ABSTRACT
BACKGROUND: The brown marmorated stink bug, Halyomorpha halys (Stål, 1855) (Hemiptera: Pentatomidae) is an invasive pest that causes economic damage on crops, decreasing fruit yield and quality. Conventional insecticides are frequently used to reduce infestations, but these are often with a limited residual effect, besides being costly and detrimental to nontarget organisms and the environment. In integrated pest management, novel strategies against H. halys are proposed, such as the use of alternative substances with an effect on insect behaviour and mobility. As one of the oldest multi-site fungicides applied against fungal pathogens and as an insecticide and acaricide to control scales and mites, sulfur is proposed here to reduce H. halys infestation in fruit orchards. RESULTS: Field experiments were performed to evaluate the effect of repeated wettable sulfur applications on H. halys in apple and pear orchards. Sulfur-induced plant phytotoxicity effects and quanti-qualitative parameters on apple fruits were also recorded. Halyomorpha halys infestation was significantly reduced in sulfur-treated compared to untreated pears and apples. Furthermore, sulfur sprays reduced fruit damage caused by H. halys. Besides, sulfur-mediated phytotoxicity such as symptoms on leaves and fruit drop were not observed. Fruit quality was not influenced by sulfur treatments. CONCLUSIONS: Wettable sulfur seems to be a promising formulation given the low phytotoxicity, considering the technical aspects for an effective use of sulfur-based products to counteract H. halys in pome fruit orchards. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
ABSTRACT
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,mass spectrometry (MS) and/or nuclear magnetic resonance (NMR) spectroscopy. As a non-destructive and non-targeted technique, NMR spectroscopy is uniquely capable of characterizing the chemical profiling of living cells and multicellular organisms. Here we use NMR spectroscopy to analyze C. 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 wild-type 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 wild type 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.
ABSTRACT
Artificial intelligence (AI)- and deep learning (DL)-based systems have shown significant progress in the field of macular disorders, demonstrating high performance in detecting retinal fluid and assessing anatomical changes during disease progression. This study aimed to validate an AI algorithm for identifying and quantifying prognostic factors in visual recovery after macular hole (MH) surgery by analyzing major optical coherence tomography (OCT) biomarkers. This study included 20 patients who underwent vitrectomy for a full-thickness macular hole (FTMH). The mean diameter of the FTMH was measured at 285.36 ± 97.4 µm. The preoperative best-corrected visual acuity (BCVA) was 0.76 ± 0.06 logMAR, improving to 0.38 ± 0.16 postoperatively, with a statistically significant difference (p = 0.001). AI software was utilized to assess biomarkers, such as intraretinal fluid (IRF) and subretinal fluid (SRF) volume, external limiting membrane (ELM) and ellipsoid zone (EZ) integrity, and retinal hyperreflective foci (HRF). The AI analysis showed a significant decrease in IRF volume, from 0.08 ± 0.12 mm3 preoperatively to 0.01 ± 0.01 mm3 postoperatively. ELM interruption improved from 79% ± 18% to 34% ± 37% after surgery (p = 0.006), whereas EZ interruption improved from 80% ± 22% to 40% ± 36% (p = 0.007) postoperatively. Additionally, the study revealed a negative correlation between preoperative IRF volume and postoperative BCVA recovery, suggesting that greater preoperative fluid volumes may hinder visual improvement. The integrity of the ELM and EZ was found to be essential for postoperative visual acuity improvement, with their disruption negatively impacting visual recovery. The study highlights the potential of AI in quantifying OCT biomarkers for managing MHs and improving patient care.
ABSTRACT
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.
Subject(s)
Biosensing Techniques , Humans , Methionine , Methionine Sulfoxide Reductases/metabolism , Oxidation-Reduction , Substrate SpecificityABSTRACT
Cardiac looping is an essential and highly conserved morphogenetic process that places the different regions of the developing vertebrate heart tube into proximity of their final topographical positions. High-resolution 4D live imaging of mosaically labelled cardiomyocytes reveals distinct cardiomyocyte behaviors that contribute to the deformation of the entire heart tube. Cardiomyocytes acquire a conical cell shape, which is most pronounced at the superior wall of the atrioventricular canal and contributes to S-shaped bending. Torsional deformation close to the outflow tract contributes to a torque-like winding of the entire heart tube between its two poles. Anisotropic growth of cardiomyocytes based on their positions reinforces S-shaping of the heart. During cardiac looping, bone morphogenetic protein pathway signaling is strongest at the future superior wall of the atrioventricular canal. Upon pharmacological or genetic inhibition of bone morphogenetic protein signaling, myocardial cells at the superior wall of the atrioventricular canal maintain cuboidal cell shapes and S-shaped bending is impaired. This description of cellular rearrangements and cardiac looping regulation may also be relevant for understanding the etiology of human congenital heart defects.
Subject(s)
Bone Morphogenetic Proteins/metabolism , Gene Expression Regulation, Developmental , Heart/embryology , Myocytes, Cardiac/metabolism , Signal Transduction , Animals , Anisotropy , Embryo, Nonmammalian/metabolism , Embryonic Development , Green Fluorescent Proteins/metabolism , Microscopy, Confocal , Morphogenesis , Organogenesis , Torque , Transcription Factors/metabolism , Wnt Proteins/metabolism , Wnt Signaling Pathway , Zebrafish/embryology , Zebrafish Proteins/metabolismABSTRACT
The aggregation of proteins into toxic conformations plays a critical role in the development of different neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Creutzfled-Jakob's disease (CJD). These disorders share a common pathological mechanism that involves the formation of aggregated protein species including toxic oligomers and amyloid fibrils. The aggregation of alpha-synuclein (αS) in PD and the amyloid beta peptide (Aß) and tau protein in AD results in neuronal death and disease onset. In the case of CJD, the misfolding of the physiological prion protein (PrP) induces a chain reaction that results in accumulation of particles that elicit brain damage. Currently, there is no preventive therapy for these diseases and the available therapeutic approaches are based on the treatment of the symptoms rather than the underlying causes of the disease. Accordingly, the aggregation pathway of these proteins represents a useful target for therapeutic intervention. Therefore, understanding the mechanism of amyloid formation and its inhibition is of high clinical importance. The design of small molecules that efficiently inhibit the aggregation process and/or neutralize its associated toxicity constitutes a promising tool for the development of therapeutic strategies against these disorders. In this accounts, we discuss current knowledge on the anti-amyloid activity of phthalocyanines and their potential use as drug candidates in neurodegeneration. These tetrapyrrolic compounds modulate the amyloid assembly of αS, tau, Aß, and the PrP in vitro, and protect cells from the toxic effects of amyloid aggregates. In addition, in scrapie-infected mice, these compounds showed important prophylactic antiscrapie properties. The structural basis for the inhibitory effect of phthalocyanines on amyloid filament assembly relies on specific π-π interactions between the aromatic ring system of these molecules and aromatic residues in the amyloidogenic proteins. Analysis of the structure-activity relationship in phthalocyanines revealed that their anti-amyloid activity is highly dependent on the type of metal ion coordinated to the tetrapyrrolic system but is not sensitive to the number of peripheral charged substituents. The tendency of phthalocyanines to oligomerize (self-association) via aromatic-aromatic stacking interactions correlates precisely with their binding capabilities to target proteins and, more importantly, determines their efficiency as anti-amyloid agents. The ability to block different types of disease-associated protein aggregation raises the possibility that these cyclic tetrapyrrole compounds have a common mechanism of action to impair the formation of a variety of pathological aggregates. Because the structural and molecular basis for the anti-amyloid effects of these molecules is starting to emerge, combined efforts from the fields of structural, cellular, and animal biology will result critical for the rational design and discovery of new drugs for the treatment of amyloid related neurological disorders.
Subject(s)
Indoles/chemistry , Neurodegenerative Diseases/metabolism , Proteins/metabolism , Humans , Isoindoles , Protein Binding , Proteins/chemistry , Structure-Activity RelationshipABSTRACT
The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.
Subject(s)
Cardiac Surgical Procedures/veterinary , Gene Expression Profiling/methods , Heart/embryology , Myocardium/chemistry , RNA, Messenger/isolation & purification , Zebrafish/embryology , Zebrafish/surgery , Animals , Animals, Genetically Modified , Cardiac Surgical Procedures/methods , Dissection/methods , Dissection/veterinary , Real-Time Polymerase Chain Reaction , Zebrafish Proteins/geneticsABSTRACT
During heart development, the onset of heartbeat and blood flow coincides with a ballooning of the cardiac chambers. Here, we have used the zebrafish as a vertebrate model to characterize chamber ballooning morphogenesis of the endocardium, a specialized population of endothelial cells that line the interior of the heart. By combining functional manipulations, fate mapping studies, and high-resolution imaging, we show that endocardial growth occurs without an influx of external cells. Instead, endocardial cell proliferation is regulated, both by blood flow and by Bmp signaling, in a manner independent of vascular endothelial growth factor (VEGF) signaling. Similar to myocardial cells, endocardial cells obtain distinct chamber-specific and inner- versus outer-curvature-specific surface area sizes. We find that the hemodynamic-sensitive transcription factor Klf2a is involved in regulating endocardial cell morphology. These findings establish the endocardium as the flow-sensitive tissue in the heart with a key role in adapting chamber growth in response to the mechanical stimulus of blood flow.
Subject(s)
Bone Morphogenetic Proteins/metabolism , Endocardium/embryology , Hemodynamics , Kruppel-Like Transcription Factors/metabolism , Morphogenesis , Zebrafish Proteins/metabolism , Animals , Cell Movement , Cell Proliferation , Endocardium/cytology , Endocardium/metabolism , Endothelial Cells/metabolism , Endothelial Cells/physiology , Kruppel-Like Transcription Factors/genetics , Signal Transduction , Vascular Endothelial Growth Factor A/metabolism , Zebrafish , Zebrafish Proteins/geneticsABSTRACT
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.
Subject(s)
Cold Temperature , Fruit/metabolism , Fruit/physiology , Hot Temperature , Metabolic Networks and Pathways , Prunus/metabolism , Prunus/physiology , Fruit/genetics , Gas Chromatography-Mass Spectrometry , Gene Expression Regulation, Plant , Metabolic Networks and Pathways/genetics , Metabolome/genetics , Metabolomics , Nitrogen/metabolism , Principal Component Analysis , Prunus/genetics , Quantitative Trait, Heritable , RNA, Messenger/genetics , RNA, Messenger/metabolism , Raffinose/metabolismABSTRACT
Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede, KikGR, Dendra and EosFP, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed ß-elimination and subsequent extension of a π-conjugated system. PCFPs and their monomeric variants are useful tools for tracking cells and studying protein dynamics, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish, chicken and mouse for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.
Subject(s)
Cell Tracking/methods , Luminescent Proteins/analysis , Zebrafish/embryology , Animals , Animals, Genetically Modified , Luminescent Proteins/chemistry , Photochemical Processes , Zebrafish/growth & developmentABSTRACT
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.
Subject(s)
Extracellular Space/metabolism , Fruit/metabolism , Plant Proteins/metabolism , Proteome , Prunus/metabolism , Amino Acid Oxidoreductases/genetics , Amino Acid Oxidoreductases/metabolism , Cell Wall/metabolism , Down-Regulation , Electrophoresis, Gel, Two-Dimensional , Fruit/cytology , Fruit/enzymology , Fruit/genetics , Gene Expression , Gene Expression Regulation, Plant , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Hot Temperature , Solanum lycopersicum/cytology , Solanum lycopersicum/enzymology , Solanum lycopersicum/genetics , Solanum lycopersicum/metabolism , Phenotype , Plant Proteins/genetics , Proteomics , Prunus/cytology , Prunus/enzymology , Prunus/genetics , RNA, Messenger/genetics , RNA, Plant/genetics , Tandem Mass Spectrometry , Up-RegulationABSTRACT
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.
Subject(s)
Fruit/growth & development , Gene Expression Regulation, Plant/physiology , Metabolome , Plant Proteins/metabolism , Prunus/growth & development , Prunus/metabolism , Amino Acids/analysis , Amino Acids/metabolism , Biological Transport , Carboxylic Acids/analysis , Carboxylic Acids/metabolism , Disaccharides/analysis , Disaccharides/metabolism , Enzymes/genetics , Enzymes/metabolism , Fruit/enzymology , Fruit/genetics , Fruit/metabolism , Gas Chromatography-Mass Spectrometry , Gene Expression Regulation, Enzymologic/physiology , Metabolic Networks and Pathways , Plant Leaves/enzymology , Plant Leaves/genetics , Plant Leaves/growth & development , Plant Leaves/metabolism , Plant Proteins/genetics , Polyphenols/analysis , Polyphenols/metabolism , Principal Component Analysis , Prunus/enzymology , Prunus/genetics , Raffinose/analysis , Raffinose/metabolism , Sucrose/analysis , Sucrose/metabolism , Sugar Alcohols/analysis , Sugar Alcohols/metabolismABSTRACT
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.
Subject(s)
Fruit/genetics , Proteomics , Prunus/genetics , Electrophoresis, Gel, Two-Dimensional , Ethylenes/metabolism , Fruit/chemistry , Fruit/metabolism , Gene Expression Regulation, Plant , Hot Temperature , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/metabolism , Prunus/chemistry , Prunus/metabolismABSTRACT
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.
Subject(s)
Carbohydrate Metabolism , Organic Chemicals/metabolism , Plant Proteins/metabolism , Prunus/enzymology , Fruit/metabolism , Plant Proteins/genetics , Proteome/genetics , Proteome/metabolism , Prunus/genetics , Prunus/metabolismABSTRACT
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.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Gene Expression Regulation, Developmental , RNA-Binding Proteins/chemistry , Zebrafish Proteins/chemistry , Amino Acid Sequence , Animals , Molecular Sequence Data , Phosphorylation , Promoter Regions, Genetic , RNA-Binding Proteins/physiology , Sequence Homology, Amino Acid , Serine/chemistry , Threonine/chemistry , Transcription, Genetic , Transcriptional Activation , Zebrafish , Zebrafish Proteins/physiologyABSTRACT
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.
Subject(s)
RNA-Binding Proteins/genetics , Zebrafish Proteins/genetics , Zebrafish/genetics , Alternative Splicing , Amino Acid Sequence , Animals , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , Embryo, Nonmammalian/metabolism , Embryonic Development , Female , Gene Expression Regulation, Developmental , Genes/genetics , Immunohistochemistry , In Situ Hybridization , Male , Molecular Sequence Data , Ovary/embryology , Ovary/growth & development , Ovary/metabolism , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA Probes/genetics , RNA Probes/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Sequence Alignment , Sequence Analysis, DNA , Zebrafish/embryology , Zebrafish/growth & development , Zebrafish Proteins/metabolismABSTRACT
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.