RESUMO
Lung cancer in East Asia is characterized by a high percentage of never-smokers, early onset and predominant EGFR mutations. To illuminate the molecular phenotype of this demographically distinct disease, we performed a deep comprehensive proteogenomic study on a prospectively collected cohort in Taiwan, representing early stage, predominantly female, non-smoking lung adenocarcinoma. Integrated genomic, proteomic, and phosphoproteomic analysis delineated the demographically distinct molecular attributes and hallmarks of tumor progression. Mutational signature analysis revealed age- and gender-related mutagenesis mechanisms, characterized by high prevalence of APOBEC mutational signature in younger females and over-representation of environmental carcinogen-like mutational signatures in older females. A proteomics-informed classification distinguished the clinical characteristics of early stage patients with EGFR mutations. Furthermore, integrated protein network analysis revealed the cellular remodeling underpinning clinical trajectories and nominated candidate biomarkers for patient stratification and therapeutic intervention. This multi-omic molecular architecture may help develop strategies for management of early stage never-smoker lung adenocarcinoma.
Assuntos
Progressão da Doença , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteogenômica , Fumar/genética , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinógenos/toxicidade , Estudos de Coortes , Citosina Desaminase/metabolismo , Ásia Oriental , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Genoma Humano , Humanos , Metaloproteinases da Matriz/metabolismo , Mutação/genética , Análise de Componente PrincipalRESUMO
Endogenous peptide hormones represent an essential class of biomolecules, which regulate cell-cell communications in diverse physiological processes of organisms. Mass spectrometry (MS) has been developed to be a powerful technology for identifying and quantifying peptides in a highly efficient manner. However, it is difficult to directly identify these peptide hormones due to their diverse characteristics, dynamic regulations, low abundance, and existence in a complicated biological matrix. Here, we summarize and discuss the roles of targeted and untargeted MS in discovering peptide hormones using bioassay-guided purification, bioinformatics screening, or the peptidomics-based approach. Although the peptidomics approach is expected to discover novel peptide hormones unbiasedly, only a limited number of successful cases have been reported. The critical challenges and corresponding measures for peptidomics from the steps of sample preparation, peptide extraction, and separation to the MS data acquisition and analysis are also discussed. We also identify emerging technologies and methods that can be integrated into the discovery platform toward the comprehensive study of endogenous peptide hormones.
RESUMO
Exposure to the physicochemical agents that interact with nucleic acids (NA) may lead to modification of DNA and RNA (i.e., NA modifications), which have been associated with various diseases, including cancer. The emerging field of NA adductomics aims to identify both known and unknown NA modifications, some of which may also be associated with proteins. One of the main challenges for adductomics is the processing of massive and complex data generated by high-resolution tandem mass spectrometry (HR-MS/MS). To address this, we have developed a software called "FeatureHunter", which provides the automated extraction, annotation, and classification of different types of key NA modifications based on the MS and MS/MS spectra acquired by HR-MS/MS, using a user-defined feature list. The capability and effectiveness of FeatureHunter was demonstrated by analyzing various NA modifications induced by formaldehyde or chlorambucil in mixtures of calf thymus DNA, yeast RNA and proteins, and by analyzing the NA modifications present in the pooled urines of smokers and nonsmokers. The incorporation of FeatureHunter into the NA adductomics workflow offers a powerful tool for the identification and classification of various types of NA modifications induced by reactive chemicals in complex biological samples, providing a valuable resource for studying the exposome.
Assuntos
Expossoma , Ácidos Nucleicos , Espectrometria de Massas em Tandem/métodos , Adutos de DNA , Fluxo de Trabalho , Software , RNARESUMO
Late blight (LB) disease is a major threat to potato and tomato production. It is caused by the hemibiotrophic pathogen, Phytophthora infestans. P. infestans can destroy all of the major organs in plants of susceptible crops and result in a total loss of productivity. At the early pathogenesis stage, this hemibiotrophic oomycete pathogen causes an asymptomatic biotrophic infection in hosts, which then progresses to a necrotrophic phase at the later infection stage. In this study, to examine how the tomato proteome is regulated by P. infestans at different stages of pathogenesis, a data-independent acquisition (DIA) proteomics approach was used to trace the dynamics of the protein regulation. A comprehensive picture of the regulation of tomato proteins functioning in the immunity, signaling, defense, and metabolism pathways at different stages of P. infestans infection is revealed. Among the regulated proteins, several involved in mediating plant defense responses were found to be differentially regulated at the transcriptional or translational levels across different pathogenesis phases. This study increases understanding of the pathogenesis of P. infestans in tomato and also identifies key transcriptional and translational events possibly targeted by the pathogen during different phases of its life cycle, thus providing novel insights for developing a new strategy towards better control of LB disease in tomato.
Assuntos
Regulação da Expressão Gênica de Plantas , Doenças das Plantas/genética , Proteoma/genética , Solanum lycopersicum/genética , Resistência à Doença , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Phytophthora/patogenicidade , Doenças das Plantas/microbiologia , Proteoma/metabolismoRESUMO
As sessile organisms, plants are exposed to diverse abiotic and biotic stresses, and thus have developed complex signaling mechanisms that orchestrate multiple stress responses. Plant peptides have recently emerged as key signaling molecules of stress responses, not only to mechanical wounding and pathogen infection but also to nutrient imbalance, drought and high salinity. The currently identified stress-related signaling peptides in plants are derived from proteolytic processing of protein precursors. Here, we review these protein-derived peptides and the evidence for their functions in stress signaling. We recommend potential research directions that could clarify their roles in stress biology, and propose possible crosstalk with regard to the physiological outcome. The stress-centric perspective allows us to highlight the crucial roles of peptides in regulating the dynamics of stress physiology. Inspired by historic and recent findings, we review how peptides initiate complex molecular interactions to coordinate biotic and abiotic stress responses in plants.
Assuntos
Adaptação Fisiológica , Genes de Plantas , Peptídeos/metabolismo , Proteínas de Plantas , Plantas , Precursores de Proteínas/metabolismo , Estresse Fisiológico , Adaptação Fisiológica/genética , Resistência à Doença/genética , Secas , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/genética , Reguladores de Crescimento de Plantas/genética , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Plantas/metabolismo , Salinidade , Transdução de Sinais , Estresse Fisiológico/genéticaRESUMO
Septins are critical for numerous cellular processes through the formation of heteromeric filaments and rings indicating the importance of structural regulators in septin assembly. Several posttranslational modifications (PTMs) mediate the dynamics of septin filaments in yeast. However, little is known about the role of PTMs in regulating mammalian septin assembly, and the in vivo significance of PTMs on mammalian septin assembly and function remains unknown. Here, we showed that SEPT12 was phosphorylated on Ser198 using mass spectrometry, and we generated SEPT12 phosphomimetic knock-in (KI) mice to study its biological significance. The homozygous KI mice displayed poor male fertility due to deformed sperm with defective motility and loss of annulus, a septin-based ring structure. Immunohistochemistry of KI testicular sections suggested that SEPT12 phosphorylation inhibits septin ring assembly during annulus biogenesis. We also observed that SEPT12 was phosphorylated via PKA, and its phosphorylation interfered with SEPT12 polymerization into complexes and filaments. Collectively, our data indicate that SEPT12 phosphorylation inhibits SEPT12 filament formation, leading to loss of the sperm annulus/septin ring and poor male fertility. Thus, we provide the first in vivo genetic evidence characterizing importance of septin phosphorylation in the assembly, cellular function and physiological significance of septins.
Assuntos
Infertilidade Masculina/genética , Septinas/genética , Motilidade dos Espermatozoides/genética , Espermatozoides/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Células HEK293 , Humanos , Imuno-Histoquímica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mutação , Fosforilação , Septinas/metabolismo , Homologia de Sequência de Aminoácidos , Serina/genética , Serina/metabolismo , Espermatozoides/ultraestruturaRESUMO
To identify endogenous peptides using MS/MS analysis and searching against a polypeptide sequence database, a non-enzyme specific (NES) search considering all of the possible proteolytic cleavages is required. However, the use of a NES search generates more false positive hits than an enzyme specific search, and therefore shows lower identification performance. In this study, the use of the sub-ranked matches for improving the identification performance of the Mascot NES search was investigated and a new scoring method was developed that considered the contribution of all sub-ranked random match probabilities, named the contribution score (CS). The CS showed the highest identification sensitivity using the Mascot NES search with a full protein database when compared to the use of the Mascot first ranked score and the delta score (DS). The confident peptides identified by DS and CS were shown to be complementary. When applied to plant endogenous peptide identification, the identification numbers of tomato endogenous peptides using DS and CS were 176.3% and 184.2%, respectively, higher than the use of the first ranked score of Mascot. The combination of DS and CS identified 200.0% and 8.6% more tomato endogenous peptides compared to the use of Mascot and DS, respectively. This method by combining the CS and DS can significantly improve the identification performance of endogenous peptides without complex computational steps and is also able to improve the identification performance of the enzyme specific search. In addition to the application in the plant peptidomics analysis, this method may be applied to the improvement of peptidomics studies in different species. A web interface for calculating the DS and CS based on Mascot search results was developed herein.
Assuntos
Algoritmos , Peptídeos/análise , Espectrometria de Massas em Tandem/métodos , Animais , Bovinos , Cromatografia Líquida/métodos , Bases de Dados de Proteínas , Escherichia coli , Humanos , Solanum lycopersicum/química , Proteínas de Plantas/análise , Coelhos , Saccharomyces cerevisiae , Ferramenta de BuscaRESUMO
Plants and pathogens are entangled in a continual arms race. Plants have evolved dynamic defence and immune mechanisms to resist infection and enhance immunity for second wave attacks from the same or different types of pathogenic species. In addition to evolutionarily and physiological changes, plant-pathogen interaction is also highly dynamic at the molecular level. Recently, an emerging quantitative mass spectrometry-based proteomics approach named data-independent acquisition (DIA), has been developed for the analysis of the proteome in a high-throughput fashion. In this study, the DIA approach was applied to quantitatively trace the change in the plant proteome from the early to the later stage of pathogenesis progression. This study revealed that at the early stage of the pathogenesis response, proteins directly related to the chaperon were regulated for the defence proteins. At the later stage, not only the defence proteins but also a set of the pathogen-associated molecular pattern-triggered immunity (PTI) and effector triggered immunity (ETI)-related proteins were highly induced. Our findings show the dynamics of the plant regulation of pathogenesis at the protein level and demonstrate the potential of using the DIA approach for tracing the dynamics of the plant proteome during pathogenesis responses.
Assuntos
Resistência à Doença/imunologia , Doenças das Plantas/imunologia , Proteoma/imunologia , Solanum lycopersicum/genética , Resistência à Doença/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/imunologia , Solanum lycopersicum/microbiologia , Doenças das Plantas/genética , Imunidade Vegetal/genética , Proteoma/genética , Proteômica/métodos , Pseudomonas syringae/imunologia , Pseudomonas syringae/patogenicidadeRESUMO
BACKGROUND: Transcriptomic sequencing (RNA-seq) related applications allow for rapid explorations due to their high-throughput and relatively fast experimental capabilities, providing unprecedented progress in gene functional annotation, gene regulation analysis, and environmental factor verification. However, with increasing amounts of sequenced reads and reference model species, the selection of appropriate reference species for gene annotation has become a new challenge. METHODS: We proposed a novel approach for finding the most effective reference model species through taxonomic associations and ultra-conserved orthologous (UCO) gene comparisons among species. An online system for multiple species selection (MSS) for RNA-seq differential expression analysis was developed, and comprehensive genomic annotations from 291 reference model eukaryotic species were retrieved from the RefSeq, KEGG, and UniProt databases. RESULTS: Using the proposed MSS pipeline, gene ontology and biological pathway enrichment analysis can be efficiently achieved, especially in the case of transcriptomic analysis of non-model organisms. The results showed that the proposed method solved problems related to limitations in annotation information and provided a roughly twenty-fold reduction in computational time, resulting in more accurate results than those of traditional approaches of using a single model reference species or the large non-redundant reference database. CONCLUSIONS: Selection of appropriate reference model species helps to reduce missing annotation information, allowing for more comprehensive results than those obtained with a single model reference species. In addition, adequate model species selection reduces the computational time significantly while retaining the same order of accuracy. The proposed system indeed provides superior performance by selecting appropriate multiple species for transcriptomic analysis compared to traditional approaches.
Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Genoma , Modelos Biológicos , Anotação de Sequência Molecular , Transcriptoma , Animais , Bactérias/genética , Ontologia Genética , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Plantas/genética , Padrões de Referência , Especificidade da EspécieRESUMO
Male infertility has become a worldwide health problem, but the etiologies of most cases are still unknown. SEPT12, a GTP-binding protein, is involved in male fertility. Two SEPT12 mutations (SEPT12(T89M) and SEPT12(D197N)) have been identified in infertile men who have a defective sperm annulus with a bent tail. The function of SEPT12 in the sperm annulus is still unclear. Here, we found that SEPT12 formed a filamentous structure with SEPT7, SEPT 6, SEPT2 and SEPT4 at the sperm annulus. The SEPT12-based septin core complex was assembled as octameric filaments comprising the SEPT proteins 12-7-6-2-2-6-7-12 or 12-7-6-4-4-6-7-12. In addition, the GTP-binding domain of SEPT12 was crucial for its interaction with SEPT7, and the N- and C-termini of SEPT12 were required for the interaction of SEPT12 with itself to polymerize octamers into filaments. Mutant mice carrying the SEPT12(D197N) mutation, which disrupts SEPT12 filament formation, showed a disorganized sperm annulus, bent tail, reduced motility and loss of the SEPT ring structure at the sperm annulus. These phenotypes were also observed in an infertile man carrying SEPT12(D197N). Taken together, our results demonstrate the molecular architecture of SEPT12 filaments at the sperm annulus, their mechanical support of sperm motility, and their correlation with male infertility.
Assuntos
Citoesqueleto/metabolismo , Infertilidade Masculina/metabolismo , Septinas/metabolismo , Motilidade dos Espermatozoides , Cauda do Espermatozoide/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Citoesqueleto/genética , Humanos , Infertilidade Masculina/genética , Masculino , Camundongos , Camundongos Mutantes , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Septinas/genéticaRESUMO
In vitro-transcribed suppressor tRNAs are commonly used in site-specific fluorescence labeling for protein and ribosome-bound nascent chains (RNCs) studies. Here, we describe the production of nonorthogonal Bacillus subtilis tRNA(cys)(Amber) from Escherichia coli, a process that is superior to in vitro transcription in terms of yield, ease of manipulation, and tRNA stability. As cysteinyl-tRNA synthetase was previously shown to aminoacylate tRNA(cys)(Amber) with lower efficiency, multiple tRNA synthetase mutants were designed to optimize aminoacylation. Aminoacylated tRNA was conjugated to a fluorophore to produce BODIPY FL-cysteinyl-tRNA(cys)(Amber), which was used to generate ribosome-bound nascent chains of different lengths with the fluorophore incorporated at various predetermined sites. This tRNA tool may be beneficial in the site-specific labeling of full-length proteins as well as RNCs for biophysical and biological research.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Bacillus subtilis/genética , Escherichia coli/genética , RNA de Transferência de Cisteína/biossíntese , RNA de Transferência de Cisteína/química , Aminoacil-tRNA Sintetases/genética , Sistema Livre de Células , Escherichia coli/metabolismo , Corantes Fluorescentes/química , Técnicas In Vitro , Modelos Moleculares , Biossíntese de Proteínas , Estabilidade de RNA , RNA Bacteriano/biossíntese , RNA Bacteriano/química , RNA Bacteriano/genética , RNA de Transferência de Cisteína/genética , Aminoacilação de RNA de TransferênciaRESUMO
Many important cell-to-cell communication events in multicellular organisms are mediated by peptides, but only a few peptides have been identified in plants. In an attempt to address the difficulties in identifying plant signaling peptides, we developed a novel peptidomics approach and used this approach to discover defense signaling peptides in plants. In addition to the canonical peptide systemin, several novel peptides were confidently identified in tomato (Solanum lycopersicum) and quantified to be induced by both wounding and methyl jasmonate (MeJA). A wounding or wounding plus MeJA-induced peptide derived from the pathogenesis-related protein 1 (PR-1) family was found to induce significant antipathogen and minor antiherbivore responses in tomato. This study highlights a role for PR-1 in immune signaling and suggests the potential application of plant endogenous peptides in efforts to defeat biological threats in crop production. As PR-1 is highly conserved across many organisms and the putative peptide from At-PR1 was also found to be bioactive in Arabidopsis thaliana, our results suggest that this peptide may be useful for enhancing resistance to stress in other plant species.
Assuntos
Peptídeos/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Solanum lycopersicum/metabolismo , Acetatos/farmacologia , Sequência de Aminoácidos , Cromatografia Líquida , Ciclopentanos/farmacologia , Resistência à Doença/efeitos dos fármacos , Resistência à Doença/genética , Resistência à Doença/imunologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Solanum lycopersicum/genética , Solanum lycopersicum/microbiologia , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Oxilipinas/farmacologia , Peptídeos/genética , Peptídeos/farmacologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Reguladores de Crescimento de Plantas/farmacologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteoma/genética , Proteoma/farmacologia , Proteômica , Pseudomonas syringae/imunologia , Pseudomonas syringae/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Mecânico , Espectrometria de Massas em Tandem , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Transcriptoma/imunologiaRESUMO
MicroRNA399-mediated regulation of the ubiquitin-conjugating enzyme UBC24/phosphate2 (PHO2) is crucial for Pi acquisition and translocation in plants. Because of a potential role for PHO2 in protein degradation and its association with membranes, an iTRAQ (for isobaric tags for relative and absolute quantitation)- based quantitative membrane proteomic method was employed to search for components downstream of PHO2. A total of 7491 proteins were identified from Arabidopsis thaliana roots by mass spectrometry, 35.2% of which were predicted to contain at least one transmembrane helix. Among the quantifiable proteins, five were significantly differentially expressed between the wild type and pho2 mutant under two growth conditions. Using immunoblot analysis, we validated the upregulation of several members in phosphate transporter1 (PHT1) family and phosphate transporter traffic facilitator1 (PHF1) in pho2 and demonstrated that PHO2 mediates the degradation of PHT1 proteins. Genetic evidence that loss of PHF1 or PHT1;1 alleviated Pi toxicity in pho2 further suggests that they play roles as downstream components of PHO2. Moreover, we showed that PHO2 interacts with PHT1s in the postendoplasmic reticulum compartments and mediates the ubiquitination of endomembrane-localized PHT1;1. This study not only uncovers a mechanism by which PHO2 modulates Pi acquisition by regulating the abundance of PHT1s in the secretory pathway destined for plasma membranes, but also provides a database of the membrane proteome that will be widely applicable in root biology research.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Transporte de Fosfato/metabolismo , Raízes de Plantas/enzimologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Retículo Endoplasmático/enzimologia , Regulação da Expressão Gênica de Plantas , Complexo de Golgi/enzimologia , Fosfatos/metabolismo , Mapeamento de Interação de Proteínas , Proteólise , Proteoma/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , UbiquitinaçãoRESUMO
Iron was previously shown to induce rapid nuclear translocation of a Myb3 transcription factor in the protozoan parasite, Trichomonas vaginalis. In the present study, iron was found to induce a transient increase in cellular cAMP, followed by the nuclear influx of Myb3, whereas the latter was also induced by 8-bromo-cyclic AMP. Iron-inducible cAMP production and nuclear influx of Myb3 were inhibited by suramin and SQ22536, respective inhibitors of the Gα subunit of heterotrimeric G proteins and adenylyl cyclases. In contrast, the nuclear influx of Myb3 induced by iron or 8-bromo-cAMP was delayed or inhibited, respectively, by H89, the inhibitor of protein kinase A. Using liquid chromatography-coupled tandem mass spectrometry, Thr(156) and Lys(143) in Myb3 were found to be phosphorylated and ubiquitinated, respectively. These modifications were induced by iron and inhibited by H89, as shown by immunoprecipitation-coupled Western blotting. Iron-inducible ubiquitination and nuclear influx were aborted in T156A and K143R, but T156D was constitutively ubiquitinated and persistently localized to the nucleus. Myb3 was phosphorylated in vitro by the catalytic subunit of a T. vaginalis protein kinase A, TvPKAc. A transient interaction between TvPKAc and Myb3 and the phosphorylation of both proteins were induced in the parasite shortly after iron or 8-bromo-cAMP treatment. Together, these observations suggest that iron may induce production of cAMP and activation of TvPKAc, which then induces the phosphorylation of Myb3 and subsequent ubiquitination for accelerated nuclear influx. It is conceivable that iron probably exerts a much broader impact on the physiology of the parasite than previously thought to encounter environmental changes.
Assuntos
Núcleo Celular/metabolismo , Ferro/metabolismo , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Trichomonas vaginalis/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Lisina/metabolismo , Dados de Sequência Molecular , Oligonucleotídeos/genética , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Ubiquitina/metabolismoRESUMO
The glutamic acid at position 100 (E(100)) in the capsid protein (CP) of Odontoglossum ringspot virus (ORSV) plays an important role in long-distance viral movement in Nicotiana benthamiana. The ORSV(E100A) mutant, which has a glutamic acid to alanine substitution, shows a loss of systemic infectivity in N. benthamiana. Transmission electron microscopy and size-exclusion chromatography assays showed that E(100) is essential for CP-CP interaction and viral particle assembly. To identify the ORSV triggering or response genes and CP-interacting proteins (CP-IP), an integrated omics approach based on next-generation sequencing and proteomics profiling was used in this study. The whole-transcriptomes of healthy and ORSV-infected leaves of N. benthamiana were analyzed, and the gene information was used to create a N. benthamiana protein database that was used for protein identification following mass spectrometry analysis. The integrated omics approach identified several putative host proteins that interact with ORSV CP(WT) and were categorized as photosystem subunits, defense-associated proteins, and cell division components. The expression pattern and CP interaction of these CP-IP were examined by semiquantitative reverse transcription polymerase chain reaction and an in vitro binding assay, respectively, to verify the in silico data. Among these proteins, a proteinase inhibitor of N. benthamiana (NbPI2) was highly associated with CP(E100A) as compared with CP(WT), and NbPI1 and NbPI2 were highly induced in ORSV-infected plants. NbPI1- and NbPI2-silenced plants (via a Tobacco rattle virus-induced gene-silencing system) did not exhibit a difference in ORSV infection. Thus, whether NbPI1 and NbPI2 play a role in plant immunity requires further investigation. In summary, the integrated omics approach provides massive and valuable information to identify the ORSV CP-IP and these CP-IP will help us to understand the movement of this virus and plant-virus interaction.
Assuntos
Proteínas do Capsídeo/metabolismo , Biologia Computacional , Nicotiana/genética , Doenças das Plantas/virologia , Proteínas de Plantas/metabolismo , Tobamovirus/metabolismo , Sequência de Aminoácidos , Proteínas do Capsídeo/genética , Genômica , Ácido Glutâmico , Modelos Moleculares , Dados de Sequência Molecular , Imunidade Vegetal , Folhas de Planta/virologia , Proteínas de Plantas/genética , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão , Alinhamento de Sequência , Análise de Sequência de DNA , Nicotiana/metabolismo , Nicotiana/virologia , Tobamovirus/genética , TranscriptomaRESUMO
Metabolite identification remains a bottleneck in mass spectrometry (MS)-based metabolomics. Currently, this process relies heavily on tandem mass spectrometry (MS/MS) spectra generated separately for peaks of interest identified from previous MS runs. Such a delayed and labor-intensive procedure creates a barrier to automation. Further, information embedded in MS data has not been used to its full extent for metabolite identification. Multimers, adducts, multiply charged ions, and fragments of given metabolites occupy a substantial proportion (40-80%) of the peaks of a quantitation result. However, extensive information on these derivatives, especially fragments, may facilitate metabolite identification. We propose a procedure with automation capability to group and annotate peaks associated with the same metabolite in the quantitation results of opposite modes and to integrate this information for metabolite identification. In addition to the conventional mass and isotope ratio matches, we would match annotated fragments with low-energy MS/MS spectra in public databases. For identification of metabolites without accessible MS/MS spectra, we have developed characteristic fragment and common substructure matches. The accuracy and effectiveness of the procedure were evaluated using one public and two in-house liquid chromatography-mass spectrometry (LC-MS) data sets. The procedure accurately identified 89% of 28 standard metabolites with derivative ions in the data sets. With respect to effectiveness, the procedure confidently identified the correct chemical formula of at least 42% of metabolites with derivative ions via MS/MS spectrum, characteristic fragment, and common substructure matches. The confidence level was determined according to the fulfilled identification criteria of various matches and relative retention time.
Assuntos
Metabolômica/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Cromatografia Líquida/métodos , Diabetes Mellitus Experimental/metabolismo , Dieta , Íons/análise , Íons/metabolismo , Metaboloma , Camundongos , RatosRESUMO
Here we demonstrate the use of strong anion-exchange fast performance liquid chromatography (FPLC) as a simple, fast, and robust method for RNA production by in vitro transcription. With this technique, we have purified different transcription templates from unreacted reagents in large quantities. The same buffer system could be used to readily remove nuclease contamination from the overexpressed pyrophosphatase, the important reagent for in vitro transcription. In addition, the method can be used to monitor in vitro transcription reactions to enable facile optimization of reaction conditions, and we have compared the separation performance between strong and weak anion-exchange FPLC for various transcribed RNAs, including the Diels-Alder ribozyme, the hammerhead ribozyme tRNA, and 4.5S RNA. The functionality of the purified tRNA(Cys) has been confirmed by the aminoacylation assay. Only the purification by strong anion-exchange FPLC has led to the enrichment of the functional tRNA from run-off transcripts as revealed by both enzymatic and electrophoretic analysis.
Assuntos
Ânions/química , Cromatografia por Troca Iônica , Cromatografia Líquida , Pirofosfatases/metabolismo , RNA/isolamento & purificação , Transcrição Gênica , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Escherichia coli/genética , Técnicas In Vitro , Pirofosfatases/genética , RNA/química , RNA Bacteriano/isolamento & purificação , RNA Catalítico/isolamento & purificação , RNA de Transferência/isolamento & purificaçãoRESUMO
The ERF (ethylene responsive factor) family is composed of transcription factors (TFs) that are critical for appropriate Arabidopsis thaliana responses to biotic and abiotic stresses. Here we identified and characterized a member of the ERF TF group IX, namely ERF96, that when overexpressed enhances Arabidopsis resistance to necrotrophic pathogens such as the fungus Botrytis cinerea and the bacterium Pectobacterium carotovorum. ERF96 is jasmonate (JA) and ethylene (ET) responsive and ERF96 transcripts accumulation was abolished in JA-insensitive coi1-16 and in ET-insensitive ein2-1 mutants. Protoplast transactivation and electrophoresis mobility shift analyses revealed that ERF96 is an activator of transcription that binds to GCC elements. In addition, ERF96 mainly localized to the nucleus. Microarray analysis coupled to chromatin immunoprecipitation-PCR of Arabidopsis overexpressing ERF96 revealed that ERF96 enhances the expression of the JA/ET defence genes PDF1.2a, PR-3 and PR-4 as well as the TF ORA59 by direct binding to GCC elements present in their promoters. While ERF96-RNAi plants demonstrated wild-type resistance to necrotrophic pathogens, basal PDF1.2 expression levels were reduced in ERF96-silenced plants. This work revealed ERF96 as a key player of the ERF network that positively regulates the Arabidopsis resistance response to necrotrophic pathogens.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Defensinas/metabolismo , Resistência à Doença , Doenças das Plantas/imunologia , Reguladores de Crescimento de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Botrytis/fisiologia , Ciclopentanos/metabolismo , Defensinas/genética , Etilenos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Análise de Sequência com Séries de Oligonucleotídeos , Oxilipinas/metabolismo , Folhas de Planta/genética , Folhas de Planta/imunologia , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes , Plântula/genética , Plântula/imunologia , Plântula/metabolismo , Fatores de Transcrição/genéticaRESUMO
High salinity has negative impacts on plant growth through altered water uptake and ion-specific toxicities. Plants have therefore evolved an intricate regulatory network in which plant hormones play significant roles in modulating physiological responses to salinity. However, current understanding of the plant peptides involved in this regulatory network remains limited. Here, we identified a salt-regulated peptide in Arabidopsis. The peptide was 11 aa and was derived from the C terminus of a cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) superfamily. This peptide was found by searching homologues in Arabidopsis using the precursor of a tomato CAP-derived peptide (CAPE) that was initially identified as an immune signal. In searching for a CAPE involved in salt responses, we screened CAPE precursor genes that showed salt-responsive expression and found that the PROAtCAPE1 (AT4G33730) gene was regulated by salinity. We confirmed the endogenous Arabidopsis CAP-derived peptide 1 (AtCAPE1) by mass spectrometry and found that a key amino acid residue in PROAtCAPE1 is critical for AtCAPE1 production. Moreover, although PROAtCAPE1 was expressed mainly in the roots, AtCAPE1 was discovered to be upregulated systemically upon salt treatment. The salt-induced AtCAPE1 negatively regulated salt tolerance by suppressing several salt-tolerance genes functioning in the production of osmolytes, detoxification, stomatal closure control, and cell membrane protection. This discovery demonstrates that AtCAPE1, a homologue of tomato immune regulator CAPE1, plays an important role in the regulation of salt stress responses. Our discovery thus suggests that the peptide may function in a trade-off between pathogen defence and salt tolerance.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Peptídeos/genética , Tolerância ao Sal , Cloreto de Sódio/farmacologia , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Regulação para Baixo , Peptídeos/química , Peptídeos/metabolismoRESUMO
A new G-quadruplex (G-4)-directing alkylating agent BMVC-C3M was designed and synthesized to integrate 3,6-bis(1-methyl-4-vinylpyridinium iodide)carbazole (BMVC) with aniline mustard. Various telomeric G-4 structures (hybrid-2 type and antiparallel) and an oncogene promoter, c-MYC (parallel), were constructed to react with BMVC-C3M, yielding 35 % alkylation yield toward G-4 DNA over other DNA categories (<6 %) and high specificity under competition conditions. Analysis of the intact alkylation adducts by electrospray ionization mass spectroscopy (ESI-MS) revealed the stepwise DNA alkylation mechanism of aniline mustard for the first time. Furthermore, the monoalkylation sites and intrastrand cross-linking sites were determined and found to be dependent on G-4 topology based on the results of footprinting analysis in combination with mass spectroscopic techniques and in silico modeling. The results indicated that BMVC-C3M preferentially alkylated at A15 (H26), G12 (H24), and G2 (c-MYC), respectively, as monoalkylated adducts and formed A15-C3M-A21 (H26), G12-C3M-G4 (H24), and G2-C3M-G4/G17 (c-MYC), respectively, as cross-linked dialkylated adducts. Collectively, the stability and site-selective cross-linking capacity of BMVC-C3M provides a credible tool for the structural and functional characterization of G-4 DNAs in biological systems.