RESUMO
Unsaturated fatty acids (UFA) play a crucial role in central cellular processes in animals, including membrane function, development, and disease. Disruptions in UFA homeostasis can contribute to the onset of metabolic, cardiovascular, and neurodegenerative disorders. Consequently, there is a high demand for analytical techniques to study lipid compositions in live cells and multicellular organisms. Conventional analysis of UFA compositions in cells, tissues, and organisms involves solvent extraction procedures coupled with analytical techniques such as gas chromatography, MS and/or NMR spectroscopy. As a nondestructive and nontargeted technique, NMR spectroscopy is uniquely capable of characterizing the chemical profiling of living cells and multicellular organisms. Here, we use NMR spectroscopy to analyze Caenorhabditis elegans, enabling the determination of their lipid compositions and fatty acid unsaturation levels both in cell-free lipid extracts and in vivo. The NMR spectra of lipid extracts from WT and fat-3 mutant C. elegans strains revealed notable differences due to the absence of Δ-6 fatty acid desaturase activity, including the lack of arachidonic and eicosapentaenoic acyl chains. Uniform 13C-isotope labeling and high-resolution 2D solution-state NMR of live worms confirmed these findings, indicating that the signals originated from fast-tumbling lipid molecules within lipid droplets. Overall, this strategy permits the analysis of lipid storage in intact worms and has enough resolution and sensitivity to identify differences between WT and mutant animals with impaired fatty acid desaturation. Our results establish methodological benchmarks for future investigations of fatty acid regulation in live C. elegans using NMR.
RESUMO
Oxidation of protein methionines to methionine-sulfoxides (MetOx) is associated with several age-related diseases. In healthy cells, MetOx is reduced to methionine by two families of conserved methionine sulfoxide reductase enzymes, MSRA and MSRB that specifically target the S- or R-diastereoisomers of methionine-sulfoxides, respectively. To directly interrogate MSRA and MSRB functions in cellular settings, we developed an NMR-based biosensor that we call CarMetOx to simultaneously measure both enzyme activities in single reaction setups. We demonstrate the suitability of our strategy to delineate MSR functions in complex biological environments, including cell lysates and live zebrafish embryos. Thereby, we establish differences in substrate specificities between prokaryotic and eukaryotic MSRs and introduce CarMetOx as a highly sensitive tool for studying therapeutic targets of oxidative stress-related human diseases and redox regulated signaling pathways.
Assuntos
Técnicas Biossensoriais , Humanos , Metionina , Metionina Sulfóxido Redutases/metabolismo , Oxirredução , Especificidade por SubstratoRESUMO
Peaches are highly perishable and deteriorate quickly at ambient temperature. Cold storage is commonly used to prevent fruit decay; however, it affects fruit quality causing physiological disorders collectively termed 'chilling injury' (CI). To prevent or ameliorate CI, heat treatment is often applied prior to cold storage. In the present work, metabolic profiling was performed to determine the metabolic dynamics associated with the induction of acquired CI tolerance in response to heat shock. 'Dixiland' peach fruits exposed to 39 °C, cold stored, or after a combined treatment of heat and cold, were compared with fruits ripening at 20 °C. Dramatic changes in the levels of compatible solutes such as galactinol and raffinose were observed, while amino acid precursors of the phenylpropanoid pathway were also modified due to the stress treatments, as was the polyamine putrescine. The observed responses towards temperature stress in peaches are composed of both common and specific response mechanisms to heat and cold, but also of more general adaptive responses that confer strategic advantages in adverse conditions such as biotic stresses. The identification of such key metabolites, which prime the fruit to cope with different stress situations, will likely greatly accelerate the design and the improvement of plant breeding programs.
Assuntos
Temperatura Baixa , Frutas/metabolismo , Frutas/fisiologia , Temperatura Alta , Redes e Vias Metabólicas , Prunus/metabolismo , Prunus/fisiologia , Frutas/genética , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica de Plantas , Redes e Vias Metabólicas/genética , Metaboloma/genética , Metabolômica , Nitrogênio/metabolismo , Análise de Componente Principal , Prunus/genética , Característica Quantitativa Herdável , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rafinose/metabolismoRESUMO
Ripening of peach (Prunus persica L. Batsch) fruit is accompanied by dramatic cell wall changes that lead to softening. Post-harvest heat treatment is effective in delaying softening and preventing some chilling injury symptoms that this fruit exhibits after storage at low temperatures. In the present work, the levels of twelve transcripts encoding proteins involved in cell wall metabolism, as well as the differential extracellular proteome, were examined after a post-harvest heat treatment (HT; 39 °C for 3 days) of "Dixiland" peach fruit. A typical softening behaviour, in correlation with an increase in 1-aminocyclopropane-1-carboxylic acid oxidase-1 (PpACO1), was observed for peach maintained at 20 °C for 3 days (R3). Six transcripts encoding proteins involved in cell wall metabolism significantly increased in R3 with respect to peach at harvest, while six showed no modification or even decreased. In contrast, after HT, fruit maintained their firmness, exhibiting low PpACO1 level and significant lower levels of the twelve cell wall-modifying genes than in R3. Differential proteomic analysis of apoplastic proteins during softening and after HT revealed a significant decrease of DUF642 proteins after HT; as well as an increase of glyceraldehyde-3-phosphate dehydrogenase (GAPC) after softening. The presence of GAPC in the peach extracellular matrix was further confirmed by in situ immunolocalization and transient expression in tomato fruit. Though further studies are required to establish the function of DUF642 and GAPC in the apoplast, this study contributes to a deeper understanding of the events during peach softening and after HT with a focus on this key compartment.
Assuntos
Espaço Extracelular/metabolismo , Frutas/metabolismo , Proteínas de Plantas/metabolismo , Proteoma , Prunus/metabolismo , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Parede Celular/metabolismo , Regulação para Baixo , Eletroforese em Gel Bidimensional , Frutas/citologia , Frutas/enzimologia , Frutas/genética , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Temperatura Alta , Solanum lycopersicum/citologia , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Fenótipo , Proteínas de Plantas/genética , Proteômica , Prunus/citologia , Prunus/enzimologia , Prunus/genética , RNA Mensageiro/genética , RNA de Plantas/genética , Espectrometria de Massas em Tandem , Regulação para CimaRESUMO
Fruit from rosaceous species collectively display a great variety of flavors and textures as well as a generally high content of nutritionally beneficial metabolites. However, relatively little analysis of metabolic networks in rosaceous fruit has been reported. Among rosaceous species, peach (Prunus persica) has stone fruits composed of a juicy mesocarp and lignified endocarp. Here, peach mesocarp metabolic networks were studied across development using metabolomics and analysis of key regulatory enzymes. Principal component analysis of peach metabolic composition revealed clear metabolic shifts from early through late development stages and subsequently during postharvest ripening. Early developmental stages were characterized by a substantial decrease in protein abundance and high levels of bioactive polyphenols and amino acids, which are substrates for the phenylpropanoid and lignin pathways during stone hardening. Sucrose levels showed a large increase during development, reflecting translocation from the leaf, while the importance of galactinol and raffinose is also inferred. Our study further suggests that posttranscriptional mechanisms are key for metabolic regulation at early stages. In contrast to early developmental stages, a decrease in amino acid levels is coupled to an induction of transcripts encoding amino acid and organic acid catabolic enzymes during ripening. These data are consistent with the mobilization of amino acids to support respiration. In addition, sucrose cycling, suggested by the parallel increase of transcripts encoding sucrose degradative and synthetic enzymes, appears to operate during postharvest ripening. When taken together, these data highlight singular metabolic programs for peach development and may allow the identification of key factors related to agronomic traits of this important crop species.
Assuntos
Frutas/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/fisiologia , Metaboloma , Proteínas de Plantas/metabolismo , Prunus/crescimento & desenvolvimento , Prunus/metabolismo , Aminoácidos/análise , Aminoácidos/metabolismo , Transporte Biológico , Ácidos Carboxílicos/análise , Ácidos Carboxílicos/metabolismo , Dissacarídeos/análise , Dissacarídeos/metabolismo , Enzimas/genética , Enzimas/metabolismo , Frutas/enzimologia , Frutas/genética , Frutas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Regulação Enzimológica da Expressão Gênica/fisiologia , Redes e Vias Metabólicas , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Polifenóis/análise , Polifenóis/metabolismo , Análise de Componente Principal , Prunus/enzimologia , Prunus/genética , Rafinose/análise , Rafinose/metabolismo , Sacarose/análise , Sacarose/metabolismo , Álcoois Açúcares/análise , Álcoois Açúcares/metabolismoRESUMO
Shipping of peaches to distant markets and storage require low temperature; however, cold storage affects fruit quality causing physiological disorders collectively termed 'chilling injury' (CI). In order to ameliorate CI, different strategies have been applied before cold storage; among them heat treatment (HT) has been widely used. In this work, the effect of HT on peach fruit quality as well as on carbon metabolism was evaluated. When fruit were exposed to 39 degrees C for 3 d, ripening was delayed, with softening inhibition and slowing down of ethylene production. Several differences were observed between fruit ripening at ambient temperature versus fruit that had been heat treated. However, the major effects of HT on carbon metabolism and organoleptic characteristics were reversible, since normal fruit ripening was restored after transferring heated peaches to ambient temperature. Positive quality features such as an increment in the fructose content, largely responsible for the sweetness, and reddish coloration were observed. Nevertheless, high amounts of acetaldehyde and low organic acid content were also detected. The differential proteome of heated fruit was characterized, revealing that heat-induced CI tolerance may be acquired by the activation of different molecular mechanisms. Induction of related stress proteins in the heat-exposed fruits such as heat shock proteins, cysteine proteases, and dehydrin, and repression of a polyphenol oxidase provide molecular evidence of candidate proteins that may prevent some of the CI symptoms. This study contributes to a deeper understanding of the cellular events in peach under HT in view of a possible technological use aimed to improve organoleptic and shelf-life features.
Assuntos
Frutas/genética , Proteômica , Prunus/genética , Eletroforese em Gel Bidimensional , Etilenos/metabolismo , Frutas/química , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Temperatura Alta , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Prunus/química , Prunus/metabolismoRESUMO
Peach (Prunus persica L. Batsch) is a climacteric fruit that ripens after harvest, prior to human consumption. Organic acids and soluble sugars contribute to the overall organoleptic quality of fresh peach; thus, the integrated study of the metabolic pathways controlling the levels of these compounds is of great relevance. Therefore, in this work, several metabolites and enzymes involved in carbon metabolism were analysed during the post-harvest ripening of peach fruit cv 'Dixiland'. Depending on the enzyme studied, activity, protein level by western blot, or transcript level by quantitative real time-PCR were analysed. Even though sorbitol did not accumulate at a high level in relation to sucrose at harvest, it was rapidly consumed once the fruit was separated from the tree. During the ripening process, sucrose degradation was accompanied by an increase of glucose and fructose. Specific transcripts encoding neutral invertases (NIs) were up-regulated or down-regulated, indicating differential functions for each putative NI isoform. Phosphoenolpyruvate carboxylase was markedly induced, and may participate as a glycolytic shunt, since the malate level did not increase during post-harvest ripening. The fermentative pathway was highly induced, with increases in both the acetaldehyde level and the enzymes involved in this process. In addition, proteins differentially expressed during the post-harvest ripening process were also analysed. Overall, the present study identified enzymes and pathways operating during the post-harvest ripening of peach fruit, which may contribute to further identification of varieties with altered levels of enzymes/metabolites or in the evaluation of post-harvest treatments to produce fruit of better organoleptic attributes.
Assuntos
Metabolismo dos Carboidratos , Compostos Orgânicos/metabolismo , Proteínas de Plantas/metabolismo , Prunus/enzimologia , Frutas/metabolismo , Proteínas de Plantas/genética , Proteoma/genética , Proteoma/metabolismo , Prunus/genética , Prunus/metabolismoRESUMO
The zinc-finger cellular nucleic acid binding protein (CNBP) is a strikingly conserved single-stranded nucleic acid binding protein essential for normal forebrain formation during mouse and chick embryogenesis. CNBP cDNAs from a number of vertebrates have been cloned and analysed. CNBP is mainly conformed by seven retroviral Cys-Cys-His-Cys zinc-knuckles and a glycine/arginine rich region box. CNBP amino acid sequences show a putative Pro-Glu-Ser-Thr site of proteolysis and several putative phosphorylation sites. In this study, we analysed CNBP phosphorylation by embryonic kinases and its consequences on CNBP biochemical activities. We report that CNBP is differentially phosphorylated by Danio rerio embryonic extracts. In vitro CNBP phosphorylation is basal and constant at early embryonic developmental stages, it begins to increase after mid-blastula transition stage reaching the highest level at 48 hours postfertilization stage, and decreases thereafter to basal levels at 5 days postfertilization. The cAMP-dependent protein kinase (PKA) was identified as responsible for phosphorylation on the unique CNBP conserved putative phosphorylation site. Site-directed mutagenesis replacing the PKA phospho-acceptor amino acid residue impairs CNBP phosphorylation, suggesting that phosphorylation may not only exist in D. rerio but also in other vertebrates. CNBP phosphorylation does not change single-stranded nucleic acid binding capability. Instead, it promotes in vitro the annealing of complementary oligonucleotides representing the CT element (CCCTCCCC) from the human cellular myelocytomatosis oncogene (c-myc) promoter, an element responsible for c-myc enhancer transcription. Our results suggest that phosphorylation might be a conserved post-translational modification that allows CNBP to perform a fine tune expression regulation of a group of target genes, including c-myc, during vertebrate embryogenesis.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Ligação a RNA/química , Proteínas de Peixe-Zebra/química , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Fosforilação , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/fisiologia , Homologia de Sequência de Aminoácidos , Serina/química , Treonina/química , Transcrição Gênica , Ativação Transcricional , Peixe-Zebra , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Here we analyse the structural organisation and expression of the zebrafish cellular nucleic acid-binding protein (zCNBP) gene and protein. The gene is organised in five exons and four introns. A noteworthy feature of the gene is the absence of a predicted promoter region. The coding region encodes a 163-amino acid polypeptide with the highly conserved general structural organisation of seven CCHC Zn knuckle domains and an RGG box between the first and the second Zn knuckles. Although theoretical alternative splicing is possible, only one form of zCNBP is actually detected. This form is able to bind to single-stranded DNA and RNA probes in vitro. The analysis of zCNBP developmental expression shows a high amount of CNBP-mRNA in ovary and during the first developmental stages. CNBP-mRNA levels decrease while early development progresses until the midblastula transition (MBT) stage and increases again thereafter. The protein is localised in the cytoplasm of blastomeres whereas it is mainly nuclear in developmental stages after the MBT. These findings suggest that CNBP is a strikingly conserved single-stranded nucleic acid-binding protein which might interact with maternal mRNA during its storage in the embryo cell cytoplasm. It becomes nuclear once MBT takes place possibly in order to modulate zygotic transcription and/or to associate with newly synthesised transcripts.
Assuntos
Proteínas de Ligação a RNA/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes/genética , Imuno-Histoquímica , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Ovário/embriologia , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sondas RNA/genética , Sondas RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismoRESUMO
Protein transport across organelles' membranes requires that precursor proteins adopt an unfolded structure in order to be translocated by the import machinery. Ferredoxin-NADP+ reductase precursor, as well as many others, acquires a tightly folded structure that needs to be unfolded before or during its import. Several steps of chloroplast protein import are not fully understood. In particular, the role of different regions of the precursor protein has not been completely elucidated. In this work, we have studied the import into chloroplasts of precursor proteins with inclusions of amino acid spacers between the transit peptide and the mature protein, and with deletions in the N-terminal region of the mature enzyme. We measured the import rate constants for these precursors and the results indicate that the distance between the transit peptide and the core of the mature protein determines the import kinetics. The longer precursors were imported into the organelle faster than the wild type form. Precursors with deletions in the N-terminal region of the mature protein also showed increased import rates compared to the wild type. Homology studies amongst all family members reveal that only chloroplastic proteins possess this region. We suggest that even if the first amino acids of the mature protein do not contribute to its overall structural stability, they condition the kinetic parameters of the import reaction. Besides, the distance between the transit peptide and the mature protein core may be modulating the import rate at which the chloroplast incorporates this protein from the cytosol.