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1.
Food Chem ; 304: 125428, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31476548

RESUMO

To protect allergic patients and guarantee correct food labeling, robust, specific and sensitive detection methods are urgently needed. Mass spectrometry (MS)-based methods could overcome the limitations of current detection techniques. The first step in the development of an MS-based method is the identification of biomarkers, which are, in the case of food allergens, peptides. Here, we implemented a strategy to identify the most salient peptide biomarkers in peanuts. Processed peanut matrices were prepared and analyzed using an untargeted approach via high-resolution MS. More than 300 identified peptides were further filtered using selection criteria to strengthen the analytical performance of a future, routine quantitative method. The resulting 16 peptides are robust to food processing, specific to peanuts, and satisfy sequence-based criteria. The aspect of multiple protein isoforms is also considered in the selection tree, an aspect that is essential for a quantitative method's robustness but seldom, if ever, considered.

2.
Food Chem ; : 125679, 2019 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-31718834

RESUMO

The interest of using LC-MS/MS as a method for detection of allergens in food is growing. In such methods, peptides are used as biomarkers for the detection and quantification of the allergens. The selection of good biomarker peptides is of high importance to develop a specific, universal and sensitive method. Biomarkers should, for example, be robust to food processing. To evaluate robustness, test material incurred with hazelnut having undergone different food processing techniques was produced. Proteins of these materials were extracted, digested and further analyzed using HRMS. After peptide identification, selection was carried out using several criteria such as hazelnut specificity and amino acid composition. Further selection was done by comparing peptide MS intensities in the different food matrices. Only peptides showing processing robustness were retained. Eventually, eight peptides coming from three major hazelnut proteins were selected as the best biomarkers for hazelnut detection in processed foods.

3.
Plant Physiol ; 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31570508

RESUMO

SNF1-RELATED PROTEIN KINASES 2 (SnRK2) are important components of early osmotic and salt stress signaling pathways in plants. The Arabidopsis (Arabidopsis thaliana) SnRK2 family comprises the abscisic acid (ABA)-activated protein kinases SnRK2.2, SnRK2.3, SnRK2.6, SnRK2.7, and SnRK2.8, and the ABA-independent subclass 1 protein kinases SnRK2.1, SnRK2.4, SnRK2.5, SnRK2.9, and SnRK2.10. ABA-independent SnRK2s act at the post-transcriptional level via phosphorylation of VARICOSE (VCS), a member of the mRNA decapping complex, that catalyzes the first step of 5'mRNA decay. Here, we identified VCS and VARICOSE RELATED (VCR) as interactors and phosphorylation targets of SnRK2.5, SnRK2.6, and SnRK2.10. All three protein kinases phosphorylated Ser645 and Ser1156 of VCS, while SnRK2.6 and SnRK2.10 also phosphorylated VCS Ser692 and Ser680 of VCR. We showed that subclass 1 SnRK2s, VCS, and 5' EXORIBONUCLEASE 4 (XRN4) are involved in regulating root growth under control conditions as well as modulating root system architecture in response to salt stress. Our results suggest interesting patterns of redundancy within subclass 1 SnRK2 protein kinases, with SnRK2.1, SnRK2.5 and SnRK2.9 controlling root growth under non-stress conditions and SnRK2.4 and SnRK2.10 acting mostly in response to salinity. We propose that subclass 1 SnRK2s function in root development under salt stress by affecting the transcript levels of aquaporins, as well as CYP79B2, an enzyme involved in auxin biosynthesis.

4.
Proc Natl Acad Sci U S A ; 116(42): 21256-21261, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31578252

RESUMO

Hydrogen peroxide (H2O2) is an important messenger molecule for diverse cellular processes. H2O2 oxidizes proteinaceous cysteinyl thiols to sulfenic acid, also known as S-sulfenylation, thereby affecting the protein conformation and functionality. Although many proteins have been identified as S-sulfenylation targets in plants, site-specific mapping and quantification remain largely unexplored. By means of a peptide-centric chemoproteomics approach, we mapped 1,537 S-sulfenylated sites on more than 1,000 proteins in Arabidopsis thaliana cells. Proteins involved in RNA homeostasis and metabolism were identified as hotspots for S-sulfenylation. Moreover, S-sulfenylation frequently occurred on cysteines located at catalytic sites of enzymes or on cysteines involved in metal binding, hinting at a direct mode of action for redox regulation. Comparison of human and Arabidopsis S-sulfenylation datasets provided 155 conserved S-sulfenylated cysteines, including Cys181 of the Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE4 (AtMAPK4) that corresponds to Cys161 in the human MAPK1, which has been identified previously as being S-sulfenylated. We show that, by replacing Cys181 of recombinant AtMAPK4 by a redox-insensitive serine residue, the kinase activity decreased, indicating the importance of this noncatalytic cysteine for the kinase mechanism. Altogether, we quantitatively mapped the S-sulfenylated cysteines in Arabidopsis cells under H2O2 stress and thereby generated a comprehensive view on the S-sulfenylation landscape that will facilitate downstream plant redox studies.

5.
Plant Physiol ; 181(2): 399-411, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31363006
6.
Nat Commun ; 10(1): 3288, 2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31337761

RESUMO

Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications.

7.
J Exp Bot ; 70(7): 1991-1995, 2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-31222306
8.
Proteomics ; 19(16): e1800435, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31241236

RESUMO

Mass spectrometry-based proteomics has been extensively used to map bacterial proteomes, which has led to a better understanding of the molecular mechanisms underlying bacterial infection and bacteria-host interactions. Quantitative proteomics using selected or parallel reaction monitoring is considered one of the most sensitive and specific quantitative MS-based approaches and has significantly advanced proteome studies of pathogenic bacteria. Here, recent applications of targeted proteomics for bacteria identification, biomarker discovery, and the characterization of bacterial virulence and antimicrobial resistance are reviewed among others. Results of such studies are expected to further contribute to improve the fight against the most common human pathogenic bacteria.

9.
Plant Cell Environ ; 42(9): 2612-2626, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31134630

RESUMO

Karrikins stimulate Arabidopsis thaliana germination, whereas parasitic weeds of the Orobanchaceae family have evolved to respond to host-exuded compounds such as strigolactones, dehydrocostus lactone, and 2-phenylethyl isothiocyanate. In Phelipanche ramosa, strigolactone-induced germination was shown to require one of the CYP707A proteins involved in abscisic acid catabolism. Here, germination and gene expression were analysed to investigate the role of CYP707As in germination of both parasitic plants and Arabidopsis upon perception of germination stimulants, after using pharmacological inhibitors and Arabidopsis mutants disrupting germination signals. CYP707A genes were up-regulated upon treatment with effective germination stimulants in both parasitic plants and Arabidopsis. Obligate parasitic plants exhibited both intensified up-regulation of CYP707A genes and increased sensitivity to the CYP707A inhibitor abscinazole-E2B, whereas Arabidopsis cyp707a mutants still positively responded to germination stimulation. In Arabidopsis, CYP707A regulation required the canonical karrikin signalling pathway KAI2/MAX2/SMAX1 and the transcription factor WRKY33. Finally, CYP707As and WRKY33 also modulated Arabidopsis root architecture in response to the synthetic strigolactone rac-GR24, and wrky33-1 exhibited a shoot hyperbranched phenotype. This study suggests that the lack of host-independent germination in obligate parasites is associated with an exacerbated CYP707A induction and that CYP707As and WRKY33 are new players involved in a variety of strigolactone/karrikin responses.

10.
Trends Biotechnol ; 37(10): 1078-1090, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31036349

RESUMO

Protein tags have been essential for advancing our knowledge of the function of proteins, their localization, and the mapping of their interaction partners. Expressing epitope-tagged proteins has become a standard practice in every life science laboratory and, thus, continues to enable new studies. In recent years, several new tagging moieties have entered the limelight, many of them bringing new functionalities, such as targeted protein degradation, accurate quantification, and proximity labeling. Other novel tags aim at tackling research questions in challenging niches. In this review, we elaborate on recently introduced tags and the opportunities they provide for future research endeavors. In addition, we highlight how the genome-engineering revolution may boost the field of protein tags.

11.
PLoS Negl Trop Dis ; 13(5): e0007416, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31125353

RESUMO

BACKGROUND: Salmonella enterica subsp. enterica contains more than 2,600 serovars of which four are of major medical relevance for humans. While the typhoidal serovars (Typhi and Paratyphi A) are human-restricted and cause enteric fever, non-typhoidal Salmonella serovars (Typhimurium and Enteritidis) have a broad host range and predominantly cause gastroenteritis. METHODOLOGY/PRINCIPLE FINDINGS: We compared the core proteomes of Salmonella Typhi, Paratyphi A, Typhimurium and Enteritidis using contemporary proteomics. For each serovar, five clinical isolates (covering different geographical origins) and one reference strain were grown in vitro to the exponential phase. Levels of orthologous proteins quantified in all four serovars and within the typhoidal and non-typhoidal groups were compared and subjected to gene ontology term enrichment and inferred regulatory interactions. Differential expression of the core proteomes of the typhoidal serovars appears mainly related to cell surface components and, for the non-typhoidal serovars, to pathogenicity. CONCLUSIONS/SIGNIFICANCE: Our comparative proteome analysis indicated differences in the expression of surface proteins between Salmonella Typhi and Paratyphi A, and in pathogenesis-related proteins between Salmonella Typhimurium and Enteritidis. Our findings may guide future development of novel diagnostics and vaccines, as well as understanding of disease progression.


Assuntos
Proteínas de Bactérias/genética , Proteoma/genética , Infecções por Salmonella/microbiologia , Salmonella enterica/genética , Salmonella paratyphi A/genética , Salmonella typhi/genética , Salmonella typhimurium/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Humanos , Proteoma/metabolismo , Salmonella enterica/metabolismo , Salmonella enterica/patogenicidade , Salmonella paratyphi A/metabolismo , Salmonella paratyphi A/patogenicidade , Salmonella typhi/metabolismo , Salmonella typhi/patogenicidade , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Virulência
12.
Nat Chem Biol ; 15(6): 641-649, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31011214

RESUMO

Clathrin-mediated endocytosis (CME) is a highly conserved and essential cellular process in eukaryotic cells, but its dynamic and vital nature makes it challenging to study using classical genetics tools. In contrast, although small molecules can acutely and reversibly perturb CME, the few chemical CME inhibitors that have been applied to plants are either ineffective or show undesirable side effects. Here, we identify the previously described endosidin9 (ES9) as an inhibitor of clathrin heavy chain (CHC) function in both Arabidopsis and human cells through affinity-based target isolation, in vitro binding studies and X-ray crystallography. Moreover, we present a chemically improved ES9 analog, ES9-17, which lacks the undesirable side effects of ES9 while retaining the ability to target CHC. ES9 and ES9-17 have expanded the chemical toolbox used to probe CHC function, and present chemical scaffolds for further design of more specific and potent CHC inhibitors across different systems.


Assuntos
Derivados de Benzeno/farmacologia , Cadeias Pesadas de Clatrina/antagonistas & inibidores , Endocitose/efeitos dos fármacos , Arabidopsis , Derivados de Benzeno/química , Cadeias Pesadas de Clatrina/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Tiofenos/farmacologia
13.
Plant J ; 99(4): 752-762, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31004550

RESUMO

Post-translational modifications (PTMs) of proteins are central in any kind of cellular signaling. Modern mass spectrometry technologies enable comprehensive identification and quantification of various PTMs. Given the increased numbers and types of mapped protein modifications, a database is necessary that simultaneously integrates and compares site-specific information for different PTMs, especially in plants for which the available PTM data are poorly catalogued. Here, we present the Plant PTM Viewer (http://www.psb.ugent.be/PlantPTMViewer), an integrative PTM resource that comprises approximately 370 000 PTM sites for 19 types of protein modifications in plant proteins from five different species. The Plant PTM Viewer provides the user with a protein sequence overview in which the experimentally evidenced PTMs are highlighted together with an estimate of the confidence by which the modified peptides and, if possible, the actual modification sites were identified and with functional protein domains or active site residues. The PTM sequence search tool can query PTM combinations in specific protein sequences, whereas the PTM BLAST tool searches for modified protein sequences to detect conserved PTMs in homologous sequences. Taken together, these tools help to assume the role and potential interplay of PTMs in specific proteins or within a broader systems biology context. The Plant PTM Viewer is an open repository that allows the submission of mass spectrometry-based PTM data to remain at pace with future PTM plant studies.

14.
Proc Natl Acad Sci U S A ; 116(17): 8597-8602, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30944225

RESUMO

In plants, postembryonic formation of new organs helps shape the adult organism. This requires the tight regulation of when and where a new organ is formed and a coordination of the underlying cell divisions. To build a root system, new lateral roots are continuously developing, and this process requires the tight coordination of asymmetric cell division in adjacent pericycle cells. We identified EXPANSIN A1 (EXPA1) as a cell wall modifying enzyme controlling the divisions marking lateral root initiation. Loss of EXPA1 leads to defects in the first asymmetric pericycle cell divisions and the radial swelling of the pericycle during auxin-driven lateral root formation. We conclude that a localized radial expansion of adjacent pericycle cells is required to position the asymmetric cell divisions and generate a core of small daughter cells, which is a prerequisite for lateral root organogenesis.

15.
Science ; 363(6433)2019 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-30898901

RESUMO

Physical damage to cells leads to the release of immunomodulatory peptides to elicit a wound defense response in the surrounding tissue. In Arabidopsis thaliana, the plant elicitor peptide 1 (Pep1) is processed from its protein precursor, PRECURSOR OF PEP1 (PROPEP1). We demonstrate that upon damage, both at the tissue and single-cell levels, the cysteine protease METACASPASE4 (MC4) is instantly and spatiotemporally activated by binding high levels of Ca2+ and is necessary and sufficient for Pep1 maturation. Cytosol-localized PROPEP1 and MC4 react only after loss of plasma membrane integrity and prolonged extracellular Ca2+ entry. Our results reveal that a robust mechanism consisting of conserved molecular components links the intracellular and Ca2+-dependent activation of a specific cysteine protease with the maturation of damage-induced wound defense signals.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/imunologia , Cálcio/metabolismo , Cisteína Endopeptidases/metabolismo , Imunomodulação , Imunidade Vegetal , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Citosol/enzimologia , Oligopeptídeos/metabolismo
16.
Sci Rep ; 9(1): 4787, 2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30886217

RESUMO

Pollens are well-known triggers of respiratory allergies and asthma. The pollen burden in today's ambient air is constantly increasing due to rising climate change and air pollution. How pollens interact with the respiratory mucosa remains largely unknown due to a lack of representative model systems. We here demonstrate how pollen proteases of Kentucky bluegrass, white birch and hazel selectively destroy integrity and anchorage of columnar respiratory epithelial cells, but not of basal cells, in both ex vivo respiratory mucosal explants and in vitro primary equine respiratory epithelial cells (EREC). In turn, this pollen protease-induced damage to respiratory epithelial cell anchorage resulted in increased infection by the host-specific and ancestral alphaherpesvirus equine herpesvirus type 1 (EHV1). Pollen proteases of all three plant species were characterized by zymography and those of white birch were fully identified for the first time as serine proteases of the subtilase family and meiotic prophase aminopeptidase 1 using mass spectrometry-based proteomics. Together, our findings demonstrate that pollen proteases selectively and irreversibly damage integrity and anchorage of columnar respiratory epithelial cells. In turn, alphaherpesviruses benefit from this partial loss-of-barrier function, resulting in increased infection of the respiratory epithelium.

17.
Nat Plants ; 5(3): 316-327, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30833711

RESUMO

The target of rapamycin (TOR) kinase is a conserved regulatory hub that translates environmental and nutritional information into permissive or restrictive growth decisions. Despite the increased appreciation of the essential role of the TOR complex in plants, no large-scale phosphoproteomics or interactomics studies have been performed to map TOR signalling events in plants. To fill this gap, we combined a systematic phosphoproteomics screen with a targeted protein complex analysis in the model plant Arabidopsis thaliana. Integration of the phosphoproteome and protein complex data on the one hand shows that both methods reveal complementary subspaces of the plant TOR signalling network, enabling proteome-wide discovery of both upstream and downstream network components. On the other hand, the overlap between both data sets reveals a set of candidate direct TOR substrates. The integrated network embeds both evolutionarily-conserved and plant-specific TOR signalling components, uncovering an intriguing complex interplay with protein synthesis. Overall, the network provides a rich data set to start addressing fundamental questions about how TOR controls key processes in plants, such as autophagy, auxin signalling, chloroplast development, lipid metabolism, nucleotide biosynthesis, protein translation or senescence.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Técnicas de Cultura de Células , Espectrometria de Massas/métodos , Fosfatidilinositol 3-Quinases/genética , Fosfoproteínas/metabolismo , Fosforilação , Plantas Geneticamente Modificadas , Mapeamento de Interação de Proteínas , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Plântula/metabolismo , Transdução de Sinais
18.
J Exp Bot ; 70(7): 2199-2210, 2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-30753577

RESUMO

During plant vascular development, xylem tracheary elements (TEs) form water-conducting, empty pipes by genetically regulated cell death. Cell death is prevented from spreading to non-TEs by unidentified intercellular mechanisms, downstream of METACASPASE9 (MC9)-mediated regulation of autophagy in TEs. Here, we identified differentially abundant extracellular peptides in vascular-differentiating wild-type and MC9-down-regulated Arabidopsis cell suspensions. A peptide named Kratos rescued the abnormally high ectopic non-TE death resulting from either MC9 knockout or TE-specific overexpression of the ATG5 autophagy protein during experimentally induced vascular differentiation in Arabidopsis cotyledons. Kratos also reduced cell death following mechanical damage and extracellular ROS production in Arabidopsis leaves. Stress-induced but not vascular non-TE cell death was enhanced by another identified peptide, named Bia. Bia is therefore reminiscent of several known plant cell death-inducing peptides acting as damage-associated molecular patterns. In contrast, Kratos plays a novel extracellular cell survival role in the context of development and during stress response.

19.
J Exp Bot ; 70(7): 2125-2141, 2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-30805604

RESUMO

Proteases are enzymes that cleave peptide bonds of other proteins. Their omnipresence and diverse activities make them important players in protein homeostasis and turnover of the total cell proteome as well as in signal transduction in plant stress responses and development. To understand protease function, it is of paramount importance to assess when and where a specific protease is active. Here, we review the existing methods to detect in vivo protease activity by means of imaging chemical activity-based probes and genetically encoded sensors. We focus on the diverse fluorescent and luminescent sensors at the researcher's disposal and evaluate the potential of imaging techniques to deliver in vivo spatiotemporal detail of protease activity. We predict that in the coming years, revised techniques will help to elucidate plant protease activity and functions and hence expand the current status of the field.

20.
J Proteome Res ; 18(1): 95-106, 2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30525648

RESUMO

The CRISPR/Cas9 revolution is profoundly changing the way life sciences technologies are used. Many assays now rely on engineered clonal cell lines to eliminate the overexpression of bait proteins. Control cell lines are typically nonengineered cells or engineered clones, implying a considerable risk for artifacts because of clonal variation. Genome engineering can also transform BioID, a proximity labeling method that relies on fusing a bait protein to a promiscuous biotin ligase, BirA*, resulting in the tagging of vicinal proteins. We here propose an innovative design to enable BioID for endogenous proteins wherein we introduce a T2A-BirA* module at the C-terminus of endogenous p53 by genome engineering, leading to bicistronic expression of both p53 and BirA* under control of the endogenous promoter. By targeting a Cas9-cytidine deaminase base editor to the T2A autocleavage site, we can efficiently derive an isogenic population expressing a functional p53-BirA* fusion protein. Using quantitative proteomics we show significant benefits over the classical ectopic expression of p53-BirA*, and we provide a first well-controlled view of the proximal proteins of endogenous p53 in colon carcinoma cells. This novel application for base editors expands the CRISPR/Cas9 toolbox and can be a valuable addition for synthetic biology.

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