scholarly journals Upregulation of Jasmonate-Inducible Defense Proteins and Differential Colonization of Roots of Oryza sativa Cultivars with the Endophyte Azoarcus sp.

2006 ◽  
Vol 19 (5) ◽  
pp. 502-511 ◽  
Author(s):  
Lucie Miché ◽  
Federico Battistoni ◽  
Sabrina Gemmer ◽  
Maya Belghazi ◽  
Barbara Reinhold-Hurek
Keyword(s):  
Oryza Sativa ◽  
Bacterial Colonization ◽  
Defense Responses ◽  
Mass Spectrometry Analysis ◽  
Compatible Interaction ◽  
Defense Proteins ◽  
Spectrometry Analysis ◽  
Rice Roots ◽  
Proteomic Approach ◽  
Pathogenesis Related

The endophyte Azoarcus sp. strain BH72 expresses nitrogenase (nif) genes inside rice roots. We applied a proteomic approach to dissect responses of rice roots toward bacterial colonization and jasmonic acid (JA) treatment. Two sister lineages of Oryza sativa were analyzed with cv. IR42 showing a less compatible interaction with the Azoarcus sp. resulting in slight root browning whereas cv. IR36 was successfully colonized as determined by nifH::gusA activity. External addition of JA inhibited colonization of roots and caused browning in contrast to the addition of ethylene, applied as ethephon (up to 5 mM). Only two of the proteins induced in cv. IR36 by JA were also induced by the endophyte (SalT, two isoforms). In contrast, seven JA-induced proteins were also induced by bacteria in cv. IR42, indicating that IR42 showed a stronger defense response. Mass spectrometry analysis identified these proteins as pathogenesis-related (PR) proteins (Prb1, RSOsPR10) or proteins sharing domains with receptorlike kinases induced by pathogens. Proteins strongly induced in roots in both varieties by JA were identified as Bowman-Birk trypsin inhibittors, germinlike protein, putative endo-1,3-beta-D-glucosidase, glutathion-S-transferase, and 1-propane-1-carboxylate oxidase synthase, peroxidase precursor, PR10-a, and a RAN protein previously not found to be JA-induced. Data suggest that plant defense responses involving JA may contribute to restricting endophytic colonization in grasses. Remarkably, in a compatible interaction with endophytes, JA-inducible stress or defense responses are apparently not important.

scholarly journals Serum-Based Proteomics Profiling in Adult Patients with Cystic Fibrosis

2020 ◽  
Vol 21 (19) ◽  
pp. 7415
Author(s):  
Hicham Benabdelkamel ◽  
Hanadi Alamri ◽  
Meshail Okla ◽  
Afshan Masood ◽  
Mai Abdel Jabar ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Multiple Reaction Monitoring ◽  
Autosomal Recessive Disorder ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Serum Proteomics ◽  
Proteomic Approach ◽  
Significant Difference ◽  
Canonical Pathways ◽  
Cf Transmembrane Conductance Regulator

Cystic fibrosis (CF), the most common lethal autosomal recessive disorder among Caucasians, is caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel gene. Despite significant advances in the management of CF patients, novel disease-related biomarkers and therapies must be identified. We performed serum proteomics profiling in CF patients (n = 28) and healthy subjects (n = 10) using the 2D-DIGE MALDI-TOF proteomic approach. Out of a total of 198 proteins identified, 134 showed a statistically significant difference in abundance and a 1.5-fold change (ANOVA, p < 0.05), including 80 proteins with increased abundance and 54 proteins with decreased abundance in CF patients. A multiple reaction monitoring-mass spectrometry analysis of six differentially expressed proteins identified by a proteomic approach (DIGE-MALD-MS) showed a significant increase in C3 and CP proteins and a decrease in APOA1, Complement C1, Hp, and RBP4proteins compared with healthy controls. Fifteen proteins were identified as potential biomarkers for CF diagnosis. An ingenuity pathway analysis of the differentially regulated proteins indicates that the central nodes dysregulated in CF subjects involve pro-inflammatory cytokines, ERK1/2, and P38 MAPK, which are primarily involved in catalytic activities and metabolic processes. The involved canonical pathways include those related to FXR/RXR, LXR/RXR, acute phase response, IL12, nitric oxide, and reactive oxygen species in macrophages. Our data support the current efforts toward augmenting protease inhibitors in patients with CF. Perturbations in lipid and vitamin metabolism frequently observed in CF patients may be partly due to abnormalities in their transport mechanism.

scholarly journals A straightforward gel-free proteomics pipeline assisted by liquid isoelectric focusing (OFFGEL) and mass spectrometry analysis to study bovine meat proteome

2020 ◽  
pp. 108201322092914
Author(s):  
Enrique Sentandreu ◽  
Claudia Fuente-García ◽  
José L Navarro ◽  
Miguel A Sentandreu
Keyword(s):  
Mass Spectrometry ◽  
Isoelectric Focusing ◽  
Quantitative Data ◽  
Ion Trap ◽  
Three Dimensional ◽  
Mass Spectrometry Analysis ◽  
Liquid Chromatography Mass Spectrometry ◽  
Protein Fractionation ◽  
Spectrometry Analysis ◽  
Proteomic Approach

Bovine sarcoplasmic sub-proteome was studied through a straightforward gel-free pipeline supported by liquid isoelectric focusing (OFFGEL) protein fractionation coupled to liquid chromatography-mass spectrometry (LC-MS) analysis. Full-MS and data-dependent MS/MS analyses were simultaneously performed by a conventional three-dimensional ion-trap addressing targeted quantitative and untargeted qualitative research, respectively. There were unambiguously identified 47 proteins distributed along 12 OFFGEL fractions assayed. Regarding intermediate- and high-abundant peptides, bulky quantitative data processing performed by MZmine 2 freeware yielded a satisfactory linearity and coefficient of variation with r2 in the 0.95–0.99 range and about 25%, respectively. Up to 41 peptides from 20 identified proteins were relatively quantified throughout OFFGEL fractions. This reliable, flexible and affordable gel-free proteomic approach could be readily implemented by industry to improve quality assessment of protein-based food products.

scholarly journals The Oligopeptide Transport System Is Essential for the Development of Natural Competence in Streptococcus thermophilus Strain LMD-9

2009 ◽  
Vol 191 (14) ◽  
pp. 4647-4655 ◽  
Author(s):  
Rozenn Gardan ◽  
Colette Besset ◽  
Alain Guillot ◽  
Christophe Gitton ◽  
Véronique Monnet
Keyword(s):  
Quorum Sensing ◽  
Streptococcus Thermophilus ◽  
Defined Medium ◽  
Mass Spectrometry Analysis ◽  
Transport Systems ◽  
Label Free ◽  
Spectrometry Analysis ◽  
Proteomic Approach ◽  
Liquid Chromatography Tandem Mass ◽  
Oligopeptide Transport

ABSTRACT In gram-positive bacteria, oligopeptide transport systems, called Opp or Ami, play a role in nutrition but are also involved in the internalization of signaling peptides that take part in the functioning of quorum-sensing pathways. Our objective was to reveal functions that are controlled by Ami via quorum-sensing mechanisms in Streptococcus thermophilus, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria. Using a label-free proteomic approach combining one-dimensional electrophoresis with liquid chromatography-tandem mass spectrometry analysis, we compared the proteome of the S. thermophilus LMD-9 to that of a mutant deleted for the subunits C, D, and E of the ami operon. Both strains were grown in a chemically defined medium (CDM) without peptides. We focused our attention on proteins that were no more detected in the ami deletion mutant. In addition to the three subunits of the Ami transporter, 17 proteins fulfilled this criterion and, among them, 7 were similar to proteins that have been identified as essential for transformation in S. pneumoniae. These results led us to find a condition of growth, the early exponential state in CDM, that allows natural transformation in S. thermophilus LMD-9 to turn on spontaneously. Cells were not competent in M17 rich medium. Furthermore, we demonstrated that the Ami transporter controls the triggering of the competence state through the control of the transcription of comX, itself controlling the transcription of late competence genes. We also showed that one of the two oligopeptide-binding proteins of strain LMD-9 plays the predominant role in the control of competence.

scholarly journals Proteomic Analysis of Liver from Human Lipoprotein(a) Transgenic Mice Shows an Oxidative Stress and Lipid Export Response

2018 ◽  
Vol 2018 ◽  
pp. 1-11
Author(s):  
Euan J. Rodger ◽  
Carolyn M. Porteous ◽  
Gregory T. Jones ◽  
Michael Legge ◽  
Torsten Kleffmann ◽  
...  
Keyword(s):  
Human Liver ◽  
Density Lipoprotein ◽  
Bioinformatic Analysis ◽  
Liver Cells ◽  
Mass Spectrometry Analysis ◽  
Two Dimensional Gel Electrophoresis ◽  
Lipoprotein A ◽  
Spectrometry Analysis ◽  
Human Liver Cells ◽  
Proteomic Approach

Background. Mouse models of hypercholesterolaemia have been used to identify arterial proteins involved in atherosclerosis. As the liver is extremely sensitive to dyslipidemia, one might expect major changes in the abundance of liver proteins in these models even before atherosclerosis develops. Methods. Lipid levels were measured and a proteomic approach was used to quantify proteins in the livers of mice with an elevated low-density lipoprotein (LDL) and the presence of lipoprotein(a) [Lp(a)] but no atherosclerosis. Results. The livers of Lp(a) mice showed an increased triglyceride but reduced phospholipid and oxidised lipid content. Two-dimensional gel electrophoresis and mass spectrometry analysis identified 24 liver proteins with significantly increased abundance in Lp(a) mice (P<0.05). A bioinformatic analysis of the 24 proteins showed the major effect was that of an enhanced antioxidant and lipid efflux response with significant increases in antioxidant (Park7, Gpx1, Prdx6, and Sod1) and lipid metabolism proteins (Fabp4, Acaa2, apoA4, and ApoA1). Interestingly, human liver cells treated with Lp(a) showed significant increases in Gpx1 and Prdx6 but not Sod1 or Park7. Conclusions. The presence of human LDL and Lp(a) in mice promotes an enhanced flux of lipids into the liver which elicits an antioxidant and lipid export response before the onset of atherosclerosis. The antioxidant response can be reproduced in human liver cells treated with Lp(a).

scholarly journals Genomic Approach for Analysis of Surface Proteins in Chlamydia pneumoniae

2002 ◽  
Vol 70 (1) ◽  
pp. 368-379 ◽  
Author(s):  
Silvia Montigiani ◽  
Fabiana Falugi ◽  
Maria Scarselli ◽  
Oretta Finco ◽  
Roberto Petracca ◽  
...  
Keyword(s):  
Chlamydia Pneumoniae ◽  
Genetic Manipulation ◽  
Respiratory Infections ◽  
Surface Protein ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Western Blots ◽  
Spectrometry Analysis ◽  
Proteomic Approach ◽  
Definition Of

ABSTRACT Chlamydia pneumoniae, a human pathogen causing respiratory infections and probably contributing to the development of atherosclerosis and heart disease, is an obligate intracellular parasite which for replication needs to productively interact with and enter human cells. Because of the intrinsic difficulty in working with C. pneumoniae and in the absence of reliable tools for its genetic manipulation, the molecular definition of the chlamydial cell surface is still limited, thus leaving the mechanisms of chlamydial entry largely unknown. In an effort to define the surface protein organization of C. pneumoniae, we have adopted a combined genomic-proteomic approach based on (i) in silico prediction from the available genome sequences of peripherally located proteins, (ii) heterologous expression and purification of selected proteins, (iii) production of mouse immune sera against the recombinant proteins to be used in Western blotting and fluorescence-activated cell sorter (FACS) analyses for the identification of surface antigens, and (iv) mass spectrometry analysis of two-dimensional electrophoresis (2DE) maps of chlamydial protein extracts to confirm the presence of the FACS-positive antigens in the chlamydial cell. Of the 53 FACS-positive sera, 41 recognized a protein species with the expected size on Western blots, and 28 of the 53 antigens shown to be surface-exposed by FACS were identified on 2DE maps of elementary-body extracts. This work represents the first systematic attempt to define surface protein organization in C. pneumoniae.

scholarly journals N-glycosylation on Oryza Sativa Root Germin-like Protein 1 is conserved but not required for stability or activity

2021 ◽  
Author(s):  
Tehseen Rubbab ◽  
Cassandra L. Pegg ◽  
Toan K. Phung ◽  
Amanda S. Nouwens ◽  
K. Y. Benjamin Yeo ◽  
...  
Keyword(s):  
Oryza Sativa ◽  
Protein Stability ◽  
Specific Activity ◽  
Bioinformatic Analysis ◽  
Mass Spectrometry Analysis ◽  
Sod Activity ◽  
Spectrometry Analysis ◽  
E Coli ◽  
Glycosylation Sites ◽  
Thermal Profiling

AbstractGermin and germin-like proteins (GLPs) are a broad family of extracellular glycoproteins ubiquitously distributed in plants. Overexpression of Oryza sativa root germin like protein 1 (OsRGLP1) enhances superoxide dismutase (SOD) activity in transgenic plants. Here, we report bioinformatic analysis and heterologous expression of OsRGLP1 to study the role of glycosylation on OsRGLP1 protein stability and activity. Sequence analysis of OsRGLP1 homologs identified diverse N-glycosylation sequons, one of which was highly conserved. We therefore expressed OsRGLP1 in glycosylation-competent Saccharomyces cerevisiae as a Maltose Binding Protein (MBP) fusion. Mass spectrometry analysis of purified OsRGLP1 showed it was expressed by S. cerevisiae in both N-glycosylated and unmodified forms. Glycoprotein thermal profiling showed little difference in the thermal stability of the glycosylated and unmodified protein forms. Circular Dichroism spectroscopy of MBP-OsRGLP1 and a N-Q glycosylation-deficient variant showed that both glycosylated and unmodified MBP-OsRGLP1 had similar secondary structure, and both forms had equivalent SOD activity. Together, we concluded that glycosylation was not critical for OsRGLP1 protein stability or activity, and it could therefore likely be produced in Escherichia coli without glycosylation. Indeed, we found that OsRGLP1 could be efficiently expressed and purified from K12 shuffle E. coli with a specific activity of 1251±70 Units/mg. In conclusion, we find that some highly conserved N-glycosylation sites are not necessarily required for protein stability or activity, and describe a suitable method for production of OsRGLP1 which paves the way for further characterization and use of this protein.

scholarly journals Three Microbial Musketeers of the Seas: Shewanella baltica, Aliivibrio fischeri and Vibrio harveyi, and Their Adaptation to Different Salinity Probed by a Proteomic Approach

2022 ◽  
Vol 23 (2) ◽  
pp. 619
Author(s):  
Anna Kloska ◽  
Grzegorz M. Cech ◽  
Dariusz Nowicki ◽  
Monika Maciąg-Dorszyńska ◽  
Aleksandra E. Bogucka ◽  
...  
Keyword(s):  
Gene Expression Regulation ◽  
Vibrio Harveyi ◽  
Dna Topology ◽  
Mass Spectrometry Analysis ◽  
Marine Microorganisms ◽  
Spectrometry Analysis ◽  
Proteomic Approach ◽  
Proteome Level ◽  
Aliivibrio Fischeri ◽  
Shewanella Baltica

Osmotic changes are common challenges for marine microorganisms. Bacteria have developed numerous ways of dealing with this stress, including reprogramming of global cellular processes. However, specific molecular adaptation mechanisms to osmotic stress have mainly been investigated in terrestrial model bacteria. In this work, we aimed to elucidate the basis of adjustment to prolonged salinity challenges at the proteome level in marine bacteria. The objects of our studies were three representatives of bacteria inhabiting various marine environments, Shewanella baltica, Vibrio harveyi and Aliivibrio fischeri. The proteomic studies were performed with bacteria cultivated in increased and decreased salinity, followed by proteolytic digestion of samples which were then subjected to liquid chromatography with tandem mass spectrometry analysis. We show that bacteria adjust at all levels of their biological processes, from DNA topology through gene expression regulation and proteasome assembly, to transport and cellular metabolism. The finding that many similar adaptation strategies were observed for both low- and high-salinity conditions is particularly striking. The results show that adaptation to salinity challenge involves the accumulation of DNA-binding proteins and increased polyamine uptake. We hypothesize that their function is to coat and protect the nucleoid to counteract adverse changes in DNA topology due to ionic shifts.

scholarly journals Mitochondrial proteomic approach reveals galectin-7 as a novel BCL-2 binding protein in human cells

2011 ◽  
Vol 22 (7) ◽  
pp. 999-1013 ◽  
Author(s):  
Christelle Villeneuve ◽  
Laurent Baricault ◽  
Ludovic Canelle ◽  
Nadia Barboule ◽  
Carine Racca ◽  
...  
Keyword(s):  
Human Cancer ◽  
Genotoxic Stress ◽  
Mass Spectrometry Analysis ◽  
Dependent Manner ◽  
Apoptosis Pathway ◽  
Spectrometry Analysis ◽  
Proteomic Approach ◽  
Mitochondrial Fractions ◽  
Intrinsic Apoptosis Pathway ◽  
Associated Proteins

Although the anti-apoptotic activity of Bcl-2 has been extensively studied, its mode of action remains incompletely understood. Deciphering the network of Bcl-2 interacting factors is necessary to better understand the key function of Bcl-2 in apoptosis initiation. To identify novel Bcl-2 mitochondrial partners, we have combined a Bcl-2 immunocapture with a mass spectrometry analysis using highly pure mitochondrial fractions isolated from human cancer cells. We identified at high confidence 127 potential Bcl-2–interacting proteins. Gene ontology mining reveals enrichment for mitochondrial proteins, endoplasmic reticulum–associated proteins, and cytoskeleton-associated proteins. Importantly, we report the identification of galectin-7 (Gal7), a member of a family of β-galactoside–binding lectins that was already known to exhibit a pro-apoptotic function, as a new mitochondrial Bcl-2 interacting partner. Our data further show that endogenous Bcl-2 coimmunoprecipitates with Gal7 and that recombinant Gal7 directly interacts with recombinant Bcl-2. A fraction of Gal7 is constitutively localized at mitochondria in a Bcl-2–dependent manner and sensitizes the mitochondria to the apoptotic signal. In addition, we show that the Bcl-2/Gal7 interaction is abolished following genotoxic stress. Taken together, our findings suggest that the binding of Gal7 to Bcl-2 may constitute a new target for enhancing the intrinsic apoptosis pathway.

Mass Spectrometry-Based Proteomic Approach in Juglans regia

2021 ◽  
Vol 885 ◽  
pp. 25-31
Author(s):  
Donatella Aiello
Keyword(s):  
Mass Spectrometry ◽  
Juglans Regia ◽  
Maldi Mass Spectrometry ◽  
Mass Spectrometry Analysis ◽  
Intact Protein ◽  
Protein Fractionation ◽  
Spectrometry Analysis ◽  
Protein Mass ◽  
Proteomic Approach ◽  
Mass Information

We proposed a MALDI mass spectrometry based approach to characterize walnut allergens. The strategy is based on the extraction of hydro-soluble tissue proteins followed by protein fractionation and mass-spectrometry analysis. Linear MALDI was adopted to evaluate the intact protein mass information and the presence of glycoproteins.

scholarly journals A Jacalin-like lectin-domain-containing protein of Sclerospora graminicola act as an apoplastic virulence effector in plant–oomycete interactions

2021 ◽  
Author(s):  
Michie Kobayashi ◽  
Hiroe Utsushi ◽  
Koki Fujisaki ◽  
Takumi Takeda ◽  
Tetsuro Yamashita ◽  
...  
Keyword(s):  
Plant Immunity ◽  
Foxtail Millet ◽  
Extracellular Fluid ◽  
Defense Responses ◽  
Mass Spectrometry Analysis ◽  
Phytophthora Palmivora ◽  
Sclerospora Graminicola ◽  
Lectin Domain ◽  
Spectrometry Analysis ◽  
Secretion Signal

SUMMARYThe plant extracellular space, including the apoplast and plasma membrane, is the initial site of plant– pathogen interactions. Pathogens deliver numerous secreted proteins, called effectors, into this region to suppress plant immunity and establish infection. Downy mildew caused by the oomycete pathogen Sclerospora graminicola (Sg) is an economically important disease of Poaceae crops including foxtail millet (Setaria italica). We previously reported the genome sequence of Sg and showed that the Jacalin-related lectin (JRL) gene family has significantly expanded in this lineage. However, the biological functions of JRL proteins remained unknown. Here, we show that JRL from S. graminicola (SgJRL) functions as an apoplastic virulence effector. We identified eight SgJRLs via protein mass spectrometry analysis of extracellular fluid from S. graminicola-inoculated foxtail millet leaves. SgJRLs consist of a Jacalin-like lectin domain and an N-terminal putative secretion signal, and SgJRL expression is induced by Sg infection. Heterologous expression of three SgJRLs with N-terminal secretion signal peptides in Nicotiana benthamiana enhanced the virulence of the pathogen Phytophthora palmivora inoculated onto the same leaves. Of the three SgJRLs, SG06536 fused with GFP localized to the apoplastic space in N. benthamiana leaves. INF1-mediated induction of defense-related genes was suppressed by co-expression of SG06536-GFP. These findings suggest that JRLs are novel apoplastic effectors that contribute to pathogenicity by suppressing plant defense responses.

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