sensitivity of intrinsically non-auxotrophic cancers to arginine deficiency are still poorly understood. We here show for the first time that arginine deficiency is accompanied by global metabolic changes and protein/membrane breakdown, and results in the induction of specific, more or less pronounced (severe vs. mild) ER stress responses in head and neck squamous cell carcinoma (HNSCC) cells that differ in their intrinsic ADT sensitivity. Combination of ADT with canavanine triggered catastrophic ER stress via the eIF2α-ATF4(GADD34)-CHOP pathway, thereby inducing apoptosis; the same signaling arm was irrelevant in ADT-related radiosensitization. The particular strong supra-additive effect of ADT, canavanine and irradiation in both intrinsically more and less sensitive cancer cells supports the rational of ER stress pathways as novel target for improving multi-modal metabolic anti-cancer therapy.


Assuntos
Canavanina/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Tolerância a Radiação/efeitos dos fármacos , Raios X , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Arginina/deficiência , Arginina/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/química , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/genética , Endorribonucleases/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , /genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Fator de Transcrição CHOP/antagonistas & inibidores , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo
5.
Cells ; 9(10)2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-33008000

RESUMO

Glioblastomas are the most frequent and aggressive form of primary brain tumors with no efficient cure. However, they often exhibit specific metabolic shifts that include deficiency in the biosynthesis of and dependence on certain exogenous amino acids. Here, we evaluated, in vitro, a novel combinatory antiglioblastoma approach based on arginine deprivation and canavanine, an arginine analogue of plant origin, using two human glioblastoma cell models, U251MG and U87MG. The combinatory treatment profoundly affected cell viability, morphology, motility and adhesion, destabilizing the cytoskeleton and mitochondrial network, and induced apoptotic cell death. Importantly, the effects were selective toward glioblastoma cells, as they were not pronounced for primary rat glial cells. At the molecular level, canavanine inhibited prosurvival kinases such as FAK, Akt and AMPK. Its effects on protein synthesis and stress response pathways were more complex and dependent on exposure time. We directly observed canavanine incorporation into nascent proteins by using quantitative proteomics. Although canavanine in the absence of arginine readily incorporated into polypeptides, no motif preference for such incorporation was observed. Our findings provide a strong rationale for further developing the proposed modality based on canavanine and arginine deprivation as a potential antiglioblastoma metabolic therapy independent of the blood-brain barrier.


Assuntos
Arginina/uso terapêutico , Canavanina/uso terapêutico , Glioblastoma/tratamento farmacológico , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Arginina/farmacologia , Canavanina/farmacologia , Linhagem Celular Tumoral , Humanos , Ratos
6.
Planta ; 252(1): 5, 2020 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-32535658

RESUMO

MAIN CONCLUSION: Nitro/oxidative modifications of proteins and RNA nitration resulted from altered peroxynitrite generation are elements of the indirect mode of action of canavanine and meta-tyrosine in plants Environmental conditions and stresses, including supplementation with toxic compounds, are known to impair reactive oxygen (ROS) and reactive nitrogen species (RNS) homeostasis, leading to modification in production of oxidized and nitrated derivatives. The role of nitrated and/or oxidized biotargets differs depending on the stress factors and developmental stage of plants. Canavanine (CAN) and meta-tyrosine (m-Tyr) are non-proteinogenic amino acids (NPAAs). CAN, the structural analog of arginine, is found mostly in seeds of Fabaceae species, as a storage form of nitrogen. In mammalian cells, CAN is used as an anticancer agent due to its inhibitory action on nitric oxide synthesis. m-Tyr is a structural analogue of phenylalanine and an allelochemical found in root exudates of fescues. In animals, m-Tyr is recognized as a marker of oxidative stress. Supplementation of plants with CAN or m-Tyr modify ROS and RNS metabolism. Over the last few years of our research, we have collected the complex data on ROS and RNS metabolism in tomato (Solanum lycopersicum L.) plants exposed to CAN or m-Tyr. In addition, we have shown the level of nitrated RNA (8-Nitro-guanine) in roots of seedlings, stressed by the tested NPAAs. In this review, we describe the model of CAN and m-Tyr mode of action in plants based on modifications of signaling pathways induced by ROS/RNS with a special focus on peroxynitrite induced RNA and protein modifications.


Assuntos
Aminoácidos/metabolismo , Lycopersicon esculentum/metabolismo , Ácido Peroxinitroso/metabolismo , Transdução de Sinais/efeitos dos fármacos , Canavanina/metabolismo , Nitratos/metabolismo , Oxirredução , Estresse Oxidativo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Plântula/metabolismo
7.
Curr Genet ; 65(5): 1199-1215, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31011791

RESUMO

When glucose is available, Saccharomyces cerevisiae prefers fermentation to respiration. In fact, it can live without respiration at all. Here, we study the role of respiration in stress tolerance in yeast. We found that colony growth of respiratory-deficient yeast (petite) is greatly inhibited by canavanine, the toxic analog of arginine that causes proteotoxic stress. We found lower amounts of the amino acids involved in arginine biosynthesis in petites compared with WT. This finding may be explained by the fact that petite cells exposed to canavanine show reduction in the efficiency of targeting of proteins required for arginine biosynthesis. The retrograde (RTG) pathway signals mitochondrial stress. It positively controls production of arginine precursors. We show that canavanine abrogates RTG signaling especially in petite cells, and mutants in the RTG pathway are extremely sensitive to canavanine. We suggest that petite cells are naturally ineffective in production of some amino acids; combination of this fact with the effect of canavanine on the RTG pathway is the simplest explanation why petite cells are inhibited by canavanine. Surprisingly, we found that canavanine greatly inhibits colony formation when WT cells are forced to respire. Our research proposes a novel connection between respiration and proteotoxic stress.


Assuntos
Canavanina/metabolismo , Respiração Celular , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/fisiologia , Aminoácidos/metabolismo , Glutamatos/metabolismo , Ácido Glutâmico/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/genética , Mutação , Nitrogênio/metabolismo
8.
Nat Commun ; 10(1): 1476, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30931940

RESUMO

Early detection and accurate monitoring of chronic kidney disease (CKD) could improve care and retard progression to end-stage renal disease. Here, using untargeted metabolomics in 2155 participants including patients with stage 1-5 CKD and healthy controls, we identify five metabolites, including 5-methoxytryptophan (5-MTP), whose levels strongly correlate with clinical markers of kidney disease. 5-MTP levels decrease with progression of CKD, and in mouse kidneys after unilateral ureteral obstruction (UUO). Treatment with 5-MTP ameliorates renal interstitial fibrosis, inhibits IκB/NF-κB signaling, and enhances Keap1/Nrf2 signaling in mice with UUO or ischemia/reperfusion injury, as well as in cultured human kidney cells. Overexpression of tryptophan hydroxylase-1 (TPH-1), an enzyme involved in 5-MTP synthesis, reduces renal injury by attenuating renal inflammation and fibrosis, whereas TPH-1 deficiency exacerbates renal injury and fibrosis by activating NF-κB and inhibiting Nrf2 pathways. Together, our results suggest that TPH-1 may serve as a target in the treatment of CKD.


Assuntos
Fibrose/metabolismo , Proteínas I-kappa B/metabolismo , NF-kappa B/metabolismo , Insuficiência Renal Crônica/metabolismo , Triptofano Hidroxilase/genética , Triptofano/análogos & derivados , Acetilcarnitina/metabolismo , Animais , Canavanina/análogos & derivados , Canavanina/metabolismo , Carnitina/análogos & derivados , Carnitina/metabolismo , Estudos de Casos e Controles , Modelos Animais de Doenças , Progressão da Doença , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Humanos , Inflamação/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Rim/citologia , Rim/efeitos dos fármacos , Rim/patologia , Metabolômica , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Taurina/metabolismo , Triptofano/metabolismo , Triptofano/farmacologia , Obstrução Ureteral
9.
Dis Model Mech ; 12(1)2019 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-30635263

RESUMO

Chorea-acanthocytosis (ChAc) is a rare neurodegenerative disease associated with mutations in the human VPS13A gene. The mechanism of ChAc pathogenesis is unclear. A simple yeast model was used to investigate the function of the single yeast VSP13 orthologue, Vps13. Vps13, like human VPS13A, is involved in vesicular protein transport, actin cytoskeleton organisation and phospholipid metabolism. A newly identified phenotype of the vps13Δ mutant, sodium dodecyl sulphate (SDS) hypersensitivity, was used to screen a yeast genomic library for multicopy suppressors. A fragment of the MYO3 gene, encoding Myo3-N (the N-terminal part of myosin, a protein involved in the actin cytoskeleton and in endocytosis), was isolated. Myo3-N protein contains a motor head domain and a linker. The linker contains IQ motifs that mediate the binding of calmodulin, a negative regulator of myosin function. Amino acid substitutions that disrupt the interaction of Myo3-N with calmodulin resulted in the loss of vps13Δ suppression. Production of Myo3-N downregulated the activity of calcineurin, a protein phosphatase regulated by calmodulin, and alleviated some defects in early endocytosis events. Importantly, ethylene glycol tetraacetic acid (EGTA), which sequesters calcium and thus downregulates calmodulin and calcineurin, was a potent suppressor of vps13Δ. We propose that Myo3-N acts by sequestering calmodulin, downregulating calcineurin and increasing activity of Myo3, which is involved in endocytosis and, together with Osh2/3 proteins, functions in endoplasmic reticulum-plasma membrane contact sites. These results show that defects associated with vps13Δ could be overcome, and point to a functional connection between Vps13 and calcium signalling as a possible target for chemical intervention in ChAc. Yeast ChAc models may uncover the underlying pathological mechanisms, and may also serve as a platform for drug testing.This article has an associated First Person interview with the first author of the paper.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Calmodulina/metabolismo , Modelos Biológicos , Miosinas/metabolismo , Neuroacantocitose/tratamento farmacológico , Neuroacantocitose/metabolismo , Saccharomyces cerevisiae/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Alelos , Substituição de Aminoácidos , Calcineurina/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Canavanina/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Endocitose/efeitos dos fármacos , Genes Supressores , Mutação/genética , Domínios Proteicos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/metabolismo , Dodecilsulfato de Sódio , Transcrição Genética/efeitos dos fármacos , Vacúolos/metabolismo
10.
Curr Genet ; 65(2): 483-492, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30244280

RESUMO

Loss of heterozygosity (LOH) in a vegetatively growing diploid cell signals irregularity of mitosis. Therefore, assays of LOH serve to discover pathways critical for proper replication and segregation of chromosomes. We screened for enhanced LOH in a whole-genome collection of diploid yeast strains in which a single gene was strongly overexpressed. We found 39 overexpression strains with substantially increased LOH caused either by recombination or by chromosome instability. Most of them, 32 in total, belonged to the category of "cell division", a broadly defined biological process. Of those, only one, TOP3, coded for an enzyme that uses DNA as a substrate. The rest related to establishment and maintenance of cell polarity, chromosome segregation, and cell cycle checkpoints. Former studies, in which gene deletions were used, showed that an absence of a protein participating in the DNA processing machinery is a potent stimulator of genome instability. As our results suggest, overexpression of such proteins is not comparably damaging as the absence of them. It may mean that the harmful effect of overexpression is more likely to occur in more complex and multistage processes, such as chromosome segregation. We also report a side finding, resulting from the fact that we worked with the yeast strains bearing a 2-micron plasmid. We noted that intense transcription from such a plasmid led to an enhanced rate of an entire chromosome loss (as opposed to LOH produced by recombination). This observation may support models linking segregation of 2-micron plasmids to segregation of chromosomes.


Assuntos
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Instabilidade Cromossômica , Cromossomos Fúngicos , Leveduras/genética , Canavanina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Deleção Cromossômica , Regulação Fúngica da Expressão Gênica , Testes Genéticos , Perda de Heterozigosidade , Mitose/genética , Espécies Reativas de Oxigênio/metabolismo , Recombinação Genética , Leveduras/metabolismo
11.
Neurotox Res ; 33(1): 15-23, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28470567

RESUMO

The implication of ß-N-methylamino-L-alanine (BMAA) in the development of neurodegenerative diseases worldwide has led to several investigations of the mechanism, or mechanisms, of toxicity of this cyanobacterially produced amino acid. The primary mechanism of toxicity that was identified is excitotoxicity, with a second possible mechanism, the misincorporation of BMAA into the primary protein structure and consequent cell damage, having been more recently reported. However, studies on excitotoxicity and misincorporation have been conducted independently and there are therefore no data available on the relative contribution of each of these mechanisms to the total toxicity of BMAA. The rat pheochromocytoma cell line PC12 is an ideal model for a study of this type, as glutamate receptor expression is modified by cell differentiation, which can be affected by exposure to nerve growth factor. In this study, the PC12 cell line was evaluated as a model to study BMAA toxicity via the two proposed mechanisms: excitotoxicity and protein misincorporation. BMAA and canavanine treatment of cultures of PC12 were evaluated for depolarization of the mitochondrial membrane. In canavanine-treated cultures, this was evident after 9 days of treatment and was attributed to the primary mechanism of canavanine toxicity, protein misincorporation. However, no membrane depolarization was observed for BMAA-treated cultures even after 21 days of continuous treatment at 500 µM. Short-term exposure to both BMAA and canavanine resulted in a slight increase in necrosis in undifferentiated cells that was prevented in canavanine-treated cultures by co-incubation with arginine, but not in BMAA-treated cultures by co-incubation with serine. A slight increase in apoptosis was observed in undifferentiated cells treated with either BMAA or glutamate, and ROS production increased in glutamate-treated cells. However, the excitotoxicity was less pronounced than reported in previous studies with neuronal cells. In contrast, apoptosis was greatly increased in both BMAA- and glutamate-treated cells after differentiation and resulting mGluR1 increase, indicating that excitotoxicity is the main, if not only, mechanism of toxicity in PC12.


Assuntos
Diamino Aminoácidos/toxicidade , Agonistas de Aminoácidos Excitatórios/toxicidade , Neurônios/efeitos dos fármacos , Diamino Aminoácidos/análise , Animais , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Arginina/metabolismo , Transporte Biológico/efeitos dos fármacos , Canavanina/análise , Canavanina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Células PC12/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo
12.
Cell Rep ; 21(10): 2978-2991, 2017 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-29212040

RESUMO

The Drosophila pharyngeal taste organs are poorly characterized despite their location at important sites for monitoring food quality. Functional analysis of pharyngeal neurons has been hindered by the paucity of molecular tools to manipulate them, as well as their relative inaccessibility for neurophysiological investigations. Here, we generate receptor-to-neuron maps of all three pharyngeal taste organs by performing a comprehensive chemoreceptor-GAL4/LexA expression analysis. The organization of pharyngeal neurons reveals similarities and distinctions in receptor repertoires and neuronal groupings compared to external taste neurons. We validate the mapping results by pinpointing a single pharyngeal neuron required for feeding avoidance of L-canavanine. Inducible activation of pharyngeal taste neurons reveals functional differences between external and internal taste neurons and functional subdivision within pharyngeal sweet neurons. Our results provide roadmaps of pharyngeal taste organs in an insect model system for probing the role of these understudied neurons in controlling feeding behaviors.


Assuntos
Proteínas de Drosophila/metabolismo , Faringe/metabolismo , Animais , Canavanina/metabolismo , Drosophila , Receptores de Superfície Celular/metabolismo , Células Receptoras Sensoriais/metabolismo , Paladar/fisiologia
13.
J Mol Microbiol Biotechnol ; 27(3): 133-146, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28456803

RESUMO

BACKGROUND: Arginine deiminase (ArcA) has been speculated to facilitate the intracellular survival of Streptococcus suis under acidic conditions. However, the physical and biological properties and function of SS2-ArcA have not yet been elucidated. METHODS: Recombinant SS2-ArcA (rSS2-ArcA) was expressed and purified using Ni-NTA affinity chromatography. Under various pH and temperature conditions, the enzymatic properties of purified rSS2-ArcA and crude native SS2-ArcA were determined. RESULTS: The SS2-arcA-deduced amino acid sequence contained a conserved catalytic triad (Cys399-His273-Glu218). The optimum temperature and pH of 47-kDa rSS2-ArcA and crude native SS2-ArcA were 42°C and pH 7.2. The rSS2-ArcA and crude native SS2-ArcA were stable for 3 h at 4 and 25°C, respectively. The pH stability and dependency tests suggested that rSS2-ArcA and crude native SS2-ArcA were functionally active in acidic conditions. The L-arginine substrate binding affinity (Km) values of rSS2-ArcA (specific activity 16.00 U/mg) and crude native SS2-ArcA (specific activity 0.23 U/mg) were 0.058 and 0.157 mM, respectively. rSS2-ArcA exhibited a weak binding affinity with the common ArcA inhibitors L-canavanine and L-NIO. Furthermore, the partial inactivation of SS2-ArcA significantly impaired the viability and growth of SS2 at pH 4.0, 6.0, and 7.5. CONCLUSIONS: This study profoundly demonstrated the involvement of ArcA enzymatic activity in S. suis survival under acidic conditions.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Hidrolases/química , Hidrolases/genética , Sorogrupo , Streptococcus suis/enzimologia , Streptococcus suis/genética , Sequência de Aminoácidos , Arginina/metabolismo , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Canavanina/antagonistas & inibidores , Clonagem Molecular , Ensaios Enzimáticos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Concentração de Íons de Hidrogênio , Hidrolases/efeitos dos fármacos , Hidrolases/metabolismo , Cinética , Ornitina/análogos & derivados , Ornitina/antagonistas & inibidores , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Streptococcus suis/metabolismo , Temperatura
14.
PLoS One ; 12(3): e0174128, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28319150

RESUMO

Saccharomyces cerevisiae (budding yeast) is a powerful eukaryotic model organism ideally suited to high-throughput genetic analyses, which time and again has yielded insights that further our understanding of cell biology processes conserved in humans. Lithium Acetate (LiAc) transformation of yeast with DNA for the purposes of exogenous protein expression (e.g., plasmids) or genome mutation (e.g., gene mutation, deletion, epitope tagging) is a useful and long established method. However, a reliable and optimized high throughput transformation protocol that runs almost no risk of human error has not been described in the literature. Here, we describe such a method that is broadly transferable to most liquid handling high-throughput robotic platforms, which are now commonplace in academic and industry settings. Using our optimized method, we are able to comfortably transform approximately 1200 individual strains per day, allowing complete transformation of typical genomic yeast libraries within 6 days. In addition, use of our protocol for gene knockout purposes also provides a potentially quicker, easier and more cost-effective approach to generating collections of double mutants than the popular and elegant synthetic genetic array methodology. In summary, our methodology will be of significant use to anyone interested in high throughput molecular and/or genetic analysis of yeast.


Assuntos
Automação Laboratorial/instrumentação , Robótica , Saccharomyces cerevisiae/genética , Transformação Genética , Automação Laboratorial/métodos , Canavanina/toxicidade , Meios de Cultura , Técnicas de Inativação de Genes , Biblioteca Genômica , Temperatura Alta , Saccharomyces cerevisiae/efeitos dos fármacos , Fatores de Tempo
15.
PLoS One ; 12(1): e0168775, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28045943

RESUMO

For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 µM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-ß-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 µM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei.


Assuntos
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Arginina/metabolismo , Lisina/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Arginina/análogos & derivados , Canavanina/metabolismo , Homoarginina/metabolismo , Humanos , Cinética , Oócitos/metabolismo , Fases de Leitura Aberta , Filogenia , Interferência de RNA , Saccharomyces cerevisiae/genética , Xenopus laevis
16.
J Biol Chem ; 292(9): 3940-3946, 2017 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-28096462

RESUMO

Glutamate recognition by neurotransmitter receptors often relies on Arg residues in the binding site, leading to the assumption that charge-charge interactions underlie ligand recognition. However, assessing the precise chemical contribution of Arg side chains to protein function and pharmacology has proven to be exceedingly difficult in such large and complex proteins. Using the in vivo nonsense suppression approach, we report the first successful incorporation of the isosteric, titratable Arg analog, canavanine, into a neurotransmitter receptor in a living cell, utilizing a glutamate-gated chloride channel from the nematode Haemonchus contortus Our data unveil a surprisingly small contribution of charge at a conserved arginine side chain previously suggested to form a salt bridge with the ligand, glutamate. Instead, our data show that Arg contributes crucially to ligand sensitivity via a hydrogen bond network, where Arg interacts both with agonist and with a conserved Thr side chain within the receptor. Together, the data provide a new explanation for the reliance of neurotransmitter receptors on Arg side chains and highlight the exceptional capacity of unnatural amino acid incorporation for increasing our understanding of ligand recognition.


Assuntos
Arginina/química , Canais de Cloreto/química , Animais , Sítios de Ligação , Canavanina/química , Drosophila melanogaster , Ácido Glutâmico/química , Haemonchus/metabolismo , Humanos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Ligantes , Mutagênese , Mutação , Neurotransmissores/metabolismo , Oócitos/citologia , Sais/química , Xenopus laevis
17.
Anticancer Agents Med Chem ; 17(2): 206-211, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-26902604

RESUMO

BACKGROUND: Due to the high level of argininosuccinate synthase (ASS), a key enzyme for the formation of arginine from citrulline, human breast cancers are often resistant to arginine deprivation therapy. An antimetabolite, Lcanavanine (L-CAV), can be incorporated into proteins in the place of arginine, disturbing protein conformation and leading to cellular death. OBJECTIVES: This study was designed to investigate the potential of L-CAV to enhance the toxicity of chemotherapeutic drugs in the human breast cancer cell line MCF-7, and determine the most favorable drug combination to exert synergistic interaction in the presence or absence of arginine in the medium. METHODS: Combination experiment based on the median-effect principle and mass-action law was conducted using constant ratios of cytotoxic agents as developed by Chou (2006). RESULTS: We observed that L-CAV enhanced the toxicity of cisplatin (CIS) and vinblastine (VIN) in MCF-7, even in the presence of L-ARG. On the other hand, L-CAV potentiated the toxicity of doxorubicin (DOX), paclitaxel (PTX), 5- fluoruracil (5-FU), and amphotericin-B (AMP-B) in cells grown in arginine deprived media. CONCLUSION: We conclude that the combination of L-CAV with CIS or VIN can potentiate the toxicity for breast cancer cells. Thus this report presents a new possibility for treating human breast cancers known to be resistant to arginine deprivation. This initial study requires further investigation in in vivo experiments and exploration of the molecular mechanism of cellular response in human breast cancer.


Assuntos
Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Mama/efeitos dos fármacos , Canavanina/farmacologia , Cisplatino/farmacologia , Vimblastina/farmacologia , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Feminino , Fluoruracila/farmacologia , Humanos , Células MCF-7 , Paclitaxel/farmacologia
18.
Oncotarget ; 7(45): 73292-73308, 2016 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-27689335

RESUMO

The moderate anticancer effect of arginine deprivation in clinical trials has been linked to an induced argininosuccinate synthetase (ASS1) expression in initially ASS1-negative tumors, and ASS1-positive cancers are anticipated as non-responders. Our previous studies indicated that arginine deprivation and low doses of the natural arginine analog canavanine can enhance radioresponse. However, the efficacy of the proposed combination in the presence of extracellular citrulline, the substrate for arginine synthesis by ASS1, remains to be elucidated, in particular for malignant cells with positive and/or inducible ASS1 as in colorectal cancer (CRC). Here, the physiological citrulline concentration of 0.05 mM was insufficient to overcome cell cycle arrest and radiosensitization triggered by arginine deficiency. Hyperphysiological citrulline (0.4 mM) did not entirely compensate for the absence of arginine and significantly decelerated cell cycling. Similar levels of canavanine-induced apoptosis were detected in the absence of arginine regardless of citrulline supplementation both in 2-D and advanced 3-D assays, while normal colon epithelial cells in organoid/colonosphere culture were unaffected. Notably, canavanine tremendously enhanced radiosensitivity of arginine-starved 3-D CRC spheroids even in the presence of hyperphysiological citrulline. We conclude that the novel combinatorial targeting strategy of metabolic-chemo-radiotherapy has great potential for the treatment of malignancies with inducible ASS1 expression.


Assuntos
Arginina/metabolismo , Canavanina/administração & dosagem , Citrulina/metabolismo , Radiação Ionizante , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Argininossuccinato Sintase/genética , Argininossuccinato Sintase/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/efeitos da radiação , Expressão Gênica , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos da radiação , Tolerância a Radiação , Esferoides Celulares , Células Tumorais Cultivadas
19.
Plant Physiol Biochem ; 103: 84-95, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26986929

RESUMO

Canavanine (CAN) is non-proteinogenic aminoacid and a structural analog of arginine (Arg). Naturally, CAN occurs in legumes e.g. jack bean and is considered as a strong allelochemical. As a selective inhibitor of inducible nitric oxide synthase in mammalians, it could act as a modifier of nitric oxide (NO) concentration in plants. Modifications in the content of endogenous reactive nitrogen species (RNS) and reactive oxygen species (ROS) influence root structure and architecture, being also under hormonal control. The aim of the work was to investigate regulation of root growth in tomato (Solanum lycopersicum L. cv. Malinowy Ozarowski) seedling by application of CAN at concentration (10 and 50 µM) leading to 50% or 100% restriction of root elongation. CAN at higher concentration led to slight DNA fragmentation, increased total RNA and protein level. Decline in total respiration rate after CAN supplementation was not associated with enhanced membrane permeability. Malformations in root morphology (shorter and thicker roots, limited number of lateral roots) were accompanied by modification in NO and ONOO(-) localization; determined mainly in peridermal cells and some border cells. Although, CAN resulted in low RNS production, addition of exogenous NO by usage of NO donors did not reverse its negative effect, nor recovery effect was detected after roots imbibition in Arg. To build up a comprehensive view on mode of action of CAN as root growth inhibitor, it was shown an elevated level of auxin. To summarize, we demonstrated several secondary mode of action of CAN, indicating its toxicity in plants linked to restriction in RNS formation accompanied by simultaneous overaccumulation of ROS.


Assuntos
Canavanina/farmacologia , Ácidos Indolacéticos/metabolismo , Lycopersicon esculentum/fisiologia , Reguladores de Crescimento de Plantas/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Germinação/efeitos dos fármacos , Lycopersicon esculentum/efeitos dos fármacos , Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/farmacologia , Oxigênio/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/fisiologia , Plântula/efeitos dos fármacos , Plântula/fisiologia
20.
Ukr Biochem J ; 88(2): 45-55, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-29227602

RESUMO

It was previously demonstrated in in vitro experiments that canavanine (Cav), a natural toxic arginine analogue of plant origin, is a promising candidate for augmenting the antineoplastic effects of arginine starvation. We demonstrated herein that recombinant human arginase, an arginine degrading enzyme, abrogated growth and significantly increased Cav cytotoxicity toward cultured L1210 murine leukemic cells. Cav co-treatment further reduced cells viability in a time-dependent manner and significantly promoted apoptosis induction. In the pilot study we also evaluated for the first time the potential toxicity of the combined arginine deprivation and Cav treatment in healthy mice. Administration of Cav alone or in combination with pegylated cobalt-containing human arginase (Co-hARG) did not evoke any apparent toxic effects in these animals, with no significant behavioural and survival changes after several weeks of the treatment. The therapeutic effects of the combination of Co-hARG and Cav were provisionally evaluated on the highly aggressive murine L1210 leukemia, which is semi-sensitive to arginine deprivation as a monotreatment. Combination of two drugs did not result in significant prolongation of the survival of leukemia-bearing mice. Thus, we have shown that the proposed combinational treatment is rather non-toxic for the animals. It has to be further evaluated in animal studies with alternative tumor models and/or drug doses and treatment modalities.


Assuntos
Antineoplásicos/farmacologia , Arginase/farmacologia , Canavanina/farmacologia , Leucemia L1210/tratamento farmacológico , Proteínas Recombinantes/farmacologia , Animais , Apoptose/efeitos dos fármacos , Arginase/sangue , Arginase/farmacocinética , Peso Corporal/efeitos dos fármacos , Canavanina/sangue , Canavanina/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quimioterapia Combinada , Humanos , Leucemia L1210/sangue , Leucemia L1210/mortalidade , Leucemia L1210/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteínas Recombinantes/sangue , Proteínas Recombinantes/farmacocinética , Análise de Sobrevida , Testes de Toxicidade Aguda
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